Balali Mood2015

Mahdi Balali-Mood
Mohammad Abdollahi Editors
Basic and
Clinical
Toxicology
of Mustard
Compounds
Basic and Clinical Toxicology
of Mustard Compounds
Mahdi Balali-Mood Mohammad Abdollahi
Editors
Basic and Clinical

Toxicology of Mustard
Compounds
Editors
Mahdi Balali-Mood Mohammad Abdollahi
Medical Toxicology Research Centre Department of Toxicology & Pharmacology
Mashhad University of Medical Sciences Tehran University of Medical Sciences
Mashhad Tehran
Iran Iran
ISBN 978-3-319-23873-9 ISBN 978-3-319-23874-6 (eBook)

DOI 10.1007/978-3-319-23874-6
Springer Cham Heidelberg New York Dordrecht London

Springer International Publishing Switzerland 2015
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Preface
Later on the use of chemical warfare agents (CWA) during World War I, all the
nations knew the extent of the tragedy and became against CWA. In spite of the
Geneva Protocol in 1925 and further chemical weapon conventions and conferences
in the last century, CWA including sulfur mustard (SM) and the nerve agents were
unfortunately used in the Iraq-Iran conflict (19831988) by the Iraqi time regime
and in the terrorist attack in Matsomoto and Tokyo metro of Japan (19941995).
SM was first synthesized by a Belgian chemist, Cesar Mansute Despretz in
1822; then a German chemist, Victor Meyer, completely described the chemical
structure of SM in 1886. It was first used during World War I in 1917. Nitrogen
mustard (NM) was initially synthesized as a CWA after World War I, but has never
been used as a chemical weapon. It is used as an anticancer medicine. Lack of sci-
entific knowledge on mustard compounds (MC) in medicine and the science of toxi-
cology have made a heavy mess of confusion among some health professionals and
scientists on the differentiation between NM and SM. The problems of NM admin-
istration and its toxic effects in patients who take this chemical as an antineoplastic
agent and also malpractice on the clinical management of patients who were exposed
to SM during the chemical wars or in occupational settings have lead us to form this
book.
There have been some little books with narrow separate subjects on NM and SM,
but a comprehensive book on Basic and Clinical Toxicology of MC as a reference
for pharmacologists, toxicologists, and health professionals who deal with different
facets of these compounds has been missing.
The main objective of this book is to provide scientific information and practical
guide on MC for the scientists and health professionals who are involved in educa-
tional activity, research, and medical care of the patients. The regulatory authorities
in different departments of Labor, environment, industries, military, and health as
well as the international governing bodies such as the UN, WHO, ILO, Red Cross,
and OPCW or the national authorities of CW conventions and military toxicologists
shall also use this book.
The first editor of this book has been in charge of the Medical Toxicology Centre
(the referral center for CWA victims during the Iraq-Iran war) of Mashhad University
v
vi Preface
of Medical Sciences since 1982. All the exposed chemical warfare victims, mainly
SM poisoning cases who were transferred to Mashhad have been under his medical
attention. He has likewise been involved in instruction and research in medical
aspects of the CWA, mainly SM exposure at the national and international stages,
giving plenary lectures at the world conferences of toxicology and published over
60 articles, book chapters, monographs, and books in this area. The second editor of
this book is also an internationally known scientist with lots of studies, publications,
and citations in the field of toxicology and pharmacology. Both editors have had
collaborations with different international organizations including WHO and
OPCW dealing with toxicological issues. The selected authors of the chapters are
highly experienced experts in the fields and have done their great efforts for the best
writing and revising the chapters under the supervision of the editors.
This volume holds 15 chapters from chemistry, history of employment, basic
pharmacology and toxicology to clinical, military, occupational, and environmental
aspects of MC. The national and international concerns on the use of MC as CWA
have also been considered. The concluding chapter written by the editors summa-
rizes the whole book content and provides expert opinion of the editors. We hope
that all students, researchers, regulators, military, security, and health professionals
who are involved in the area of toxicology specially CWA will benefit from this
book.
Understanding and kind supports of our families who encouraged us to work
hard at home on this book project are highly appreciated. We are of course very
grateful to the authors of the chapters for their hard work during writing and making
several revisions. We would also wish to thank the Springer publisher specially Ms
Manika Power (right away moved out of Springer) and Ms Rosie Daniel, who
kindly cooperated with this book project.
We would welcome any comments and feedback from the experts in the field to
help us improve the future editions.
Mahdi Balali-Mood, MD, PhD

Medical Toxicology Research Centre, Faculty of Medicine,
Mashhad University of Medical Sciences, Mashhad, Iran
Mohammad Abdollahi, Pharm D, PhD

Department of Toxicology and Pharmacology,
Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
Editor Biography
Mahdi Balali-Mood was awarded BSc 1st class Hon of chemistry in 1963 and then
MD in 1970 from Tehran University. After his medical military service and training
in internal medicine/clinical toxicology, he was appointed as an assistant professor in
clinical toxicology and head of his newly established Poisoning Treatment Center
(PTC) of Mashhad University in 1974. Mahdi was awarded a scholarship from the
Ministry of Science of Iran and did his PhD in Clinical Pharmacology & Toxicology
at Edinburgh University Medical School in 19781981. He was then working as a
lecturer in this department until winter 1982, when decided to return to Mashhad to
advance his established PTC, as the chemical war gas attack of Iraqi army against
Iranian troops was his main concern.
Mahdi was promoted to associate professor and full professor of Medicine and
Clinical Toxicology, Mashhad University Medical Sciences (MUMS) in 1984 and
1988, respectively.
He has served as a Clinical Toxicology Adviser to the International Programme on
Chemical Safety (IPCS) and the World Health Organization (WHO), 1989 to date.
He has also been a member of the IPCS Programme Advisory Committee since 2000.
Prof. Balali-Mood was a founding member and the first President of Iranian
Society of Toxicology and was also elected twice as the President of Irantox between
vii
viii Editor Biography
1989 and 2001. He has been a member of the Iranian Academy of Medical Sciences
since its establishment in 1990.
Mahdi with collaboration of the other internationally recognized medical toxi-
cologists founded the Asia-Pacific Association of Medical Toxicology (1989) and
served as the first Vice-President and then as the President for 8 years (19942001).
He was elected as a Permanent Member of the Academy of Sciences for Developing
World (TWAS) in 1997. He was a member of Scientific Advisory Board of the
Organization for Prohibition of Chemical Weapons (OPCW) 20042011.
His other achievements include co-founder and Director, Medical Toxicology
Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences,
1988 to date; Editor-in-Chief, Scientific Journal of Birjand University of Medical
Sciences, 2002 to date; and Associate Editor and editorial board member of many
national and international medical journals.
His research areas are Clinical Toxicology of Chemical Warfare Agents,
Organophosphorous Pesticide Poisonings, Heavy Metals, Drug Abuse/Overdosage,
Epidemiology of Poisonings, Occupational and Environmental Toxicology, and
Natural Toxins including snake and spider bites.
Prof. Balali-Mood was awarded several national and international prizes on
Teaching, Research, and Medical Care including the prizes in medical care of chemi-
cal war veterans and research from the Presidents of IR Iran in 2003 and 2011, respec-
tively. He supervised more than100 theses for MSc, PhD, PharmD, MD, and specialities
in clinical medicine, mostly on CWA. Mahdi is the author/editor of 29 books/mono-
graphs/chapters and 147 articles and 289 short papers and abstracts. His recent books
on Basic and Clinical Toxicology of Organophosphorus Compounds and on Biological
Toxins and Bioterrorism were published by Springer in 2014 and 2015, respectively.
Editor Biography ix
Mohammad Abdollahi (MA) acquired a PharmD in 1988 from the University of

Tehran and then finished a PhD in Toxicology and Pharmacology in 1994 from the
Tehran University of Medical Sciences in 2001. MA completed his postdoctoral
training in Mechanistic Toxicology in the School of Pharmacy of the University of
Toronto. In the interim, he contributed in a key meta-analysis study in the School of
Medicine, University of Toronto. MA has obtained the honor of full professorship
of Tehran University of Medical Sciences (TUMS) since the second half of 2002.
He has chaired the Department of Toxicology and Pharmacology at the Faculty of
Pharmacy, TUMS since 2005. So far, MA has contributed in authoring more than
650 papers in prestigious journals and authoring 48 book chapters and editing 11
books. MA has been listed among top scientists of ESI/ISI and OIC Member States.
According to Google Scholar, current H-index, total citations, and i10 index of MA
are 64, 15,500, and 340, respectively. Total citations of MA in the books are more
than 1000. MA is the Editor-in-Chief of two TUMS prestigious journals published
by Springer BMC and Elsevier. MA has cooperated with some key international
organizations such as OPCW (Organization for Prohibition of Chemical Weapons)
as a Scientific Advisory Board in the Netherlands (since 2012), WHO (World
Health Organization) as a Member of Guideline Developing Group for Prevention
of Lead Poisoning in Switzerland (since 2011), COPE (Committee on Publication
Ethics) as a Council Member in the UK (since 2013), World Library of Toxicology
as the Country Correspondent (since 2008), IAS (Islamic-World Academy of
Sciences) as a Fellow (since 2007), International Society of Pharmacoeconomics
and Outcomes Research (ISPOR) Iran Chapter as a Founder/Board of Directors
(since 2013), and Asian Council of Science Editors as the Board of Directors (since
2014). In addition to more than ten prestigious national awards, MA has received
the prominent award of IAS-COMSTECH in 2005 in the field of Pharmacology &
Toxicology. The main research interests of MA are Mechanistic and Environmental
Toxicology, Evidence-Based Medicine, and Pharmacology. MAs contribution to
this field is attested by an extensive array of citations in papers and books. MA has
uncovered the critical mechanistic connections between the toxicity of chemicals
and the etiology of human diseases.
Contents
1 Chemistry of Mustard Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Mahmood Sadeghi and Beeta Balali-Mood
2 History of Use and Epidemiology of Mustard Compounds . . . . . . . 29
Leila Etemad, Mohammad Moshiri, and Mahdi Balali-Mood
3 Basic Pharmacology and Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Sara Mostafalou and Mohammad Abdollahi
4 Clinical Pharmacology and Toxicology of Mustard Compounds . . . 63
Adel Ghorani-Azam and Mahdi Balali-Mood
5 Delayed Complications and Long-Term Effects of SM Poisonings:
Experience of Iran-Iraq War . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Emadodin Darchini-Maragheh, Peter G. Blain, and
Mahdi Balali-Mood
6 Upper Respiratory Complications of Sulfur
Mustard (SM) Poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Ramin Zojaji and Morteza Mazloum Farsi Baf
7 Lower Airway Complications of Sulfur Mustard Exposure . . . . . . . 171
Mostafa Ghanei and Amin Saburi
8 Dermatologic Aspects of Sulfur Mustard Exposure . . . . . . . . . . . . . 213
Masoud Maleki and Pouran Layegh
9 Ocular Injury by Mustard Gas; Early and Late Complications . . . 253
Nasser Shoeibi, Mojtaba Abrishami, and Alireza Eslampoor
10 Immunological and Hematological Complications of
Sulfur Mustard Poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Bamdad Riahi-Zanjani and Mahmoud Mahmoudi
xi
xii Contents
11 Psychiatric Complications of Sulfur Mustard (SM) Poisoning . . . . 291

Mohammad Reza Fayyazi Bordbar, Farhad Faridhosseini,
and Ali Saghebi
12 Genotoxicity, Teratogenicity and
Mutagenicity of Sulfur Mustard Poisoning . . . . . . . . . . . . . . . . . . . . 317
Effat Behravan and Mitra Asgharian Rezaee
13 Verification of SM Exposure in Biological Samples . . . . . . . . . . . . . . 349
Dirk Steinritz and Horst Thiermann
14 Occupational and Environmental Mustard Exposure,
Prevention and Chemical Weapons Convention . . . . . . . . . . . . . . . . 359
Slavica Vucinic, Branka Djurovic, and Biljana Antonijevic
15 Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Mahdi Balali-Mood and Mohammad Abdollahi
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
Contributors
Mohammad Abdollahi, PharmD, PhD Department of Toxicology and

Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
Mojtaba Abrishami, MD Eye Research Center, Farabi Eye Hospital,
Tehran University of Medical Sciences, Tehran, Iran
Biljana Antonijevic Faculty of Pharmacy, University of Belgrade, Belgrade,
Serbia
Morteza Mazloum Farsi Baf Faculty of Medicine, Mashhad Branch,
Islamic Azad University, Mashhad, Iran
Mahdi Balali-Mood Medical Toxicology Research Centre, Faculty of Medicine,
Beeta Balali-Mood MoodBioPharm, London, UK
Effat Behravan Medical Toxicology Research Center, School of Medicine,
Peter G. Blain Medical Toxicology Centre, Newcastle University, Newcastle,
UK
Mohammad Reza Fayyazi Bordbar Department of Psychiatry, Psychiatry and
Behavioral Sciences Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran
Emadodin Darchini-Maragheh Medical Toxicology Research centre, Faculty of
Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Branka Djurovic Institute of Occupational Medicine, Military Medical
Academy, Medical Faculty/University of Defense, Belgrade, Serbia
Alireza Eslampoor, MD, FICO Eye Research Center, Mashhad University
of Medical Sciences, Mashhad, Iran
xiii
xiv Contributors
Leila Etemad Pharmaceutical Research Center, Mashhad University of Medical

Sciences, Mashhad, Iran
Farhad Faridhosseini Department of Psychiatry, Psychiatry and Behavioral
Sciences Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Mostafa Ghanei Chemical Injuries Research Center & Faculty of Medicine,
Baqiyatallah University of Medical Sciences, Tehran, Iran
Adel Ghorani-Azam Faculty of Medicine, Medical Toxicology Research Centre,
Pouran Layegh Dermatology Department, Cutaneous Leishmaniasis Research
Center, Mashhad University of Medical Sciences, Mashhad, Iran
Ghaem Hospital, Mashhad, Iran
Mahmoud Mahmoudi Department of Immunology and Allergy, Immunology
Research Center, School of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran
Masoud Maleki Dermatology Department, Cutaneous Leishmaniasis
Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Emam Reza Hospital, Mashhad, Iran
Mohammad Moshiri Pharmacodynamy and Toxicology Department,
Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
Health Insurance Research Office, Mashhad Branch, Health Insurance
Research Office, Armed Forces Insurance Organization of Islamic
Republic of Iran (Mashhad Branch), Tehran, Iran
Sara Mostafalou, PharmD, PhD School of Pharmacy, Ardabil University
of Medical Sciences, Ardabil, Iran
Mitra Asgharian Rezaee Department of Toxicology and Pharmacology,
Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
Bamdad Riahi-Zanjani Medical Toxicology Research Center,
School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Amin Saburi Chemical Injuries Research Center & Faculty of medicine,
Mahmood Sadeghi Medical Toxicology Research Center, School of Medicine,
Ali Saghebi Department of Psychiatry, Psychiatry and Behavioral Sciences
Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Nasser Shoeibi, MD Eye Research Center, Mashhad University of Medical
Contributors xv
Dirk Steinritz Bundeswehr Institute of Pharmacology and Toxicology, Munich,

Germany
Horst Thiermann Bundeswehr Institute of Pharmacology and Toxicology,
Munich, Germany
Slavica Vucinic National Poison Control Centre, Military Medical Academy/
Medical Faculty, University of Defense, Belgrade, Serbia
Ramin Zojaji Mashhad Branch, Otorhinolaryngology Department,
Islamic Azad University, Mashhad, Iran
Arya Teaching Medical Hospital ENT Department, Islamic Azad Medical
University, Mashhad, Iran
Chapter 1
Chemistry ofMustard Compounds
MahmoodSadeghi andBeetaBalali-Mood
Contents
1.1 Introduction.................................................................................................................... 2
1.1.1 Commonly Used Chemical Warfare Agents....................................................... 3
1.1.2 Nitrogen.............................................................................................................. 3
1.1.3 Sulfur.................................................................................................................. 7
1.2 Mustards......................................................................................................................... 7
1.2.1 Nitrogen Mustard................................................................................................ 7
1.2.2 Sulfur Mustards................................................................................................... 8
1.3 Applications ofMustard Compounds............................................................................. 8
1.3.1 Medicinal Uses................................................................................................... 10
1.4 Synthesis......................................................................................................................... 10
1.4.1 Synthesis ofSulfur Mustard............................................................................... 10
1.4.2 Synthesis ofNitrogen Mustard Compounds....................................................... 13
1.5 Physical Properties......................................................................................................... 15
1.5.1 Spectroscopic andPhysical Properties ofSulfur Mustard.................................. 15
1.6 Chemical Properties........................................................................................................ 17
1.7 Analysis andDetection ofSulfur Mustard..................................................................... 19
1.8 Interactions withBiological Molecules.......................................................................... 19
1.8.1 Interactions withDNA andMode ofCytotoxicity.............................................. 19
1.8.2 Interaction withImidazole.................................................................................. 20
1.8.3 Nitrogen Mustard Interactions andMechanism ofActions................................ 21
1.9 Decontamination ofSulfur Mustard Compounds........................................................... 21
1.10 Antidotes forSulfur Mustard.......................................................................................... 22
1.11 Conclusion...................................................................................................................... 23
Glossary................................................................................................................................... 24
References................................................................................................................................ 24
M. Sadeghi
Medical Toxicology Research Center, School of Medicine, Mashhad University of Medical
e-mail: sadeghi.mahmud@yahoo.com; sadeghim923@mums.ac.ir
B. Balali-Mood (*)
MoodBioPharm, London, UK
e-mail: beetabalali@gmail.com; beeta@moodbiopharm.com
Springer International Publishing Switzerland 2015 1

M. Balali-Mood, M. Abdollahi (eds.), Basic and Clinical
Toxicology of Mustard Compounds, DOI10.1007/978-3-319-23874-6_1
2 M. Sadeghi and B. Balali-Mood
Abstract The two main categories of mustard compounds are sulfur mustards and
nitrogen mustards. Sulfur mustard was the first vesicant chemical weapon used. Its first
widespread use was recorded in the World War One. After a number of sporadic mili-
tary attacks, another widespread use of sulfur mustard occurred in the Iran-Iraq war.
Nitrogen mustard derivatives are used in chemotherapy. HN-1, HN-2, HN-3 are the
most important forms of nitrogen mustards. Nitrogen mustard HN-2 is chlormethine
(Mechlorethamine) and has been used for treatment of multiple cancer diseases such as
Hodgkins disease. Sulfur mustard has the chemical name bis(2-chloroethyl) sulfide
and the IUPAC name 1-chloro-2-(2-chloroethylsulfanyl) ethane. It is also known as
mustard, mustard gas, HD or Yperite. The compound is highly reactive and has carci-
nogenic, cytotoxic and powerful vesicant characteristics. Mustard gas was first synthe-
sized from the reaction of ethylene and sulfur dichloride (Levinstein process) through
an electrophilic addition mechanism. Later, it was prepared by the reaction of thiodi-
glycol with phosphorus trichloride (Meyer reaction) in a substitution reaction. Finally,
reaction of concentrated hydrochloric acid (HCl) and thiodiglycol resulted in the pro-
duction of sulfur mustard. Pure mustard is a viscous, colorless and odorless liquid
which evaporates slowly in the atmosphere. Cytotoxicity of sulfur mustard stems from
the formation of electrophilic species called sulfonium cation upon nucleophilic attack.
This transient cation then readily reacts with macromolecules of DNA, RNA and pro-
teins or with water to form the corresponding hydroxyl compounds. DNA Cross-
linking of guanine by sulfur mustard and its interaction with imidazole are well studied.
Nitrogen mustard (NM) and sulfur mustard (SM) slightly differ in properties. Like
sulfur mustard, nitrogen mustard compounds are also alkylating agents and are reactive
compounds that covalently bind to nucleophilic groups such as amine, carboxyl, sulf-
hydryl and imidazole moieties in DNA, RNA and proteins. Decontamination of SM
can be achieved via hydrolysis in presence of aqueous solutions of sodium hypochlo-
rite and or chloramine-T; in which HD decomposes into thiodiglycol non-poisonous
product. No specific antidote for SM poisoning has been introduced. However, some
formulations have been introduced as effective skin decontaminants.
Keywords Sulfur mustard Nitrogen mustards HN-1 HN-2 HN-3

Mechlorethamine Synthesis Reaction Physical characteristics Chemical prop-
erties Mechanism of cytotoxicity DNA-crosslinking Aziridinium ion
1.1 Introduction
The misuse of toxic substances has been intertwined with chemistry research
throughout history. But the large scale use of chemical agents in conflicts began
with World War I when chlorine, phosgene and mustard gas were used by both sides
of the conflict (Maras 1979). This process continued with synthesis of Soman,
Tabun, Sarin and VX.By 1993 many countries signed the Chemical Weapons
Convention and the production of chemical agents was ceased. However, since then
1 Chemistry ofMustard Compounds 3
a number of terrorism attacks that involved the use of chemical warfare agents have
been reported worldwide (Kort 2010).
Sulfur mustard was the first blister agent used as a chemical weapon (Romano
etal. 2007); a highly reactive compound with carcinogenic, cytotoxic and powerful
vesicant characteristics. Sulfur mustard has been widely used since the World War I
in different attacks. Victims of sulfur mustard have suffered from devastating acute
and chronic health impairments. Mustard gas became known as the King of Battle
Gases for its high number of resulting casualties. There is no specific antidote for
injuries of this chemical agent. From a chemical point of view, it readily reacts with
sulfhydryl and imidazole groups of bio-molecules. The two main categories of mus-
tard compounds are sulfur mustards and nitrogen mustards. Chemistry of sulfur mus-
tard shows high boiling point, low volatility, high penetrability, high chemical stability
and high specific gravity; and is the class of mustard compounds used in conflicts.
The properties of nitrogen mustards are only slightly different from those of sulfur
mustards. However, exposure to nitrogen mustards may be more immediately toxic
than exposure to sulfur mustard (Dacre and Goldman 1996). Derivatives of nitrogen
mustard compounds have been extensively studied and applied in the treatment of
cancers. In the next sections of this chapter, the chemical properties of sulfur and
nitrogen mustard, their structures, synthesis and related interactions are addressed.
1.1.1 Commonly Used Chemical Warfare Agents
Toxic chemical compounds in munitions/devices causing death or harm to human

beings are considered chemical warfare agents (CWA). The chemical warfare agents
are highly toxic agents and organized into several categories, consisting of nerve
agents, blister agents or vesicants, choking agents and blood agents. Vesicants like
sulfur mustards, nitrogen mustards and Lewisite are a class of CWAs. They cause
severe skin irritations and painful burns. The most common CWAs are depicted in
Table 1.1. Each class of chemical agents in Table1.1 shows the chemical com-
pounds that share similar structural features and to some extent similar properties.
For example, all the vesicants are persistent, or organophosphorous-based nerve
agents are toxic cholinesterases inhibitors. One or two replacements in the substitu-
ents bond to P=O of an organophosphorus nerve agent results in formation of new
compounds. Amongst blister and nerve agents, vesicant sulfur mustard is the only
agent with reported carcinogenicity in animal studies (Agents etal. 1999).
1.1.2 Nitrogen
Nitrogen, element 7 with the symbol N, is the first member of group 5A (or group
15) in the periodic table. The electronic configuration of nitrogen is: [He], 2s2, 2p3.
Nitrogen has common valences of 3 and 5. It accounts for 78% of the atmosphere.
4
Table 1.1 The most common chemical warfare agents

Chemical name Common name Class Formula Structure
Izopropylmethylphosphonofluoridate Sarin Nerve agent C4H10FO2P O
P O
H 3C F CH3
H3C
Pinacolylmethylphosphonofluoridate Soman Nerve agent C7H16FO2P O
H3C P
O F
H3C CH3
H3C
CH3
Cyclohexylmethylphosphonofluoridate Cyclosarin Nerve agent C7H14FO2P O
P O
H3C F
Ethyl N,N-dimethylphosphoroamidocya Tabun Nerve agent C5H11N2O2P CH3

nidate O
N
P CH3
H3C O
N
M. Sadeghi and B. Balali-Mood
O-ethyl, S-2-diisopropylaminoethyl VX Nerve agent C7H18NO2PS H3C
methylphosphonothiolate O
CH3
P H3C
O
S CH3
N
CH3
H3C
Bis(2-chloroethyl)sulfide Sulfur mustard Vesicant C4H8Cl2S S
Cl Cl
Bis(2-chloroethylthioethyl)ether O-mustard Vesicant C8H16Cl2OS2 S S
1 Chemistry ofMustard Compounds
Cl O Cl
1,2-Bis(2-chloroethylthio) ethane Sesquimustard Vesicant C6H12Cl2S2 Cl S
S Cl
Tris-(2-chloroethyl)amine Nitrogen mustard Vesicant C6H12Cl3N Cl
N
Cl Cl
2-Chlorovinyldichloroarsine Lewisite Vesicant C2H2AsCl3 Cl
As
Cl Cl
Chlorine Chlorine Choking agent Cl2 Cl Cl
(continued)
5
6
Table 1.1(continued)
Carbonyl dichloride Phosgene Choking agent CCl2O Cl
O
Cl
Cyanogen chloride CK Blood agent CClN Cl N
Hydrogen cyanide Prussic acid Blood agent HCN H N
Nitrogen molecules form an inert gas with the formula N2. Nitrogen is a component
of amino acids and nucleic acids and is commonly found in a variety of foods, fertil-
izers and explosives. Nitrates, nitrites, amines, azides and azos are amongst preva-
lently existing nitrogen containing compounds (Heather 2005).
1.1.3 Sulfur
Sulfur, element 16 with the symbol S, is the second member of the group 6A (or
group 16) and has the electronic configuration: [Ne], 3s2, 3p4. Common valances of
sulfur are 2, +4 and +6. The infamous smell that sulfur is commonly associated
with is that of hydrogen sulfide (H2S). Sulfur, in its elemental form reacts with all
metals except gold and platinum, forming sulfides. Sulfide and sulfate as well as
sulfur in its elemental form are all found in nature. Sulfur compounds include sul-
fates, sulfites, sulfides and organosulfurs such as thiols, sulfonates and sulfonamides.
The latter class comprises of synthetic compounds and posses antibacterial capabili-
ties. In the late 1930s a newly adopted approach to combating microbial infection
using Sulfanilamide, a member of the sulfonamides family of compounds lead to
acute renal failure. This incident known as the sulfanilamide disaster was caused by
the solvent medium of the drug and triggered a series of studies that molded the dis-
cipline of modern toxicology (Klaassen 2007). The main commercial applications of
sulfur are in the synthesis of sulfuric acid and fertilizers. Sulfur is also used in pro-
duction of gunpowder, matches, insecticides and fungicides (Ede 2006).
1.2 Mustards
1.2.1 Nitrogen Mustard
Nitrogen mustard was never used in combats. However, it was initially intended to
be used as a chemical warfare as its mechanism of action and symptoms are similar
to those of sulfur mustard. Nitrogen mustard was soon excluded in chemical weap-
ons production programs. Instead, nitrogen mustard compounds have been used in
chemotherapy (Hanna etal. 1963; Brunton etal. 2007; Saha etal. 2013).
Nitrogen mustards (NMs) are colorless to pale yellow, oily liquids that evaporate
slowly. Most nitrogen mustard compounds are derivatives of the following four key
compounds: HN-1, HN-2, HN-3 and Iso- Pr-N(EtCl)2. Pr and Et refer to propyl and
ethyl groups, respectively. HN-1 was the first of the HN series to be synthesized.
HN-2 was the second compound to be developed. The synthesis of which was fol-
lowed by that of HN-3. HN-2 has a fruity odor at high concentrations, and a soapy
or fishy odor at low concentrations. HN-1 has a faint fishy or musty dour. It evapo-
rates slowly and is mildly persistent (Leikin etal. 2007). Nitrogen mustard HN-3 is
odorless when pure and is the most stable and most toxic of the HN series (Hoenig
Cl
Cl
CH3
CH3
CH3 N
N N CH3
N
Cl Cl Cl Cl Cl Cl Cl
HN1 HN2 HN3 Iso-Pr-N(EtCl)2
Fig. 1.1 Nitrogen mustards
2002; Hoenig 2007). Fig.1.1 shows the structures of nitrogen mustards. Their phys-
ical properties are listed in Table1.5.
1.2.2 Sulfur Mustards
Sulfur Mustard (SM) with the chemical name bis(2-chloroethyl) sulfide or bis(-
chloroethyl) sulfide and molecular formula C4H8Cl2S (Tables1.1 and 1.2) is a poi-
sonous chemical agent belonging to the blister/vesicant class of compounds.
Receiving its most common name from its smell, mustard is also called mustard gas,
1,1-thiobis(2-chloroethane) and 2,2-chlorodiethylsulfide, Lost (derived from
Wilhelm Lommel and Wilhelm Steinkopf who developed a method for mass produc-
tion of mustard gas for German army in 1916); HS (Hun stuff); HD (distilled sulfur
mustard); Schwefel-lost; yellow cross liquid, Senfgas and Yperite which was used in
Ypres in world war I (Balali-Mood etal. 2014). Although referred to as mustard gas,
the compound is in liquid state at room temperature. The IUPAC name for the com-
pound ClCH2CH2SCH2CH2Cl is 1-chloro-2-(2-chloroethylsulfanyl) ethane. It has
been categorized as Group 1carcinogen to humans by the International Agency for
Research on Cancer (IARC) and was listed as a toxic chemical in Schedule I by the
first Annual Report on Carcinogens and Chemical Weapons Convention (1980). It is
a severe irritant and vesicant of skin, eyes and lungs and was formerly used as a war
gas (Balali-Mood etal. 2005; BalaliMood and Hefazi 2006).
Sulfur mustard and other members of its family of compounds are linear mole-
cules and have similar structures. Sulfur Mustard and examples of compounds in the
sulfur mustard family are shown by some examples in Table1.2.
1.3 Applications ofMustard Compounds
As chemical weapons, chlorine and phosgene spread in the wind and therefore
dilute in the air. Sulfur Mustard, on the other hand, is persistent in the environment
and as such an effective battle gas. The other members of mustard compounds fam-
ily- nitrogen mustards- have been used as antineoplastic drugs. Mechanism of
action and symptoms of nitrogen mustards (also known as N-mustards) closely
Table 1.2 Examples of sulfur mustard compounds
Chemical name Common name Formula Structure
Bis(2-chloroethyl)sulfide Sulfur mustard C4H8Cl2S Cl Cl
1 Chemistry ofMustard Compounds
S
Bis(2-chloroethylthioethyl)ether O-mustard C8H16Cl2OS2 Cl O Cl
S S
1,2-Bis(2-chloroethylthio) ethane Sesquimustard C6H12Cl2S2 Cl S
S Cl
2-Chloroethyl chloromethyl sulfide C3H6Cl2S Cl
S Cl
Bis(2-chloroethylthio)-methane C5H10Cl2S2 Cl Cl
S S
Bis(2-chloroethylthiomethyl)-ether C6H12Cl2OS2 Cl Cl
S O S
9
resemble those of sulfur mustard. The research studies carried out in 1946 showed
that nitrogen mustard can reduce tumor growth in mice. However, several side
effects have been reported in patients that were treated with nitrogen mustard. These
include low blood cell counts leading to potential infections, anemia; nausea and
vomiting as well as hair loss (Daniel 2011).
1.3.1 Medicinal Uses
Chemotherapy dates back to the 1940s and the use of nitrogen mustards and antifo-
late (folic acid antagonist) drugs (Chabner and Roberts 2005; DeVita and Chu 2008).
The aim of Milton Winternitz at Yale and two other pharmacologists, Alfred Gilman
and Louis Goodman was to investigate potential therapeutic effects of chemical war-
fare agents. Mustard was noted to destroy lymphocytes and lymphoid tissues of labo-
ratory animals. Also, they recognized in their research that nitrogen mustards analogs
of mustard gas -could have potential chemotherapeutic effects in the treatment of
lymphosarcoma. Based on their experiments sulfur mustard was less suitable for
cancer treatment (Liebow and Waters 1959). In the presence of nitrogen mustard,
however, the tumors shrunk and in cases, by the end of the treatment, they had disap-
peared. Reports on the clinical use of chlormethine (mechlorethamine) in lympho-
mas provided stimulus for the preparation of other drugs and resulted in development
of the field of anticancer chemotherapy (Lu and Mahato 2009).
Derivatives of nitrogen mustard are used for treatment of multiple cancer diseases
such as Hodgkins disease (cancer of the lymph nodes) and remain potent chemo-
therapeutic agents for malignant diseases (Ullmann and Bohnet 2003). Application
of nitrogen mustards as effective anti-cancer agents is based on their ability of induc-
ing apoptosis. Nitrogen mustard HN2 (Mechlorethamine) was found useful for che-
motherapy (Gilman 1963). Analogs of HN2 have various therapeutic applications as
anti-cancer drugs. Cyclophosphamide, Chlorambucil, Melphalan and Ifosfamide are
also amongst nitrogen mustards with anticancer activities (Pratt 1994).
Some other applications of nitrogen mustards like causing convulsions in animal
studies or effects on influenza virus have been reported in earlier publications (Graef
etal. 1948; Rose and Gellhorn 1947).
1.4 Synthesis
1.4.1 Synthesis ofSulfur Mustard
Chemists Csar-Mansuete Despretz and Alfred Riche both reported the synthesis
of sulfur mustard from the reaction of ethylene and sulfur dichloride in 1822 and
1854, respectively (Tuorinsky 2008). Later, the reaction conditions for the synthe-
sis of sulfur mustard were optimized by Levinstein; a dye manufacturer in England
and was called the Levinstein process. Empirically, ethylene may also react with
disulfur dichloride (sulfur monochloride) to produce sulfur mustard (Malhotra
etal. 1999).
SCl2 + 2CH 2 = CH 2 S ( CH 2 CH 2 Cl )2

In the Levinstein process sulfur mustard is attained by the reaction of ethylene mol-
ecule with sulfur dichloride. This electrophilic addition to ethane happens in two
steps and sulfur mustard forms by addition of a second molecule of alkene to the
intermediate 2-chloroethylsulfenyl chloride:
SCl2 + CH 2 = CH 2 ClCH 2CH 2SCl

ClCH 2CH 2SCl + CH 2 = CH 2 S ( CH 2CH 2Cl )2

SCl2 undergoes anti addition to the olefinic bond i.e. two substituents are added to
opposite sides of the double bond. In 1860, Frederick Guthrie used the above
method to synthesize the compound and reported minor irritation (vesicant/blister-
inducing effects) while Despeterez did not. Guthrie also reported the mustard like
odor of the product (Freemantle 2014).
Viktor Meyer first prepared pure sulfur mustard in 1886 by the reaction of thiodi-
glycol with phosphorus trichloride in a two stage synthetic process (Pechura and
Rall 1993; Tuorinsky 2008). Germany used the high yielding method of Meyer to
manufacture mustard during World War I:
3S ( CH 2CH 2OH )2 + 2PCl3 3S ( CH 2CH 2Cl )2 + 2H 3PO3

Here, the diol-compound is chlorinated with PCl3. Thiodiglycol was itself prepared
by the reaction of 2-chloroethanol (ethylene chlorohydrin) with aqueous solution of
potassium sulfide:
2ClCH 2 CH 2 OH + K 2S S ( CH 2 CH 2 OH )2 + 2KCl

Meyer prepared 2-chloroethanol needed for the above process from the reaction of
ethylene (which was synthesized by Meyer from ethanol) and carbon dioxide (CO2)
with calcium hypochlorite Ca(ClO)2 known as chlorine powder or bleach powder.
Finally in 1913, Hans Thacher Clarke replaced phosphorus trichloride (PCl3)
with concentrated hydrochloric acid (HCl) in the reaction used by Victor Meyer
(Puskar 2011). Steinkoff and Ludin also repeated this preparation pathway
using concentrated hydrochloric acid as the chlorinating agent (Lundin and
Institute 1991).
S ( CH 2CH 2OH )2 + 2HCl S ( CH 2CH 2Cl )2 + 2H 2O

The treatment of bis(2-hydroxyethyl) sulfide with hydrogen chloride (above) was

used in preparation of sulfur mustard for warfare applications.
In 1945 the photochemical preparation of SM was reported through the addition
of H2S to vinyl chloride:
2CH 2 = CHCl + H 2S S ( CH 2CH 2Cl )2

The mechanism of the photoaddition of hydrogen sulfide to olefinic double bond is a
chain reaction involving free radical intermediates. The reaction occurs in the pres-
ence of peroxides (organic compounds of the type ROOR) which are initiators; they
act as a source of radicals necessary to get the chain reaction started. The oxygen
oxygen bond of the peroxide is relatively weak and breaks homolytically, giving two
alkoxy radicals. Heat or 2800 UV irradiation easily breaks the O-O bond of perox-
ides. Alkoxy radical abstracts a hydrogen atom from hydrogen sulfide producing HS
radical. Once HS radical becomes available, the propagation step of the chain reac-
tion begins. This radical is added to the alkene by a pi-bond cleavage forming a new
free radical that easily separates a hydrogen atom from H2S molecule. Initiation and
propagation steps of the radical addition mechanism are depicted below:

Peroxides Alkoxyradical ( Rad )
Chain initiation
Rad + H 2S Rad : H + H S

H S + CH 2 = CHCl HSCH 2 CHCl

Chain propagation

HSCH 2 C HCl + H 2S HSCH 2 CH 2 Cl + H S
In the propagation phase, during the addition of mercaptans to double bonds, the sulf-
hydryl group bonds to the carbon with greater number of hydrogen atoms to give the
more stable radical. In a similar way, the second hydrogen bonded to sulfur atom can
be replaced and finally the sulfur mustard product is formed (Vaughan and Rust 1942).
The purification of the crude compound can be achieved through three different
methods namely vacuum distillation, steam distillation, and solvent extraction
(Gates and Moore 1946).
1.4.1.1 Bis(2-Chloroethyl) Polysulfides
Levinstein mustard gas composes a considerable amount of bis(2-chloroethyl) poly-

sulfides which have high sulfur content and have the general formula Sx(CH2CH2Cl)2.
These polysulfides vary in composition and consist of bis(2-chloroethyl) disulfide,
trisulfide, and pentasulfide. The composition of the polysulfide depends on the con-
dition of the reaction, particularly on the temperature and the rate of addition of
ClCH2CH2SSSCH2CH2Cl ClCH2CH2SSSCH2CH2Cl
Fig. 1.2 Structures of bis(2-chloroethyl) tri and penta sulfides
ethylene. The more elevated temperatures produce greater yield of SM and higher
sulfur content in the polysulfide. The trisulfide compound has three sulfur atoms
which are held in a linear skeleton and the pentasulfide has the similar structure with
two dative bonds to the central S atom (Fig.1.2). Disulfide preparations are of spe-
cial value in the synthesis of mustard gas; and pentasulfide compound is observed
in the hydrolysis of sulfur mustard. The trisulfide molecule easily takes up two
additional sulfur atoms to yield the pentasulfide compound (Fuson etal. 1946).
1.4.2 Synthesis ofNitrogen Mustard Compounds
Nitrogen mustards were synthesized and investigated during the 1930s. The most
important nitrogen mustards were ethyl-bis(-chloroethyl) amine (HN1), methyl-
bis(-chloroethyl) amine (HN2), tris(-chloroethyl) amine (HN3) and isopropyl-
bis(-chloroethyl) amine (Tuorinsky 2008).
The most practical method for preparation of nitrogen mustards is from the cor-
responding hydroxy compounds. The ethanolamine is chlorinated with thionyl
chloride (SOCl2), resulting in the synthesis of the above mentioned nitrogen mus-
tards (Cope etal. 1946). For instance HN3 can be prepared as follows:
N ( CH 2 CH 2 OH )3 + 3SOCl2 N ( CH 2 CH 2 Cl )3 + 3SO 2 + 3HCl

Alternatively, phosphorous trichloride can be used instead of thionyl chloride.
Phosphorous trichloride, phosgene and hydrochloric acid are other alternative
reagents that can replace thionyl chloride.
Ethylene oxide reacts with primary amines forming a mixture of mono and di-
ethanolamines. Optimum conditions, as described in the equation below, can direct
this reaction to yield the diethanolamine product. Preparation of N-methyl-2,
2-dichlorodiethylamine (nitrogen mustard HN2) can be done by condensation of
gaseous ethylene oxide with aqueous methylamine (25%w/v), and treating the
resultant N-methyldiethanolamine with thionyl chloride (Abrams etal. 1949). The
product is then purified and separated from water, methylamine and mono- ethanol-
amine by distillation.
80 C CH3
1500 rpm CH3
O 1h N SOCl2 N
H3C NH2 HO OH
+ Cl Cl
Another synthetic route is to prepare alkanolamines without employing ethylene

oxide. Alkanolamine can be converted to nitrogen mustard in a chlorination step. A
developed method for preparing HN1 initiates with formaldehyde and hydrogen
cyanide (HCN) and follows the below steps (Et stands for ethyl group):
1. HCN + HCHO CH 2 OHCN

2. CH 2 OHCN + CH 2 ( OEt )2 EtO CH 2 OCH 2 CN + EtOH
3. 2 EtO CH 2 OCH 2 CN + 4 H 2 ( EtO CH 2 OCH 2 CH 2 )2 NH + NH 3
4 . 2 ( EtO CH 2 OCH 2 CH 2 )2 NH + Et 2SO 4 + Na 2 CO3
( EtO CH 2 OCH 2 CH 2 )2 NEt + Na 2SO 4 + CO 2 + H 2 O
5. ( EtO CH 2 OCH 2 CH 2 )2 NEt + HCl + EtOH
( HOCH 2 CH 2 )2 NEt. HCl + 2CH 2 ( OEt )2
The final diethanolamine derivative is the precursor for preparation of nitrogen mus-
tard HN1. In the above reactions, formaldehyde cyanohydrin is formed in step one.
This is then followed by hydrogenation (step 3) and alkylation (steps 2 & 4). Methyl-
bis(-hydroxyethyl) amine as the precursor of HN2 can be prepared by hydrogena-
tion of diethanolamine in the presence of formaldehyde (Cope etal. 1946).
Synthetic methods for preparation of nitrogen mustard analogs have also been
reported. Figure1.3 shows the structures of these synthetic analogs. The synthesis
as depicted in Fig.1.4, involves alkylation via nucleophilic substitution followed by
a chlorination step (Bons etal. 2013). The aim of continuous research on nitrogen
mustard compounds is in part to attain more potent & less cytotoxic analogues of
nitrogen mustard compounds. From a therapeutic viewpoint the presence of chloro-
ethyl chains can lead to DNA alkylation.
Bendamustine with the trade name Treanda (Fig.1.5) is a nitrogen mustard ana-
log used for the treatment of patients with chronic lymphocytic leukemia.
H3C CH3 H3C CH3 H3C CH3 H3C CH3
N N N N
N N
N N N
N N N
N N N
H H2N N N N N
H H2N N
H
HO
Fig. 1.3 Examples of nitrogen mustard analogs

Cl Cl
HO OH
Cl N
N
N nucleophilic N
N substitution N N
N chlorination
N R N N
R N R N N H
H H
Fig. 1.4 Preparations of analogs of nitrogen mustard
Fig. 1.5Chemical Cl
structure of Bendamustine;
HO
a nitrogen mustard analog
used for the treatment of O
chronic lymphocytic N N
leukemia Cl
N
CH3
Containing a mechlorethamine group and a benzimidazole heterocyclic ring with

a butyric acid substituent, this alkylating agent was first synthesized in Germany
(1963) and later received approval from FDA (1963) for the treatment of
non-Hodgkins lymphoma which induces more DNA double-strand breaks than
other alkylating drugs (Teichert etal. 2005).
1.5 Physical Properties
1.5.1 Spectroscopic andPhysical Properties ofSulfur Mustard
Pure mustard is a viscous, colorless and odorless liquid. In contrast, the warfare
agent is intentionally used in its impure form so that it would be effectively acces-
sible at lower temperatures. In its impure form, sulfur mustard is usually yellow to
brown in color and has a sweet odor. Mustard with impurities has a lower melting
point and is highly hydrophobic (has a high lipid permeability). Having a density of
1.27g/cm3 mustard gas is heavier than the air and settles on the ground as an oily
liquid.
As the temperature increases, the vapor pressure increases and the density (spe-
cific gravity) decreases. The viscosity of the compound is 0.459 poise and increases
at lower temperatures.
SM is soluble in oils and other common organic solvents such as ethanol, carbon
tetrachloride, acetone and benzene and it readily dissolves in alcohols. Solubility in
water is in marked contrast to organic solvents. Around 20C, 0.06% of mustard

dissolves in water. The physical characteristics of sulfur mustard are shown in
Table1.3 (Kehe and Szinicz 2005).
SM has low volatility which makes it a persistent substance. Volatility is directly
related to vapor pressure and as the temperature elevates the volatility increases. For
mustard, volatility is 610mg/m3 at 20C; but it is 831mg/m3 at 25C.
Freezing point depressants are used in developing munitions. In laboratory test-
ing chlorobenzene, nitrobenzene, benzene and tetrachloroethane have all brought
down the freezing point of SM (naturally 14C) when added in 2530% propor-
tions. In industrial mustard, however those proportions of solvents will diminish the
efficacy of mustard as a chemical warfare through dilution. Therefore, the com-
pound is mixed with Lewisite in order to increase its volatility. The persistency is
raised through increasing the viscosity by the use of polymer additive. Such form of
mustard with high viscosity by means of the addition of polymers was synthesized
during the Second World War and was the first known example of a thickened chem-
ical warfare agent (Malhotra etal. 1999).
Spectral data related to sulfur mustard compound (Malhotra etal. 1999) have
been summarized in Table1.4.
The purity of a sulfur mustard sample obtained from the US Army was deter-
mined 97.5% by NMR spectroscopy in which 500mL aliquots of sulfur mustard
solution (2mM HD in D2O containing 0.17M NaCl) were transferred into 600MHz
NMR spectrometer tubes. NMR spectral data for HD indicated that the influence of
solvent on chemical shift is relatively small. Furthermore, other deuterated organic
solvents (like CDCl3, CD2Cl2,C2D5OD and deuterated hexane) besides D2O for sul-
fur mustard spectroscopy, showed that SM is much less stable in water than the
other organic solvents referring to sulfur mustard instantaneous hydrolysis in water
(Logan and Sartori 2002).
Table 1.3 Physical properties of sulfur mustard

Appearance Oily liquid
Color Yellow
Odor Garlic mustard onion
No odor in distilled compound
Molecular weight (g/mol) 159.08
Density (g/cm3, 2025C) 1.27
Solubility in water (mg/L, 2025C) 0.5mg/L
Freezing point (C) 14.4
Boiling point (C at 760mmHg) 217
Vapor pressure (mm Hg, 25C) 0.11
Volatility (mg/m3, 25C) 831
Viscosity (poise at 20C) 0.459
Stability & persistence Hydrolysis in water to thiodiglycol and hydrochloric
acid
Table 1.4 IR, NMR spectroscopy & mass spectrometry data of sulfur mustard compound
IR (cm1) 2960 C-H stretching
1440 S-C-H
700 C-C1
H-NMR (ppm) 3.6 C2 proton
Solution in CDCl3 2.9 C1 proton
C-NMR (ppm) 43 C2
Solution in CDCl3 34 C1
Mass spectrometry m/z Relative densities
63 262.1
109 1000.0
111 341.5
158 230.4
160 157.4
162 33.40
There is a direct correlation between the boiling points and molecular weights.
As shown in Table1.5, as the molecular weight of nitrogen mustards increase, so
does the boiling point (Spencer etal. 2010).
HN1 is more volatile and less persistent than sulfur mustard and has only a fifth
of sulfur mustards potency as a vesicant. HN2 has not been used as a war gas
mainly due to its instability. Nevertheless, HN3 is less volatile and more persistent
than HD and has equal vesicant effects. For isopropyl-bis(-chloroethyl) amine the
toxicological potencies are inferior to those of HN1 and HN3. HN3 is considered
the potential substitute agent for HD (Cope etal. 1946).
1.6 Chemical Properties
Key features in the chemical properties of the sulfur mustard group of compounds
include lone electron pairs on sulfur atom and their side chains. Sulfur Mustard can
undergo an intramolecular cyclization. As shown in Fig.1.6 the unpaired electrons
on sulfur initiate a nucleophilic attack in which chlorine atom leaves the molecule;
resulting in formation of electrophilic species called sulfonium cation. The transient
cation then reacts with macromolecules of DNA, RNA and proteins or with water to
form the corresponding hydroxyl compounds. In oxidation the central atom (S) is
subjected to the interactions that lead to the formation of sulfoxide, sulfone and
sulfonium compounds. Chemistry of SM, its nucleophilic sulfur atom and the cyclic
sulfonium cation formation is discussed in further details in Sect.1.8.1.
In the presence of heat (>100C), sulfur mustard produces toxic fumes of sulfur
oxide, chlorine and hydrochloric acid (Lewis 2008).
Nitrogen mustard and sulfur mustard slightly differ in properties. Nitrogen
mustard compounds are also alkylating agents and are reactive compounds that
18
Table 1.5 Physical properties of nitrogen mustards

Vapor
Molecular pressure
weight (g/ Boiling Freezing Density (mmHg at Volatility
Common names Formula mol) point (C) point (C) (g/cm3) 25C) (mg/m3)
HN-2a Mustargen; mechlorethamine; C5H11NCl2 156.06 75 60 1.12 0.427 3580 (25C)
Chlormethine; mustin;
N, N-bis(2-chloroethyl)
methylamine
HN-1b Bis(2-chloroethyl)ethylamine; C6H13NCl2 170.08 194 34 1.09 0.25 1520 (20C)
2, 2 -dichlorotriethylamine
isopropyl-bis(- C7H15NCl2 184 - 13.7 1.05 0.13 1290 (25C)
chloroethyl)
aminec
HN-3d Nitrogen lost; trichlormethine; C6H12NCl3 204.53 256 3.7 1.23 0.0109 121 (25C)
Trimustine; tris(2-chloroethyl)
amine;
Tris(beta-chloroethyl)amine;
2, 2, 2- trichlorotriethylamine
a
Nitrogen mustard HN-2 has a fruity odor at high concentrations and a soapy or fishy odor at low concentrations
b
HN-1 has a faint fishy or musty odor. It evaporates slowly and is mildly persistent
c
Iso-Propyl-N(Ethylchloro)2 has the structure of nitrogen mustards. The compound has the least toxic effects
d
Pure HN-3 is odorless and is the most stable and most toxic of the HN series. HN3 is considered the potential substitute agent for HD
Fig. 1.6Intramolecular
..
cyclization of sulfur S SN1 Cl Cl
Cl ..
mustard Cl S+
..
episulfonium ion
covalently bond to nucleophilic groups such as amine, carboxyl, sulfhydryl and

imidazole moieties in DNA, RNA and proteins.
1.7 Analysis andDetection ofSulfur Mustard
Analytical methods such as Thin Layer Chromatography (TLC) (Sass and Stutz
1981; Stanford 1967), Gas Chromatography (GC) (Albrio and Fishbein 1970;
Stankov etal. 2004), Gas chromatographymass spectrometry (GC-MS) (Mazurek
etal. 2001; Boyer etal. 2004), High Performance Liquid Chromatography (HPLC)
(Hallowell etal. 1986; Bossle etal. 1984) and Liquid Chromatography/Mass
Spectrometry (LC/MS) (Rohrbaugh and Yang 1997; DAgostino etal. 1998) have
been employed for the extraction and analysis of sulfur mustard and its metabolites.
Reversed phase HPLC has been used for detection of SM in water at 200nm (UV
detector). This quantitative technique showed that the maximum of 5mg/L of SM
could be dissolved in water (Raghuveeran etal. 1993). Pronase digestions of albu-
min after exposure to sulfur mustard will produce a hydroxyethylthioethyl adduct-
a quantitative biomarker which can be measured via isotope dilution tandem mass
spectrometry (Andacht etal. 2014). Combination of solid-phase micro-extraction
with gas chromatography uses thiodiglycol detection, thiodiglycol from the hydro-
lysis of SM, in natural samples of air, water and soil (Popiel and Sankowska 2011).
1.8 Interactions withBiological Molecules
Mustard agent reacts with a large number of biological molecules in different resi-
dues of bio molecules such as sulfhydryl, imidazole, amino and carboxylate groups.
Very reactive solfonium intermediate tends to permanently alkylate the guanine
base in DNA strands which leads to cell death or cancer.
1.8.1 Interactions withDNA andMode ofCytotoxicity
The basis of understanding of the mode of cytotoxic action of SM was based on the
administration of the sulfur-35-labeled mustard gas; indicating the reaction of alkyl-
ating agent (sulfur mustard) with the DNA.The proposed mechanism initiates with
an intermolecular cyclization of SM which leads to formation of an intermediate ion

through the SN1 mechanism (Fig.1.6). This intermediate molecule reacts with elec-
tron-rich parts of macromolecules, such as the sulfhydryl (SH) and amine (NH2)
groups. Accordingly, alkylates nucleic acids and proteins; resulting in impaired cell
homeostasis and eventually cell death. There are also evidences showing that these
alkylated groups to a small degree are removed from deoxyribonucleic acid (Balali-
Mood etal. 2005; Crathorn and Roberts 1966).
Both in vitro and in vivo studies indicate involvement of carboxyl groups in reac-
tion to SM.Sodium salts of acetic, hippuric, salisylic, citric, succinic and diethyl-
barbituric acids produce esters of thiodiglycol in reaction with SM at pH8in
aqueous solutions. Consequently, based on the reactions of acetate, hippurate,
citrate and succinate, the carboxyl group of aspartic and glutamic acids which exist
in proteins is responsible for interactions with SM molecules. This reaction can also
happen with steartate which is a saturated fatty acid with a carboxylate ending
(Moore etal. 1946).
The alkylating site of DNA for SM attack is preferentially the guanine base of
nucleotides. Mustard can react with two guanine moieties in adjacent base pairs. This
is due to the fact that SM has two chloroethyl groups and as such it is bifunctional.
Reaching an additional guanine achieves inter and intra-strand DNA cross-linking.
Cross-linking in a pair of guanines lying in opposite strands of the DNA molecule is
inter-strand DNA cross-linking and in two guanines that lie in the same strand is
intrastrand DNA cross-linking (Ball and Roberts 1972; Walker 1971). Figure1.7
demonstrates DNA Cross-linking of guanine by sulfur mustard at the imidazole site.
Similar to sulfur mustard, nitrogen mustard is also an alkylating agent so can add
an alkyl group (CnH2n+1) to DNA molecule. SM can also react with RNA, proteins
and phospholipids. However SMs alkylating effects on DNA has received greater
attention and has been more extensively studied.
1.8.2 Interaction withImidazole
Sulfur mustards reaction with histidine; an imidazole-based amino acid demon-

strates an interactive target with proteins. The metabolite from this interaction can
be analyzed in urine as an imidazole derivative. The alkylation of histidine occurs,
H O O H
N N
H2N NH2
S
N N+ N+ N
N N
H3C CH3
Fig. 1.7 Cross-linking of guanine by sulfur mustard

for example, in albumin and hemoglobin (Flora etal. 2004). In a 1989 report by
R.S.Brown some thiomethylimidazole compounds have been proposed to be capa-
ble of scavenging episulfonium ions produced during the hydrolysis of sulfur mus-
tard. A nucleophilic reaction is the mechanism of this effect. So these could be
assumed as decontamination materials (Brown 1989). Another interaction with
imidazole can be seen in the reaction with guanine (Fig.1.7).
1.8.3 N
itrogen Mustard Interactions andMechanism
ofActions
Nitrogen mustards covalently bind to DNA, crosslinking two strands and preventing
cell duplication. They bind to the N7 nitrogen on the DNA base guanine. HN2
which is also a bifunctional alkylating agent is cytotoxic due to its ability to intro-
duce cross-links into the genome (Pratt 1994; De Alencar etal. 2005).
Nitrogen mustards form aziridinium ion intermediate of intramolecular displace-
ment of the chloride of the nitrogen atom. This reactive intermediate ion then alkyl-
ates DNA on the N-7 of the guanine base (Fig.1.8). Nitrogen mustard residue forms
the second quaternary aziridinium ion via a second cyclization. A second attack
brings out the next alkylation step and results in the formation of inter-strand cross-
links (Fig.1.7).
These kinds of lesions affect cells to undergo apoptosis via p53 and other signal-
ing molecules which scan the genome for defects (Ruff and Dillman 2008).
The second chloroethyl arm may not necessarily react with a second guanine and
reacts with water instead, in which case, water acts as a weak nucleophile and the
product will be a monoalkylated guanine unit (Muniandy etal. 2010).
1.9 Decontamination ofSulfur Mustard Compounds
The best way to detoxify sulfur mustard is to destroy it irreversibly. Incineration is

considered as the preferred way of destructing the chemical stockpiles. Another
alternative for mustard gas decontamination is its hydrolysis process to non-toxic
N
NH
O H
R N
R N N NH2
H NH2
Cl H3C N+
N Cl N+ N
Cl Cl
R
N
H
Fig. 1.8 Formation of aziridinium ion and bonding to guanine

dechlorinated products. Unlike poor solution in water, mustard agent can easily be
dissolved in many organic solvents, in aqueous solutions of detergents and soaps or
in alkali media (hydrolysis at pH value of 810.5). By hydrolysis, mustard agent
decomposes into thiodiglycol non-poisonous product. For hydrolysis and oxidation
the following reagents have been proposed: sodium hypochlorite, chloramine-T,
Halazone, ozone, alkaline solution of sodium bicarbonate and hydrogen peroxide.
Thiodiglycol and the sulfonium salts have been reported to be the hydrolysis prod-
ucts. Its dissolution and hydrolysis are simultaneous. Temperature is effective on the
rate of HD hydrolysis in the presence of NaOH, so that at 80 and 90C it is very fast
and more than 30 times the rate at 30C.An empirical analysis used biodegradation
as one approach for sulfur mustard decontamination after the hydrolysis step. Bis(2-
chloroethyl) sulfide forms thiodiglycol after hydrolysis; and then an input of O2,
KH2PO4 and NH4Cl in an alkaline medium of NaOH produces a biomass formula of
C, H, O, N, P and S elements by biodegradation. Na2SO4, NaCl and CO2 are also
formed which are all non-toxic substances (Harvey etal. 1997; Price and Bullitt
1947).
ClCH 2 CH 2SCH 2 CH 2 Cl hydrolysis

HOCH 2 CH 2SCH 2 CH 2 OH biodegrad
ation
boimass
Calcium hypochlorite known as bleaching powder or chlorine powder with the for-
mula Ca(ClO)2 and chloramines have a violent reaction with sulfur mustard.
Potassium permanganate (KMnO4) and nitric acid can also destroy sulfur mustard.
These are all strong oxidizing agents. Nitrogen mustards show slow reaction with
these agents in comparison to sulfur mustard. Therefore, sodium hydroxide, metal-
lic sodium and chlorinated phenols are applicable chemicals for neutralizing nitro-
gen mustards (Ledgard 2007).
1.10 Antidotes forSulfur Mustard
No specific antidote for SM injury has been introduced. On the battlefield, the
first measure is to distance the victim from the contaminated area. After taking
off all the clothes and removing mustard gas from the skin, the body is washed
with soapy water followed by treatment with chemical neutralizers. Exposed per-
son can be treated with Povidon Iodine; the ointment is especially effective in the
first twenty minutes following exposure (Wormser etal. 1997). Also 7,8-diphenyl-
1,3,4,6-tetrachloro-2,5-diaminoglycoluril (also known as S-330 or M-5) has
been introduced as an effective skin decontaminant during the initial critical
exposures (Shih etal. 1999). N,N-dichloro-bis(2,4,6-trichlorophenyl) urea is a
hydrophilic decontamination formulation and has been used as decontaminant
against mustard. It is claimed that is equally effective as framycetin as a decon-
taminant and wound healer against vesicant-induced skin injury.
P-chloroperbenzoic acid and disinfectant chloramine-T detoxify sulfur mustard

to crystalline sulphoxide and sulphone. Decontaminants Fullers earth (clay),
Ambergard and BDH spillage granules have been studied for their possible effec-
tiveness against the mustard dermal administration by measuring the skin absorp-
tion rates. Investigations in skin decontamination and therapy pointed that the
potency of the Canadian RSDL (Reactive Skin Decontaminant Lotion) a decon-
tamination product for removing or neutralizing CWAs was statistically better
than Fullers earth against skin injury induced by SM (Chilcott etal. 2001; Taysse
etal. 2007; Lomash and Pant 2014; Kumar etal. 2013). Several topical antibiot-
ics and antimicrobials can be used for management of damaged skin. For exam-
ple silver nitrate 0.5% can protect the skin against bacteria and yeast-like
organisms (Tuorinsky 2008).
1.11 Conclusion
Mustard compounds are generally classified into two major groups: sulfur mus-
tard and nitrogen mustards. Mustard compounds have been used as chemical
warfare agents and also as therapeutic anti-cancer drugs. Mustard compounds
are alkylating agents. They covalently bond to nucleophilic sites of amine, car-
boxyl, sulfhydryl and imidazole in biomolecules. Since its initial synthesis in
the nineteenth century, sulfur mustard has been used many times in battles as a
chemical warfare agent. Bis(2-chloroethyl) sulfide has been produced by many
different synthetic routes such as electrophilic additions of alkenes, chlorination
of thiodiglycols by nucleophilic substitutions and photochemical reactions.
However, not all of the applied synthetic routes have proved successful in large
scale production of stockpile. Mustard lethality has reportedly been lower than
that of the gaseous chlorine and phosgene but the immense psychological toll
and the heavy casualties make SM a very obnoxious chemical. Various anti-gas
ointments and powders had been employed on the battlefield with varying
degrees of success. However no specific antidotes for injuries of this chemical
agent have been identified.
Nitrogen mustards have shown to be effective on cells that are actively proliferat-
ing, including the lymphoid tissue and bone marrow. Nitrogen mustards in low
doses inhibit DNA synthesis in cultured mammalian cells more rapidly and to a
greater extent than it inhibits RNA or protein synthesis. Therefore, in the modern
chemotherapy treatments, nitrogen mustards are used in treatment of both Hodgkins
and Non-Hodgkins disease and also for lung and breast cancers but they impose
adverse side effects.
Current research on sulfur mustard focuses on ways to decontaminate the com-
pound and on discovering effective substances that can better neutralize this toxic
chemical. Research is also ongoing on the development of antidotes for sulfur mus-
tard casualties. Studies on nitrogen mustard group of compounds mostly lie under
the antineoplastic and anticancer drug research efforts.
Glossary
Alkylation Reaction involving a transfer of an alkyle group from one

molecule to another for instance to a DNA molecule which
can consequently result in cell death.
Antidotes Medicines for counteracting/neutralizing the harmful
effects of a poison
Carcinogenicity Genetic alterations such as DNA strand breaks and
unscheduled DNA synthesis that may lead to cancer
development
Chemical Properties The ability to undergo changes that alter a materials com-
position and are observed during a reaction
CWA: chemical
warfare agents Toxic chemical compounds in munitions/devices causing
death or harm to human beings.
Cytotoxic Being toxic to cells which may result in cell death
Decontamination Ways of detoxifying hazardous chemicals using specific
compounds
Exposure Being subjected to radiation or chemicals with potentially
harmful effect.
Nitrogen Mustards A family of mustard compounds with the central atom of
nitrogen. Nitrogen mustards derivatives are used as che-
motherapy drugs
Physical Properties Properties that account for identifying substances. And are
observed without changing the composition of matter
SM: Sulfur Mustard A toxic and vesicant chemical warfare agent which is
highly reactive and forms large blisters on the exposed
skin
Spectroscopy Study of the absorption and emission of light and matter.
Spectral data shed light on the structure of compounds and
can also be used in the analysis of a known analyte in a
matrix
Synthesis Production of chemical compounds
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Chapter 2
History of Use and Epidemiology
Leila Etemad, Mohammad Moshiri, and Mahdi Balali-Mood
Contents
2.1 Introduction .................................................................................................................... 30
2.2 Synthesis and Development of Sulfur Mustard ............................................................. 31
2.2.1 Different Names of Sulfur Mustard After Synthesis ......................................... 31
2.3 Use of Sulfur Mustard During the Wars ........................................................................ 32
2.3.1 World War One (WWI) ...................................................................................... 32
2.3.2 Rif War in Morocco (19211926)...................................................................... 33
2.3.3 Italian-Ethiopian War (19351936) ................................................................... 33
2.3.4 EGYPT-Yaman War (19631967) ..................................................................... 34
2.3.5 World War II (19391945) ................................................................................. 34
2.3.6 Japan-China War (19391945) .......................................................................... 35
2.3.7 Iraq Iran War (19811988) ............................................................................. 35
2.3.8 Other SM Exposure During the Conflicts .......................................................... 37
2.4 Disposal.......................................................................................................................... 40
2.5 Nitrogen Mustard (NM) ................................................................................................. 41
2.5.1 Chemotherapy .................................................................................................... 41
2.6 Conclusions and Recommendations .............................................................................. 42
Glossary................................................................................................................................... 43
References ............................................................................................................................... 44
L. Etemad
Pharmaceutical Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran
e-mail: Etemadl@mums.ac.ir
M. Moshiri
Pharmacodynamics and Toxicology Department, Faculty of Pharmacy, Mashhad University
of Medical Sciences, Mashhad, Iran
Health Insurance Research Office, Armed Forces Insurance Organization of Islamic Republic
of Iran (Mashhad Branch), Tehran, Iran
e-mail: Moshirimo@gmail.com
M. Balali-Mood, MD, PhD (*)
Medical Toxicology Research Centre, Faculty of Medicine, Mashhad University of Medical
e-mail: BalaliMoodM@mums.ac.ir

Toxicology of Mustard Compounds, DOI 10.1007/978-3-319-23874-6_2
30 L. Etemad et al.
Abstract Chemical warfare agents (CWAs) are synthetic chemicals that have toxic
effects on plants, animals and humans. Sulfur mustard (SM) is one of blister agents
which is synthesized by Belgian chemist Cesar Mansute Despretz in 1822 for the
first time. Victor Meyer, a Germania chemist, in 1886, completely described the
chemical structure of SM. In the World War One, German army used SM for the first
time against British soldiers in a field near Ypres Belgium. Nitrogen mustard (NM)
was initially synthesized as a CWA, but has never been used as a chemical weapon.
Different analogues of NM were made during the early twentieth century and some
of them have been prescribed as chemotherapeutic medications. Spain was the first
government that used SM against the Rif rebellion civilian in 19211926. Mussolini
also ordered the Italian army to use SM against unprotected Ethiopian forces and
civilian population in 19351936. Through 19631967, the Egyptian air force used
CWAs and SM in Yaman. Although a large amount of SM was made during the
World War II, fortunately it was not used during that war. Iraqi army used SM and
other CWAs against Iranian forces and Iranian and Kurdish civilian in 19831988.
The result of repeated Iraqs chemical attacks during the 8 years of war was above
100 thousand casualties, of which almost 5000 were died. It was estimated that
more than half of the chemical casualties were due to SM poisoning, but 32,000 of
them have medical records and around 30,000 of them are now suffering from the
delayed toxic effects of SM. The most tragic use of SM was the chemical
bombardment of the city of Sardasht (a city in the northwestern border of Iran with
Iraq) in spring of 1987 and Halabja (a Kurdish town in Iraq) massacre in 1988.
Keywords Sulfur Mustard Nitrogen Mustard Chemical Warfare Agents War

History Iraq-Iran War Word War One Chemotherapy Blister Agents
2.1 Introduction
Chemical warfare agents (CWAs) are synthetic chemicals that have toxic effect on
plants, animals and humans (Sidell et al. 2007; Marrs et al. 1996). CWAs can be
widely dispersed as a gas, liquid or solid forms. They are capable of killing, injuring
or incapacitating enemy personnel (Moshiri et al. 2012; Geoghegan and Tong 2006).
Chemical agents from plant extracts that poisoned individuals have been used as
warfare since historical times. According to the documentary evidence, the earliest
successful use of chemical agents in the war occurred in 600 B.C. when Athenians
contaminated the water supplies during the siege of Kirhha by Helleborus root.
CWA were widely used during the World War One (WWI) (Chauhan et al. 2008).
The French were the first to use tear gasses including ethyl bromoacetate and chlo-
roacetone, in the WWI (Black 2010; Balali Mood et al. 2014a).
CWAs are divided into four major categories: nerve, blistering, choking, and
blood agents (Chauhan et al. 2008). Blistering agents or vesicants cause severe eye,
skin and lung injuries (Chauhan et al. 2008; Balali Mood et al. 2014b). The name
blistering come from its ability to cause painful water blisters which resemble
severe burns (Chauhan et al. 2008). Blister agents are used to decreases the oppo-
2 History of Use and Epidemiology of Mustard Compounds 31
nents ability to fight rather than to kill, although exposure to high concentrations
can be fatal. The blistering agent that has been used since the WWI is sulfur mustard
(SM) (Balali Mood et al. 2014b).
2.2 Synthesis and Development of Sulfur Mustard
Belgian chemist Cesar Mansute Despretz (17981863), In 1822, produced foul-

smelling liquid for the first time, when he combined ethene and sulfur dichloride, but
he never describe the irritating properties of his SM made compound (Kehe and Szinicz
2005; Organisation for the Prohibition of Chemical Weapons 2015; Sun and Zheng
2012; Pearson 1993; Balali-Mood and Hefazi 2005a; Khateri 2013; Ellison 2007).
In 1854, the Frenchman Alfred Riche (18291908) made SM by combining of
chlorine and diethyl, he also did not described any adverse physiological properties
(Kehe and Szinicz 2005; Anonymous 2013; Duchovic and Vilensky 2007). However,
its injurious properties had not been known until 1860 by Frederick Guthrie
(Organisation for the Prohibition of Chemical Weapons 2015; Duchovic and
Vilensky 2007; Sun and Zheng 2012).
Victor Meyer, a Germania chemist, In 1886, completely described the chemical
structure of SM (Kehe and Szinicz 2005). He produced thiodiglycol from reaction
of aqueous potassium sulfide with 2-chloroethanol and chlorinated with phosphorus
trichloride (Duchovic and Vilensky 2007). This compound was more pure and had
more severe adverse health effects. As one of Meyers assistant presented symptoms
of SM intoxication, he tested the effect of the compound on rabbit and it died
(Duchovic and Vilensky 2007). Albert Niemann also reported blister-forming prop-
erties of SM, in 1886 (Duchovic and Vilensky 2007).
Hans Thacher Clarke, English chemist who worked with Emil Fischer in Berlin,
in 1913 changed in Meyers recipe by replacing hydrochloric acid instead of phos-
phorus trichloride (Duchovic and Vilensky 2007). Due to dermal contact by SM, he
was hospitalized for 2 months and his coworker, Fisher, reported this accident to
German Chemical Society and Germany government that he found a chemical
weapon (Duchovic and Vilensky 2007). History of sulfur mustard synthesis and
development events were summarized in Table 2.1.
2.2.1 Different Names of Sulfur Mustard After Synthesis
The German named SM as LOST in 1916; combination the first two letters of the
last name of two scientists; Wilhelm Lommel and Wilhelm Steinkopf. These two
chemists were working in Fritz Haber laboratory at the Kaiser Wilhelm Institute;
when they established an industrial method for large-scale production of SM for
Imperial German Army (Anonymous 2013; Peano and Bernardi 2015). SM is also
named Yperite (Ypres, the place of the first military use of SM in Belgium), and
yellow cross, because German shells with vesicants were labeled with a yellow
cross (Sun and Zheng 2012).
32 L. Etemad et al.
2.3 Use of Sulfur Mustard During the Wars
2.3.1 World War One (WWI)
During the latter part of WWI on 12 July 1917, German army used LOST for the
first time against British soldiers in a field near Ypres Belgium (Table 2.2) (Peano
and Bernardi 2015; Ellison 2007; Hurst et al. 2007). SM was also named Yperit
because of this event (Peano and Bernardi 2015; Kehe and Szinicz 2005). Germany
fired more than 1 million SM missiles, during 10 days against Allied troops (Balali-
Mood and Hefazi 2005a). A large number of Allied soldiers suffered from the eyes
and lung injuries and most of them underwent chronic complication 3040 year
after exposure (Organisation for the Prohibition of Chemical Weapons 2015; Khateri
Table 2.1 History of sulfur mustard synthesis and development events

Year Scientist Development events
1822 Cesar Mansute Reaction of sulfur dichloride and ethane no irritating properties
Despretz
1854 Frenchman Alfred Predicting by chlorine and diethyl sulfide
Riche
1860 Frederick Guthrie Irritating properties
1886 Victor Meyer Completely described the chemical structure 2-chloroethanol +
potassium sulfide + phosphorus trichloride fatal in animal test
1886 Albert Niemann Blister forming properties
1913 Hans Thacher Replaced phosphorus trichloride by hydrochloric acid
Clarke
1913 Emil Fischer Reported Belistering property
Table 2.2 Brief history of sulfur mustard uses in conflicts between 1917 and 2015
Year (s) Against
1917 (WWI) Germany French (at Ypres)
1919 United Kingdom Red Army of Russian
19211927 Spain and France Rif insurgents in Morocco
1930 Italy Libya
1934 and 19361937 Soviet Union Xinjiang, China
19351940 Italy Abyssinia (now Ethiopia)
1939 Poland Germany
WWII Germany Poland
WWII Germany Soviet Union
19371945 Japan China
19631967 Egypt North Yemen
19811988 Iraq Iran
1988 Iraq Kurds on Halabja
1995 and 1997 Sudan Insurgents in the civil war
2013). German army used SM against the second French Army, for the second time
in WWI (Hilmas et al. 2008; Duchovic and Vilensky 2007).
The Allies had been able to use SM at Cambrai, France, in November 1917 after
they captured the German SM shells (Peano and Bernardi 2015). One year later,
Britania used his own SM, in September 1918, during Hindenburg Line (Peano and
Bernardi 2015; Hilmas et al. 2008; Duchovic and Vilensky 2007).
2.3.2 Rif War in Morocco (19211926)
One of the first governments that used CWA against civilian has been Spain that
applied SM against the Rif rebellion (Javier 2006). Rif War (192126) was a con-
flict between Spanish colonial forces (later assisted by France) and Moroccan
Berbers of the Rif mountainous region led by Muhammad Abd el-Krim (Balfour
2002; Jernigan et al. 2002). On the first part of war, Berbers used the guerrilla war-
fare and defeated Spanish force and could capture Spanish weapons (Balfour 2002).
Through 3 weeks, 800010,000 Spanish soldiers were killed (Kehe and Szinicz
2005). Then the Spaniards extensively used SM against Rif civilian and troops in
1924 (Anonymus 2014). This happened 1 year before time that the protocol of
Prohibition of the Use in War of Asphyxiating, Poisonous or other Gases, and of
Bacteriological Methods of Warfare; as the Geneva Protocol; had been signed
(Anonymus 2014). The applied CWA were produced in National Chemical Factory
of Spain at La Maraosa near Madrid (Balfour 2002). Hugo Stoltzenberg; a German
chemist who later became a citizen of Spain, had great help in the production of
these compounds (Anonymus 2014).
The main targets of Spain CWAs attacks were civilian populations, markets, and
rivers (Anonymus 2014; Balfour 2002). For the first time on November 27, 1921,
these events were revealed in Francophone newspaper which was published in
Tangier (Balfour 2002). Then, the usage of SM by Spanish government was described
in an article called Cartas de un soldado (Letters of a soldier) on August 16, 1923 in
a Spanish newspaper La Correspondencia de Espaa (Balfour 2002). Hidalgo de
Cisneros, a pilot of Farman F60 Goliath aircraft, wrote in his autobiographical book
that in the summer of 1924 he dropped a 100-kg over Rif people (Balfour 2002).
2.3.3 Italian-Ethiopian War (19351936)
Mussolini ordered that around 200,000 Italian soldiers to attack the Ethiopian on 3
October 1935 and then second Italiano-Abyssinian War started. They could not
develop as much as they suggested. Italian ignored the Geneva Protocol, that had
been singed 7 years earlier (on 26 December 1925), and used poison gas and large-
scale aerial bombardment (Khateri 2013; Wikipedia 2015; Anonymous 2013;
SMART 1997). The massive air attacks with SM, were done against unprotected
34 L. Etemad et al.
Ethiopian forces and civilian population. About 150,000 SM casualties were

reported. The Italian attack, contaminated agricultural land and destroyed Ethiopian
villages (Anonymous 2013; Balali-Mood and Hefazi 2005b; Wikipedia 2015).
Ethiopian had named this event terrible rain that burned and killed (Barker 1971).
Italian also attacked the Red Cross and the Red Crescent hospitals (Kehe and Szinicz
2005; Barker 1971). The Italian tried to justified their performances and ignoring
Geneva Protocol by stating that Ethiopians had tortured their Captives and killed
wounded soldiers (SMART 1997).
2.3.4 EGYPT-Yaman War (19631967)
The Egyptian air force used CWA in Yemen for 5 years (196367). They applied CW
against Yamanian royalists who sheltered in inaccessible caves of Yamanes North
Mountains that conventional warfare was not effective. Egyptian army used mixed of
CWAs such as tear gas (CN), SM and phosgene as an asphyxiant (Shoham 1998). The
CWA attacks induced approximately 1500 fatalities and 1500 injuries (Hadden 2012).
Cairo decided to increase the power of CWA when they assumed to attack Israel in
the Yom Kippur War, in 1973. Egypt tried to raise its technology and knowledge of
CWAs synthesis and try to improved CWAs quality and power (Shoham 1998). Ain-
Shams University, the Central Military Chemical Laboratories of the Egyptian Army,
Egyptian National Research Center and the Technical University of Budapest in
Hungary and some institutions and laboratories in the Unit States and West Germany
studied on organophosphorus nerve agents, glycolates BZ, nitrogen and SM (Shoham
1998). The CWA which produced in the institutes and factories were filled into mines,
artillery shells, mortar bombs, rockets and aerial bombs (Shoham 1998).
2.3.5 World War II (19391945)
Large amounts of SM was made during WWII (Balali-Mood and Hefazi 2005b;
Anonymous 2013). Only Germany had produced and accumulated around 25,000 t
of SM and 2000 t of nitrogen mustard (John Aa et al. 2002). In 8 September 1939,
a polish officer and his troops destroyed a bridge and blocked a road near Jaso. This
combat induced two deaths and 12 injured German soldiers. Berlin did not do any
retaliation activities (Anonymous 2013).
On 2 December 1943, U.S. ship; SS John Harvey, which had warfare and sulfur
mustard grenades, on harbor of Bari, Southern Italy, was bombarded and hit and
sunk by German airplane. A part of shipment sank in water and other parts exploded
and fired which induced 83 death and nearly 600 human injuries. Due to highly clas-
sified of this shipment, the U.S. Army killed all injured who could not treated prop-
erly (Kehe and Szinicz 2005; Balali-Mood and Hefazi 2005a; Wikipedia 2015).
British and U.S. Armies tested SM on Australian Army volunteers in Queensland in

19431944 (Goodwin 1998).
2.3.6 Japan-China War (19391945)
The Imperial Japanese Army in 1939 applied SM and Lewisit gases in conflict
against Chinese Kuomintang and Communist (Wikipedia 2015; Khateri 2013). The
Japanese forces abandoned several barrels of SM in a building at urban Qiqihar in
1945 when they exited from china (Sun and Zheng 2012). On August 4, 2003, 5 SM
barrels leaked and injured 43 person (39 males and 5 females) and killed one patient
due to multiple organs failure (Sun and Zheng 2012; Hurst et al. 2007). The victims
suffered from ocular lesions, cutaneous blisters and pulmonary injuries (Sun and
Zheng 2012).
2.3.7 Iraq Iran War (19811988)
In 1881, Iraq began a widespread offensive attack against Iran and occupied large
areas of Iran in a short time (Javed 2001; Ahmadi et al. 2010). But moving forward
in Irans territory didnt follow the preplanned programs, and lots of Iraqi armys
parts crippled, stopped moving forward, and took a defensive position (Yekta 2012).
After the freedom of Khorramshahr (a city in Suth west Iran) in 1883, and the
Iraqis primary withdrawal, the Iraqi army in order to stop the attacks and moving
forward of Iranian troops used tear gas (CS), and yellow rain (Javed 2001; Balali
Mood et al. 2014a). Then Iraq found the power of chemical facilities against Iranian
troops attacks and progress. Therefore, Iraq trained distinct and regular chemically
military units as the pattern of chemical units of the Soviet Union, that had a special
position in Iraqi army (Cordesman and Wagner 1990).
According to The Los Angeles Times, Iraqis in order to crush the Iranians troops
army organization in night raids used SM sporadically, since December 1982.
On 8 August 1983, Iraqi army targeted different places in western Iran
including Haj-Omran, Shiveh Roush, Tamarchin, Doyeraj river and its sur-
rounding area by artillery and aircraft, with nauseous and vesicant gas, later
diagnosed as SM. On these battles, around 300 combatants were poisoned. In
addition, on the 23rd of August, Qamtareh heights (Chamartheh) were targeted
by artillery and vesicatory gases caused the death of three people and injuring
of 200 people (Iranian Revolutionary Guards of the Islamic Revolution 1985;
Yekta 2012).
In October 1983 during the Iran war operation of Valfajr 4 in the public area of
Sulaymaniyah and the Panjwin region, Iraqi forces attacked the positions occupied
by Iranian with SM. As a result, around 300 combatants were injured, which by that
36 L. Etemad et al.
time was unprecedented (Yekta 2012). A few days later, in November 1983, Iraqi
army attacked the Iranian troops and even the civilians with SM on a large area of
west Iran. Apart from the military areas, the civilians of Marivan, Sardasht (3rd of
October), Bane (8th of November) cities and neighboring villages were under attack
(Javed 2001; Security Council of the United Nations 21 April 1988).
In a report that a few days after the incident was transmitted, Islamic Republic
News Agency stated more details on how the event took place (Islamic Republic
News Agency 1983; Security Council of the United Nations 21 April 1988):
At 17:00 h on 16 November 1983, while the Iraqi regime had suffered another
defeat in the Valfajr 4, area targeted the area between Schiller the mountain range by
four of its planes that were armed with chemical weapons. This time, another type
of chemical weapons, was tested by Iraq.
Four days after the onset of Iran war Khaybar operation in south of Iran; on 7th
March 1984, Iraq began a massive and widespread chemical attack against Iranian
forces (Iranian Revolutionary Guards of the Islamic Revolution 1985). In the first
48 h of the attack by the explosion of over 100 mustard gas bombs; about 500 t of
SM was dropped on Iranian forces. As a result 1100 people were injured that 150 of
whom were in critical condition (Yekta 2012). Up to 27th of March a large number
of chemical attacks with SM were done, the most important of them were per-
formed on 9th March 1984 and 14 March 1984. On 9th March 1984, 543 Iranian
soldiers were poisoned, mostly in Majnoon islands (Yekta 2012; Javed 2001). On 14
March 1984, a group of Esfehan Jihad volunteers; who were taking a rest, were
under attack with SM that poisoned all of them (Yekta 2012).
On 12 March 1985, Iraqi army attacked Majnoun Islands with nerve gas and then
mustard gas in large scales (Yekta 2012). In one of the Irans authorities letters to
the United Nations Organization, it is noted that in the first week of chemical attack
(from 13 to 20 March 1985), 77 bombs, 23 rockets, and 639 chemical cannonball
were shot to Iran that resulted in 2231 injuries and 32 deaths (Javed 2001). On April
23, 1985 after sending a group of patients with SM poisoning to European hospitals
for treatment, the second UN report on Iraqs use of these bombs was presented
(Yekta 2012; Security Council of the United Nations 21 April 1988).
In winter 1986, another Iran war operation (Karbala 4) was performed in south-
ern region and on 26th December 1986, Abadan was under the chemical attack with
SM by Iraqi forces. The attack had left 2000 injured combatants that were mostly
mild SM poisoning (Yekta 2012; Javed 2001).
After the Karbala 5 operation in Shalamcheh by the Iranian Army, Iraq army
performed extensive chemical attacks by SM for 2 months (Security Council of the
United Nations 21 April 1988). The number of moderate and severe SM poisoned
patients was around 3000, with the sum of mild injuries it was around 7000 chemi-
cal casualties. And within a month, 170 people died from exposure to SM (Yekta
2012). On 31th December 1986, Iraqi army attacked Sumar field hospital. As the
result of this chemical attack with SM 400 injuries and 20 deaths were reported.
Some of the chemical injured combatants were sent to Paris Saint Antoine Hospital
(Yekta 2012; Javed 2001; Cordesman and Wagner 1990). Before this event, the
other Iranian military hospitals were chemically bombarded by Iraq, too. For
example, the bombing of Fatima field hospital on February 27, 1986 caused the
hospital to be unusable for weeks, because it was polluted chemically by SM
(Javed 2001; Security Council of the United Nations 21 April 1988; Iranian
Revolutionary Guards of the Islamic Revolution 1985).
During the war, Iraqi army used numerous chemical and possibly biological factors
separately or in combination (Balali-Mood et al. 2013, 2014c). It was estimated that
more than half of the Iraq-Iran war chemical casualties were due to SM poisoning, but
32,000 of them have medical records and around 30,000 of them are now suffering from
the delayed toxic effects of SM. (Lvarsson et al. 1992; Security Council of the United
Nations 21 April 1988; Javed 2001; Yekta 2012). The Acting Permanente Representative
of I.R.Iran in united nations sent a letter to security council of UN, which is preserved as
S/19816 document of UN, as a report on Iraqi Chemical and biological attacks against
Iranian troops and people (Security Council of the United Nations 21 April 1988).
2.3.7.1 Sardasht
The most tragic use of mustard gas was the chemical bombardment of the city of
Sardasht (Khateri et al. 2004; Security Council of the United Nations 1986, 30 June
1987a, Jul 30 1987b). In the spring of 1987, the Iraqi army released four 250-kg
mustard gas bombs on Sardasht, a small Kurdish town in northwestern Iran (Security
Council of the United Nations 30 June 1987a). An estimated of 4500 innocent civil-
ians were exposed to SM and over one-third of them developed moderate to severe
medical complications that require hospitalization (Security Council of the United
Nations 1986, 30 June 1987a). Many suffered from injuries from the explosions in
addition to their exposure to SM (Security Council of the United Nations 30 June
1987a; Mansour Razavi et al. 2012). Sulfur mustard attracts of Iraqi army against
Iranian troops and civilians in 19831988 were described in Table 2.3.
The Iraqi troops also used SM and the nerve agent sarin alongside at large
scales in 1988 against the Kurdish population of Halabja in north of Iraq, which
made a terrible massacre. This chemical attack killed between 3200 and 5000
people and poisoned 700010,000 more, most of them were civilians. Thousands
more died of the complications and delayed toxic effects of both NAs and SM
some years later.
2.3.8 Other SM Exposure During the Conflicts
The Karenni people of Burma were presented symptoms of SM when the Burmese
military used CWA against them (Hurst et al. 2007).
Since January 2011, there have been concerns on the prabable use of the chemi-
cal weapons in some Syrian cities reported by the media (Marsh 2011; Al Jazeera
2011; Yan 2012). Especially, the Military analysts suggested that Syria may have
one of the largest word stockpiles of CWA. Although, Foreign Ministry spokesman
38 L. Etemad et al.
Table 2.3 Sulfur mustard attracts of Iraqi army against Iranian troops and civilians in
19831988
Number
Place Date Means of victims
Shiveh Rash 8 Aug 1983 Aircraft 24
Haaj Omraan 8 Aug 1983 Aircraft
Piranshahr 8 Aug 1983 Aircraft 10
Tamr Chin 9 Aug 1983 Aircraft 30
Ghamtareh heights 14 Aug 1983 Artillery 203
Badamjan Village 23 Oct 1983 Aircraft 30
Bayenjan Village 28 Oct 1983 Artillery
Shatte Ali 26 Feb 1984 Aircraft
Hour Alhoveyzeh 27 Feb 1984 Aircraft 1100
Majnoon 10 Mar 1984 Aircraft 543
Majnoon 11 Mar 1984 Aircraft 20
Road between Kiyand Dasht and Shate Ali 15 Mar 1985 Aircraft
North Port of Shate Ali 15 Mar 1985 Aircraft 738
AE Rahr Fath 15 Mar 1985 Artillery
Khaibar Bridge 15 Mar 1985 Aircraft 17
Avaleh jazireh 15 Mar 1985 Aircraft
Ghamareh bani hashem Emergency center 15 Mar 1985 Aircraft 112
Keibar Bridge 15 Mar 1985 Aircraft
Majnoon Island 16 Mar 1985 Aircraft 70
South west of Majnoon Island 18 Mar 1985 Aircraft
South of Majnoon Island 18 Mar 1985 Aircraft 111
Ashioura Emergency center 18 Mar 1985 Aircraft
Hamid Base 8 Apr 1985 Aircraft 1110
Majnoon island 9 Apr 1985 Artillery 5
Abaadan city 26 Jan 1986 Aircraft 11
Fow Basreh road 13 Feb 1986 Aircraft 8500
Abaadan 13 Feb 1986 Artillery 20
Abaadan 13 Feb 1986 Aircraft 11
Jofeir 15 Feb 1986 Aircraft 20
Abaadan 16 Feb 1986 Aircraft
Khosroaabaad 16 Feb 1986 Aircraft 6
Valfajr operational theatre 23 Mar 1986
Mehran Amir road 24 Apr 1986 Aircraft 150
Mehran -salehabad 25 May 1986 Aircraft 150
Bomoud Cheikh salah 4 Sep 1986 Artillery 125
Khoramshahr 25 Dec 1986 Aircraft
Khoramshahr- shalamcheh 25 Dec 1986 Artillery
Khoramshahr- shalamcheh 25 Dec 1986 Aircraft 1160
Abaadan Khoramshahr 26 Dec 1986 Artillery
(continued)
Table 2.3 (continued)

Number
Abaadan Khoramshahr 26 Dec 1986 Aircraft
Abaadan Down to Minu Sq. 29 Dec 1986 Aircraft
Abaadan Down to Minu Sq. 29 Dec 1986 Artillery
West front Infirmary 31 Dec 1986 300
Soumar (4 KM away) 31 Dec 1986 Aircraft
Soumar (4 KM away) 31 Dec 1986 Aircraft
Soumar 31 Dec 1986 Aircraft
Minu Island 2 Jan 1987 Artillery
Khoramshahr road 10 Jan 1987 Aircraft 3000
Khoramshahr road 10 Jan 1987 Artillery
Khoramshahr (North west) 11 Jan 1987 Artillery
Khoramshahr (North west) 11 Jan 1987 Aircraft
Around Martyr Dezfuly road 12 Jan 1987 Aircraft
Around Martyr Dezfuly road 12 Jan 1987 Artillery
Lower Mearaj road 13 Jan 1987 Artillery
Lower Mearaj road 13 Jan 1987 Aircraft
West of Shalamcheh 14 Jan 1987 Aircraft
West of Shalamcheh 14 Jan 1987 Artillery
West of Shalamcheh 14 Jan 1987 Mortar shell
Martyr Amaani road 15 Jan 1987 Artillery
Martyr Amaani road 15 Jan 1987 Aircraft
East of Jaasem river 16 Jan 1987 Artillery
Logestic roads 21 Jan 1987 Artillery
Logestic roads 21 Jan 1987 Aircraft
Martyr Ahmad Gholzari road 24 Jan 1987 Aircraft
Military equipment workshop 25 Jan 1987 Mortar Shell
Military equipment workshop 25 Jan 1987 Aircraft
Along Arvand road 27 Jan 1987 Artillery
Khoramshahr 28 Jan 1987 Aircraft
Artillery emplacement No. 5 (Nasr1 29 Jan 1987 Artillery
Operation)
Artillery emplacement No. 5 (Nasr1 29 Jan 1987 Mortar shell
Operation)
Karbalaa 5 Operational theatre 1 Feb 1987 Artillery
Karbalaa 5 Operational theatre 1 Feb 1987 Aircraft
48 Fath Motor Pool 2 Feb 1987 Artillery
Karbalaa 5 Operational theatre 6 Feb 1987 Aircraft 50
Two sides of Khramshahr road 6 Feb 1987 Aircraft
South Eastern of Majnoon Island 14 Mar 1987 Aircraft 640
North of Majnoon Island 16 Mar 1987 Aircraft
Beginning of Khaibar Bridge 16 Mar 1987 Aircraft
40 L. Etemad et al.

Number
Martyr Rajaai road 16 Mar 1987 Aircraft
Around of Kosar headquarters 16 Mar 1987 Aircraft 241
Karbalaa 8 Operational theatre 7 Apr 1987 Artillery
Karbalaa 8 Operational theatre 7 Apr 1987 Aircraft 6
Karbalaa 8 Operational theatre 8 Apr 1987 Aircraft
Solimaniyeh and Arbil (Iraq) 16 Apr 1987 Aircraft 450
Four areas on sardasht 28 June 1987 Aircraft 8025
Ghallehvash Village in Sardasht 28 June 1987 Aircraft 132
Extracted from S/19816 document of United Nation
of Syria said: All the stocks of these weapons that the Syrian Arab Republic pos-
sesses are monitored and guarded by the Syrian army. These weapons are meant to
be used only and strictly in the event of external aggression against the Syrian Arab
Republic (Yan 2012). On 27 September 2013, the OPCW accepted a demilitariza-
tion plan for Syrias CWA. As of 23 June, 2014, all of Syrias CWA have been ren-
dered permanent and removed from Syria. The United States, began neutralizing
600 t of SM and other CWA, on 7 July, 2014 (OPCW 2014).
2.4 Disposal
After WWII the most of German SM was sunk into Baltic Sea. In the Bornholm
region, through 19662002, about 700 chemical weapons were discovered which
majority contained SM mixed with a thickener, Sprhbchse 37 (Albright
2011). The barrels have been leaked and induced sea water contaminated. The
sea water could not hydrolysis SM released from weapons and SM has been
found in concentration of 2.4 mg/kg (dry sediment) at least in one sample of
water, the least dose of SM could induce blister is 6 g/cm2 (John Aa et al. 2002).
Contact to the sea water could form burns sings (Anonymous 2013; Albright
2011; John Aa et al. 2002). The Danish and Swedish coasts have been contami-
nated by CWAs and the fisherman workers of fishing port in south Sweden and
Denmark.
About 1700 t of lewisite and 1700 t SM containing bombs were dumped in
Mediterranean sea of St. Rafaela of France (Albright 2011). Due to health problem
induced by leaked SM, France established an automated factory to dispose of war-
fare contain SM. Also there are some shells containing SM which were drown in
ocean near Belgium based on conventional method of SM ruining that were drown
on sea water in WWI.
William Brankowitz, an assistant project manager in the U.S. Army Chemical
Materials Agency, reported in 1998 that the US Army dumped its Chemical weapons
in at least 26 sites of ocean. The US army dumped about 64 million pounds of SM

and nerve agents into ocean. However US congress, in 1972, has banned disposition
of CWA in ocean (Albright 2011).
In February 2005, a disposal factory have established on Edgewood Area of
Aberdeen Proving Ground in Maryland that had been buried about 1500 t of SM. The
biggest SM stockpile in US with more than 6000 t CWAs was in Deseret Chemical
Depot in Utah. This storage is managed by the US Armys Chemical Materials
Agency (USACMA) and destruction of this storage has been started since 2006.
Disposal operations in Oregon, Indiana, Utah, Arkansas and Alabama have been
managed by USACMA (The U S Army Chemical Materials Activity (CMA) 2015).
SM has no industrial use (Balali Mood et al. 2014b), but there are some reports
of occupational exposure to SM (Graham and Schoneboom 2013). In 2009, a min-
ing investigation on Chinchilla, Queensland found Howitzer shell contain SM that
had been hidden under ground by US Army after WWII (Fette 2012). In 2010, a
crewman of a ship pooled up some shells from waters of south of Long Island,
New York, which were dumped in WWI. He presented with skin and respiratory
symptoms of SM and thus hospitalized (The Associated Press 2010). The plastic
manufactures works also might be exposed to SM or NM, such as Plaquemine,
Louisiana in 1996 (Rosemond et al. 2003; Iyriboz 2004).
2.5 Nitrogen Mustard (NM)
Nitrogen mustard (NM) has been made by replacement the sulfur atom with a nitrogen
atom (Bouziane et al. 1998). It is much less toxic and less potent vesicant than SM,
however more delicate due to its less odorous (Peano and Bernardi 2015; Opresko et al.
1998). Mechanism and clinical manifestation of NM is similar to SM (Organisation for
the Prohibition of Chemical Weapons 2015; Saladi et al. 2006). During 19411943
American and Germany try to synthesis NM (Ellison 2007; Organisation for the
Prohibition of Chemical Weapons 2015). As NM is not as stable for storage as SM,
there is lesser inclined to it and there is little verified use of NM as a CWA (Organisation
for the Prohibition of Chemical Weapons 2015). Italians used NM in Sirtica (Libya)
and Ethiopia, in 1930 and 1936 respectively (Peano and Bernardi 2015).
2.5.1 Chemotherapy
NM can suppress bone marrow cell lines (Balali Mood et al. 2014b). However, the
effects of MCs on blood cells and bone marrow had been known since 1919
(Krumbhaar and Krumbhaar 1919), this fact had not been used for chemotherapy
until several years later. In the 1940s, scientific secret of US chemical weapons
program implied the hypothesis on usefulness of NM for chemotherapy. They found
that NM killed the white blood cells and has a suppressive effect on bone marrow.
42 L. Etemad et al.
Therefore, they suggested that NM could be effective in treatment of lymphoma, a

cancer of white blood cells (Nutritional Biochemistry Inc 2013; Saladi et al. 2006).
In 1946, two American pharmacologist, Gilman and Philips, published an article on
antiproliferative action of NM on tumor cells and its cytotoxicity effects. They syn-
thesized NM to find a compound similar to SM, because they were convinced that
SM may be an effective treatment against cancer, but very low dose of SM caused
death in trailed animals (Gilman and Philips 1946).
The Mechlorethamine (mustargen) was the first usable alkylating agent for can-
cer chemotherapy which has been made by remodeling of SM molecular structure.
The alkylating agents reacted with DNA and stop the cell replication by alkylating
the nitrogen of nucleotide (Bouziane et al. 1998; Peano and Bernardi 2015).
Different analogues of NM have now been prescribed for treatment of various can-
cer diseases including lymphoma and leukemia.
2.6 Conclusions and Recommendations
Mustard compounds were initially synthesized as CWAs. SM was first synthesized

by Cesar Mansute Despretz in 1822. Victor Meyer, also prepared and described the
chemical structure of SM in 1886. German army used SM for the first time against
British soldiers and then Allied forces also used it against the German army.
Nitrogen mustard (NM) was initially synthesized as a CWA, but has never been
used as a chemical weapon. Different analogues of NM have been prescribed as
chemotherapy for cancers. Spain was the first government that used SM against the
civilian in 19211926. Italian army used SM against unprotected Ethiopian forces
and civilian population in 19351936. The Egyptian air force used CWAs including
SM in Yaman in 19631967. Iraqi army used SM against Iranian forces and Iranian
and Kurds in large scale between 1983 and 1988. It was estimated that more than
half of the 100,000 Iranian chemical casualties were due to SM poisoning, and
around 30,000 of them are now suffering from the delayed toxic effects of SM. The
most tragic use of SM was the chemical bombardment of the city of Sardasht (a city
in the northwestern border of Iran with Iraq) in spring of 1987 and Halabja (a
Kurdish town in Iraq) massacre in 1988.
SM has been the most widely used CWA since the early last century. It is still a
big threat and likely to be used again in a war or even in a terrorist attack. Therefore,
all security authorities of the countries and international organizations, particularly
the UN and OPCW should be aware and take all preventive measures in this respect.
The OPCW has played an active role in implementation of chemical weapon con-
vention since its establishment in 1997. It is hoped that further activities of this
organization and cooperation of the state parties will provide a world free of chemi-
cal weapons.
Glossary
Aerial bomb Is a type of explosive weapon intended to travel through the air with
predictable trajectories, usually designed to be dropped from an aircraft.
Allies Were the countries that opposed the Axis powers together during the
Second World War. The Allies promoted the alliance as seeking to stop German,
Japanese and Italian aggression.
Artillery shells Is a payload-carrying projectile which, as opposed to shot, con-
tains an explosive or other filling.
Chemotherapy Is a category of cancer treatment that uses chemical substances,
especially one or more anti-cancer drugs that are given as part of a standardized
chemotherapy regimen.
CWA: chemical warfare agents A chemical substance whose toxic properties
are used to kill, injure or incapacitate human beings.
Ethiopian Officially known as the Federal Democratic Republic of Ethiopia, is a
country located in the Horn of Africa.
Exposure The condition of being subjected to something, as to infectious agents,
extremes of weather, radiation, or chemical agent which may have a harmful effect.
Geneva Protocol The Protocol for the Prohibition of the Use in War of
Asphyxiating, Poisonous or other Gases, and of Bacteriological Methods of
Warfare is a treaty prohibiting the use of chemical and biological weapons in
international armed conflicts.
Iraq Iran war (19811988) Is a conflict that started by Republic of Iraq offence
to Islamic Republic of Iran at September 1980 and lasting to August 1988. It was
initially referred to in English as the Gulf War.
Intoxication An abnormal state that is essentially a poisoning.
Lymphoma Is the name applied to a group of blood cell tumors that develop from
lymphatic cells.
Mines It is an explosive device concealed under or on the ground and designed to
destroy or disable enemy targets, ranging from combatants to vehicles and tanks,
as they pass over or near it.
Nitrogen mustard (NM) Is cytotoxic chemotherapy agent similar to mustard gas
and can induce blister. Although their common use is medicinal, in principle
these compounds can also be deployed as chemical warfare agents.
Rif The Rif is a mainly mountainous region of northern Morocco.
Rif War (192126) Was a conflict between Spanish colonial forces (later
assisted by France) and Moroccan Berbers of the Rif mountainous region led by
Muhammad Abd el-Krim.
Mortar bombs Is a weapon that fires explosive projectiles known as (mortar)
bombs at low velocities, short ranges, and high-arcing ballistic trajectories.
Rockets Is a missile, spacecraft, aircraft or other vehicle that obtains thrust from
a rocket engine.
44 L. Etemad et al.
Sardasht Is a city in and the capital of Zeydun District, in Behbahan County,

Khuzestan Province, Iran.
Security counsel of UN Is one of the six principal organs of the United Nations
and is charged with the maintenance of international peace and security as well
as accepting new members to the United Nations and approving any changes to
its United Nations Charter.
SM: Sulfur mustard A class of related cytotoxic and vesicant chemical warfare
agents with the ability to form large blisters on the exposed skin and in the
lungs.
Toxicity The degree to which a substance can damage an organism.
World War One (WWI) It is also known as the First World War or the Great War,
was a global war centred in Europe that began on 28 July 1914 and lasted until
11 November 1918.
World War II (19391945) Also known as the Second World War, was a global
war that lasted from 1939 to 1945.
Yellow rain Is airborne substance that was alleged to have been used in biological
attacks and contains trichothecene mycotoxin (a poison produced by fungi that
was known to have potential as a biological weapon).
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Chapter 3
Basic Pharmacology and Toxicology
Sara Mostafalou and Mohammad Abdollahi
Contents
3.1 Introduction .................................................................................................................... 50
3.2 Pharmaco- and Toxico- Kinetics .................................................................................... 51
3.2.1 Sulfur Mustard ................................................................................................... 51
3.2.2 Nitrogen Mustard ............................................................................................... 55
3.3 Mechanism of Action..................................................................................................... 56
3.3.1 DNA Damage ..................................................................................................... 56
3.3.2 Thiol Depletion .................................................................................................. 58
3.3.3 Inflammation ...................................................................................................... 58
3.3.4 Disrupted Calcium Homeostasis ........................................................................ 59
3.3.5 Apoptosis ........................................................................................................... 59
3.4 Conclusion ..................................................................................................................... 60
Glossary .................................................................................................................................. 60
References ............................................................................................................................... 61
Abstract Sulfur mustards are well absorbed through inhalational, dermal, and ocular
contacts and tend to distribute mostly to the lungs, liver, and kidneys. DNA and protein
adducts are the main metabolites of sulfur mustards which are mainly excreted in the
urine along with unchanged compounds. Sine nitrogen mustards have never been used
as chemical warfare, their kinetic information are mostly related to those which have
been used as chemotherapeutic agents. Upon absorption through intravenous or oral
administration, nitrogen mustards are rapidly converted to their reactive metabolites and
distributed so that the highest concentration can be found in bone marrows. Mono-
alkylation of guanine at N7 and then N3 respectively give the main DNA adducts of
S. Mostafalou, PharmD, PhD

School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran
M. Abdollahi, PharmD, PhD (*)
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical
Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
e-mail: Mohammad.Abdollahi@UToronto.Ca; Mohammad@TUMS.Ac.Ir

50 S. Mostafalou and M. Abdollahi
nitrogen mustards. In an aqueous environment, mustard compounds convert to very

active electrophilic metabolites which can attack nucleophilic groups in the structure of
cellular macromolecules. DNA alkylation is known as the main mechanism by which
mustard compounds exert their both toxic and therapeutic effects. They can also alkyl-
ate other nucleophils, most notably thiol groups in the structure of proteins, leading to
excessive production of reactive oxygen species in the cell. Following the disruption of
such functional macromolecules, a series of maladaptive responses are activated,
including excessive production of reactive oxygen species and inflammatory cytokines,
metabolic imbalance in energy production, elevated release of calcium into the cytosol
from intracellular and extracellular sources, and consequently the expression of enzymes
involved in necrotic or apoptotic cell death pathways.
Keywords Pharmacokinetics Toxicokinetics DNA alkylation Thiol adduct

Cell cycle arrest Apoptosis Inflammation
3.1 Introduction
Mustard compounds are a class of cytotoxic chemicals with the ability to alkylate
DNA, which are known for their application as vesicant chemical warfare agents
forming large blisters on the exposed skin and as chemotherapeutic agents. The
soldiers in World War I were the ones who were first faced with sulfur analogs of
these chemicals as warfare agents and used the name mustard because of the
smell, although there is no similarity with mustard oil (allyl isothiocyanate) which
interestingly is a vesicant, too. Mustard compounds fall into two subtypes; sulfur
mustards and nitrogen mustards, both are classified as Schedule 1 Substances
within the Chemical Weapons Convention (CWC) meaning that their production
and use is restricted. Use of chemical warfare was prohibited by the Geneva
Protocol of 1925, and latter Chemical Weapons Convention of 1993 prohibited the
production, development, stockpiling, and sale of such chemicals (Chemical
Weapons Convention-OPCW).
Sulfur mustards were mainly of interest for military purposes and their different
combinations have been extensively used as a chemical warfare agent in several
wartimes (Razavi et al. 2012). Sulfur mustard (SM) is the most famous member and
representative of its group which has a purity 97 % and is somehow called distilled
mustard. Though, the name mustard gas is frequently used for this chemical, in
pure form and in the ambient temperature, it is a colorless and an oily liquid. The
impure form, as used in warfare agents, have a yellow-brown color and an odor
resembling garlic or horseradish. SM was originally named by German as LOST
because of the two German scientists, Lommel and Steinkopf, who developed a
method to produce SM for Imperial German Army in 1916. In 1917 during World
War I, SM was used by the German army against British soldiers in Ypres, Belgium
for the first time, so the other name Yperite was given to the SM. At that time, SM
3 Basic Pharmacology and Toxicology 51
was the most effective chemical warfare agent, so it was known as the King of the
Battle Gases. Then after, SM was used in several major battlefields, and a number
of non-battlefield exposures have also been reported due to dumped munitions and
shells (Maynard 2007).
Nitrogen mustards initially drew attention and were specifically developed as
warfare agents during World War II, but unlike sulfur mustards, they were never
used in the battlefields and their common use is as chemotherapeutic drugs.
Mechlorethamine, known as (HN-2) or mustine was the first chemotherapeutic
agent started to be used for treatment of lymphoma in 1942 (Gilman 1963). Because
of toxic effects, it is not used anymore and the other safer developed nitrogen mus-
tards including chlorambucil, cyclophosphamide, ifosfamide, melphalan, uramus-
tine, and bendamustine are currently used for chemotherapy of cancer (Mattes et al.
1986). Spiromustine is a combination of a nitrogen mustard compound (HN-1) and
hydantoin developed to readily pass through the blood brain barrier for more effi-
cient treatment of brain tumors (Peng et al. 1975). Trichlormethine or trimustine
(HN-3) was initially developed as chemical warfare and still remains anywhere for
this purpose.
However, the number of victims for mustards has been recorded the most among
all kinds of chemical warfare and there is still no efficient antidote for treatment of
mustard casualties. Considering that at least a dozen countries currently have mus-
tards in their arsenals creates enough concern for medical care services to search
new approaches for more efficient treatment of casualties with a mechanistic focus
on toxicology and pharmacology of these chemicals. The nomenclature, molecular
formula, and chemical structure of mustard compounds are brought in Table 3.1.
The physico-chemical properties of mustard compounds are also shown in Table 3.2.
3.2 Pharmaco- and Toxico- Kinetics
3.2.1 Sulfur Mustard

3.2.1.1 Absorption
Skin, eyes, and respiratory tract are the main routes of exposure to mustards whose
oily nature causes them to persist longer on the exposed surface of the body, giving
more time for exerting local effects or systemic absorption. High lipid solubility of
mustards, further, facilitates their passage through cell membrane leading to the high
rate of absorption (ATSDR 2003). Mustard compounds can be absorbed through the
skin, eyes, respiratory and GI tract. Both vapor and liquid forms of the SM can pen-
etrate through the skin with a rate estimated to be 14 g/cm2/min at 25 C. However,
the rate of dermal penetration of SM is dependent on the dose, temperature, humid-
ity, and thickness of the skin. The base of the hair shaft or hair follicle, which have a
thinner epithelial tissue are suitable for more absorption of SM applied cutaneous
(Young and Bast 2009). It has been estimated that around 80 % of total dose applied
52
Table 3.1 Nomenclature, molecular formula, and chemical structure of mustard compounds (www.chem.sis.nlm.nih.gov/chemidplus)
Compound CAS number Molecular formula Chemical structure Synonyms

Bis-(2-chloroethyl) sulfide 505-60-2 C4-H8-Cl2-S S HD, agent HD, mustard gas, yperite,
CI CI
LOST, Sulfur mustard
1,2-Bis-(2-chloroethylthio)- 3563-36-8 C6-H12-Cl2-S2 CI S Q, agent Q, sesquimustard,

S CI
ethane 1,8-dichloro-3,6-dithiaoctane
Bis-(2-chloroethylthioethyl)- 63918-89-8 C8-H16-Cl2-O-S2 CI O CI T, agent T, O-mustard, 2-2-Di(3-

S S
ether chloroethylthio)-diethyl ether
2-Chloroethyl chloromethyl 2625-76-5 C3-H6-S-Cl2 S CI Ethane,1-chloro-2-[(chloromethyl)thio]-

CI
sulfide
Bis-(2-chloroethylthio)- 63869-13-6 C5-H10-Cl2-S2 CI CI HK, Bis(2-chloroethylthiomethyl)

S S
methane ether
Bis-1,3-(2-chloroethylthio)- 63905-10-2 C7-H14-Cl2-S2 S S 1,9-Dichloro-3,7-dithianonane

CI CI
n-propane
Bis-1,4-(2-chloroethylthio)- 142868-93-7 C8-H16-Cl2-S2 S CI 1,4-Bis (2-chloroethylthio) butane

CI S
n-butane
Bis-1,5-(2-chloroethylthio)- 142868-94-8 C9-H18-Cl2-S2 S S Pentane, 1,5-bis((2-chloroethyl)thio)-

CI CI
n-pentane
Bis-(2- 63918-90-1 C6-H12-Cl2-o-S2 S O S Ethane,1,1(oxybis(methylenethio))

CI CI
chloroethylthiomethyl)-ether bis(2-chloro-
S. Mostafalou and M. Abdollahi
Compound CAS number Molecular formula Chemical structure Synonyms
Bis-(2-chloroethyl) 538-07-8 C6H13Cl2N CI CI HN-1, Nitrogen mustard (HN-1),
N
ethylamine Ethylbis(2-chloroethyl)amine
Bis-(2-chloroethyl) 51-75-2 C5H11Cl2N CI CI HN-2, Nitrogen mustard (HN-2),

methylamine N Mechlorethamine, Chlormethine,
Mustine
Tris-(2-chloroethyl)amine 555-77-1 C6H12Cl3N CI CI HN-3, Nitrogen mustard (HN-3),

N
Trichlormethine, Trimustine,
2,2,2-Trichlorotriethylamine
3 Basic Pharmacology and Toxicology
CI
53
Table 3.2 Physico-chemical properties of mustard compounds (www.chem.sis.nlm.nih.gov/

chemidplus)
Physical properties SM HN-1 HN-2 HN-3
Melting point (C) 13.5 3.40E + 01 6.00E + 01 4.00E + 00
Boiling point (C) 216 194 87
log P (octonal- water) 2.410 2.02 0.91 2.270
Water solubility (mg/L) 684 160 1.20E + 04 1600
Vapor pressure (mm Hg) 0.11 0.25 65.1 0.011
Henrys law constant (atm-m3/mole) 3.37E-0.5 3.36E-04 8.48E-08 1.85E-06
Atmospheric OH rate constant (cm3/ 7.82E-12 1.59E-11 8.39E-12 1.07E-11
molecule-s)
to the skin evaporates while 10 % absorbs systemically and 10 % remains at the site
of exposure that is responsible for local effects. The rate of dermal absorption can
reach up to 90 % when applied by occlusion with an exposure duration of 6 h
(Hambrook et al. 1993). When administered by inhalation, SM is absorbed 7090 %
through the mucous membrane of the nasal system (Papirmeister et al. 1984).
3.2.1.2 Distribution
Upon entering into the blood, SM highly tends to bind hemoglobin and then gluta-
thione (Hambrook et al. 1993). Because of high lipid solubility, SM and its metabo-
lites can be widely distributed in the body after intravenous or percutaneous
exposure. Regardless of the route of exposure, the equilibrium between blood and
tissue levels of SM is established 5 min after systemic absorption. Maximum levels
can be detected in the lung, liver, and kidney, however, postmortem and in vivo stud-
ies has indicated that SM can be efficiently distributed to the other organs including
fat, brain, muscle, spleen, adrenals, bone marrow, cerebral fluid, and abdominal
skin. Within 5 min after ocular application, SM is shown to be concentrated in the
cornea, but lesser extent can also be found in the iris, lens, and conjunctiva (Axelrod
and Hamilton 1947).
3.2.1.3 Metabolism
In aqueous conditions, SM undergoes intramolecular cyclization producing ethyl-

ene episulfonium ion which is a hyperactive compound and tends to react with
electron rich molecules such as SH and NH2. The main metabolic pathway is
hydrolysis by which SM is converted to thiodiglycol and then s-oxidation create
sulfoxide and sufone. These products are finally conjugated and excreted mainly in
the urine. The major urinary metabolites include glutathione-bis-chloroethyl sulfide
conjugates (45 %), thiodiglycol plus its conjugates (14.4 %), sulfone conjugates
(7 %) and minute amounts of cysteine-bis-(-chloroethyl) sulfone which is
produced under the effect of -lyase on cysteine. It has been estimated that urinary
concentration of thiodiglycol reaches to the peak on post-exposure day 4 and can be
detected in the urine up to 2 weeks. It has the first-order elimination kinetics with a
half-life 1.2 days. Since active metabolites of SM are capable to react with nucleo-
philes found in the structure of DNA and glutathione, some DNA adducts can also
be detected in the urine like N7-(2-hydroxyethylthioethyl)-2-deoxyguanosine and
2-deoxyguanosine derivatives of N7-HETE-guanine (TOXNET 2013).
3.2.1.4 Elimination
The main route of elimination of SM is urinary with the first order pattern and its
metabolites sometimes detected in the urine for up to 3 months. In an experiment on
the rat, urine and feces half-lives of SM were estimated 1.4 and 1.6 days, respec-
tively (TOXNET 2013).
3.2.2.1 Absorption
Most nitrogen mustards, used as chemotherapeutic agents, are administered through

intravenous and oral routes. Cyclophosphamide and ifosfamide are well absorbed
orally, while melphalan has an incomplete rate of oral absorption. Chlorambucil is
also adequately absorbed through oral route (Brunton and Parker 2008).
3.2.2.2 Distribution
Nitrogen mustards are highly reactive and combine rapidly with proteins, DNA, or
other molecules. Therefore, little time after exposure, nitrogen mustards or their
reactive metabolites may not be found in biological fluids or tissues. Nitrogen mus-
tards are rapidly disappearing from blood and low levels can be found in tissues,
among which bone marrow has shown the highest concentration (TOXNET 2004).
The maximum half-life for melphalan and chlorambucil has been estimated 90 min
(Brunton and Parker 2008).
3.2.2.3 Metabolism
Regarding nitrogen mustards, there is limited information mostly related to HN-2

which has been used as a chemotherapeutic drug. Cyclophosphamide is con-
verted to the active metabolites, 4-hydroxycyclophosphamide and its acyclic tau-
tomer aldophosphamide by CYP2B in the liver. Ifosfamide is also hydroxylated
and activated by CYP3A4 but with a slower rate. The main excretory metabolites
of chlorambucil are phenyl acetic acid mustards (Brunton and Parker 2008). The
main metabolites of nitrogen mustards are produced from their hydrolysis in
aqueous environment. Nitrogen mustards HN-1, HN-2, and HN-3 tend to be
hydrolyzed to the related ethanolamines including N-ethyldiethanolamine,
N-methyldiethanolamine, and triethanolamine, respectively. There is no report
on the metabolites derived from conjugation of nitrogen mustards with glutathi-
one. Like sulfur mustard, nitrogen mustards also form protein and DNA adducts.
HN-2 can covalently bind to cysteine-34 residue of albumin and histidine resi-
dues in hemoglobin. Experimentally, HN-2 can give DNA adducts derived from
mono-alkylation of guanine at N7 and adenine at N3, and from crosslinking of
guanine to guanine or guanine to adenine. Mono-alkylation of guanine at N7
gives the major DNA adduct, N-[2-(hydroxyethyl)-N-(2-(7-guanyl) ethyl]
methylamine, whose ratio to that of adenine N3 is 86:14 (TOXNET 2004).
3.2.2.4 Elimination
Ethanolamine metabolites produced from hydrolyzing nitrogen mustards seem to

be excreted unconjugated. Urinary excretion of ethanolamines up to 2 days post-
exposure was recorded less than 0.1 % of the administered dose for HN-1 and HN-2,
and about 0.3 % for HN-3 (TOXNET 2004).
3.3 Mechanism of Action
Although much is known about mustards, the exact mechanism by which these
compounds exert their toxicity is not fully understood.
In an aqueous environment, mustard compounds undergo intramolecular cycli-
zation by eliminating a chloride ion on the basis of nucleophilic substitution reac-
tions, and produce intermediate metabolites known to be responsible for cytotoxicity
of these compounds. Sulfur mustard and nitrogen mustard give, respectively, episul-
fonium ion and immonium ion which are reactive and strongly tend to bind cova-
lently to nucleophilic molecules in the cell. Nucleophiles such as nitrogen in the
bases of nucleic acids (DNA and RNA), and sulfur in the SH groups of proteins are
attractive targets for mustard alkylating action which can result in the formation of
DNA and protein adducts (Fig. 3.1).
3.3.1 DNA Damage
Among cellular macromolecules, DNA is the most sensitive target for attack by
mustard compounds so that mustards have become famous as DNA alkylating
agents. DNA damaging effects have been studied in more detail for nitrogen

CI +
S S
CI CI Ethylensulfonium ione
Intramolecular CI
cyclization, 1st
Sulfur mustard
O
N
NH
N
Guanine (in DNA)
N NH2
O + O
S CI S
+ +
CI N NH N NH
Intramolecular
N N NH2
cyclization, 2nd N N NH2
O
N
HN
DNA adduct
H2N N N
Guanine (in DNA)
O O
S +
+ N
N NH
HN
N N N NH2
H 2N N
Single or double strand DNA cross-link
Fig. 3.1 Mustard induced formation of DNA adduct and single/double strand DNA cross-link
mustards which have had chemotherapy application, as well. Sulfur and nitrogen
mustards have two chains, each can undergo cyclization reaction which is needed
for alkylation, so they are dysfunctional alkylating agents. This property allows
them to form cross-link between DNA strands, plus forming adducts. As mentioned
earlier, for sulfur mustard, the major adducts are resulted from monoalkylation of
guanine at N7 (60 %), monoalkylation of adenine at N3 (16 %), and bifunctional
alkylation of guanine at N7 in the form of interstrand or intrastrand cross-link
(16 %). Adenine and guanine are purine bases whose alkylation lead to their removal
from DNA by the enzymes endonucleases or spontaneously. The produced apurinic
sites are attracting substrate for attack by the other specific enzymes, apurinic endo-
nucleases, resulting in DNA breaks, which further are a good target for exonucle-
ases (Papirmeister et al. 1985). These events arrest cell cycle at G1 to give time for
repairing DNA and on the other hand, activate the DNA repair system. The main
DNA repair enzyme is poly (ADP-ribose) polymerase (PARP) which uses nicotin-
amide adenine dinucleotide (NAD+) as the cofactor. Activation of this enzyme can
lead to depletion of NAD+, a situation in which NAD+ dependent step of glycoly-
sis, catalyzed by glyceraldehyde 3-phosphate dehydrogenase, is inhibited. Thus,
formation of pyruvic acid as the first entry into the citric acid cycle in glucose
metabolism is prevented and the rate of ATP production decreases. In order to com-
pensate this situation, the other metabolic pathway, hexose monophosphate shunt
(pentose phosphate pathway) which uses NADP+ as the cofactor, is activated.
Activation of this pathway has been shown to be associated with the release of some
protease enzymes like plasminogen activator which can damage structural proteins
and trigger inflammatory cascades eventuating in blister formation (Schnyder and
Baggiolini 1980).
3.3.2 Thiol Depletion
Another hypothesis for the mechanism of mustard cytotoxicity came from their
ability to alkylate the functional thiol groups. Thiol groups are reducing agents
which exist in the cells at a concentration around 5 mM. Glutathione is the main
cellular thiol, which defends cell against the damaging effects of free radicals par-
ticularly, reactive oxygen species. Glutathione reduces oxidant elements by donat-
ing an electron and being itself oxidized to glutathione disulfide. Glutathione
reductase is an enzyme responsible for reducing back glutathione by using NADPH
as the electron donor. Depletion of cellular glutathione reservoir due to alkylation
by mustard compounds can lead to excessive amounts of oxygen radicals in the cell.
Reactive oxygen species can react with phospholipids in the cell membrane and
produce lipid peroxides which in turn initiate chain reactions of lipid peroxidation
leading to cell membrane breakdown (Vijayaraghavan et al. 1991). In addition to
glutathione, there are some antioxidant enzymes like superoxide dismutase, cata-
lase and glutathione peroxidase, which defend cells against the damaging effects of
free radicals. Alkylation of SH groups in the structure of this enzyme can result in
their dysfunction in defending cell against free radicals and consequently oxidative
stress. Dermal application of sulfur mustard has been found to decrease the activity
of these enzymes in internal organs such as blood cells, platelets, spleen, brain, and
liver (Husain et al. 1996).
3.3.3 Inflammation
In oxidative imbalanced states, activation of inflammatory cascades is expected to

present, as many inflammatory mediators are involved in production of free radicals
and reactive oxygen species. Regarding mustard compounds, there has been much
evidence of elevated levels of cytokines and secondary inflammatory response in
different organs. Therefore, inflammation has been proposed as a mechanism by
which these chemicals exert their toxic effects. TNF-, IL-1, IL-1, IL-6, and IL-8
are the main inflammatory cytokines reported to be increased with exposure to mus-
tard compounds in different studies. It has also been shown that sulfur mustard
activates NF-B pathway and mitogen activated protein kinase (MAPK) cascades
including extracellular signal-regulated kinase (ERK), Jun-N terminal kinase
(JNK), and p38 MAPK (Kehe et al. 2009).
3.3.4 Disrupted Calcium Homeostasis
Imbalanced oxidative state causes calcium influx into the cytoplasm from both
extracellular and intracellular (endoplasmic reticulum) sources. Alkylation of
sulfhydryl groups in the structure of proteins responsible for calcium transloca-
tion (calcium stimulated, magnesium dependent ATPase) can also disrupt cal-
cium homeostasis. Increased calcium concentration stimulates the enzyme
phospholipase A2 which releases arachidonic acid from the cell membrane and
eventually breakdown cell membrane. Sulfur mustard was reported to induce a
series of these events including raised calcium concentration, activated phos-
pholipase A2, and release of arachidonic acid (Ray et al. 1995). On the other
hand, calcium-calmodulin complex can activate nitric oxide synthase (NOS)
leading to the production of nitric oxide. In this regard, reactive nitrogen species
like peroxynitrite (ONOO) have presented an essential role in mustard induced
cytotoxicity (Korkmaz et al. 2006).
3.3.5 Apoptosis
It has been proposed that alkylating mustards exert their cytotoxic effects through
induction of apoptosis which can be secondary to the arousal of mentioned cas-
cades particularly DNA damage. The tumor suppressor gene, p53, also known as
the guardian of the genome is activated in DNA damaged situation in order to
prevent mutation. In the activated form, p53 induce cell cycle arrest at G1 in
order to give time for fixing DNA damage by activating repair enzymes. If the
repair mechanism can not save the cell, p53 provokes programmed cell death,
apoptosis, to discard the damaged cell. Nitrogen mustard has been reported to
induce apoptosis in association with activated p53 and cell cycle arrest at G1
(Bhatia et al. 1995). Regarding SM, several reports implicate on the activation of
both extrinsic (death receptor) and intrinsic (mitochondrial) pathways responsi-
ble for apoptosis. SM has been shown to up-regulate the members of the death
receptor pathway of apoptosis, including Fas receptor/Fas ligand and TNF-. In
case of mitochondrial pathway, SM has been reported to increase permeability of
mitochondrial outer membrane, releases cytochrome c and activate caspase
downstream (Kehe et al. 2009). Involvement of calcium-calmodulin pathway in
SM-induced apoptosis and terminal differentiation of keratinocytes has also been
documented (Rosenthal et al. 1998).
3.4 Conclusion
Mustard compounds are known as vesicant agents among which sulfur mustards have
been used in chemical warfare and the other group, nitrogen mustards, have never been
used in the battle field. Some nitrogen mustards have medicinal use as chemotherapeu-
tic agents in treatment of neoplastic diseases. Because of low vapor pressure and high
lipophilic property, sulfur mustards tend to persist near the ground state which make
them suitable as a chemical warfare agent. When exposed, sulfur mustards are well
absorbed through inhalational, dermal, and ocular routes and distributed mainly toward
the lungs, kidneys, and liver. The main route of absorption for medicinal nitrogen mus-
tards is through intravenous and oral administration. The reactive electrophilic metab-
olites of mustards attack the nucleophilic molecules in the body, such as SH and
guanine in the structure of proteins and DNA, respectively. DNA alkylation and subse-
quent alterations in cell homeostasis and growth is known as the same cytotoxic mech-
anism for both pharmacological and toxicological applications of mustard compounds.
DNA damage induced hyper activation of DNA repair enzymes, depletion of cellular
thiol content, disturbed homeostasis of calcium ion in the cell, expression of inflamma-
tory cytokines, and arousal of apoptosis cascade have been proposed and somehow
confirmed as the mechanisms by which mustard compounds exert their effects.
However, some aspects of mustards cytotoxic mechanism need more clarification in
order to find clues toward development of newer approach for both antineoplastic
application and antidotal management of poisoning with mustard compounds.
Glossary
ATP Adenosine triphosphate

ATSDR Agency for Toxic Substances and Disease Registry
CWC Chemical weapon convention
CYP Cytochrome P450
ERK Extracellular signal-regulated kinases
IL-1 Interleukin-1 alpha
IL-1 Interleukin-1 beta
IL-6 Interleukin-6
IL-8 Interleukin-8
JNK c-Jun N-terminal kinases
MAPK Mitogen activated protein kinase
NAD Nicotinamide adenine dinucleotide
NADP Nicotinamide adenine dinucleotide phosphate
NOS Nitric oxide synthase
OPCW Organization for the Prohibition of Chemical Weapons
PARP Poly (ADP-ribose) polymerase
SM Sulfur mustard
TNF- Tumor necrosis factor alpha
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after cutaneous application of 35S-sulphur mustard in rat and comparison with human blood
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Papirmeister B, Gross CL, Meier HL, Petrali JP, Johnson JB (1985) Molecular basis for mustard-
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sulfur mustard in Iranian veterans. Daru 20(1):51
Rosenthal DS, Simbulan-Rosenthal CM, Iyer S, Spoonde A, Smith W, Ray R, Smulson ME (1998)
Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a
Ca2 + calmodulin and caspase-dependent pathway. J Invest Dermatol 111:6471
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Chapter 4
Clinical Pharmacology and Toxicology
Adel Ghorani-Azam and Mahdi Balali-Mood
Contents
4.1 Introduction .................................................................................................................... 64
4.1.1 Sulfur Mustard ................................................................................................... 65
4.2 Clinical Pharmacology of Mustard Compounds ............................................................ 66
4.2.1 Routes of Exposure ............................................................................................ 66
4.2.2 Distribution ........................................................................................................ 68
4.2.3 Metabolism ........................................................................................................ 69
4.2.4 Mechanism of Action......................................................................................... 69
4.3 Therapeutic Uses of Mustard Compounds ..................................................................... 72
4.3.1 Sulfur Mustard ................................................................................................... 72
4.4 Acute Toxic Effects of MCs........................................................................................... 75
4.4.1 The Skin ............................................................................................................. 75
4.4.2 Respiratory System ............................................................................................ 76
4.4.3 The Eyes............................................................................................................. 77
4.4.4 Oral and Gastrointestinal Tract Injury ............................................................... 77
4.4.5 Nervous System Injury....................................................................................... 77
4.4.6 Renal Dysfunction ............................................................................................. 77
4.4.7 Hematological Effects and Immunotoxicity ...................................................... 78
4.5 Clinical Manifestations of SM Poisoning ...................................................................... 78
4.5.1 Dermatologic Symptoms ................................................................................... 79
4.5.2 Gastrointestinal Symptoms ................................................................................ 79
4.5.3 Respiratory Symptoms ....................................................................................... 80
4.5.4 Acute Effects in the Eyes ................................................................................... 80
4.6 Chronic Intoxication of Mustard Gas ............................................................................ 80
4.7 Delayed Toxic Effects of Mustard Gas .......................................................................... 82
4.7.1 Long-Term Dermal Complications .................................................................... 82
4.7.2 Delayed Respiratory Effects .............................................................................. 83
4.7.3 Delayed Eyes Effects ......................................................................................... 83
4.7.4 Reproductive System ......................................................................................... 84
A. Ghorani-Azam M. Balali-Mood, MD, PhD (*)

Medical Toxicology Research Center, Faculty of Medicine,
e-mail: mbalalimood@hotmail.com

64 A. Ghorani-Azam and M. Balali-Mood
4.7.5 Cardiac Associated Complications .................................................................... 84

4.7.6 Genotoxicity....................................................................................................... 84
4.7.7 Carcinogenicity .................................................................................................. 85
4.8 Treatments of SM Poisoning.......................................................................................... 86
4.8.1 New Therapeutic Approaches ............................................................................ 88
4.9 Conclusion ..................................................................................................................... 90
Glossary .................................................................................................................................. 90
References ............................................................................................................................... 91
Abstract Sulfur mustard (SM) and nitrogen mustard (NM) are the two types of
mustard compounds (MCs). SM has mainly been used as a chemical warfare agent
(CWA), but NM has been administered as an anti-cancer drug. MCs are alkylating
agents and initially synthesised for military purposes as a chemical blistering agent.
However, they were also used as medications in the treatment of several diseases
such as psoriasis and variety of cancers. MCs, especially SM can cause acute and
chronic toxicities, particularly acute toxic effects and complications in the eyes,
lungs, kidneys, skin and other vital organs in human. Several therapeutic strategies
have been proposed so far to treat these complications, but no specific antidote has
been introduced for these health problems. Conventional medical treatments with
antioxidants e.g. N-acetyl cysteine and sodium thiosulfate and some anti-
inflammatory drugs such as corticosteroids have been used, but their effects are not
satisfactory. Nowadays, some new therapeutic strategies such as antimicrobial pep-
tides, gene and stem cell therapy, and herbal medicines have been proposed for the
treatment of SM complications.
In this chapter, we have reviewed clinical pharmacology of NM and acute, chronic
and late clinical complications of SM poisoning in all affected organs. In addition, we
have discussed conventional treatment as well as some new therapeutic approaches.
Keywords Sulfur mustard Nitrogen mustard Acute effects Chronic Poisoning

Delayed toxicity Mustard gas Intoxication
4.1 Introduction
There are two types of mustard compounds (MCs), sulfur mustard (SM) and nitro-
gen mustard (NM). MCs are not part of or related to the mustard plant. The name
mustard refers to the smell of these agents. Since MCs actively cause blistering and
tissue damage, they are categorized as vesicant or blistering agents. These terms are
also used by the Organization for Prohibition of Chemical Weapons (OPCW).
Producing MC particularly SM is simple and cheap, it is thus the most applicable
and potential candidates as a chemical weapon for military use and terrorism
(Balali-Mood and Hefazi 2005a).
4 Clinical Pharmacology and Toxicology of Mustard Compounds 65
Sulfur mustard (SM), generally known as mustard gas is an alkylating and blistering
agent with chemical formula of C4H8Cl2S and molecular weight of 159.08 g/mol.
SM with an IUPAC name of bis (2-chloroethyl) sulfide contains two chloroethyle
linked to a sulfur atom, which is not naturally found in the environment. It can be
easily produced by a combination of ethylene with sulfur chloride in a chemical
reaction known as Levinstein process. SM is also generally known as H agent in
military term, which refers to undistilled mustard with impurities. HD or HS is
considered as pure SM. Its color may differ depending on the type and purity, but it
is generally a white brown and oily chemical with melting and boiling point of
around 14.4 and 217.5 C, respectively (Balali-Mood and Hefazi 2005a). As a
result, it turns to solid in temperature below 14 C. Therefore, SM is a good choice
for use as chemical weapon in warm or hot geographical regions, because at higher
temperature it decomposes to highly reactive and toxic sulfur and chlorine radicals,
which may intensify the toxic effects. Under normal circumstances, SM can remain
up to 2 days in the environment, but may last several months under very cold condi-
tions (Balali-Mood et al. 2008).
SM is a hazardous chemical warfare agent (CWA) that has numerous acute and
long-term toxic effects on respiratory, the eyes, skin, CNS, cardiovascular, hema-
tology and immune system. In addition, SM causes histopathological changes in
mucous membrane and cells of these vital organs (Balali-Mood et al. 2008;
Lewisite 1993).
Due to its lipophilic nature, SM is readily absorbed by tissues and cells. In recent
decades, SM has been clinically used to treat psoriasis and other skin diseases (Illig
et al. 1979), but currently it has no further clinical application. Both liquid and its
volatile forms are blistering and may be used as a chemical warfare in battlefield.
Because SM is lipid soluble and, predominantly vaporous, it can enter the body by
inhalation, via the skin, the eyes and even by ingestion of SM contaminated food
(Malhotra et al. 1999; Ganesan et al. 2010). Although SM is a powerful irritant
chemical; nevertheless, length and frequency of exposure are major factors influ-
encing the intensity of health impacts (Poursaleh et al. 2012).
Nitrogen mustard (NM) is another potential chemical warfare agent with high struc-
tural similarity to SM. There are several types of nitrogen mustards (HN-1, HN-2,
HN-3), which are mainly differ in substitution on the 3rd position of nitrogen.
Although NMs are harmful chemical materials with great potential as a CWA, there
is no report of using them in a combat (Keyes 2005). This is mainly due to the fact
that the storage of NM is rather hard. While SM is dominantly used in the war,
nitrogen derivatives such as mustargen have some peaceful applications in pharma-

cology and using as anticancer agents in chemotherapy. Historical experience of
exposure showed that MCs destructively reduce the number of lymphocytes (Hirsch
2006). Similar to SM, NM is a nonspecific alkylating and blistering agent, which
was first introduced in early 1930 by chemically modifications of SM (Pechura and
Rall 1993). Later, systemic administration of a NM compound known as mustine
was successfully used in treatment of lymphoma as a new cancer chemotherapeutic
agent at Yale University (Goodman et al. 1946). It was the first chemical, which was
used clinically in treatment of tumor cells, but now due to excessive toxicity it is no
longer commonly used. However, other NM compounds have been developed,
which are now widely used as chemotherapeutic agents. Anti-mitotic and anti-
cancer properties of MCs particularly NMs are mostly mediated by alkylation of
N-7 atom of guanine residue on DNA strands, which leads apoptosis in affected
cells (Polavarapu et al. 2012).
4.2 Clinical Pharmacology of Mustard Compounds
Several factors such as length of time, frequency and severity of the exposure, and
other environmental factors such as presence of alkalies in biological environments,
atmospheric conditions, temperature and wind determine the level of exposure and
therefore the level of toxicity. For example, the toxicity of mustard gas increases by
rising the temperatures, whereas it may decrease in rainy weather. Other biological
characteristics affecting pharmacodynamic and pharmacokinetic of MCs are gen-
der, age, immune and genetically susceptibility of individuals (Ghabili et al. 2011).
4.2.1 Routes of Exposure
Effects of MCs depend mostly on the amount, duration, frequency and the route of
exposure. Therefore, the route of exposure may determine the type and severity of
subsequent acute and chronic effects and complications. Route of exposure undeni-
ably determines the rate of absorption, biotransformation, and metabolism of these
agents. As expected, inhalation, eye contact, oral and dermal exposure are major
entry sites of SM, whereas oral and injection are the normal route of entries of NM
(Xu et al. 2014).
4.2.1.1 Inhalation
Inhalation is an important entry site of toxic gas like SM especially when it is used
in battlefields. This will happen when the toxic agents are in small size, or vaporous.
SM is readily absorbed in the upper respiratory tract when inhaled and affect the
lungs which may lead to pulmonary edema in severe acute intoxication. In 2014, a
new SM vapor inhalation exposure system was designed and used to evaluate accu-
rate effects of controlled inhaled doses of SM on respiratory system (Perry et al.
2015). Inhalation of mustard gas may result in severe nasal epithelial degeneration.
The lower respiratory system is less affected by SM, because it is highly reactive
and affects more the upper airways (Weber et al. 2010). Death from exposure to
mustard gas usually occurs due to respiratory obstruction. But, acute mortality due
to exposure to SM is very rare. This is partly due to the protective effects of a tissue
plasminogen activator which prevents airway cast obstruction and improves respira-
tory function (Veress et al. 2015). Air way obstruction due to the inhalation of SM
may occur due to formation of fibrin casts. Hence, fibrin-degrading and plasminogen-
activating capabilities of the airways are inhibited in response to mustard agents
leading to air way obstruction (Rancourt et al. 2014).
4.2.1.2 Eye Contact
Eyes are the most susceptible target organs and are at higher risk of contact to for-
eign toxicants. SM in both gas and liquid phase can pass through corneal surface,
and cause damage to the eyes and distort it leading to temporary blindness. Exposure
to mustard gas especially through the eye contact leads to structural and functional
changes such as persistent epithelial lesions and progressive corneal degeneration in
ocular surface (McNutt et al. 2013).
4.2.1.3 Oral Ingestion
Ingestion of SM is another possible site of entry, but no animals or humans data are
available about toxicokinetic studies in oral exposure. Food and water contaminated
by SM is the most important route of intoxication when orally exposed with mus-
tard agents. SM may be absorbed in oral cavity, esophagus and the lower gastroin-
testinal tract (Balali-Mood et al. 2008). On the other hand, cyclophosphamide
mostly known as Cytoxan is an active form of NM for oral administration. But,
because it is remarkably more toxic than azathioprine and methotrexate as immuno-
suppressive and anti-cancer agent, respectively; therefore, NMs are limitedly used
in oncology (Ben-Ari 2004).
4.2.1.4 Dermal Exposure
Solubility in water and lipid is the most important factor influencing the absorption
of mustard agents from the skin. Because of lipophilicity, MCs particularly SM are
absorbed and penetrate the skin. Its liquid or saturated form can penetrate human
skin at a rate of 14 mg/cm2/min at 21 C. Nevertheless, it was shown that only
10 % of SM can be absorbed by contact to the skin (Renshaw 1947). Any increase
in ambient temperature causes increased penetration to the skin and cells, because
the rate of its hydrolysis reaction is increased by rising temperature. Therefore, the
rate of diffusion is proportional to concentration in situ (absorbed dose), tempera-
ture and humidity. In another word, skin damages are more severe in humid and
warm condition (Balali-Mood and Hefazi 2005a, b).
It is reported that approximately 20 % of the SM is absorbed through the skin and
approximately 70 % of this chemical remains and is concentrated in the epidermis.
Long-term effects of exposure to SM can cause skin pruritus, edema, inflammation,
and skin cell death. Various cutaneous lesions such as itching burning and erythema
even in unexposed areas, which may remain for several years have been observed
(Ghanei et al. 2010). Animal experiments in guinea pigs have shown histopatho-
logic changes including programmed cell death, necrosis, blister formation, and
delayed re-epithelialization (Smith et al. 1995). Structural changes in hair follicles
and sebaceous glands are other major complications resulted from dermal exposure
of mustard agents which can lead to follicular and interfollicular epithelial damage
in hairless mice (Joseph et al. 2014). It is suggested that these structural changes are
associated with inflammation and up-regulation of several pro-inflammatory media-
tors such as myeloperoxidase and cyclooxygenase-2, deposition of collagen in the
dermis, hyperplasia, hyperkeratosis and DNA damage (Joseph et al. 2011).
Similar to SM, NM can also cause skin damage such as increased epidermal
thickness, epidermal-dermal separation, epidermal denuding, parakeratosis, hyper-
keratosis, necrosis, edema, and hyperplasia. These cutaneous histopathological
changes induced by exposure to NM are similar to those reported from SM expo-
sure (Tewari-Singh et al. 2014).
4.2.2 Distribution
Diffusion and influx of toxic materials into the body depends mostly on the adhesive
strength of the chemical to the exposed era, solubility and lipophilicity, the value of
the diffusion coefficient, physical state and its biological reactivity. Regardless of the
route of exposure, when MC enters the body, they are distributed among various tis-
sues. Later they are concentrated in the liver, kidneys, and lungs. Studies showed that
after intravenous (IV) injection of SM into a rabbit, most of it may excrete from the
body after 72 h (Boursnell et al. 1946). Radioisotope labelling of SM also showed
that 8090 % of SM absorbed in the body are excreted mainly in the urine (Davison
et al. 1961). Lethal dose (LD50) and other toxicological parameters for mustard gas
vary among different species, but LD50 is reported about 100 mg/m3 for 10 min when
inhalation is the major route of exposure (Somani and Babu 1989). Also it is known
that percutaneous administration of SM is more effective than the other routes such
as oral and subcutaneous. These findings have been confirmed by histopathological
examination of the liver, spleen, and lungs (Vijayaraghavan et al. 2005). Identification
of urinary metabolites exhibited that SM is actively involved in metabolic processes
including oxidation, hydrolase and conjugation (Black et al. 1992).
Intravenous injection of [14C] SM (10 mg/kg) in rats showed that its distribution
is rather fast, and the radiolabelled SM appears in the liver, kidney, lungs, intestine,
and stomach. But, a small percent of SM are detected in fat tissues, indicated that
this tissue does not actively involved in metabolism, biotransformation or storage of
mustard agents (Maisonneuve et al. 1994). Animal studies on inhalation of 14C-SM
vapor in rats and cutaneous exposure in hairless guinea pigs showed that Inhaled
SM is rapidly distributed throughout the body within 2 h after exposure. But, data
revealed that dermal exposure has rather limited distribution to blood and kidneys
and the majority (>90 %) of SM in guinea pigs remained in the skin. However, the
rate of uptake is greater in the first 2 h post exposure, also in both type of exposure,
urine is the major route of excretion (Benson et al. 2011). It was reported that there
were similar SM absorption rates in different parts of the skin (Logan et al. 1999).
4.2.3 Metabolism
Metabolism, biodegradation, and biological actions of the mustard agents depend

on their reactivity and the type of chemical reactions they involve. Formation of
heterocyclic ions by the interaction between the nucleophilic central atom and the
negative inductive effect of the chlorine atoms in the side chains is of the greatest
chemical importance. The first step in the hydrolysis of SM is the formation of
transient cyclic sulphonium cation, and then the cation reacts quickly with water to
produce hydroxyl compounds. Oxidising agent can also react with SM to form
sulphoxide or sulphone (Malhotra et al. 1999). According to aforementioned, mus-
tard agents can react with alcohol, thiol, and amine groups; therefore, it can interact
with carbohydrates and hydroxyl groups on protein and peptides side chains, phos-
pholipids or nucleotides containing amine groups in the structure of DNA and
RNA. Due to the lack of human data on metabolic pathways of mustard agents, no
trustful information is available on metabolism, biotransformation and toxicokinet-
ics of mustard compounds. But animal studies suggested that conjugation with glu-
tathione is more important than hydrolysis in metabolism of MC (Roberts and
Warwick 1963).
4.2.4 Mechanism of Action
Mustard agents are chemically active and react with large number of biological
macromolecules. SM irreversibly alkylates nucleic acids and proteins, specifically
purine bases of DNA (Shakarjian et al. 2010). Numerous mechanistic pathways
have been proposed for SM induced injuries, but DNA crosslinking, nicotinamide
adenine dinucleotide (NAD) depletion, and inactivation of sulphydryl-containing
proteins and peptides are the most plausible mechanism underlying the toxicity of
SM. Production of a reactive sulphonium ion may be a critical step in all of the
mechanism described. This ion has chemical affinity to nucleophiles such as amines
and nitrogen atoms of RNA and DNA bases and sulfur atoms in SH-groups of pro-
teins and peptides. Below, some important mechanistic pathways of mustard agents
are described.
4.2.4.1 Inflammatory Response
Many inflammatory factors including interleukin (IL)-1, IL-1, IL-6, IL-8,

granulocyte-monocyte colony stimulating factor, chemokines growth-regulated
oncogene (GRO), leukotriene B4, monocyte activating protein-1 (MAP-1), and
TNF- are produced in response to SM exposure. Studies showed that increasing
the level of these mediators regardless of exposure to mustard agents, cause similar
symptoms as observed in SM induced injuries (Ruff and Dillman 2007). Also,
expression of some important inflammatory mediators such as IL-8, MAP-1, IL-1
beta, and GRO leads to erythema; therefore, inhibition of these modulators and their
mechanistic pathways may limit the injuries. It was shown that the expression of
TNF- alters human epidermal keratinocyte sensitivity to SM induced cell death
(Qabar et al. 2005).
NF-kappa B is suggested to be involved in inflammatory response and cytokine
production resulted from exposure to MC. Evidences also suggest that NF-B is
responsible for SM-induced cell death (Baeuerle and Henkel 1994). Several hours
after exposure to sulfur mustard, NF- B is activated and modulated the release of
cutaneous inflammatory factors, but p53 is activated through the phosphorylation of
Ser-15 residue a few minutes after exposure to SM which is mostly involved in
apoptosis and tumor suppression (Minsavage and Dillman 2007).
It was also shown that SM may activate and phosphorylate several molecular
targets including transcription factors (TFs) through the induction of other signaling
molecules such as p38 MAP kinase and p90 ribosomal S6 kinase (p90RSK). The
activated TFs regulate the expression of inflammatory cytokines, which are involved
in SM-induced inflammatory cytokine production (Dillman et al. 2004). Hence,
inhibiting these regulators and inflammatory modulators such as p38 may theoreti-
cally reduce inflammatory cytokine production in response to SM-induced tissue
injuries. Findings showed that SB203580, a specific inhibitor of pyridinyl imidazole
in the p38-MAPK signaling pathway reduces the production of inflammatory mod-
ulators such as IL-6, IL-8, TNF- and IL-1 (Dillman et al. 2004).
4.2.4.2 DNA Alkylation
DNA crosslink is an important mechanism of intoxication with mustard agents, and

it is considered as the primary initiator of the cellular response that leads to the clini-
cal symptoms (Papirmeister et al. 1969). It is proposed that serine proteases and
matrix metalloproteases are involved in SM induced tissue injury. Poly (ADP-
ribose) polymerase (PARP) is a family of proteins involved in a number of cellular
processes including DNA repair and apoptosis. Activation of PARP due to DNA
damage leads to NAD+ depletion and thus induction of hexose monophosphate
shunt, which finally results in enhanced synthesis and release of proteases. These
proteases that include the digestive enzymes are responsible for necrosis, cell death,
cutaneous injuries and other cutaneous pathogenesis (Xu et al. 2006). There are
other mechanisms such as apoptosis proposed for the pathogenesis of mustard
agents. Molecular events such as nuclear factor-B (NF-B), tumor protein p53,
mitogen-activated protein kinase p38, PARP, Fas, calcium, and calmodulin are
actively involved in the molecular mechanisms of SM induced cell death, inflamma-
tion, and other pathogenesis.
4.2.4.3 NAD+ and ATP Depletion
ADP ribosylation of nuclear proteins is a common post-translational process in

eukaryotic cells in which poly (ADP-ribose) synthetase transfers ADP-ribose from
Nicotinamide adenine dinucleotide (NAD) to acceptor proteins. ADP ribosylation is
involved in numbers of biological processes including DNA repair of defected DNA,
and gene expression (Grube and Burkle 1992). ADP-ribosylation is catalyzed by
PARPs and use NAD as substrate; therefore, increasing level of ADP-ribosylation
would lead to a drop in cellular level of NAD+ and ATP. Hence, NAD+ depletion may
lead to blister formation through the activation of signal for the PARP-mediated cell
death (Alvarez-Gonzalez et al. 1986). According to aforementioned, exposure to any
chemicals including alkylating agents which cause DNA damage may lead to skin
damage. But, it should be noted that PARP inhibitors do not necessarily reduce the
symptoms of vesication and skin injuries, and may not be used as antidote in SM
induced skin injury (Mol et al. 1991). Nevertheless, data shows that necrosis in lym-
phocytes due to exposure to SM can be reduced by PARP inhibitors (Meier and
Millard 1998).
4.2.4.4 Ca Ion Calmodulin Signaling Pathway
Some studies have shown that possibly Ca2+- calmodulin signaling pathway is
involved in intoxication of some toxicants such as SM (Simbulan-Rosenthal et al.
2006). Increasing the level of cytosolic calcium and hemostasis imbalance of this
ion due to damage in Ca2+ channels is thought to have critical role in SM induced
fibroblasts and keratinocytes toxicity (Hua et al. 1993; Mol and Smith 1996).
Studies showed that mustard agents particularly SM reduces cell responsiveness to
some extracellular signals which give raise the intracellular Ca2+ levels. Increasing
the level of intracellular Ca2+, in part leads to induction of apoptosis markers such
as p53, suppression of Bcl-2, and activation of caspase-3 (Rosenthal et al. 1998;
Rosenthal et al. 2000). It was shown that calmodulin antisense RNA prevents the
activation of caspase-3 and other proapoptotic protein and proteolytic factors, which
are only caused by Ca2+- calmodulin signaling pathway. This means that, the
induction of caspase-3 is not affected when a signaling pathway other than Ca2+-
calmodulin in involved (Ruff and Dillman 2007).
4.2.4.5 Oxidative Stress
Production of reactive oxygen and nitrogen species is suggested to contribute in

pathophysiology and the early effects of SM intoxication. Reactive species may
aggravate DNA breakage and modification of other macromolecules. These modifi-
cations are shown to be associated with DNA damage and necrosis (Chien et al.
2004). Lipid peroxidation also occurs due to the formation of high reactive oxygen
species leading to glutathione and NAD depletion.
4.3 Therapeutic Uses of Mustard Compounds
Previously, SM has been used for the treatment of malignant tumors as a potent
chemotherapeutic agent, but due to its toxic effects on human organs, SM have now
been used as a topical ointment for the treatment of psoriasis and mycosis fungoides
only in low concentrations (Illig et al. 1979; Aghaei and Moradi 2010). No further
findings on the therapeutic effects of this compound have been reported so far.
Mechlorethamine or Mustargen (HN2) is a type of NM which has non-military use

and is prescribed as a chemotherapeutic agent (Ganesan et al. 2010). Bendamustine
a drug with chemical similarity to nitrogen mustards is an alkylating agent which is
recently used to treat disease such as non-Hodgkins lymphoma, chronic lympho-
cytic leukemia (Cheson and Rummel 2009). NM was the first immunosuppressive
alkylating agent that was used for the treatment of renal disease. Adrenal corticoste-
roids such as prednisone have been widely used as an effective therapy for most
children with the idiopathic nephrotic syndrome, but a small percentage of patients
have not responded to this therapy. For this purpose, NM (HN-2, mechlorethamine)
therapy has been used during 19501970 in treating children with nephrotic syn-
drome unresponsive to corticosteroids. No significant complication has been
reported to the HN-2 therapy except germinal aplasia and a small abscess at the site
of infusion. Gastrointestinal complications have also been controlled by prior treat-
ment with Prochlorperazine (Fine et al. 1976). High dose of NM has been widely
used to treat many un-operable cancers in last several decades (Duff et al. 1961).
But, clinical reports during 19501970 showed that beside some helpful effects,
NM therapy of nasopharyngeal carcinoma and other malignant disease cause fatal
cerebral toxicity in patients leading to death (Clifford et al. 1965).
4.3.2.1 Mechlorethamine
Mechlorethamine (HN2) which known as mustine, is classified as an alkylating

agent and is used for cancer chemotherapy. HN2 has some beneficial effects in
patients with Hodgkin disease and other lymphomas. NM and its related compounds
were the first used cancer chemotherapeutic drugs which could effectively suppress
the immune system. NMs were also successfully applied to treat other non-cancer
diseases such as rheumatoid arthritis, systemic lupus erythematosus, and Wegener
granulomatosis (Ben-Ari 2004).
4.3.2.2 Cyclophosphamide
Cyclophosphamide generally known as Cytoxan is another orally active form of

NM that is used alone or in combination with other medications to treat cancer and
non-cancer disease both in adults and in children (Yule et al. 2004). It is used in
combination with corticosteroids for remission and preserving kidney function in
people with severe lupus nephritis (Ben-Ari 2004). Since Cytoxan is approved by
the FDA in 1959, it is used in combination with other drugs to treat Hodgkins and
non-Hodgkins lymphoma and other types of cancers (Stolzenbach and Garbrecht
1979). A study showed that cyclophosphamide does not inhibit cancer cell growth
immediately after administration, its anticancer properties are mediated by deacti-
vation or reduction of nucleotidyltransferases activity almost 48 h post-administration
(Wheeler and Alexander 1969).
Other NM compounds which are used for treatment of cancers include chloram-
bucil, uramustine, ifosfamide, melphalan, and bendamustine (Mattes et al. 1986).
4.3.2.3 Chlorambucil
Chlorambucil had been previously used as a bifunctional alkylating agent in the

treatment of neoplastic and autoimmune diseases, systemic lupus erythematous,
acute and chronic glomerular nephritis, nephrotic syndrome (Kalita et al. 2014;
Detke et al. 1980). Cytotoxic anticancer properties of chlorambucil may be medi-
ated by alkylating and consequently down-regulating the transcription of a histone
gene, which results in the inhibition of cancer cell growth, especially chronic
myelogenous leukemia and malignant B Lymphocytes without considerable toxic-
ity to the normal cells (Chou et al. 2008). Another possible mechanism of action for
chlorambucil is the adduct formation and then inhibition of DNA double-strand
break damage repairing through the prevention of DNA-dependent protein kinase

(Amrein et al. 2007).
4.3.2.4 Uramustine
Uramustine or uracil mustard with the chemical formula of C8H11Cl2N3O2 is an

alkylating agent, which is typically used as a chemotherapeutic drug to treat lym-
phatic malignancies such as non-Hodgkins lymphoma. Similar to other alkylating
agents, uramustine acts through its DNA binding properties (Baraldi et al. 2002). It
is suggested that antitumor properties of uramustine may be due to its selective
interaction with DNA sequences at specific regions (Hartley et al. 1988).
4.3.2.5 Ifosfamide
Ifosfamide is known as an antitumor drug for the treatment of malignant lymphoma

and osteogenic sarcoma, which is converted to its active form after being metabo-
lized by cytochrome P450 (Maki 2012). Nonetheless, beside its antitumor proper-
ties, it may cause neurotoxicity and nephrotoxicity due to bisalkylating action
(Storme et al. 2009; Rodriguez et al. 1982). Recently, it has been shown that these
side effects can be reduced by making some modification in the structure of ifos-
famide (Storme et al. 2009). Two studies also showed that subcutaneous continuous
infusion of ifosfamide and its analogs in patients with progressive cancer has more
efficacies with no significant local toxicity (Cerny et al. 1990, 1991).
4.3.2.6 Melphalan
Melphalan or L-phenylalanine NM has long been administered as a potent thera-

peutic strategy in the treatment of diseases such as neoplastic meningitis, multiple
myeloma and primary systemic amyloidosis (Drivsholm and Videbaek 1966;
Sanchorawala et al. 2002; Friedman et al. 1994). It was first introduced in 1953 and
successfully used to treat cancer (Drivsholm and Videbaek 1966). The use of mel-
phalan is limited, because it is dose dependently associated with side effects such as
diarrhea, and myelosuppression (Kuhne et al. 2008).
4.3.2.7 Bendamustine
Bendamustine mostly known as ribomustin is a bifunctional alkylating agent that

can be considered as an effective therapeutic agent for clinical use (Anastasia et al.
2014). Due to its multiple unique mechanistic signaling pathways, this agent is
often employed in treatment of several diseases such as Hodgkin lymphoma, indo-
lent non-Hodgkins lymphoma and chronic lymphocytic leukemia (Cheson and
Rummel 2009; Brugger and Ghielmini 2013; Haddad et al. 2014). Moreover, a
report revealed that bendamustine can induce complete remission in patients with
indolent B-cell non-Hodgkin lymphoma compared with other conventional drugs
such as rituximab (Kalaycio 2009). Due to its potential antimetabolite properties
and satisfactory toxicity profile, bendamustine is the drug of choice, especially in
elderly patients (Montillo et al. 2010).
4.4 Acute Toxic Effects of MCs
As previously described, MCs are extremely hazardous chemicals, and exposure to

these compounds may cause the irritation of eyes, and skin with general action on the
respiratory, nervous, cardiovascular, and digestive systems, causing tearing of the eyes,
anorexia, salivation, respiratory distress, vomiting, and cardiac distress. Depending on
the circumstances and considering all effective internal and external factors including
the dose and length of exposure, necrosis of the skin and mucous membranes of the
respiratory system, bronchopneumonia, lesions in intestinal mucosa, leucopenia, con-
vulsions with systemic distress, and death may occur (Dacre and Goldman 1996).
SM is a toxicant which may disrupt a variety of cellular functions. As a well-
known disreputable chemical warfare agent, SM is an antimitotic, mutagenic, carci-
nogenic, teratogenic and cytotoxic agent (Wheeler 1962). Although exposure to
mustard compounds, particularly SM is not usually fatal, but depending on the
length of time and severity of the exposure, many short and long term complications
may occur (Malhotra et al. 1999). The late complications of SM poisoning in the
eyes, respiratory system and the skin are mainly due to local effects of SM (Etezad-
Razavi et al. 2006).
Several studies in vitro and on different species of animal models have shown that
SM is a mutagenic agent (Fox and Scott 1980), carcinogenic and a developmental neu-
rotoxicant. But a few data (Sanjarmoosavi et al. 2012) has been yet found to show the
adverse effects of MCs on teratogenicity and the reproductive system of both human
and animal. Lack of these data is partly due to the fact that no study was conducted on
women since men are at higher risk of exposure to the chemical warfare agents.
A report showed that soon after exposure to mustard gas, patients mostly experi-
ence eye and throat irritation and breathing difficulties. Other symptoms such as
itching, erythema, blisters on the skin, photophobia, eyelid edema generally known
as swollen eyelids, coughing, dyspnea and hemoptysis may develop within 1 or 2
days (Willems 1989).
4.4.1 The Skin
The skin is the largest organ, and consists almost 18 % of the total body mass.
Anatomically, the skin is divided into three layers including the epidermis, dermis,
and subcutaneous. SM penetrates the skin and causes severe skin damage, cytotox-
icity and necrosis at sites of absorption. It is also readily absorbed through hair
follicle. Stratum corneum is a layer of the epidermis mostly found in palms, and
areas of the skin that contain this protective layer are protected from chemical
agents and infections. But there are other factors that determine permeability of an
agent to the skin. Atomic and chemical structures play a crucial role in the perme-
ability and so the toxicity of a compound. In mustard gas poisoning, having chlorine
atoms in the structure of mustard gas is necessary for skin damage. Also, it is shown
that introduction of methylene groups between two sulfur atoms give rise to the
toxic effects of SM on the skin (Malhotra et al. 1999).
SM induced dermal injuries are classified as follow (Balali-Mood et al. 2008):
erythematous form
pigmentary exfoliation
superficial vesicular to bullous form
bullous necrotisation
deep necrotising non-bullous form
allergic and toxic contact reactions of the skin
According to above mentioned and classification of the dermis layers, the sever-
ity of skin injury is classified according to layer involved in skin damaged. Also, the
thickness of skin varies in different parts; hence, the penetration rate differs in
different parts of the skin. For example, the thickness of stratum corneum is low on
the scrotum, flexor surfaces of the forearms, axillae, and around the eyes. The epi-
dermis can be regenerated every 4575 days in normal cells, but mustard agents,
particularly SM disrupts normal cell proliferation in the basal layer of the epidermis
by inhibiting the cell regeneration. Also, the cells on the basal membrane lose their
integrity due to exposure to SM leading to the destruction of cell membranes and
blister formation ((IMC) 1993). Development of cutaneous cancers is linked with
chronic and delayed toxicity of sulfur mustard ((IMC) 1993).
Blister formation on the skin generally begins on the second day after exposure,
and continues for up to 2 weeks. Although the time of onset of visible cutaneous
effects is related to dosage, but exposure to large quantities of mustard gas may
yield an immediate response and causes vesication and necrosis of the skin in 12 h
((IMC) 1993). The level of prostaglandin E (PGE) also increases in skin homoge-
nates 8 h after exposure to mustard agents (Dachir et al. 2004). Prostaglandin E
which is the most abundant prostaglandin in the human body, has numbers of bio-
logical actions, but mostly contributes to inflammatory pain. Therefore, any thera-
peutic agent that inhibit the synthesis of PGE may serve as novel therapeutics for
the treatment of pain and inflammation (Pulichino et al. 2006).
4.4.2 Respiratory System
The most important affected organ due to mustard gas poisoning is pulmonary system.
Damage to the respiratory mucosa is the second most important toxic effect after skin
blisters which were observed in the Iranian veterans with SM poisoning (Balali-Mood
and Hefazi 2005b). The SM veterans suffer mostly from coughing, wheezing,
hypoxemia, and dyspnea. Also findings showed that hyperinflation, pulmonary hyper-
tension and bronchiectatic lesions are commonly observed pulmonary injuries.
4.4.3 The Eyes
Due to the lipophilic nature of mustard gas and its binding affinity to the lipid layer,
eyes are the most vulnerable part of the body to SM exposure. Although corneal lim-
bus is the most vulnerable part of the eyes, due to lack of stratum corneum, other
internal parts of the eyes are also affected by mustard gas. Photophobia, tearing of the
eyes, delayed keratitis, and pre-limbal hyperpigmentation were also observed. Eye
contact to high doses of mustard gas in the SM veterans may lead to a chronic injury
known as mustard gas keratopathy (MGK). This secondary keratopathy consists of
persistent epithelial lesions, corneal neovascularization, and progressive corneal
degeneration. Centripetal endothelial injury and endothelial cytotoxicity is other
acute toxicity in animal model following exposure to SM vapors (McNutt et al. 2013).
4.4.4 Oral and Gastrointestinal Tract Injury
Ingestion of SM-contaminated food and water can cause gastrointestinal (GI) tract
mucosal necrosis, membrane damage, abdominal pain, and bloody diarrhea
(Malhotra et al. 1999). Chemical burns of the GI tract may also occur following
exposure to mustard gas. Most important clinical manifestations on the gastrointes-
tinal system of mustard gas reported in the Iranian veterans were nausea, vomiting,
hematemesis, abdominal pain, and dyspnoea (Balali-Mood and Hefazi 2006).
4.4.5 Nervous System Injury
Nervous system is not severely affected by mustard agent. However, heavy expo-
sure to SM can cause central nervous system (CNS) excitation leading to convul-
sions and death in the field (Balali-Mood and Hefazi 2005b). A study on the Iranian
veterans showed that some patients have convulsions during the early stages of the
severe intoxication (Balali-Mood and Navaeian 1986).
4.4.6 Renal Dysfunction
Interstitial nephritis, glomerulosclerosis, and nephrosclerosis are most common

kidney abnormality found in patients exposed to SM (Kazemzadeh et al. 2014).
Human urologic data showed that exposure to high dose of mustard gas may cause
urinary calculi, recurrent urinary tract infections, benign prostatic hypertrophy

(BPH), and kidney failure (Soroush et al. 2009). Animal studies demonstrated that
serum level of uric acid increases in mice following exposure to SM (Kumar and
Vijayaraghavan 1998).
4.4.7 Hematological Effects and Immunotoxicity
Because the distribution and transport of chemicals in the body is facilitated by the
blood, this tissue will have essential role in the intoxication of sulfur mustard. Any
alteration in the hematological factors may result in severe clinical consequences.
Results of a study showed that leukocytosis is the most common hematological
complication within the first few days after exposure to SM, but there is a signifi-
cant reduction in the WBC count during the first week after exposure (Mahmoudi
et al. 2005). Moreover, cytokine and other hematological parameters including the
percentages of reticulocytes, total counts of RBC, the percentages of monocytes
and CD3(+) T-lymphocytes, IgM, complement component 3 (C3), the absolute
level of alpha(1), alpha(2) and beta globulins and Intercellular Adhesion Molecule
1 (ICAM-1) are significantly higher in the blood samples of the veterans exposed
to mustard gas. On the other hand, the level of total protein, albumin, IL-1, IL-8
and TNF are relatively low in patients with SM intoxication (Mahmoudi et al.
2005; Riahi-Zanjani et al. 2014). Other biochemical factors including cholesterol,
triglyceride, and gamma-glutamyl transferase (GTT) activity are higher in patients
exposed to SM (Keramati et al. 2013). A significant reduction of white blood cell
count was reported in mustard gas intoxication by all routes of exposure
(Vijayaraghavan et al. 2005). Bone marrow suppression is reported in patients
exposed to high dose of SM, which may subsequently result in hemorrhage, ane-
mia and other hematological complications. Immunotoxicity and hematological
complications of SM has also been shown in experimental animal model (Gold and
Scharf 1995; Husain et al. 1996).
4.5 Clinical Manifestations of SM Poisoning
Skin, respiratory tract, bone marrow and ocular irritation are the main risk of acute
poisoning when exposed to SM vapors. Unlike Lewisite, which may cause immedi-
ate and painful reaction, no early reaction is reported in SM intoxication, and symp-
toms do not appear for some hours unless irritation of eyes and respiratory tract that
may be manifested by coughing and eye irritation. The main symptoms and signs of
exposure to mustard gas are not immediate and typically develop over a period of
several days ((IMC) 1993). Clinical symptoms such as nausea, eye irritation, irrita-
tion of the mucosal membrane, coughing, sneezing and vomiting were reported that
had occurred after couple of hours after exposure. But, lethal concentrations may
lead to convulsions, coma and death even 1 h after exposure. However, mortality due
to acute exposure to mustard gas is rare, and may possibly occur after a couple of
days to weeks (Ivarsson et al. 1992). Pulmonary complications due to mustard gas
exposure are the most important cause of death (Balali-Mood and Hefazi 2005a, b).
4.5.1 Dermatologic Symptoms
The most important dermatological symptoms due to exposure to SM are chronic

dryness, itching, loss of hair in the exposed skin surface, burning sensation, and
increased sweating (Balali-Mood and Hefazi 2006). Other major complications
reported in Iranian veterans include blister formations, erythema, macular and pop-
ular rashes, vesicles and skin wounds (Ghasemi-Boroumand et al. 2007). Plaques,
angioedema, hypo and hyper pigmentations, scaling, neurosis and skin atrophy are
other important manifestations in the skin. Depending on the grade of intoxication,
excoriation, telangiectasia, cherry angiomas, acne form lesions, melanocytic nevi,
seborrhea dermatitis, vitelligo, keratosis pillars, actinic keratosis, and various basal
cell cancers may also be observed as a results of SM intoxication (Razavi et al.
2012). Different sizes of blisters may be seen in direct SM skin exposure of patient
as shown in Fig. 4.1.
4.5.2 Gastrointestinal Symptoms
The gastrointestinal symptoms of acute phase in SM intoxication during the first

week after exposure in Iranian veterans were nausea (47 %), vomiting (42 %),
anorexia (40 %), abdominal pain (35 %), diarrhea (15 %), melena (7 %), and
hematemesis (5 %) (Noorbakhsh and Balali-mood 1994). Endoscopy and physical
examination show that acute esophagitis and gastritis may be diagnosed in patients
who had ingested contaminated foods. Erosion and blisters as common irritations in
the pharynx, esophagus, and stomach may also be observed in these patients. But,
Fig. 4.1 Different sizes of

blisters in a hand of Iranian
soldier 3 days after direct
SM skin exposure during
the Iraq Iran war (An
unpublished slide of the
corresponding authors
collections that was taken
with permission of the
patient under his care)
common GI chronic symptoms 2 months to 6 years after exposure were nausea

(45 %), anorexia (42 %), abdominal pain (38 %), hematochezia (12 %), hemateme-
sis (5 %), gastritis (12 %), duodenitis 8 %), and duodenal ulcer (1 %) (Noorbakhsh
and Balali-mood 1994).
4.5.3 Respiratory Symptoms
Coughing, expectoration, dyspnea, hemoptysis, wheezing, crackles, stridor and

hypoxemia are major SM-induced respiratory clinical manifestations (Balali-Mood
et al. 2005b, 2011). The most important toxicity on respiratory system in Iranian
veterans intoxicated with SM included obstruction of upper airways, chronic bron-
chitis, bronchiectasis, asthma, emphysema, stenosis of large airways and pulmonary
fibrosis (Ghasemi-Boroumand et al. 2007). Other pathological effects on the respi-
ratory system were chronic laryngitis, thickening of the bronchial walls, hoarseness
of voice, tracheobronchitis, recurrent respiratory infections, acute respiratory fail-
ure, laryngeal carcinoma, and lung cancer (Razavi et al. 2012). Also, pulmonary
edema was the main cause of death among the soldiers who died a few days after
exposure to SM (Freitag et al. 1991).
4.5.4 Acute Effects in the Eyes
Eyelid edema, limbal ischemia, limbal pigment loss, blepharospasm, chemosis,

conjunctivitis and retina ulcer were the most frequent ocular manifestations due to
SM poisoning. Blurred vision, visual impairment, scarring and neovascularization
of the anterior chamber has also been reported among Iranian veterans. Other ocular
complications include corneal opacity, keratitis, uveitis, corneal melting, conjuncti-
valization, perforation, and blindness in rare cases (Ghasemi-Boroumand et al.
2007; Razavi et al. 2012). A picture taken from a left eye of a patient taken 5 days
after SM exposure during the Iraq Iran war that revealed blepharospasm, chemosis,
keratitis with severe eye vision deficit is shown in Fig. 4.2.
4.6 Chronic Intoxication of Mustard Gas
Chronic toxicity of SM intoxication often occur when manual workers who are
chemically exposed to mustard agents in factories that produce these agent (Balali-
Mood et al. 2008). Although the chronic and delayed toxicity of mustard gas typi-
cally occur in the affected organs of acute toxicity, but some chronic and long-term
complications may also occur in organs which are less affected in acute intoxica-
tions (Panahi et al. 2013; Amirzargar et al. 2009).
Fig. 4.2 A picture taken

from a left eye of a patient
taken 5 days after SM
exposure during the Iraq
Iran war, showing
blepharospasm, chemosis,
keratitis with severe eye
vision deficit (An
unpublished slide of the
corresponding authors
collections that was taken
with permission of the
patient under his care)
Because the skin is the largest organ exposed to these chemical agent, it is sup-
posed to be more susceptible to these compounds than any other organs. The most
common skin complications of SM toxicity are abnormal skin pigmentation and dry
skin (Balali-Mood et al. 2005a, b). On the other hand, some findings showed that
even after decades of exposure to SM, severe long-term respiratory disease such as
obstructive pulmonary disease (COPD), lung fibrosis and can occur. Other chronic
respiratory disorder due to SM poisoning include bronchitis, bronchiectasis, asthma
and interstitial fibrosis (Balali-Mood et al. 2005b).
Liver and other internal organs, which are less likely to be directly affected by
mustard gas are mostly involved in chronic and delayed toxicity (Kazemzadeh et al.
2014). It seems that the liver is also affected organ in chronic SM poisoning
(Vijayaraghavan et al. 2005). Liver function evaluation of Iranian veterans with
mustard gas poisoning showed that chronic hepatitis and steatosis were the most
common pathologies (Kazemzadeh et al. 2014).
Vascular tortuosity, chronic conjunctivitis, corneal vascularization, corneal thin-
ning, corneal opacity, corneal epithelial defect and cornea melting are considered as
the most common long-term complications of the eyes due to exposure to mustard
gas (Javadi et al. 2005; Balali-Mood et al. 2005b).
Although thrombocytopenia, anemia and leukocytosis are the most common
hematological complications soon after exposure to SM, long-term studies on
Iranian veterans 1620 years after exposure revealed no significant hematological
abnormalities. Total counts for WBC and RBC, percentages of monocytes and
CD3+ T-lymphocytes, the level of IgM, absolute levels and percentages of 2 and
globulins and C3 levels were slightly high in the intoxicated patients compared to
the count in healthy controls (Mahmoudi et al. 2005). Depression of the cell-medi-
ated immunity is reported as an important long-term effect of SM poisoning. Also,
the reduction of helper T cells and increased level of T suppressors may result from
chronic immunotoxicity of SM intoxication (Zandieh et al. 1990). A report showed

that natural killer cells are impaired in patients with occupational exposure to SM
(Yokoyama 1993). Studies on Iranian veterans also revealed that the percentage of
natural killer cells almost 10 years after exposure to mustard gas is significantly
lower than that of the healthy individuals (Ghotbi and Hassan 2002). Also it is
shown that immunotoxicity and hematological complications are mainly due to sys-
temic toxicity (Balali-Mood et al. 2005a).
4.7 Delayed Toxic Effects of Mustard Gas
According to the studies conducted on chemically injured war victims, the most
common late clinical complications due to exposure to SM were observed in the
respiratory tract, neuropsychiatrics systems, the skin, and eyes with the frequency
of 78 %, 45 %, 41 %, and 36 %, respectively (Balali-Mood 1992; Balali-Mood and
Navaeian 1986). Delayed toxicity of mustard agents typically refers to genotoxicity,
carcinogenicity and adverse reproductive effects. Due to their alkylating potential,
MCs are mutagenic and genotoxic materials (Fox and Scott 1980).
4.7.1 Long-Term Dermal Complications
Dry skin, burning sensation, itching, atrophy and hypo and hyper pigmentation in
the skin are the most important late dermatologic complications of exposure to mus-
tard gas (Hefazi et al. 2006). Some important skin injuries in Iranian veterans
exposed to mustard gas include hyperpigmentation, erythematous popular rash, dry
skin, multiple cherry angiomas, atrophic scar, hypopigmentation, hair loss and
hypertrophy. As shown in Figs. 4.3 and 4.4, other microscopic skin changes include
epidermal atrophy, hyperkeratosis, basal membrane hyperpigmentation, non-
specific dermal fibrosis, melanocytes and melanosomes within epidermis and
Fig. 4.3 Orthokeratotic

hyperkeratosis, and
atrophy of appendices
(Light Microscopy:
10 10, Courtesy of Prof.
Tabatabei, MUMS, Iran)
Fig. 4.4 Basal membrane

hyperpigmentation with
mononuclear infiltration
(Light Microscopy:
20 10, Courtesy of Prof.
Tabatabei, MUMS, Iran)
increased collagen fibers and mononuclear inflammatory cells within dermis

(Balali-Mood et al. 2005b).
4.7.2 Delayed Respiratory Effects
Coughing, expectoration, dyspnea, and hemoptysis are the most important and
common symptoms of delayed respiratory complications. Other important long-
term clinical findings in patients intoxicated with SM are wheezing, crackles and
stridor (Balali-Mood et al. 2005b). Moreover, lung cancer and pneumonia is usually
common cause of death from exposure to mustard gas (Manning et al. 1981). The
results of a study conducted in Britain during 19611940 on mustard gas producers
showed that the incidence of death from laryngeal cancer in this population has
increased (Easton et al. 1988). Infection of the airways that leads to bronchopneu-
monia can also lead to death. A report showed that respiratory illness may worsen
over time, whereas ocular lesions and skin damages may be declined (Hefazi et al.
2005). It was also reported that the severity of bronchiectasis exacerbates in long-
term follow-up (Balali-Mood and Hefazi 2006).
4.7.3 Delayed Eyes Effects
Itching, burning sensation, red eyes, blurred vision, vision loss, photophobia and
tearing are the most common recorded delayed eyes symptoms (Naderi et al. 2014).
Corneal thinning, neovascularization and epithelial defects are other major ocular
complications 1620 years after initial exposure to SM (Etezad-Razavi et al. 2006).
Study on 500 male patients intoxicated with mustard gas 15 years after initial expo-
sure in the IraqiIran conflict (19801988) showed that the patients may exhibit
different grade of ocular complications (Ghassemi-Broumand et al. 2004).
4.7.4 Reproductive System
Animal data showed that exposure to SM may cause damage to the reproductive
system in mice due to the inhibition of spermatogenesis (Graef et al. 1948).
Although, due to high regenerative capacity of germ cells, it is theoretically sug-
gested that the performance of reproductive system or the fertility may not be
affected (Balali-Mood et al. 2008). However, studies on both humans and animals
showed that as long-term complications of mustard gas can lead to impaired sper-
matogenesis even decades after exposure (Safarinejad 2001). A study on the SM
veterans showed that semen indices are significantly lower in patients exposed to
SM during Iran-Iraq conflict compared to normal healthy individuals. Therefore,
SM can be considered as a gonadotoxic warfare agent (Amirzargar et al. 2009).
4.7.5 Cardiac Associated Complications
Clinical findings suggest that the incidence of coronary artery disease (CAD) and
angiographic changes may increase due to exposure to SM. The myocardium of
patients with mustard gas poisoning is pale and seems do not work properly, but no
heart abnormalities are reported in these patients. Other cardiac associated anoma-
lies such as left ventricular (LV) diastolic abnormality, myocardial perfusion and
dilated cardiomyopathy may also observed in patients exposed to high dose of mus-
tard gas (Rohani et al. 2010).
4.7.6 Genotoxicity
Also, because most of the complications occur several years after exposure to mustard
agents, it is now suggested that genotoxicity and epigenetic abnormalities may be
involved in delayed toxic effects of mustard gas. For example, sister chromatid
exchanges in the peripheral lymphocytes are reported in patients exposed to SM
(Aasted et al. 1987). Varieties of enzymes are involved in epigenetic changes includ-
ing DNA methyltransferases (DNMTs), histone acetyl transferases (HATs) and his-
tone deacetylases (HDACs), which cause chromatin changes leading to altered gene
expression (Miremadi et al. 2007; Kunak et al. 2012). Exposure to mustard gas is also
shown to be associated with almost 400 transcriptional changes, which may lead to
DNA damage, cell cycle arrest, cell death and inflammation (Jowsey and Blain 2014).
In a study of Behravan et al. (2013), shorter telomere length and increased level
of marker of oxidative stress in SM exposed veterans were found. They concluded
that the findings confirm delayed genotoxicity of SM in patients over 25 years after
single exposure to SM.
4.7.7 Carcinogenicity
Based on numerous evidences, it is obvious that mustard gas is a potent car-

cinogen in humans (Watson et al. 1989). It may causes cancer of the lung and
cancer of the larynx (Easton et al. 1988). It is suggested that the carcinogenicity
of mustard gas may be mediated by a genotoxic mechanism of action that
involves DNA alkylation, loss of DNA repairing potency, mutations, and induc-
tion of chromosomal alterations ((IARC) 1999). Studies on workers of the
Ohkunojima poison gas factory show that exposure to mustard gas is associated
with high incidence of mutation, chromosome abnormality, sister chromatid
and cytogenetic changes and missing Y chromosomes (Shakil et al. 1993).
Therefore, it is suggested that exposure to mustard gas in long-term may lead
to incidence of malignant tumors such as respiratory tract cancer and leukemia
(Yanagida et al. 1988).
Because the mechanism of action in acute and delayed toxicity may differ,
clinical manifestations may also differ in SM induced toxicity. Based on the doc-
uments that have been mostly obtained by the studies on the Iranian chemical
warfare victims, some major clinical features of SM intoxication are summarized
in Table 4.1.
Table 4.1 Major clinical features of SM intoxication in acute, chronic and delayed toxicity
Major clinical features
Organs Acute Chronic Delayed
Skin Blister formation, Dryness, pigmentation hyper pigmentation,
vesication popular rash, atrophic
scar erythematous
Eyes Tearing of the eyes, Chronic conjunctivitis, Blurred vision, visual
corneal corneal thinning, impairment, keratitis,
neovascularization corneal opacity uveitis
Respiratory system Coughing, wheezing, Asthma, bronchiectasis, Coughing,
bronchiectatic lesions obstructive pulmonary expectoration,
disease dyspnea, and
hemoptysis,
pneumonia
Gastrointestinal Mucosal necrosis, Nausea, abdominal
system bloody diarrhea, pain, anorexia
vomiting, nausea
Immune system Depression of Reduction of natural
immunity, reduction of killer cells
natural killer cells
Hematological Leukocytosis, Thrombocytopenia, Anemia
system hemorrhage anemia
Neuro-psychiatric Convulsions
disorders
4.8 Treatments of SM Poisoning
It was shown that SM may cause irritations in a few minutes after exposure, but no
specific treatment is still available (Sidell et al. 1997). Physical blister removal,
removing the contents of the blisters in the skin, and administering topical antibiot-
ics have been widely used for the treatment of cutaneous SM injuries. Although,
therapeutic potentials of several anti-inflammatory drugs, antioxidants, protease
inhibitors and antiapoptotic compounds for SM induced injuries have been investi-
gated; no significant progress for producing an efficient antidote has been obtained
so far (Gu 2014; Poursaleh et al. 2012).
The healing rate of wounds induced by mustard gas is considerably slow, and typi-
cal treatment is almost symptomatic and supportive (Rice 2003). Effectiveness of
anti-inflammatory drugs on SM induced skin injuries has been confirmed in an animal
model. Data showed that anti-inflammatory drugs such as Adexone can relieve pain
and reduce inflammation and the level of prostaglandin E. Researches also showed
that non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (Voltaren)
do not have such therapeutic effects on SM induced skin damages, but combination of
these drugs may potentially increases the chance of healing (Dachir et al. 2004).
Pharmacologically, no specific antidote is known for SM poisoning, but as previ-
ously described, the treatment to SM toxicity is typically symptomatic and support-
ive (Graham et al. 2005). Nonetheless, medical management of SM poisoning
includes primary and secondary prevention (Poursaleh et al. 2012). Primary preven-
tion is typically the promotion of healthcare through first-aid protection, control of
pain and potential antidotal treatment with 100500 mg sodium thiosulphate per
kilogram body weight (Balali-Mood and Hefazi 2005b). Secondary prevention
includes treatment with antibiotics and conventional therapies. Depending on the
organ involved in acute SM poisoning, several medical management may be applied.
For example, bronchodilators, corticosteroids, immunosuppressive agents, antibiot-
ics, and oxygen therapy is often used in SM induced lung injuries (Vojvodic et al.
1985). Also several agents such as cysteine, sodium citrate, promethazine, heparin
and vitamin E are shown to have protective effects against SM induced lung inju-
ries. But, removing the victims from the contaminated areas is always the first step
in treatment of veterans exposed to mustard gas.
To prevent further pollution, decontamination of clothes, skin, hair, and eyes
with proper solution is required prior to therapy of SM intoxication. Decontamination
efficiency of N, N-dichloro-bis (2, 4, 6-trichlorophenyl) urea (CC-2) and Fullers
earth (BPC standard) combination is shown in dermal intoxication in animal model
(Kumar et al. 2013). For skin decontamination, great amounts of water and 0.5 %
hypochlorite solution is recommended. For treating eyes, it should be noted that
prior to decontamination and washing, the eyes should not be bandaged.
Although the main medical management in SM poisoning is almost standard first
aid treatment, A study showed that thiol containing compounds such as N-acetyl
cystine (NAC) and dimercapto succinic acid (DMSA) can have protective effects in
SM induced injuries (Balali-mood and Anari 1989). Animal studies also suggest
that some antioxidant and anti-inflammatory agents such as sodium thiosulfate,
N-acetyl-L-cysteine, nicotinamide, nicotinic acid, promethazine, dexamethasone,

prednisone, and vitamin E may have supportive effects and decrease tissue damage
(Vojvodic et al. 1985; Dabney et al. 1991). Using poly (ADP-ribose) polymerase
inhibitors, calmodulin antagonists and Ca2+ chelators are also suggested for primary
care in lung injuries (Merat et al. 2003).
(a) The skin
Nowadays extensive research program has started to provide new medical
managements and therapeutic strategies for cutaneous pathology and blister
formation caused by mustard gas poisoning. Because of the most important
complication followed by SM exposure is cutaneous and respiratory injury,
development of topical skin protectants and medical management for wound
healing is of great interest and importance. Several months is often needed to
cure skin injuries due to mustard gas exposure. Epithelium, dermal collagen
and other connective tissues are affected and do not have normal functions in
SM poisoning (Graham et al. 2009). Similar to other skin injuries such as severe
burning and diabetic foot ulcers, debridement particularly laser debridement of
lesion and blister is usually a successful strategy for treating SM induced der-
mal injury prior to any pharmacological treatment. Another report showed that
the lesion is significantly lower when debridement is performed with trypsin-
linked gauze than surgical or laser debridement (Eldad et al. 1998). From phar-
macological aspect, compounds that are used to treat skin lesions, should
increase the oxygen supply to the tissue, and stimulate epithelium to grow. For
example, amino-Plex is considered as a nutritive cosmeceutical product that
increase oxygen in cells, improve glucose transportation, stimulate collagen
formation, and promote angiogenesis (Graham et al. 2009).
Treatment of skin lesions in Iranian veterans for the chemical burns, using
sulfadiazine in hydroxyl propylmethyl cellulose and furacine showed some
beneficial effects. It is recommended to avoid the use of wet-to-dry dressings,
and the wound is better to keep in moist condition. Unless for primary dress-
ings, which shall be remained for at least a week, secondary bandages is ought
to be changed repeatedly (Momeni et al. 1992).
Experiences on treatment of dermal injuries from SM poisoning have shown
that even extremely extensive skin damage can be cured if the interfering of the
infection is eliminated. Thus, the proper use of antibiotics is a critical step in
management of the SM poisoning. Below some important drugs are tabulated
(Balali-Mood et al. 2008; Vojvodic et al. 1985; Dabney et al. 1991). Different
medications that were used for SM treatment is summarized in Table 4.2.
(b) Respiratory system
Management of SM-induced pulmonary injuries mainly include conservative
and protective treatments such as inhalation corticosteroids therapy. Because oxi-
dative stress and chronic inflammation is considered as the causality of lung injury,
every therapeutic agent such as N-Acetyl Cysteine (NAC) that increase cellular
anti-oxidant supply may be beneficial for treating lung injuries. Recently it is
shown that nebulized morphine has beneficial effects on dyspnea, cough, respira-
tory rate, and heart rate in patients exposed to mustard gas (Shohrati et al. 2012).
Table 4.2 Different pharmaceuticals were used to treat SM induced injuries

Class of drug Drug name
SM scavengers Sodium thiosulphate, N-acetyl-L-cysteine, 4-Methyl-2-
mercaptopyridine-1-oxide, Dimercaprol
Anti-oxidants and inhibitors of Vitamin-A, Vitamin-C, Vitamin-E, Hydroxyethyl, Rutoside
lipid peroxidation
Anti-inflammatory Octyl homovanillamide, Indomethacin
Protease inhibitor 1-(40-Aminophenyl)-3-(4-chlorophenyl) urea, N-(0-P)-L-
Ala-L-Ala-benzy ester hydrate
4.8.1 New Therapeutic Approaches
4.8.1.1 Corticosteroids
Corticosteroids as a type of anti-inflammatory drugs are used in the treatment of

patients with SM-induced respiratory illnesses such as asthma or chronic obstruc-
tive pulmonary disease. This steroid is usually applied in combination with non-
steroidal anti-inflammatory drugs and antibiotics as a protective therapy. Studies
show that this method of therapy ameliorate SM-induced skin injury (Dachir et al.
2004). Also, it is reported that corticosteroid inhalers specially fluticasone in com-
bination with long-acting beta 2-agonists (salmetrol) may be effective in treatment
of patients with chronic bronchiolitis (Ghanei et al. 2007).
4.8.1.2 Roxithromycin
Roxithromycin is a macrolide antibiotic that is typically used to treat respiratory

and urinary tract and skin infections. Recently, it is shown that roxithromycin may
have inhibitory effects on the cytotoxicity and inflammation in human bronchial
and tracheal epithelial cells caused by exposure to SM (Gao et al. 2007). Also,
roxithromycin significantly decrease the expression of pro-inflammatory cytokines
including IL-1, IL-6, IL-8 and TNF at both the protein and the mRNA level.
Therefore, down-regulation of pro-inflammatory cytokines at DNA and protein
level may play an important role in designing new therapeutic agents (Weinberger
et al. 2011; Gao et al. 2007).
4.8.1.3 Iodine
Studies on an animal model showed that some agent such as iodine/povidone may
be a potential antidote for the skin in SM induced poisoning (Wormser 1991).
Treatment with provodine iodine (PI) may also protect from ulceration and vesica-
tion (Wormser et al. 1997). Other therapies for SM induced skin damage include
glucose-saline treatment and sodium thiosulphate (Sugendran et al. 2013).
4.8.1.4 Recombinant Protein Technology
Recombinant DNA and protein technology may open a gate to design and develop
new drugs in the future. For example, animal studies showed that recombinant
human erythropoietin (rhEPO) and recombinant human granulocyte colony stimu-
lating factor (rhG-CSF) stimulate the growth of the erythrocyte, reticulocyte and
leucocyte (Cai et al. 2004). Keratinocyte suspension and stem cell technology may
also develop and be used in epidermal wound healing of SM poisoning (Schmidt
et al. 2013; Henemyre-Harris et al. 2008).
4.8.1.5 Epigenetic and Gene Therapy
Because, most of SM-induced toxicity is mediated through DNA damage; hence,

epigenetic and gene therapy may be a new and valuable treatment modality in the
treatment of SM poisoning. Epigenetic drugs may be used alone or in combination
with other conventional drugs including antioxidants and anti-inflammatory agents
to treat both acute and delayed SM-induced toxicity (Kunak et al. 2012).
4.8.1.6 Biologically Active Peptides
Antimicrobial peptides (AMPs) are novel type of antibiotics, which are thought to
have key role in designing antibiotics and anti-inflammatory drugs in the near future
(Asoodeh et al. 2012). Because a number of unusual peptides have been found in the
blood of animals that treated with iodine, it is deduced that biologically active pep-
tides may play critical role in the treatment of infection, and inflammation and dis-
orders caused by oxidative stress (Brodsky et al. 2008). Also AMPs are a major part
of the innate immune defense at the ocular surface and protect the epithelia of cor-
nea and conjunctiva from microbial invasion (Garreis et al. 2010). AMPs as new
sources of antibacterial are also involved in healing processes of the skin disease
such as psoriasis, burns and wounds (Sorensen et al. 2003). Many antibacterial and
anti-inflammatory and regulatory peptides have been reported so far, have good
potential as antibacterial agents (Asoodeh et al. 2014). Recent studies suggest that
peptide-drug conjugation may enhance the efficacy of drugs through different
mechanistic pathways such as decreasing drug efflux via biological pumps (Fonseca
and Kelley 2011).
4.8.1.7 Cell Death Suppressing
New therapeutic strategy may also rely on the preventing cell death and therefore
vesication by inhibiting apoptosis with suppressing the death receptor (DR) or by
administration of reducing biomolecules such as Niacinamide, Nicotinamide and
Glutathione as potential inhibitors of cell death and promoters of cell survival
(Keyser et al. 2014). A study showed that the SM-induced apoptosis pathway is via
the Fas response; hence, caspase-3 activation can be inhibited by FasR siRNA and
FasR antagonistic antibody (Keyser et al. 2013).
4.8.1.8 Herbal Medicine
Curcumin as medicinal herb is the yellow bioactive ingredient of Curcuma longa,

which is shown to improve SM-induced chronic pruritus in Iranian patients (Panahi
et al. 2012a). Pruritus is the most common chronic skin complication of mustard
gas. This bioactive compound has anti-inflammatory effects and can reduce serum
concentrations of interleukins (IL) 6 and 8 (Panahi et al. 2012b). Therefore, it is
thought that curcumin may be a potential drug to treat cutaneous complications.
Moreover, it is shown that Nigella sativa has protective effect lung inflammation in
guinea pigs exposed to SM, it has also effects on tracheal responsiveness (Hossein
et al. 2008).
4.9 Conclusion
Acute, chronic and delayed toxic effects of SM have been extensively studied, par-
ticularly on Iranian veterans. Despite the large number of studies, the mechanistic
pathways of SM intoxication in cellular level, as well as clinical pharmacology and
toxicology of MCs are less understood. In this chapter, toxic effects of mustard gas
on different organs, particularly skin, lung, eyes and kidney have been comprehen-
sively reviewed. In addition, long-term hematological complication, neurological
impairment, and other delayed immunotoxicity have been discussed. Data shows
that, SM-induced pulmonary toxicity, ocular irritation, and skin disease are the most
affected organs of SM poisoning. The most important and plausible mechanistic
pathways of SM intoxication include DNA alkylation, inflammatory response, ATP
depletion, and oxidative stress.
It is recommended to conduct comprehensive studies both in vitro and in vivo to
elucidate the exact mechanism of SM induced toxicity and to find new therapeutic
modalities by using most recent technology.
Glossary
Alkylating agent A molecule that transfer an alkyl group to target molecule,

which acts as anticancer drugs by inhibiting DNA replication and transcription.
Biotransformation Chemical modification of a compound in the body of living
organism.
Bronchiectasis A pulmonary disease in which some parts of lung airways is

enlarged mainly due to inflammation or infection.
Calmodulin A calcium binding protein in eukaryotic cells that regulates biologi-
cal activities of calcium dependent proteins in many cellular processes.
Chemical warfare agent (CWA) A chemically toxic substance that are used as
chemical weapons in battlefield.
Corneal conjunctivalization The presence of goblet cells in the corneal
epithelium.
Epigenetic therapy The use of drugs or epigenome-influencing techniques to
treat diseases.
Keratitis Inflammation of the cornea.
Lipophilicity Ability of a chemical compound to dissolve in lipids and non-polar
solvents.
Nitrogen mustard (NM)\ A cytotoxic chemotherapy agents similar to sulfur
mustard.
Organization for Prohibition of Chemical Weapons (OPCW) An international
organization for preventing the use of chemical warfare agents and collaborating
with many academics to find suitable therapy for medical conditions caused by
these agents.
Pharmacodynamics Studies effects of a drug to the body.
Pharmacokinetic Studies effects of the body to a drug.
Steatosis The process in which lipids are accumulated in the liver cells.
Sulfur mustard (SM) Also known as mustard gas, is a cytotoxic, alkylating and
blistering chemical warfare agents.
Teratogenicity The property or capability of producing congenital anomalies.
Tracheobronchitis Inflammation of the bronchi.
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Chapter 5
Delayed Complications and Long-Term
Effects of SM Poisonings: Experience
of Iran-Iraq War
Emadodin Darchini-Maragheh, Peter G. Blain, and Mahdi Balali-Mood
Contents
5.1 Introduction .................................................................................................................... 102
5.1.1 Brief History of Creation and Usage ................................................................. 102
5.1.2 Chemical Structure and Properties in Brief ....................................................... 103
5.1.3 Brief Mechanism of Action and Metabolism..................................................... 104
5.1.4 Relative Toxicity in Brief ................................................................................... 104
5.2 Reminiscence of the Iran-Iraq War ................................................................................ 105
5.3 Delayed Complications of SM Poisoning ...................................................................... 106
5.3.1 Distribution of Delayed SM Complications in Various Organs ......................... 106
5.3.2 Delayed Respiratory Complications .................................................................. 107
5.3.3 Delayed Ophthalmologic Complications ........................................................... 111
5.3.4 Delayed Dermal Complications ......................................................................... 112
5.3.5 Delayed Neuropsychiatric Complications ......................................................... 113
5.3.6 Delayed Immuno-hematological Complications ............................................... 116
5.3.7 Other Delayed Complications ............................................................................ 117
5.4 Clinical Management of Delayed SM Complications,
According to Iranian Experiences .................................................................................. 119
5.4.1 Management of Respiratory Complications ...................................................... 119
5.4.2 Management of Ocular Complications .............................................................. 122
5.4.3 Management of Skin Complications.................................................................. 122
5.5 Conclusion and Recommendations ................................................................................ 123
Glossary .................................................................................................................................. 124
References ............................................................................................................................... 127
E. Darchini-Maragheh
Medical Toxicology Research centre, Faculty of Medicine,
P.G. Blain
Medical Toxicology Centre, Newcastle University, Newcastle upon Tyne, UK
e-mail: mbalalimood@hotmail.com

102 E. Darchini-Maragheh et al.
Abstract Among the weapons of mass destruction, Chemical Warfare Agents

(CWAs) are one of the most brutal created by humankind in comparison with biological
and nuclear warfare. Sulfur mustard (SM) which has the sobriquet King of the Battle
Gases has been the most widely used chemical weapons during the wars. SM was the
most destructive chemical weapon used during the World War one (WWI). Thereafter,
it remained the chemical weapon of choice in modern tactile warfare, as evidenced by
widely use during the Iran-Iraq war. Acute and long-term incapacitating properties of
SM, in combination with the lack of an antidote, significant environmental persis-
tence, and relative ease of manufacturing, still kept it a potential agent for both military
and terrorist use. Delayed complications of SM exposure can still be observed in sev-
eral thousands of Iranian victims of the Iran-Iraq war. Delayed complications of SM
have been reported in several organs, however, the most common delayed complica-
tions have been observed in the respiratory tracts of Iranian chemical veterans. Also,
the skin lesions as well as the eye disorders have been observed in most of Iranian
exposed veterans in the delayed phase of intoxication. This chapter reviewed type,
severity and distribution pattern of long-term effects of SM poisoning in different
organs among as well as long-term clinical managements and treatments of complica-
tions, according to the experimental and Iranian studies and experiences.
Keywords Chemical warfare Sulfur mustard Poisoning Complications Long-

term effects Delayed toxic effects
5.1 Introduction
5.1.1 Brief History of Creation and Usage
Sulfur mustard (SM) is a toxic alkylating chemical warfare agent (CWA) which was
the most widely-used CWA in the past century (Balali-Mood et al. 2005a). Despretz
was the first to prepare SM at around 1822 (Balali-Mood and Hefazi 2005b). Pure
SM was synthesized in 1886 by Victor Meyer through the reaction of thiodiglycol
with phosphorus trichloride (Balali-Mood and Balali-Mood 2009). However, SM
for use in warfare was produced by what is known as the Levinstein process -the
reaction of ethylene with sulphur dichloride- before the World War One (WWI)
(Pechura and Rall 1993; Prentiss 1937).
SM is Known by different names such as Yperite (Ypres was the site of its first
military use in Belgium), Lost (acronym of the German chemists Lommel and
Steinkopf), yellow cross (German shells which were marked with yellow cross and
meant skin damaging agent) and HD (military code of distilled sulfur mustard that
is approximately 96 % pure). SM has been remained the chemical weapon of choice
in modern tactile warfare and also earned the sobriquet King of the Battle Gases
(Balali-Mood and Hefazi 2005b, 2006; Balali-Mood et al. 2005a, 2008; Mandel and
Gibson 1917; Prentiss 1937; Sidell et al. 1997; Vijayaraghavan et al. 2009).
5 Long-term effects of SM poisoning in Iranian veterans 103
The first military use of SM was in summer 1917 during German attacks in a
field near Ypres, Belgium. In the September of that year, more than one million SM
shells were fired at Allied troops by Germans (Borak and Sidell 1992). After that,
SM was extensively used throughout the WWI by both sides of the war between
1914 and 1918. It is reported that over 1,200,000 soldiers were poisoned with SM
during the war and about 400,000 of them needed long-term medical care (Mansour
Razavi et al. 2012; Mousavi et al. 2009; Prentiss and Fisher 2007; Shadboorestan
2012). In the US army, out of the total of 36,956 chemically injured soldiers,
27,711(75 %) were poisoned by SM gas, and according to contamination control
unit of British army, out of the total of 160,970 chemical warfare victims, 124,752
(77.5 %) were due to SM poisoning (Mansour Razavi et al. 2012). In aggregate, it
is estimated that SM caused about 80 % of the chemical casualties in the WWI
(Noort et al. 2002). After WWI, the Geneva Protocol was promulgated in 1925 and
widespread campaign was formed to ban chemical warfare (Alexander 1947).
There is sparse documents of SM use in the 1930s by Italy against Ethiopia, by
Poland against Germany and by Japan against China (Feister 1991). There is no evi-
dence of deliberate SM attack during the World War II (WWII), the so-called
Unfought Chemical War. As the only recorded evidence, in December 1943, an
Allied ship carrying large scales of SM and other munitions was attacked by German
troops and exploded in the harbor of Bary, Italy (Feister 1991; Borak and Sidell 1992).
Dispersing agent caused more than 600 casualties (Alexander 1947). After WWII,
accusation of chemical attacks have been common, of which, SM attacks of Egyptian
forces in Yemen (19631967) seems to be better supported than most (Institute 1975).
The greatest military use of SM, however, was happened during 19801988
throughout Iran-Iraq war. Widespread chemical war gas attacks by the Iraqi army
against the Iranian combatants and civilians (and later against member of its Kurdish
population in Iraq) left more than 100,000 military and even civilian casualties
(Khateri 2001; Balali-Mood 2011), as well as 25,000 mortalities (Darchini-
Maragheh et al. 2012).
The most recently, outstanding reports of chemical military attacks including
nerve agents and probably mustard compounds has been released in summer 2013 in
the Syria (Shea 2012).
5.1.2 Chemical Structure and Properties in Brief
There are two groups of mustards: SM (C4H8Cl2S) (2,2-dichlorethyl sulfide; HD),

which is a vesicant CWA, and nitrogen mustard tris-(chloroethyl) amine, which was
found to be unsuitable as a munition but is currently a useful chemotherapeutic
agents known as Mustargen (Gupta 2009).
SM is a heavy oily liquid that is clear or straw colored while pure but dark while
crude. The molecular weight is 159.08, freezing point 14.45 C, boiling point 215
227 C and specific gravity is 1.27 (Mousavi et al. 2009). It is fully soluble in organic
solvents, fuels and lubricants, but barely soluble in water (0.07 % at 10 C) (1993;
Ghanei et al. 2003b; Wattana and Bey 2009). It evaporates at 15 C and in warm tem-
peratures becomes less stable, so its vapor form increases, and at night it sediments
because of decreased temperature (Safarinejad et al. 2001). SM is heavier than water
when it is in the form of liquid and also heavier than air when it is in the form of vapor
or gas (Wattana and Bey 2009). SM vapor has marked penetrating power in porous
cloths and food and plants and can easily penetrate into the cell membrane of wood,
leather, plastic (plastic breathing masks) and rubber, but metal, glass and glazed tiles
are resistant against penetration (Davis and Aspera 2001; Ghasemi Boroumand and
Amiri 2008; Safarinejad et al. 2001). Low volatility beside low solubility in water
leads to long persistence of the compound in the environment (Okumura et al. 1996).
5.1.3 Brief Mechanism of Action and Metabolism
After absorption, SM undergoes intramolecular cyclization which leads to formation

of ethylene episulphonium ion intermediate (Gilman and Philips 1946). The cyclic
intermediate reacts with and alkylates a wide variety of electron-rich biological mol-
ecules (Feister 1991; Wheeler 1962). It can attack and break the DNA and RNA at
specific nucleotides. The major alkylating site of nucleic acids is the nitrogen residue
of guanine in mammalian origin (Timmis 1960; Trams et al. 1961). The result is
manifested in chromatid aberration and inhibition of DNA, RNA and protein synthe-
sis. SM can also react with proteins and phospholipids, however, the consensus had
been made that it is a DNA alkylating agent, which has an effective role in delayed
healing (Ball and Roberts 1972; Crathorn and Roberts 1966; Walker 1971). Cell
death from DNA cross-linking does not occur until the cell undergoes division and
DNA replication phase. Thus, at higher cellular exposure, the acute damages of skin
and mucous membranes are probably generated by other noted mechanisms. Other
potential mechanisms that may be involved in acute damages are nicotinamide ade-
nine dinucleotide (NAD) depletion and also inactivation of sulfhydryl-containing
proteins and peptides, such as glutathione. Glutathione is critical in reducing reac-
tive oxygen species in the cell and thought to be preventive in peroxidation and loss
of membrane integrity (Eklow et al. 1984; Rankin et al. 1980). In addition to cell
death, many adverse effects on cells can be presumed such as alkylating effects,
mitosis inhibition, mutagenesis and carcinogenesis (Foroutan 1997).
5.1.4 Relative Toxicity in Brief
Although there are more potent CWAs than SM, such as the nerve agents, which
have been known as lethal agents, the toxicity of SM as incapacitating agent is
much more important than its capacity to kill, in terms of lethal dose 50 % (LD50).
In other words, compared with the nerve agents, SM has relatively lower acute lethal
toxicity, but more long-term incapacitating effects (Balali-Mood and Navaeian 1986;
Ballantyne et al. 2000). Reported fatality rates due to SM intoxication in the WWI
and Iran-Iraq conflict were 2 % and 34 %, respectively (Pechura and Rall 1993).
Intravenous LD50 figures in mice and rats are 8.6 and 3.3 mg/kg, respectively
(Balali-Mood and Navaeian 1986). The LD50 in human being is between 200 mg
(when swallowed) and 45 g when applied to the bare skin over a long exposure
time (Balali-Mood and Hefazi 2005a). The respiratory lethal dose is estimated at
1500 mg.min/m3 of C(t); the product of concentration [mg.m3] and exposure dura-
tion [min] (Balali-Mood and Hefazi 2005a; Dacre and Goldman 1996).
5.2 Reminiscence of the Iran-Iraq War
In the 25 conflicts during the twentieth century, 72 million deaths were reported, of
which nearly half of the deaths occurred during the armed conflicts of 19871997
(Zargar et al. 2007). Several features of Iran-Iraq war made it unique among the
conflicts of modern times. It was the longest warfare attacks of the twentieth cen-
tury. Neither the WWI nor the WWII lasted 8 years. Iran-Iraq war developed the
pattern of its own: constrained, episodic action, interspersed with bouts of feverish
fighting. In addition, this war did not lead to a sharp division between the two super-
powers. The war was one of the bloodiest as well as the most costly. The direct and
indirect damage caused by the war is put at an astronomical figure of US $ 1190
billion (Hiro 1991).
According to the official Islamic Republic historiography, the Iran-Iraq war
began on August 22, 1980, when Iraqi forces conducted a surprise invasion of
Iranian territory.
The first use of SM in this war was in November 1980 when Iraqi troops attacked
Susangerd (a city of Iran in the border with Iraq). Despite of the International con-
ventions on prohibition of using CWAs, extensive chemical attacks were continued
by Iraqi troops particularly in 19831988 which brought the number of chemical
attacks over 30 during the war. It is estimated that more than 1800 metric tonnes of
mustard gas was used against Iran. Some catastrophic chemical attacks were in
Majnoon island (February 1984), Hawizah Marsh (March 1985), Sumar/Mehran
(October 1987), Al-Faw (February 1986 and April 1988) and in many west border
cities of Iran. A chemical war attack in March 1988 in Halabja, a Kurdish town in
Iraq, caused rapid deaths from exposure to SM and other CWAs, including sarin
(Balali-Mood and Balali-Mood 2008; Darchini-Maragheh et al. 2012; Mousavi
et al. 2013). It was reported that over 5000 Kurdish civilians were killed in the
Iranian-occupied village of Halabja during the mentioned chemical attack (Balali-
Mood and Hefazi 2005b). The last SM attack by Iraqi troops was in July 1988 at the
south central border of Oshnavieh.
Iran accepted ceasefire on 20 July 1988 and the war stopped on 20 August 1988.
The Iraqi army attacked the rebellious Kurds in the north of Iraq by CWA because
of having actively co-operated with Iran during the war (Balali-Mood and Abdollahi
2013). At the end of the war, 398,500 injured individuals as well as 52,000 chemical
Table 5.1 An estimate of the number of Iranian morbidities and mortalities due to chemical
attacks during Iran-Iraq war
The number of Iranians exposed to chemical 1,000,000 people
weapons during the war
The number of Iranians who received medical care 100,000 people
during their exposures to chemical gases
Iranians killed by the immediate effects of 5500 (3500 people by nerve agents and
chemical agents 2000 people by mustard gas)
Total Iranian mortalities due to chemical warfare 25,000
agents during the war
Iranians veterans who exposed to chemical agents 40,00070,000 people
(registered and not registered)
Iranian civilians who exposed to chemical agents 35,000 people
(registered and not registered)
warfare victims (SM and nerve agents) needed medical and health care follow-up
(Salamati et al. 2013; Zargar et al. 2007). In aggregate, More than 100,000 chemical
causalities as well as 25,000 chemical mortalities have been recorded (Khateri
2001; Darchini-Maragheh et al. 2012). Even after 20 years of war, about 40,000
Iranian veterans have complains of delayed effects of SM poisoning (Balali-Mood
and Balali-Mood 2009; Balali-Mood and Hefazi 2005b; Ghanei and Adibi 2007).
Khateri et al. stated that there were 34,000 mustard gas injured veterans in 2002.
However, many chemically poisoned patients had been excluded based on their
inclusion and exclusion criteria (Khateri et al. 2003). Zargar et al. (2007) mentioned
that there were 398,587 veterans who needed long- term follow- up during the war;
of which, 52,195 (13 %) were chemically injured victims (Zargar et al. 2007).
Number of Iranian veterans and civilians exposed to CWAs during the Iraq-Iran war
were summarized in Table 5.1.
Despite passing 25 years after the ceasefire, the chemical war victims are one of
the main health challenges in Iran that unfortunately leads to deaths due to compli-
cations of SM poisoning. An estimate of the number of Iranian morbidities and
mortalities due to chemical exposures during Iran-Iraq war are presented in Table 5.1
(Ghanei et al. 2003a; Salamati et al. 2013).
5.3 Delayed Complications of SM Poisoning
5.3.1 Distribution of Delayed SM Complications in Various

Organs
Effects of SM on body organs are divided into acute and chronic/delayed phases.
While the term chronic complications is referred to occupational exposure,
delayed or late complications seems to be more suitable for long-term SM
effects following battle-field exposure (Balali-Mood and Hefazi 2006).
In delayed phase of SM intoxication, incidence of organ involvement, have been

reported differently in various Iranian soldiers and also at different time intervals.
As the first report of delayed toxic effects of SM poisoning, Balali-Mood (1984)
evaluated 236 Iranian SM victims 228 months after exposure and delayed SM
complications were as follows: respiratory complications in 78 %, CNS in 45 %,
dermatologic complications in 41 % and eye complications in 36 % of cases (Balali-
Mood 1984). Balali-Mood (1992) evaluated delayed toxic effects of SM on differ-
ent organs of 1428 Iranian chemical victims 39 years after exposure and reported
the most SM complications in the respiratory tracts (90 %), skin (88 %), the eyes
(78 %), neural system (71 %), gastrointestinal system (55 %), genitalia (52 %) and
hematopoietic system (38 %) (Balali-Mood 1992). Holisaz et al. (2003) in a study
on 100 Iranian chemical victims, reported dermatologic and ophthalmic complica-
tions in 94 %, pulmonary in 75 %, hematologic complications in 10 % and GI
complications in 5 % of the victims (Holisaz et al. 2003). According to Khateri and
co-workers (2003), the pulmonary, dermatologic and ophthalmic complications
were the most common organ delayed complications among 34,000 SM victims
(including from mild to severe intoxication). Balali-Mood et al. (2005a) described
late toxic effects of SM poisoning in a group of 40 severely intoxicated Iranian vet-
erans 1620 years after exposure. The most commonly affected organs were lungs
(95 %), peripheral nerves (77.5 %), the skin (75 %) and the eyes (65 %) (Balali-
Mood et al. 2005a). More recently, Namazi and colleagues (2009) studied 134
patients with delayed complications of SM poisoning and reported the lungs
(100 %), the skin (82.84 %) and the eyes (77.61 %) as the most frequent affected
organs (Namazi et al. 2009). Distribution of SM delayed complications in different
organs were listed in Table 5.2, according to several studies in Iran.
5.3.2 Delayed Respiratory Complications
Respiratory problems are the greatest cause of long-term disability among Iranian
veterans with combat-exposure to SM gas. Khateri et al. (2003) in a study con-
ducted on 34,000 Iranians who were exposed to SM, reported that 14,450 (42.5 %)
of them were suffering from respiratory problems (Khateri et al. 2003). Respiratory
complications exacerbate over time while cutaneous and ocular injuries tend to
either alleviate or remain invariable (Balali-Mood and Hefazi 2005b; Balali-Mood
and Hefazi 2006; Ghanei and Adibi 2007; Khateri et al. 2003). Comparison of acute
and late toxic effects of SM poisoning in 77 Iranian CWA victims indicated that
dermal complications tend to decrease, eye lesions do not change significantly, and
respiratory complications generally deteriorate over the years (Zarchi et al. 2004).
Even those veterans who had not developed acute symptoms of SM (sub-clinical
exposure) may suffer from late respiratory complications (Ghanei and Adibi 2007;
Ghanei et al. 2004a).
In the long-term phase of SM intoxication, a triad of cough, expectoration and
dyspnea has been found as the most respiratory symptoms among Iranian SM
Table 5.2 Distribution of delayed complications of SM poisoning in various organs based on

several studies in Iran
Publication Case Distribution of
Author(s) year Population numbers complications Ref.
Balali- 1984 Veterans 236 Respiratory tract Balali-Mood
Mood et al. (228 (78 %), CNS (45 %), (1984)
month after skin (41 %), eyes
exposure) (36 %)
Shirazi and 1987 Veterans 77 Lungs (58 %), eyes Shirazi and
Balali- (2 years (46 %), skin (38 %) Balali-Mood
Mood after (1988)
exposure)
Balali- 1992 Veterans 1428 Lungs (90 %), skin Balali-Mood
Mood (39 years (88 %), eyes (78 %), (1992)
after neural system (71 %),
exposure) gastrointestinal system
(55 %), hematopoietic
system (38 %)
Khateri 2003 Veterans, 34,000 Lungs (42.5 %), eyes Khateri et al.
et al. (1320 civilians (mild to (39 %), skin (24.2 %) (2003)
years after severe
exposure) exposure)
Holisaz 2003 Veterans 100 Skin (94 %), eyes Holisaz et al.
et al. (1420 (94 %), lungs (75 %), (2003)
years after hematopoietic system
exposure) (10 %),
gastrointestinal system
(5 %)
Balali- 2005 Veterans 40 Lungs (95 %), Balali-Mood
Mood et al. (1620 peripheral nerves et al. (2005b)
years after (77.5 %), skin (75 %),
exposure) eyes (65 %)
Etezad- 2006 Veterans 40 Lungs (95 %), skin Etezad-
Razavi et al. (1620 (90 %), eyes (65 %) Razavi et al.
years after (2006)
exposure)
Ghasemi- 2008 Civilians 600 Lungs (45.8 %), eyes Ghassemi-
Boroumand (19 years (37.7 %), skin Broumand
et al. after (31.5 %) et al. (2008)
exposure)
Namazi 2009 Veterans 134 Lungs (100 %), skin Namazi et al.
et al. (1722 (82.84 %), eyes (2009)
years after (77.61 %)
exposure)
Zojaji et al. 2009 Veterans 43 Lungs (95 %), Zojaji et al.
(1722 peripheral nerves (2009)
years after (77 %), skin (73 %),
exposure) eyes (68 %)
veterans (Balali-Mood 1992; Balali-Mood and Hefazi 2006; Darchini-Maragheh

et al. 2011). Generalized wheezing is the most objective finding in delayed phase of
respiratory complications (Balali-Mood and Balali-Mood 2009). Crackles, club-
bing, decreased lung sounds and cyanosis have been also reported as other common
objective findings (Balali-Mood 1992; Balali-Mood and Hefazi 2006; Ghanei and
Adibi 2007; Razavi et al. 2013a).
Spirometry is a valuable diagnostic tool for evaluation of pulmonary impairment
during regular follow-ups of SM victims (Hefazi et al. 2005). Pulmonary function
testing (PFT) had been revealed more obstructive pattern than restriction (Balali-
Mood 1992). Although some investigators notice that obstructive pattern is still the
most common spirometric finding among SM poisoned veterans, it seems that
restrictive pattern has been increased over the years and reported as dominant pat-
tern of spirometry among SM patients in more recent studies (Balali-Mood and
Hefazi 2005b; Balali-Mood et al. 2005a; Darchini-Maragheh et al. 2011; Ghanei
and Adibi 2007; Ghanei et al. 2004a). Emad and Rezaian (1997) in a respiratory
survey of 197 Iranian veterans 10 years after a heavy SM exposure, reported the
diversity of the effect of SM on respiratory pattern according to possible lung
fibrosis over the years based on spirometric findings and lung biopsies (Emad and
Rezaian 1997).
Chest radiography has been shown an increased bronchovascular markings,
hyperinflation, pneumonic infiltration, bronchiectasis and radiologic evidence of
pulmonary hypertension (Bagheri et al. 2003; Balali-Mood and Hefazi 2006; Bijani
and Moghadamnia 2002; Ghanei and Adibi 2007; Ghanei et al. 2004b). However,
such radiography is not sensitive enough for detection of delayed respiratory com-
plications among SM victims. High Resolution Computed Tomography (HRCT) is
imaging modality of choice in diagnosis of SM pulmonary complications (Bagheri
et al. 2003; Bakhtavar et al. 2008; Balali-Mood et al. 2005a; Emad et al. 1995;
Balali-Mood et al. 2011). An HRCT study in delayed phase of SM poisoning among
Iranian veterans revealed that a series of delayed destructive pulmonary sequelae
such as chronic bronchitis (58 %), asthma (10 %), bronchiectasis (8 %), large air-
way narrowing (9 %), and pulmonary fibrosis (12 %) were developed (Emad and
Rezaian 1997). Furthermore, a respiratory survey of 40 severely SM intoxicated
Iranian veterans (2005), reported main delayed respiratory complications as chronic
obstructive pulmonary disease (COPD) (35 %), bronchiectasis (32.5 %), asthma
(25 %), large airway narrowing (15 %), pulmonary fibrosis (7.5 %), and simple
chronic bronchitis (5 %) (Hefazi et al. 2005).
As evidenced by a long-term follow-up study of 40 SM veterans conducted by
Balali-Mood and co-workers (2005a), both the severity and frequency of bronchiec-
tatic lesions tend to increase over the time (Balali-Mood et al. 2005a). Bronchiectasis
usually begins bilaterally in the lower lobes of the lungs and then has cephalic pro-
gression. Direct effects of SM on bronchial wall mucosa as well as recurrent respi-
ratory infections among SM veterans are known to be responsible for development
of bronchiectasis (Ghanei and Adibi 2007).
Hypoxemia and hypercapnia are observed in severe cases of bronchitis and in
bronchiectatic lesions leading to pulmonary hypertension and core pulmonale in
severe stages of the complications (Balali-Mood et al. 2005a; Ghanei et al. 2004a;
Hosseini et al. 1998).
In the study of Ghanei et al. (2006a) on 300 symptomatic SM patients, 45.6 %
had various degrees of air trapping. The study reported air trapping and tracheo-
bronchomalacia as common delayed sequelae in SM exposed patients and hypoth-
esized that SM may affect both small and large airways (Ghanei et al. 2006a).
Furthermore, in an HRCT study of 50 Iranian patients with delayed respiratory
complications of SM, air trapping (76 %), bronchiectasis (74 %) and mosaic paren-
chymal attenuation (72 %) were reported as the most frequent findings and revealed
the diagnosis of bronchiolitis obliterans (BO) (Ghanei et al. 2004b). This was also
proved by a later pathologic study (Beheshti et al. 2006). In a cross-sectional study
conducted by Beheshti and colleagues (2006) on 23 patients with late complications
of SM, main respiratory complications were diagnosed as air trapping (76 %) and
bronchiectasis (74 %). It was also stated in the report that in nine lung biopsies out
of 14, histopathological changes were diagnosed as BO (Beheshti et al. 2006).
Although, this diagnosis should be corroborated by further histopathological stud-
ies, BO seems to be one of the main underlying pulmonary diseases in delayed SM
intoxication and depends on host response rather than a dose response manner
(Ghanei et al. 2008a).
Bronchoscopic appearance of airway mucosa has been reported to be a combina-
tion of erythema, chronic inflammatory changes and mucosal thickening in most of
SM patients (Ghanei and Adibi 2007). Broncho-Alveolar Lavage (BAL) fluid analy-
sis of SM patients has been revealed an ongoing local inflammatory process result-
ing in the development of pulmonary fibrosis, years after initial exposure (Emad and
Rezaian 1997). Diffusing capacity of the lungs can be used as an objective monitor
of the degree of lung fibrosis in SM patients and also as a good predictor of progno-
sis (Balali-Mood and Balali-Mood 2009). BAL fluid analysis of SM patients has
been revealed increased inflammatory cells even more than two decades after SM
exposure (Beheshti et al. 2006; Emad and Rezaian 1999; Sohrabpour et al. 1988).
Increased neutrophil as well as eosinophil counts have been reported in BAL fluid
analysis, which is more common in asthmatic respiratory conditions (Beheshti et al.
2006; Ghanei et al. 2005a). Inflammatory pattern of BAL analysis have been reported
to be neutrophil dominant in some previous studies (Ghanei et al. 2007). Typical SM
exposed patients have normal values of albumin and immunoglobulin (Ig) in the
BAL fluid. However, those who were diagnosed as asthma show an increased IgG
level (Ghanei et al. 2005a). Aghanouri and colleagues (2004) reported increased
levels of transforming growth factor 1 (TGF-1) as well as TGF-1 receptors, in
BAL fluid of SM-exposed patients compared with non-exposed individuals and con-
cluded that since TGF-1 can cause BO changes and is substantially increased in
BAL aspirates and target tissue of SM patients, the role of BO as the main underly-
ing pathology in mustard lung becomes evident (Aghanouri et al. 2004).
It is well known that SM is a mutagenic alkylating agent. In vitro studies, it have
been shown that mustard is both mutagenic and carcinogenic. Human data from
WWI battlefield exposure and among chemical factory workers, who have pro-
longed exposure with mustard compounds, reported increased risk of pulmonary
carcinoma. However, figures failed to make a strong case, and there is controversy
around a carcinogenic effect after a single low or high dose exposure (Ghanei and
Harandi 2007). Also, there are no substantial reports regarding this issue on Iranian
patients. Sparse cases of bronchogenic carcinoma have already been reported in
Iranian veterans (Balali-Mood 1992; Zojaji et al. 2004). Thus, long-term follow-up
is required to discover the incidence of lung carcinogenicity in such patients.
5.3.3 Delayed Ophthalmologic Complications
The eyes have the most sensitivity organ to SM which is attributed to several ocular
features. The aqueous-mucous surface of the cornea and conjunctiva, as well as
higher turnover rate and intense metabolic activity of corneal epithelial cells make
remarkable hypersensitivity in the event of SM exposure (Etezad-Razavi et al. 2006;
Namazi et al. 2009).
In the study of Namazi et al. (2009) on 134 Iranian SM veterans (2009), burning
sensation, photophobia, red eye and itching were the most common delayed eye
complications (Namazi et al. 2009). Balali-Mood and colleagues (2005a), through
ophthalmologic examination of 40 SM intoxicated Iranian veterans, reported sub-
jective eye complications in almost all the patients which were recorded as itching
(42.5 %), burning sensation (37.5 %), photophobia (30 %), tearing (27.5 %), prema-
ture presbyopia with reading difficulties (10 %), ocular pain (2.5 %) and foreign
body sensation (2.5 %). Common objective findings were found in the following
order: chronic conjunctivitis (17.5 %), peri-limbal hyperpigmentation (17.5 %),
corneal thinning (15 %), vascular tortuosity (15 %), limbal ischaemia (12.5 %), cor-
neal opacity (10 %), corneal vascularization (7.5 %) and corneal epithelial defect
(5 %) (Balali-Mood et al. 2005a). Ghasemi et al. (2008) studied 367 chemical war
victims of Sardasht, Iran and reported that photophobia and ocular surface discom-
fort (burning, itching and redness) were the most significant symptoms, while, bul-
bar conjunctival abnormality and limbal tissue changes were the most slit-lamp
findings among the victims (Ghasemi et al. 2008).
Although most of early ocular complications of SM exposure such as lacrima-
tion, edema, discharge and even blindness usually recover after a few days to weeks,
a kind of delayed ulcerative keratopathy may develop, leading to permanent resid-
ual effects (Etezad-Razavi et al. 2006). This usually occurs 1520 years after the
initial injury and starts with a sudden onset of photophobia, tearing and decreasing
vision (Javadi et al. 2007). It is characterized by corneal thinning, corneal opacifica-
tion, neovascularization, and corneal epithelial deficiency advances after a symptom-
free period (Balali-Mood et al. 2005a; Etezad-Razavi et al. 2006). In acute stages of
ulcerative keratitis, the limbal region frequently presents a marbled appearance in
which porcelain-like areas of ischaemia are surrounded by blood vessels with irreg-
ular diameters. Then, vascularized scars of the cornea are covered with crystal and
cholesterol deposits, leading to worsening of opacification, recurrent ulcerations,
and sometimes corneal perforation (Balali-Mood and Hefazi 2006). Opacification is
seen in lower and central portions of cornea, whereas the upper part is almost pro-
tected due to eyelids (Balali-Mood and Balali-Mood 2009; Balali-Mood et al.
2008). Lesions were surprisingly recurring even after corneal transplantation (Javadi
et al. 2005). Etezad-Razavi (2006) noticed delayed ulcerative keratitis, as a delayed
objective finding in 15 % of the patients which in comparison to a 0.51 % inci-
dence of delayed keratitis observed in the WWI SM casualties, was significantly
higher (Etezad-Razavi et al. 2006). Interestingly, the severity of the initial exposure
and duration of the ophthalmic symptoms is directly related to the likelihood of later
keratopathy (Ghasemi et al. 2009).
In a recent cross-sectional study on 40 severely intoxicated Iranian veterans with
delayed complications of SM exposure (2013), retinal electrophysiological evalua-
tions including electroretinography (ERG) and electrooculography (EOG) were
performed. The study, as the first report on the SM-induced delayed-onset func-
tional retinal changes, showed a general reduction of retinal photoreceptor function
in delayed phase of SM exposure. This effect involves both cone and rod photore-
ceptors in terms of amplitude and implicit time. These findings among SM veterans
showed that SM intoxication also have long-term complications on the eyes neuro-
logic tissues such as retina (Darchini-Maragheh et al. 2013).
5.3.4 Delayed Dermal Complications
The lipophilic nature of SM and high affinity of the skin for lipophilic substances,
make the skin an appropriate transporting system for this agent. Acute skin injury
with SM without vesicle formation is almost always followed by a complete heal-
ing (Balali-Mood and Balali-Mood 2009; Balali-Mood and Hefazi 2005b). In con-
trast, blisters and necrotic wounds cause permanent residual effects. Most of
delayed cutaneous skin lesions are on the site of blisters at the acute phase of SM
poisoning. Furthermore, previously injured sites have been reported to be sensitive
to subsequent mechanical injury and showed recurrent blistering after mild injury
(Fekri et al. 1992).
Balali-Mood in the first report of delayed SM skin complications 2 years after
exposure among 236 Iranian veterans declared hyperpigmentation (34 %), hypopig-
mentation (16 %), and dermal scar (8 %) as the most common findings. The most
common skin complaint among these patients was itching followed by a burning
sensation and desquamation (Balali-Mood and Navaeian 1986). Several years later,
pruritus was still the most common subjective finding (Balali-Mood et al. 2005a;
Panahi et al. 2008). Balali-Mood et al. (2005a) and Panahi et al. (2008) reported
hyperpigmentation and xerosis as the most frequent objective findings 1620 years
after SM exposure. Fekri et al. (1992) compared cutaneous lesions of 500
SM-exposed Iranian veterans with unexposed veterans. Significant association was
reported between SM exposure and late skin lesions such as severe dry skin, hyper-
and hypopigmentation, local hair loss, eczema, and chronic urticaria. Moreover,
higher incidence of vitiligo, psoriasis, and discoid lupus erythematosus was reported
among SM poisoned patients. In the study of Hefazi and colleagues (2006), delayed
cutaneous complications of SM poisoning 1620 years after exposure among
Iranian veterans, the main objective findings were hyperpigmentation (55 %), dry
skin (40 %), multiple cherry angiomas (37.5 %), atrophy (27.5 %), and hypopig-
mentation (25 %).
Emadi et al. (2008) in a study on 800 war veterans 1420 years after SM intoxi-
cation noticed that most of the patients (93 %), showed non-specific skin disorders,
while only 5 % developed scars with different patterns principally at the sites of
previous MG-induced skin injuries (Emadi et al. 2008).
Scarring, results from connective tissue hypertrophy and dysregulated fibroblast
activity during wound repair. It can be incapacitating, especially in the genital area
(Momeni et al. 1992). In a cross-sectional study on 43 SM Iranian patients con-
ducted by Layegh and colleagues (2011), the main cutaneous complain was itching
(23.30 %). The most common clinical diagnosis was multiple Cherry angioma
(72.1 %), which were significantly more common in SM-exposed group than in the
controls. Significant lower skin moisture and lipid content in the SM exposed
veterans compared with control group was also reported, thus, decreased function
of stratum corneum and lipid production was considered as a delayed SM skin effect
(Layegh et al. 2011).
Histopathological examination of skin biopsies has been revealed non-specific
findings such as epidermal atrophy, keratosis, and basal membrane hyperpigmenta-
tion. Non-specific fibrosis and melanophages have also been observed within the
dermis (Balali-Mood et al. 2005a; Fekri et al. 1992; Hefazi et al. 2006).
Sparse case reports of skin malignancies have been reported up to now and no
casual connection has been firmly stablished (Emadi et al. 2012). It could be con-
cluded that cutaneous malignancies appear to be a late uncommon consequence of
SM exposure (Firooz et al. 2011). However, it may need a longer period of time for
a malignancy to occur.
The skin hyper and hypo-pigmentaions of three patients with skin delayed com-
plications of SM poisoning around 2 years after exposure are illustrated in Figs. 5.1,
5.2, and 5.3.
5.3.5 Delayed Neuropsychiatric Complications
In a study conducted by Namazi and colleagues (2009) on 134 patients with long-
term complications of SM poisoning, the most common neurological complications
were headache (26.86 %), epilepsy (16.42 %), vertigo (11.94 %), and tremor
(4.48 %) (Namazi et al. 2009). In a survey of delayed neurological complications of
SM poisoning (2012), sensory nerve impairments, including paresthesia (88.3 %),
hyperesthesia (72.1 %) and hypoesthesia (11.6 %) were the most commonly
observed clinical complications. Fatigue (93 %), paresthesia (88.3 %) and headache
(83.7 %) were the most common subjective findings, while hyperesthesia (72.1 %)
was the most objective finding.
Fig. 5.1 Skin hyper and

hypo-pigmentations of the
neck of a patient around 2
years after SM exposure
during the Iraq-Iran war
(Unpublished slide of a
SM veteran under Prof.
Balali-Moods medical
care, taken with permission
of the patient)

hypopigmentation of the
thorax of a patient around
2 years after SM exposure
during the Iraq-Iran war
(Unpublished slide of a
SM veteran under Prof.
Balali-Mood medical care,
taken with permission of
the patient)

hypopigmentation of the
low back of a patient
around 2 years after SM
exposure during the
Iraq-Iran war (Unpublished
slide of a SM veteran
under Prof. Balali-Mood
medical care, taken with
permission of the patient)
Sparse delayed neurological complications of SM that were reported are

mostly discussing about peripheral neuropathies and neuromuscular lesions.
Electromyography (EMG) and Nerve Conduction Velocity (NCV) findings
showed abnormal pattern in seven SM patients (16.3 %) out of twelve patients
who had the clinical indication for the experiments. NCV disrupted patterns were
symmetric in both upper and lower extremities. Three patients had pure sensory
polyneuropathy and four patients had sensory-motor distal polyneuropathy of
axonal type. EMG pathologies contained chronic polyphasic motor unit action
potential (MUAP) in distal tested muscles (Darchini-Maragheh et al. 2012).
Balali-Mood et al. (2005a) reported 77.5 % peripheral neuropathy in 43
SM-intoxicated Iranian veterans with more sensory than motor nerve dysfunc-
tions. It was also concluded that although late complications of SM are usually
because of its direct toxic effect, neuromuscular complications are probably the
result of systemic toxicity (Balali-Mood et al. 2005a). Even after electrophysio-
logical procedures, approximately 50 % of polyneuropathies remain unrevealed
(Darchini-Maragheh et al. 2012).
Exposure to CWA is an extreme traumatic event that has long-lasting adverse
consequences on mental health. Long-term psychological symptoms of SM vic-
tims appear to be more related to the trauma caused by the war itself rather than
SM poisoning per se. Strong association between physical illnesses and psychiat-
ric disorders in chemical warfare survivors has been reported (Mansour Razavi
et al. 2012). In addition, exposure to war, adverse physical health consequences
and also fear of the future CWA exposure, represent an additive effect for involved
and persistent mental health (Hashemian et al. 2006). Disorders of emotion
(98 %), memory (80 %), behavior (80 %), attention (54 %), consciousness (27 %)
and thought process (14 %) were reported in 70 SM patients 35 years after expo-
sure (Balali-Mood 1986). Hashemian et al. (2006) reported in a cross-sectional
study on long-term psychological impact of chemical warfare on a civilian popu-
lation of Kurdish ethnicity, compared with individuals exposed to warfare, those
exposed to warfare and chemical weapons were at higher risk for lifetime Post-
Traumatic Stress Disorder (PTSD), increased anxiety and depressive symptoms
(Hashemian et al. 2006). Roshan et al. (2013) compared 367 SM exposed civil-
ians from Sardasht, Iran with matched control group and reported significantly
more somatization, obsessive-compulsive, depression, anxiety and hostility
among exposed civilians. In addition, significant differences between the two
groups were reported regarding the Global Severity Index (GSI) and Positive
Symptom Distress Index (PSDI). Razavi et al. (2014) in a review of articles has
described long-term common psychiatric complications of SM exposure. The fre-
quency of emotional problems was (98 %), memory impairment (80 %), behav-
ioral abnormalities (80 %), social performance disturbances (10.73 %), anxiety
(1865 %), insomnia (13.63 %), low concentration (54 %), severe depression
(646 %), personality disorders (31 %), thought processing disturbances (14 %),
seizures (6 %), psychosis (3 %), based on reviewing valid published articles.
Lifetime and current PTSD have been reported as 859 % and 233 % in the lit-
erature, respectively (Razavi et al. 2014). Vafaee and Seidy (2004) showed that
the frequency of depression in physically injured victims was two times more
than the control group and in chemically injured victims was two times more
frequent than physically injured victims (Vafaee and Seidy 2004). Functional
aphonia, photophobia, and dry eyes have also been previously reported in Iranian
SM victims (Balali-Mood et al. 2005a).
5.3.6 Delayed Immuno-hematological Complications
SM can cause long-term effects on hematologic and immune system in patients with
moderate and severe intoxication. While leukopenia and anemia were reported to be
major acute hematological variations following SM exposure, the total red blood cell
(RBC) count as well as hematocrit (Hct) level is higher than expected in long-term
phase, due to hypoxemic status of SM patients as a result of respiratory problems.
Long-term follow-up of Iranian veterans showed a significant increase in the per-
centage of the reticulocyte counts (Keramati et al. 2013). White Blood Cell (WBC)
count is higher among SM exposed patients which is more attributed with recurrent
respiratory infections in these patients rather than direct effects of SM on the bone
marrow (Mahmoudi et al. 2005). Decrease in both cell-mediated and humoral immu-
nity have been reported several years after exposure with SM among Iranian veter-
ans (Ghotbi and Hassan 2002). Balali-Mood and colleagues (2005) reported
long-term hematological and immunological complications of 40 patients with
delayed complications of SM poisoning as follows: Total WBC and RBC counts as
well as HCT level were significantly higher in SM group. The percentages of mono-
cytes and CD3+ lymphocytes were significantly higher, while the percentage of
natural killer cells was significantly lower in the SM patients. Serum IgM and C3
levels were significantly higher in the patients in comparison with the controls
(Balali-Mood et al. 2005a). Riahi-Zanjani and colleagues (2014) reported lower lev-
els of IL-1, IL-8 levels and TNF among SM poisoned Iranian veterans compared
with a control group, but levels of other assayed cytokines including IL-2, -4, -5, -6,
-10, -12, IFN and TNF were not significantly different between the two groups.
Keramati and colleagues (2013) in a study on 42 Iranian SM-exposed and a control
group, reported higher reticulocytes as well as lower total protein and albumin levels
in veterans compared to the controls. In addition, significant increase of serum lipids
and gamma-glutamyl transferase activity were also reported in the patients. In a
study on 40 Iranian veterans with late complications of SM 1620 years after expo-
sure conducted by Mahmoudi and colleagues (2005), the percentages of monocytes
and CD3+ T-lymphocytes were significantly lower in the patients. CD16 + 56 posi-
tive cells were significantly higher in patients than in the control group. IgM and C3,
as well as absolute levels of 1, 2 and globulins were also significantly higher in
the patients (Mahmoudi et al. 2005). Hassan and Ebtekar (2002) demonstrated
increased levels of IgG and IgM even 8 years after exposure to SM compared to the
controls. Decreased number of natural killer cells (CD45+/CD56+) plus higher
activity of natural killer cells (CD56+/CD25+) was also reported as long-term

immunological complication of SM (Ghotbi and Hassan 2002). Impaired immunity,
especially in the number of B and T lymphocytes, as a long-term complication of
SM exposure could be responsible for increased risk of recurrent infections among
SM victims (Balali-Mood 1986).
5.3.7 Other Delayed Complications
Knowledge about long-term mustard-induced cardiotoxic effects reveals possible

relationship between SM and heart diseases. Ventricular diastolic abnormalities
have been reported as late cardiac complication and were much more frequent than
the ventricular systolic abnormalities in the literature (Gholamrezanezhad et al.
2007; Pishgoo et al. 2007; Rohani et al. 2010). Veterans exposed to SM have been
shown lower functional capacity, reduced right ventricular function and elevated
pulmonary artery pressure compared to the control group (Shabestari et al. 2013).
Regarding to the respiratory disorders in the veterans, as one of the most common
long-term SM complications, which can lead to the well-known cor-pulmonale, role
of cardiac performance in occurrence of this phenomenon remains to be clear (Emad
and Rezaian 1997).
Gholamrezanezhad et al. (2007), in scintigraphic myocardial perfusion scans of
22 veterans with late complications of SM exposure during the Iran-Iraq war,
declared that patterns of myocardial perfusion in case group was completely differ-
ent from the controls and was resembled to either coronary artery disease or mild
cardiomyopathic changes. It was also noted that both dilated right ventricular cham-
ber and ischemia were significantly more prevalent among SM patients
(Gholamrezanezhad et al. 2007). Shabestari et al. (2011) in a study on 40 mustard-
poisoned patients, reported coronary artery ectasia as the most finding of conven-
tional angiography with a prevalence of 22.5 % versus 2.2 % in the control group. It
was concluded that coronary ectasia occurs approximately 11 times more frequently
in SM poisoned veterans, as a delayed complication. Karbasi-afshar and colleagues
(2013) compared conventional angiography findings of Iranian veterans with late
complications of SM exposure with unexposed grou and reported significantly
higher incidence of atherosclerotic lesions among the SM patients, compared to the
control group (Karbasi-Afshar et al. 2013).
Few studies are available regarding the urogenital and reproductive complica-
tions of SM, thus data on this issue are both lacking and contradictory. Soroush
et al. (2009) in a survey of 289 Iranian male veterans, reported history of urinary
calculi in 17.3 %, recurrent urinary tract infections (UTI) 8.7 %, Benign Prostatic
Hyperplasia (BPH) 1.7 % and kidney failure in 0.7 % of the patients. In delayed
phase of SM intoxication, the main target of gonadal effect injury is spermatogen-
esis (Panahi et al. 2013). Three years after SM exposure during Iran-Iraq war, infer-
tile victims showed almost total atrophy of the seminiferous epithelium and intact
interstitial cells. In addition, the infertile azoospermic in SM victims appeared to

have a Sertoli cell only pattern in the testicular biopsy (Safarinejad 2001). Several
years later, these findings were confirmed by Amirzargar and colleagues (2009)
(Amirzargar et al. 2009). Balali-Mood (1992) reported significantly diminished
sperm count among SM-exposed veterans in comparison to unexposed militants,
39 years post-exposure (Balali-Mood 1992). Azizi et al. (1995) also described
reproductive effects of SM on Iranian veterans following battlefield exposure. The
sperm count was less than 3 million cells/mL and the FSH level was higher compar-
ing with that of normal men (Azizi et al. 1995). In contrast, results of another study
by Ghanei et al. (2004c) failed to have an association between long-term infertility
and SM exposure in residents of Sardasht, Iran. Although studies resulted in contro-
versial findings, it seems that serum levels of the reproductive hormones are within
the normal range in SM-exposed men several years post-exposure (Panahi et al.
2013). Ahmadi et al. (2014) reported the prevalence of sexual dysfunction in Iranian
chemically injured veterans as 65.9 % as opposed to 33.0 % in non-chemically
injured veterans. The most commonly affected domain in both groups was erectile
dysfunction.
SM is considered as a suspected carcinogen CWA due to ability of chromatid
aberration and inhibition of DNA, RNA and protein synthesis and thus classified as
a carcinogen agent. Behravan and colleagues (2013) measured DNA breaks using
single-cell microgel electrophoresis technique under alkaline conditions (Comet
assay) after 25 years of SM exposure in Iranian veterans, and reported significant
higher lymphocyte DNA damage in SM-exposed individuals compared with a
matched control group (Behravan et al. 2013). In addition, Point mutations of p53
consistent with SM-induced DNA damage have been observed in some Iranian vic-
tims with lung cancer (Hosseini-khalili et al. 2009). Although former reports are
available on excessive occurrence of malignancies after the WWI and in high-dose
occupational exposures, there are sparse studies reporting higher occurrence of
malignancies among chemical victims of Iran-Iraq war. Bronchogenic carcinoma,
as well as carcinoma of the nasopharynx, thyroid cancer, adenocarcinoma of the
stomach, acute myeloblastic and lymphoblastic leukemia, have been case reported
in Iranian SM veterans (Balali-Mood 1992; Balali-Mood and Hefazi 2005a; Ghanei
and Vosoghi 2002; Zojaji et al. 2009).
In a group of 500 Iranian SM-exposed patients compared with 500 unexposed
soldiers 18 years post-exposure, only three cases with malignancies were found
among the exposed veterans. Although no such cases occurred in the unexposed
group, there was no significant correlation between cancer occurrence and exposure
to SM (Gilasi et al. 2006). Therefore, as quantitative risk assessment cannot be
developed from the available data, long-term follow-up is required to discover the
incidence of carcinogenicity among Iranian SM victims.
As SM distributes systematically, it may affect several body organs. Iran is within
the few countries faced several massive high-dose SM exposures. Thus, the litera-
ture should be made in Iran, and it seems a must for the Iranian scientists to investi-
gate all other possible effects of SM.
5.4 Clinical Management of Delayed SM Complications,

According to Iranian Experiences
5.4.1 Management of Respiratory Complications
Respiratory complications are the most common cause of long-term disability

among SM-exposed Iranian veterans. As noted in Khateri et al. study (2003), among
34,000 Iranian SM victims, 42.5 % were suffering from respiratory problems. In
moderate to severe SM exposure nearly all suffering from delayed respiratory com-
plications of SM poisoning. Thus, most studies on clinical management of SM poi-
soning in chronic phase are focused on respiratory problems.
Physical therapies Physical therapies are important for chronic pulmonary dis-
eases, in which SM lung injuries are not exception. Respiratory physiotherapy
rehabilitation are postural drainage of sputum and chest percussion and vibration
applied by devices during deep breathing (Razavi et al. 2013b).
Mucolytic agents N-Acetyl Cysteine (NAC) as a mucolytic and antioxidant agent
could be effective in the treatment and control of couphing due to SM. NAC
improves PFT, quality of life and could also reduce bronchial infections and exac-
erbations (Ghanei et al. 2008b). NAC is a potent antioxidant agent that acts as a
pro-drug for cysteine and glutathione. It may produce effects by preventing the
release of inflammatory mediators in different lung conditions. Particularly, could
be effective in the treatment and control of clinical conditions in COPD patients as
it interacts with inflammatory processes underlying the pathophysiology of COPD
(Ghanei et al. 2008b).
Ghanei and colleagues (2008b) determined the effects of NAC on SM-induced
bronchiolitis obliterans, as a delayed lung complication in Iranian victims. After 4
month of follow-up, dyspnea, wake-up dyspnea and cough significantly improved
compared with the control group. Spirometric components were also significantly
improved in NAC group compared to the placebo group. It was noted that 1200 mg
oral NAC per day can be used in treating bronchitis and bronchiolitis in SM patients
(Ghanei et al. 2008b). Shohrati et al. (2008) in a clinical trial conducted on 144
Iranian SM victims with BO, found that administration of NAC (1800 mg daily) for
4 months can significantly improve clinical conditions as well as PFT indices
(Shohrati et al. 2008).
Bronchodilators Combined agents including a beta agonist like salbutamol and an
anticholinergic such as ipratropium bromide has long been prescribed to improve
lung functions in patients with moderate and severe SM poisoning (Sohrabpour
et al. 1996; Balali-Mood and Navaeian 1986). Bronchodilators can also be applied
in SM-victims with increased airway hyper-reactivity. In a study by Ghanei et al.
(2007), two regimens of combination inhaler therapy on amount of reversibility of
chronic bronchiolitis in SM exposed patients were studied. Patients received either
combination form of fluticasone propionate and salmetrol or beclomethasone and
salbutamol inhaler. Respiratory symptoms and PFT indices were improved in both
groups after 12 months of follow-up. It was concluded that inhaled corticosteroids
beside long-acting 2-agonists are effective in treatment of chronic bronchiolitis as
a late complication of exposure to SM (Ghanei et al. 2007).
Corticosteroids Inhaled corticosteroids are widely used in treatment of delayed
lung complications due to SM poisoning. Altered lung function in SM-induced lung
injury causes accumulation of inflammatory cells in the respiratory tract and pro-
duction of inflammatory mediators suggests that steroids play a key role in the treat-
ment (Yaraee et al. 2009). In addition, oral corticosteroids are used only in patients
of respiratory exacerbation. On the other hand, since SM complications are contrib-
uted to higher morbidities rather than mortalities, long-term prescription of oral
corticosteroids may increases the complications and have no effect on patients sur-
vival. Therefore, although inhaled corticosteroids are significantly effective in this
setting, maintenance and long-term oral corticosteroids consumption should be con-
sidered for only very severe cases.
In a study on 65 Iranian SM veterans with chronic bronchitis, patients were
divided into two categories. Intravenous treatment group who received intravenous
methylprednisolone acetate 500 mg daily for 6 months and oral treatment group
who received oral prednisolone 1 mg/kg daily for 6 months. It was revealed that,
there was significant improvement in PFT indices of both groups in approximately
half of the patients over the study period. There was no difference between the pulse
corticosteroid versus oral corticosteroid therapy in the patients (Ghanei et al. 2005b).
However, ineffectiveness of corticosteroids in airway reversibility in more than
50 % of mustard poisoned cases, may imply the absence of active eosinophilic
inflammation in these patients. It was previously reported that mustard lung injury,
is a neutrophil dominated inflammatory disease and thus oral corticosteroids may
not have a considerable effect (Ghanei and Harandi 2007).
Nonsteroidal anti-inflammatory agents Reports indicate that, use of nonsteroi-
dal anti-inflammatory drugs may be effective in late SM lung complications (Razavi
et al. 2013b). It has been shown that administration of NAC can also reduce the
inflammation phenomena in the lungs (Ghanei et al. 2008b).
Macrolides Macrolide antibiotics are effective in reducing SM-induced overpro-
duction of pro-inflammatory cytokines and mediators, as well as improving the
degenerated chemotactic and phagocytotic functions of monocytes following SM
exposure. Macrolides may lead to improvement of apoptotic material in the airway
and thus cause reduced airway inflammation due to SM inhalation (Poursaleh et al.
2012). As non-eosinophilic (neutrophil mediated) inflammation is relatively com-
mon in mustard lung patients, the macrolides are one of best candidate to play their
anti-inflammatory role. In chronic bronchitis and bronchiolitis, as late SM lung
complications, administration of a 6-month combination of clarithromycin and
NAC has been recommended (Ghanei and Harandi 2007).
Gamma interferon Transforming growth factor 1 (TGF-b1) substantially
increased in BAL aspirates and target tissues of SM exposed patients and thus plays
a fundamental role in the pathogenesis of progressive inflammatory and fibrotic

diseases such as idiopathic pulmonary fibrosis and BO (Aghanouri et al. 2004).
Therefore, it can be proposed to treat the victims with gamma interferon which
reduce TGF-. Short-term administration of oral and intravenous corticosteroids for
exacerbated forms is recommended and when therapeutic response is negative,
gamma interferon may be effective (Ghanei et al. 2005b). It could also have benefi-
cial in post-lung transplant patients (Poursaleh et al. 2012).
The IFN-1b is a bioengineered form of interferon gamma that acts as a biologic
response modifier through stimulation of the human immune system. It was shown
in SM- poisoned Iranian victims with long-term lung complications received
6-month treatment with IFN-1b plus a low-dose prednisolone revealed an improve-
ment in the PFT indices (Ghanei et al. 2006b; Panahi et al. 2005).
It is presumed that gamma interferon response to treatment in SM lung patients
can be attributed to the down regulating effects on TGF-b1 (Aghanouri et al. 2004;
Ghanei et al. 2006b).
Other therapeutic choices Regarding to the pathogenesis of the disorder (oxidant-
antioxidant imbalance) in long-term SM-induced lung injuries, certain antioxidants
in reducing chronic pulmonary complications could play a therapeutic role (Shohrati
et al. 2008, 2010). Curcuminoids are phytochemicals with remarkable anti-
inflammatory properties that are derived from dried rhizomes of the plant Curcuma
longa L. (turmeric). It was shown in a randomized double-blind clinical trial there
was a great effect of curcuminoids vs. placebo in modulating inflammatory media-
tors included IL-6, IL-8, TNF, TGF, substance P and CRP (Panahi et al. 2014).
Thymus vulgaris essence (thyme) was reported not to have any effect in improve-
ment of respiratory symptoms in patients with chemical bronchitis among SM vic-
tims in Kurdistan, Iran (Razavi et al. 2013b). Magnesium ion has several well-known
effects on the respiratory system and can be applied to SM lung patients suffering
from asthma. Magnesium ion stabilizes mast cells, relaxes smooth muscles in the
respiratory system leading to bronchial dilation and decreases bronchial responsive-
ness in tracheobronchial tree (Razavi et al. 2013b).
Pulmonary artery hypertension is a delayed complication of SM poisoning
which can lead to progressive right heart failure and death (Shabestari et al.
2013). Sildenafil as an approved drug for treatment of pulmonary artery hyper-
tension has been shown to decrease pulmonary artery pressure in SM victims
(Razavi et al. 2013b). Therapeutic effects of these compounds have yet to be
confirmed. In advanced cases, lung transplantation may be indicated, but since
SM victims have long-term survival, this method is not usually indicated (Razavi
et al. 2013b).
SM lung complications is supposed to be different from one patient to another
due to various internal factors such as healthy status, underlying diseases, genetic
tendency, etc. as well as external factors such as toxicities, duration and frequency
of exposure, emergent and follow-up medical care, co-exposures and smoking.
Therefore, we recommend making decisions case by case to choose suitable therapy
in this setting.
5.4.2 Management of Ocular Complications
Management of delayed ocular complications of SM is difficult and requires an

overwhelming long-term follow-up. To date, no definite treatment for the delayed
keratitis caused by SM has been confirmed. Therapy initially is symptomatic and
includes measures to address tear deficiency and ocular surface instability.
Preservative-free artificial tears, therapeutic contact lenses, immunosuppressive
drugs such as azathioprin, temporary or permanent punctal occlusion, blepharor-
rhaphy, and tarsorrhaphy can be used according to the severity of keratitis (Balali-
Mood 1992; Balali-Mood and Hefazi 2005a). A limited course of topical steroids
may be used to control recurrent episodes of superficial inflammation, keratitis, or
limbal inflammation. Some sort of surgical intervention are ultimately required in
the vast majority of victims. Corneal argon laser photocoagulation has limited suc-
cess in the prevention of corneal vascularization. Keratoplasty, has also proven inef-
fective since the limbal blood supply is poor in these patients (Balali-Mood and
Hefazi 2005a; Javadi et al. 2011).
The outcomes of penetrating keratoplasty (PK) in delayed-onset mustard gas
keratitis in 22 eyes indicated a clear graft in 77.3 % of cases, but it failed in 22.7 %
after 41 months and subepithelial or endothelial graft rejection, or both, developed
in 50 % of cases (Javadi et al. 2007).
In a study of 175 eyes of 90 cases with delayed SM complications (2011), 41.1 %
of limbal stem cell deficiency necessitating stem cell transplantation. Limbal stem
cell transplantation techniques were living-related conjunctival-limbal allograft
(lrCLAL) and keratolimbal allograft (KLAL). Corneal transplantation techniques
were PK and lamellar keratoplasty (LK). In terms of clinical outcomes and graft
survival rates after 101 months of follow-up, it was concluded that limbal and cor-
neal abnormalities as delayed eye complications of SM poisoning can be managed
best by KLAL and LK, respectively (Javadi et al. 2011).
5.4.3 Management of Skin Complications
Management of skin complications in chronic phase is almost symptomatic. Skin

healing proceeds by re-epithelialization starting from skin adnexae, as well as via-
ble epidermis at the border of the lesion. In delayed phase skin management, local
emollients and systemic antihistamines can improve skin dryness and reduce itch-
ing. These drugs beside topical corticosteroids are currently the most administered
medications for chronic skin lesions and pruritus due to dermal complications of
SM poisoning. Frequent taking shower and bath should be discouraged as well as
continuous use of sunscreen lotions applied for prevention of hyper-pigmented
lesions. Contractures rarely occur with chemical burns caused by SM (Balali-Mood
and Hefazi 2005b, 2006). As chronic cutaneous complications of SM intoxication
could be categorized as a form of atopic dermatitis, corticosteroids are widely used
as effective therapeutic approach, however, continuous and long-term application is

associated with the incidence of several side effects (Panahi et al. 2012b).
In the study conducted by Panahi et al. (2007), a phenol 1 % and menthol 1 %
combination showed significant therapeutic effect for pruritus and other skin lesions
due to SM skin exposure, in comparison to placebo. In another randomized clinical
trial, compared topical pimecrolimus with betamethasone in the treatment of pruri-
tus and chronic skin lesions due to SM exposure, topical pimecrolimus was as effec-
tive as topical betamethasone in controlling long-term skin lesions of SM exposure
(Panahi et al. 2008). In a double-blind control trial which compared the safety and
efficacy of doxepin (10 mg/day) and hydroxyzine (25 mg/day) in the treatment of
chronic pruritus due to exposure to SM in Iranian veterans, severity of pruritus
decreased by 80 % in the hydroxyzine group and 75 % in the doxepin group, after 4
weeks of treatment, both had equivalent results in controlling the symptoms of
patients (Shohrati et al. 2007). Doxepin cream 5 % was also reported to have equal
efficacy with betamethasone cream 0.1 % to control pruritus caused by SM (Panahi
et al. 2011). Higher improvement of skin clinical symptoms have been reported by
IFN- (50 g/m2) subcutaneously three times per week versus betamethasone valer-
ate topical cream 0.1 % every night in the treatment of SM-induced chronic skin
complications (Panahi et al. 2012a, b).
Overall, there is no specific treatment for the delayed toxic effects and complica-
tions of SM in different body organs, so the main therapeutic approach is symptom-
atic and supportive therapy. These patients receive many drugs for the management
of multiple organ diseases. Thus, they are at high risk for drug interactions and
adverse drug reactions. Given the range of chronic health effects of SM, patients
should be managed by a clinical toxicology expert in the field or by a multidisci-
plinary clinical teams of specialists. Financial, social, and cultural support as well
as health education to maintain in a good life style is also of great importance.
Spirituality and religious practice may also an effective approach among Iranian
veterans to cope with their chronic illness complications (Ebadi et al. 2009). Besides,
reassurance and supportive love care of the veterans in the family as well as in the
society, are very important in their health management.
There is still paucity of information regarding the medical management of toxic
effects of SM poisoning, a subject which greatly challenges toxicologists and
health-care specialists. Research cannot be stopped until we completely eradicate
the threat of this agent from the military and civilian worlds.
5.5 Conclusion and Recommendations
The Iran-Iraq war caused hundreds of thousands deaths and injured, millions of
displaced, and billions of dollars cost. CWAs were frequently used by Iraqi troops
during Iran-Iraq war and Iran faced several massive high-dose SM exposures during
the imposed war. More than 100,000 chemical causalities as well as 25,000 chemi-
cal mortalities have been recorded in Iran. Even after three decades of the war,
around 40,000 Iranian veterans have complains of delayed effects of SM poisoning.

As SM is a potent incapacitating CWA, some toxic effects of SM poisoning in
intoxicated patients persist for their entire life.
Iranian veterans still suffering from delayed complications of SM exposure in
different body organs of which the lungs, eyes and skin are the three major involved
organs in delayed SM effects. Respiratory complications are the greatest cause of
long-term disability among Iranian veterans which exacerbate overtime.
Furthermore, neuropsychiatric, reproductive, urogenital, immuno-hematological
and cardiac complications as well as cellular damage and carcinogenicity alongside
with many other delayed complications have been studied and reported among
Iranian SM veterans. Thus, veterans need life-long medical and nursing care. As
there is no specific treatment for delayed toxic SM effects, the main therapeutic
approach is symptomatic and supportive therapy. Financial, social, and cultural sup-
port as well as reassurance and supportive love care of the veterans in the family and
society are also important beside medical therapy.
Although there is no conclusive evidence of SM use since the establishment of
the Organization for the Prohibition of Chemical Weapons (OPCW) in 1993, the
threat of SM use as well as other CWAs for a possible chemical war and or terrorism
is still exist. Therefore, preparedness on the management of SM exposure is
required. Health professionals should learn more and be updated on the possible
abuse of any of CWAs particularly SM poisoning. Poison centers and medical toxi-
cologists are playing important roles in training, preparedness and leadership.
Glossary
Anemia A reduction in the number of circulating erythrocytes or in the quantity

of hemoglobin
Aphonia Complete loss of phonation due to organic disease of the larynx or to
nonorganic (i.e., psychogenic) causes
Asthma A form of bronchial disorder with three distinct components: respiratory
hypersensitivity, airway inflammation, and intermittent airway obstruction. It is
characterized by spasmodic contraction of airway smooth muscle, wheezing, and
dyspnea
Atherosclerosis A thickening and loss of elasticity of the walls of arteries that
occurs with formation of atherosclerotic plaques within the blood vessels
Atrophy Decrease in the size of a cell, tissue, organ, or multiple organs
Axon Nerve fibers that are capable of rapidly conducting impulses away from the
neuron cell body
Azoospermia A condition of having no sperm present in the ejaculate (semen).
Bronchiectasis A disease in which there is permanent enlargement of parts of the
airways of the lung
Bronchiolitis obliterans Inflammation of the bronchioles leading to an obstruc-
tive lung disease. Characterized by fibrous granulation tissue with bronchial exu-
dates in the lumens. Clinical features include a nonproductive cough and dyspnea
Carcinogenicity The ability to produces cancer

Cherry angioma Also called capillary angioma, De Morganss spots, and senile
angioma. A small, bright red, clearly circumscribed vascular tumor on the skin.
More than 85 % of people over 45 years of age have cherry angiomas on their
skin
Clubbing The rounding of the ends and swelling of fingers found in people with
lung disease
Conjunctiva The mucous membrane that covers the posterior surface of the eye-
lids and the anterior pericorneal surface of the eyeball
Core pulmonale Hypertrophy and dilation of the right ventricle of the heart, gen-
erally caused by chronic disease and malfunction of the lungs. This condition
can lead to heart failure
Cornea The transparent anterior portion of the fibrous coat of the eye consisting
of five layers and serves as the first refracting medium of the eye
Crackles Abnormal noise, heard on auscultation over any part of the respiratory
tract
CWA Chemical warfare agents: a chemical substance whose toxic properties
are used to kill, injure or incapacitate human beings
Cyanosis A physical sign causing bluish discoloration of the skin and mucous
membranes. It is caused by a lack of oxygen in the blood and could be associated
with cold temperature, heart failure, lung diseases or something else
Discoid lupus erythematosus A chronic form of cutaneous lupus erythematosus
in which the skin lesions mimic those of the systemic form but in which systemic
signs are rare. It is characterized by the presence of discoid skin plaques show-
ing varying degrees of edema, erythema, scaliness, follicular plugging, and skin
atrophy. The condition typically involves the face and scalp, but widespread dis-
semination may occur
Dysphonia An impairment in the ability to produce voice sounds using the vocal
organs
Dyspnea Shortness of breath or breathlessness or feelings associated with
impaired breathing
Ectasia The condition of an anatomical structures being dilated beyond normal
dimensions
Eosinophil Granular leukocytes with a nucleus that usually has two lobes, con-
nected with threads of chromatin and cytoplasm, containing coarse, round gran-
ules that are uniform in size and stainable by eosin
Epilepsy A disorder characterized by recurrent episodes of paroxysmal brain dys-
function due to a sudden, disorderly, and excessive neuronal discharge that may
be manifested as episodic impairment or loss of consciousness, abnormal motor
phenomena, psychic or sensory disturbances, or perturbation of the autonomic
nervous system
extremes of weather, radiation, or chemical agent which may have a harmful
effect
Hyperaesthesia Increased sensitivity to cutaneous stimulation due to a dimin-
ished threshold or an increased response to stimuli
Hypercapnia A clinical manifestation of abnormal increase in the amount of car-

bon dioxide in arterial blood
Hyperinflation Excessive inflation or expansion, as of the lungs
Hypoesthesia Absent or reduced sensitivity to cutaneous stimulation
Hypoxemia Relatively absence of oxygen in one or more tissues
Immunoglobulin Multi-subunit proteins which function in immunity. They are
produced by B lymphocytes from the immunoglobulin genes. They are com-
prised of two heavy and two light chains with additional ancillary polypeptide
chains depending on their isoforms. They are divided by the amino acid sequence
of their heavy chains into five classes: Ig A, Ig D, Ig E, Ig G, and Ig M and vari-
ous subclasses
Intoxication An abnormal state that is essentially a poisoning
Ischemia A hypoperfusion of the blood through an organ or tissue caused by a
pathologic constriction or obstruction of its blood vessels, or an absence of blood
circulation
Lacrimation The secretion of tears, especially in excess.
Lethal dose 50 The dose amount of poisonous or toxic substance or dose of ion-
izing radiation required to kill 50 % of the tested population
Leukopenia Decrease in number of leukocytes
Melanophage A histiocyte that contains phagocytized melanin
Mitosis A type of cell nucleus division by means of which the two daughter nuclei
receive identical complements of the number of chromosomes of the somatic
cells of the species
Mutagenicity The ability of a chemical or physical agent to cause permanent
changes in DNA
Nerve agents Any of several highly toxic organophosphorus compounds, devel-
oped as chemical warfare agents because of their ability to inhibit cholinesterase
Neutrophil A granular leukocyte having a nucleus with three to five lobes, con-
nected with threads of chromatin and cytoplasm, containing very fine granules
and stainable by neutral dyes
Paraesthesia Subjective cutaneous sensations (e.g., cold, warmth, tingling, pres-
sure, etc.) that are experienced spontaneously in the absence of stimulation
PFT Pulmonary function test
Photophobia Abnormal sensitivity of the eyes to light. This may occur as a
manifestation of eye diseases, migraine, subarachnoid hemorrhage, meningitis,
depression and other mental disorders
Presbyopia Known as Old eye: the normal decreasing elasticity of the crystalline
lens that leads to loss of accommodation and the eyes ability to focus on close
subjects.
Pruritus An intense itching sensation that produces the urge to rub or scratch the
skin to obtain relief.
Psoriasis A common genetically determined, chronic, inflammatory skin disease
characterized by rounded erythematous, dry, scaling patches. The lesions have
a predilection for nails, scalp, genitalia, extensor surfaces, and the lumbosacral
region
Pulmonary fibrosis A process in which normal lung tissues are progressively

replaced by fibroblasts and collagen causing an irreversible loss of the ability to
transfer oxygen into the bloodstream via pulmonary alveoli
Pulmonary hypertension Increased vascular resistance in the pulmonary circu-
lation, characterized by increased pressure in the pulmonary artery. It could be
secondary to heart diseases or lung diseases
SM Sulfur mustard: a class of related cytotoxic and vesicant chemical warfare
agents with the ability to form large blisters on the exposed skin and in the lungs
Spermatogenesis The process of germ cell development in the male from the
primordial germ cells, through the mature haploid spermatozoa
Spirometry A test using an instrument called a spirometer, for measurement of
the breathing capacity of the lungs, such as in pulmonary function test
Toxicity The degree to which a substance can damage an organism
Tracheobronchomalacia A congenital or acquired condition of underdeveloped
or degeneration of cartilage in the trachea and the bronchi. This results in a
floppy non-rigid airway making patency difficult to maintain
Tremor Cyclical movement of a body part that can represent either a physiologic
process or a manifestation of disease
Vertigo An illusion of movement, either of the external world revolving around
the individual or of the individual revolving in space, in any plane
Vitiligo A disorder consisting of areas of macular depigmentation, commonly on
extensor aspects of extremities, on the face or neck, and in skin folds. Age of
onset is often in young adulthood and the condition tends to progress gradually
with lesions enlarging and extending until a quiescent state is reached
Wheezing A high-pitched whistling sound associated with labored breathing. It is
most common in exhaling and occurs when an individual tries to breathe deeply
through air passages that are narrowed or filled with mucus
Xerosis Abnormal dryness, as of the eye, skin, or mouth
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Chapter 6
Upper Respiratory Complications of Sulfur
Mustard (SM) Poisoning
Ramin Zojaji and Morteza Mazloum Farsi Baf
Contents
6.1 Introduction .................................................................................................................... 136
6.1.1 Brief History of Mustard Gas Usage as a Chemical Weapon ............................ 137
6.1.2 Chemical and Physical Properties ...................................................................... 137
6.1.3 Routes of Entry and Types of SM Poisoning ..................................................... 138
6.2 Upper Respiratory Tract................................................................................................. 138
6.2.1 Anatomy and Physiology ................................................................................... 139
6.3 Clinical Features ............................................................................................................ 142
6.3.1 Early Clinical Features of the Upper Respiratory
Tract After Mustard Gas Exposure .................................................................... 143
6.3.2 Late Clinical Features of the Upper Respiratory
Tract After Mustard Gas Exposure .................................................................... 144
6.3.3 Linkage of the Early and Late Toxic Effects of SM .......................................... 150
6.4 Upper Respiratory Tract Cancer .................................................................................... 150
6.5 Molecular Mechanisms Involved in Toxic Effects of Mustard
Gas in the Upper Respiratory Tract ............................................................................... 152
6.6 Diagnostic Approaches to the SM Induced Injuries in Upper Respiratory Tract .......... 155
6.6.1 High Resolution Computed Tomography (HRCT) ............................................ 155
6.6.2 Laryngoscopy ..................................................................................................... 156
6.6.3 Video Laryngoscopy and Stroboscopy .............................................................. 157
6.6.4 Speech Evaluation .............................................................................................. 158
6.7 Experimental Study of SM -Induced Upper Respiratory
Tract Diseases in the Animal Models ............................................................................ 158
6.7.1 Acute Effects ...................................................................................................... 158
6.7.2 Chronic Effects .................................................................................................. 159
R. Zojaji, MD (*)
Otorhinolaryngology Department, Mashhad Branch, Islamic Azad University, Mashhad, Iran
Arya Teaching Medical Hospital ENT Department, Islamic Azad Medical University,
Golestan 5, East GolestanStr, Jahanbany Street, Mashhad, Iran
e-mail: raminzojaji@yahoo.com
M. Mazloum Farsi Baf, MD
Faculty of Medicine, Mashhad Branch, Islamic Azad University, Mashhad, Iran
e-mail: mmazloomfarsibaf@yahoo.com

136 R. Zojaji and M. Mazloum Farsi Baf
6.8 Clinical Management of SM-Induced Damages in the Upper Respiratory Tract .......... 160
6.8.1 Management of Acute Phase of Poisoning ........................................................ 160
6.8.2 Management of Chronic Phase of Poisoning ..................................................... 161
6.8.3 Treatments .......................................................................................................... 162
6.9 Conclusion and Recommendations ................................................................................ 163
Glossary .................................................................................................................................. 163
References ............................................................................................................................... 164
Abstract Various chemical agents have been used as a war weapon. Sulfur mus-
tard (SM) due to its low cost, easy access and easy manufacture and storage are
the most wildly used warfare agents in the world. It was used widely during Iraq-
Iran conflict against Iranian troops. SM is a potent alkylating blistering agent that
causes low mortality, but it could incapacitate a large number of soldiers in the
war. SM exposure may occur in occupational or war exposure. SM can be absorbed
from skin, eye, mouth and respiratory and the gastrointestinal systems. Among
these organs, respiratory tract and skin are the main susceptible organs for SM
intoxication and injury. Upper and lower respiratory tract may be affected by SM,
however the acute and chronic effects of SM in upper respiratory tract has been
less studied and most of studies have focused on lung injuries induced by SM. This
study reviewed early and late clinical features and complications of SM in upper
respiratory tract as well as its molecular mechanism of action and treatment.
Keywords Upper respiratory tract Sulfur mustard Poisoning Complications

Inflammation Sinusitis Laryngoscopy Thyroid cancer
6.1 Introduction
The main chemical agents that have been used as weapons can be categorized into
four groups of choking, blistering, blood, and the nerve agents. The well known
chemical warfare agents (CWA) in these groups are as follows: chlorine and phos-
gene as choking agents, sulfur mustard and lewisite as blistering, hydrogen cyanide
as a blood agent, tabun, sarin and soman as the nerve agents.
Sulfur mustard (SM) conventionally known as mustard gas is a potent alkylating
and blistering agent with the chemical formula of (ClCH2CH2)2S. It was first dis-
covered by a German chemist in 1822 (Despretz 1822) and then was synthesized by
Guthrie and Niemann separately in 1860 (Guthrie 1860; Niemann 1860). Acute
mortality due to SM exposure is low (about 24 %) and the required doses of SM
for acute mortality following gas inhalation is about 50 times greater than the fatal
dose for Nerve agents. Only intoxication with very high doses of SM (inhalation
and dermal exposure) can cause mortality in humans over 1 h after exposure. Death
usually occurs because of suffocation due to respiratory damage (Marshall 1987;
Maynard et al. 1991).
6 Upper Respiratory Complications of Sulfur Mustard (SM) Poisoning 137
However, SM incapacitating ability is more important than its lethality because

it could incapable large numbers of soldiers in the war.
The other mustard compound, is nitrogen mustard which has remarkable vesi-
cant effect and its mechanism of action, pharmacology, toxicology and symptom-
atology are similar to SM. Nitrogen mustard has been used successfully as an
antimitotic and anticancer medication for several decades, but has never been used
as a CWA (Saladi et al. 2006; Kehe et al. 2008).
Due to its very simple synthesis, easy accessibility, and low-cost production, SM
has been the most widely distributed and used warfare agent in the world (WHO
1970; Szinicz 2005; Balali 1984; Graham et al. 2009). SM disperses as a vapor,
aerosol, or in liquid droplets in the environment and remains active for a long time
and thus is a threat not only for the war troops, but also for the civilians and animal
life (Graham et al. 2005).
6.1.1 Brief History of Mustard Gas Usage as a Chemical Weapon
Since the discovery, SM has been used as a toxic CWA against human beings in
different combats. SM as a chemical warfare agent was used first by the German
army in the World War I against French and the other allied troops in 1917. Since
then, despite the Geneva protocol (1925) that banned application of these agents
against human being, they have been used in many battles. SM was used by Italy in
1936 against Ethiopian troops, by Poland against Germany in 1939, by Egypt
against Yemeni civilians in the mid-1960s (WHO 2004) and extensively used by
Iraqi army against Iranian troops and even civilians during 19831988 in Sardadht
(Iran) and Kurdish city of Halabja (Leikin et al. 2007; Papirmeister et al. 1991).
6.1.2 Chemical and Physical Properties
SM is synthesized from chemical reaction between ethylene and sulfur chloride

[SCl2 + 2 C2H4 (ClCH2CH2)2S] or by combination of 2, 2-dihydroxyethyl sulfide
with HCl gas [(HO-CH2CH2)2S + 2HCl (ClCH2CH)2S + 2H2O].
It is a viscous and oily colorless (in pure form) or yellow or brown (when mixed
with other chemicals) liquid at room temperature, with slight garlic or horseradish
type odor. The melting point of pure SM is 14 C (57 F) and it catalyzed at 218 C
(424.4 F) before boiling. By spraying and explosion of bomb, SM becomes aero-
solized and dispersed in the air. SM vapors are heavier than air and spread along the
ground, accumulate and remain in poorly-ventilated and low-lying areas for a long
time. SM vapors have marked penetrating power and it can penetrate usual cloth,
leather, wood and paint on metallic surfaces but metal, glass and glazed tiles are
resistant against it. Liquid SM is heavier than water but its droplets float on water
surfaces in the contaminated areas.
SM is highly soluble in the fat and organic solvents, which contributes to its
rapid absorption from the skin and mucosal membranes. It is poorly soluble in water
but when it contacts with water, it hydrolyses to thiodiglycol and hydrochloric acid
which are different toxic chemicals (Rosemond et al. 2003).
SM primarily dispersed in liquid or vapor form and due to its persistency
can stay in the ground and water for a long time especially in cold environment.
Therefore, its exposure may occur later after the attack (Balali-Mood and
Hefazi 2005a, b).
6.1.3 Routes of Entry and Types of SM Poisoning
SM can be absorbed in the body through dermal, oral, respiratory, ocular and the
gastrointestinal routes (Ketabchi 1998). SM poisoning may occur during occupa-
tional or non-occupational exposures. Occupational exposure may happen in mus-
tard gas industry workers, medical staff caring for SM exposed patients and in a
research lab personnel using this agent without proper protection. Non-occupational
exposure may occur during working on the soil previously contaminated with SM
and in a battle that SM is used (ATSDR 2003).
SM is highly lipophilic and easily penetrates into the skin and mucosal mem-
branes and provides high bioavailability (Drasch et al. 1987). Warm and moist
membranes absorb more SM and consequently are most vulnerable to hazardous
effects of this agent. Also, in hot and humid weather, SM action increases.
Considering the above mentioned conditions, respiratory tract, the eyes and skin are
the main vulnerable organs for SM intoxication. Inhalation is the major route of
exposure that causes respiratory and systemic toxicity (Hefazi et al. 2005).
In dermal route, about 80 % of SM evaporates from the skin and only 20 % of
SM penetrates the skin within 10 min (Kehe et al. 2000).
In the respiratory route, after inhalation, SM passes through upper airways
before reaching the lung tissue. During this passage, most of SM vapor is removed
by the upper airways and a small amount reaches the alveoli. Absorption of mus-
tard gas by the upper airways, results in damages in these airways while protect-
ing the lung tissue from the destructive effects of SM. The upper airway mucosa
has natural cooling capacity and absorbs the heat of gases before reaching the
trachea. Thus, the heat of SM can severely damage the upper airways mucosa
6.2 Upper Respiratory Tract
Understanding of the anatomy and physiology of upper respiratory tract is required

to understand of the injuries and complications of mustard gas in this system.
6.2.1 Anatomy and Physiology
Respiratory system is divided into the upper and lower respiratory tract. Upper
respiratory tract begins from external nares of nose and mouth that continues to the
larynx (Fig. 6.1). The lower respiratory tract begins after the larynx to the alveoli.
During passing the inspiratory airflow through the upper respiratory tract, large par-
ticles are filtered and the air is moistened and warmed and is delivered to the lower
respiratory tract.
Functionally, respiratory system is divided into the conducting and the gas
exchanging parts. The nose, mouth, pharynx, larynx, trachea and bronchi are vari-
ous parts of conducting airways. The respiratory bronchioles and alveoli compose
the gas exchanging part of the lungs. Thus, upper respiratory tract is a part of con-
ducting airways in the respiratory system (Gaga et al. 2001).
Nose is the first part of upper airways which is divided into two cavities by nasal
septum. These two cavities again at the nasopharynx level join together and form a
unique airway. Nasal vestibules are the most anterior parts of the nasal cavity. They
are narrowing towards the main nasal cavity in junction with the main nasal cavity
making the narrowest part of the airways, which is called nasal valve. Nasal vesti-
bules are enclosed by the cartilages of nose and are covered by stratified squamous
epithelium and contain hairs (vibrissae) and sebaceous glands. The small hairs of
vestibules act as a filter and remove any large dust particles in the inspirated air.
These short stiff hairs are exceedingly sensitive to certain mechanical stimuli and
respond immediately to the stimulation with itching and sneeze, protecting and
notifying (Gaga et al. 2001).
Sphenpidal sinus
Frontal sinus
Superior meatus Cribriform plate
of ethmoid bone
Middle meatus
Superior concho
Pharyngeal tonsil
Opening of Middle concha
Inferior concha
pharyngotympanic Vestibule
(auditory) tube
Inferior meatus
Nasopharynx Nostril
Posterior nasal aperture Hard palate
Soft plate
Uvula Tongue
Lingual tonsil
Palatine tonsil
Epiglottis
Fauces
Hyyoid bone
Oropharynx
Thyroid cartilage
Laryngopharynx of larynx Laryngeal
Vestibular fold Cricoid cartilage cartilages
Vocal fold
Esophagus
Thyroid gland
Tranchea
Fig. 6.1 Anatomy of upper respiratory tract

In the main nasal cavity, there are three bony structures protruding from the lat-
eral wall on each side which are known as the nasal turbinates or conchae. Inferior,
middle and superior turbinates increase the surface of the nose whereas at the same
time narrow the lumen. These structures facilitate close contact of inhaled air with
the nasal mucosa, and promote humidification and warming of the air. Also, nasal
turbinates by air conditioning and shaping the nasal airway provide air flow turbu-
lence and increase deposition and trapping of the particles on to the nasal mucosa.
Therefore, the air that is delivered to the lower airways is filtered and conditioned
(Mygind et al. 1990).
The olfactory region has been placed in the upper part of the nasal cavity.
Paranasal sinuses are air-filled spaces located within the bones of the skull and
face around the nasal cavity (Fig. 6.2). They communicate with nasal cavity and
provide voice resonance and possibly heat and cold insulation (Blanton and
Biggs 1969).
The surface of the paranasal sinuses is covered with ciliated pseudostratified
columnar epithelium.
Formation of paranasal sinuses begins in the fetus by excavation of bone and air-
filled spaces from the nasal cavity. This process continues and completes after birth
during the course of growth and maturity. Four paranasal sinuses in human are max-
illary, sphenoid, ethmoid and frontal sinuses that maxillary sinuses are the largest
sinuses (Rhys Evans 1987).
Frontal
Ethmoidal
Sphenoidal
Maxillary
Fig. 6.2 Anatomy of nasal sinuses

The next part of the upper airways after the nose is the pharynx. This structure
consists of the nasopharynx, oropharynx, and hypopharynx. The nasopharynx
begins from the choanae down to the lower margin of the soft palate. The orophar-
ynx, which is located behind the oral cavity extends from the soft palate to the tip of
the epiglottis inferiorly. The hypopharynx extends from the upper margin of the
epiglottis to the lower border of the cricoids cartilage, serving as the channel from
the oropharynx to the laryngeal inlet and esophagus (Kimoff 2005).
The openings of the Eustachian tubes, the adenoids and the tonsils are located in
the pharynx.
The pharynx is involved in both the digestive and respiratory tracts and directs
the food to the oesophagus and to the stomach and the air to the trachea and lungs.
The last part of the upper respiratory tract after the pharynx is the larynx. The
larynx after the nasal valve is the second narrowest part of the airway. It is the organ
of phonation and acts as a valve that protects the lower airways and the lungs
(Fig. 6.3). The vocal cords and several cartilages are located in the larynx. The larg-
est cartilage found in the larynx is the thyroid cartilage, which produces Adams
Apple prominence on the front of the neck. Another cartilage is the epiglottis that
lies on top of the larynx and prevents entrance of the food to the trachea during
swallowing. The laryngeal mucosa is loosely bound to the supporting cartilage
(Gaga et al. 2001).
Below the larynx, lower respiratory tract begins with trachea which is supported
by irregular rings of cartilage that are incomplete dorsally. These cartilages prevent
the trachea from collapsing during the rise of intrathoracic pressure. The trachea at
Posterior
Corniculate cartilage
Glottis
Cuneiform cartilage
False vocal cord
Vocal cord
Epiglottis
Root of tongue
Anterior
Fig. 6.3 Anatomy of Larynx

its distal end bifurcates to the two main bronchi. The main bronchi also are divided
and keep branching and make smaller airways. From the trachea to the alveoli 823
generations of airways may exist. The cartilaginous rings yet are present in the main
bronchi but they are scarce in the small and more distal airways while no cartilage
is found in the bronchioles. The conducting airways end at the terminal bronchioles.
After these, respiratory bronchioles and alveoli are present that constitute the gas
exchanging unit of the lung (Gaga et al. 2001).
6.3 Clinical Features
Since SM is very lipophilic, it can easily penetrate epithelial tissues and cause
marked local damage as well as severe systemic intoxication (Kehe and Szinicz
2005). SM has bidirectional effects; a direct effect via inhalation, and an indirect
effect by recirculation. Studies using whole-body autographic with S35-labeled SM
have shown increased radioactivity in the nasal region after percutaneous or intrave-
nous administration (Clemedson et al. 1963).
The eyes, nasal mucosa, throat, pulmonary tract, and skin are the most com-
monly affected sites of body by SM. These organs are the main targets for direct
toxic effects of SM (Ghanei et al. 2006b).
As SM is dispersed in the form of aerosol or vapor (Borak and Sidell 1992), it
enters the body by inhalation and the first contact area of inhaled toxins with
respiratory tract is the nasal and oral mucosa. Most of the SM is absorbed in the
upper airways and little reaches the lung parenchyma and alveoli. This mecha-
nism protects the lung tissue against toxic effects of SM but causes upper airway
diseases. SM deeply affects respiratory tract from its initial contact area of nasal
and oral cavity to the vulnerable surfaces of distal respiratory targets of the pul-
monary tree.
The special nature of the respiratory system mucosal membranes, the rapid turn-
over of its epithelium, the large surface area of the respiratory tract and the oily
nature and persistency of the SM (Vander et al. 1998), all cause susceptibility of the
respiratory tract to the toxic effects of SM (Graham and Schoneboom 2013).
Epithelial cells of respiratory tract are extremely vulnerable to the toxic effects
of mustard gas.
The main injures of respiratory damage by SM are sloughing of the epithelial
cells and increases in production of the secretions in the entire respiratory tract.
These changes cause nasal discharge, bronchiolar obstruction, and even broncho-
spasm. These events may interfere with gas exchange at the alveolar level, which
can result in hypoxia, hypercarbia, and respiratory and metabolic acidosis (Borak
and Sidell 1992; Kehe and Szinicz 2005; Haber 1986).
Effects of SM in the respiratory tract from the nasal mucosa to the terminal bron-
chioles are dose dependent (Hefazi et al. 2005; Balali-Mood and Hefazi 2006) and
the inhalation dose depends on the respiration rate; the higher respiratory rate, the
higher inhalation doses (Maynard 2007). Severity of intoxication can be different
based on the age, duration and frequency of exposure, gas concentration and quan-
tity, environmental temperature, and the use of protective equipments.
SM can induce early (acute) and late (chronic) complications in the upper respi-
ratory tract (Rowell et al. 2009; Bijani and Moghadamnia 2002).
6.3.1 Early Clinical Features of the Upper Respiratory Tract

After Mustard Gas Exposure
The data about early effects of SM on upper airways are scares and there is only one
study available in this regards, is from the Iraq-Iran battle (Kehe et al. 2009).
Potent acids, alkalies, mustard gas, phenols, cresols orphosphorus can cause
chemical burns.
Characteristics of burn injury in the upper respiratory tract are different from
those in the bronchus and lung parenchyma. Chemical burn complications in the
upper airway usually develop late with tracheal stenosis after a symptom-free
period, unlike the lower respiratory tract injury, which manifests soon after burns
(Yang et al. 1999). Symptoms of burn injury in the upper respiratory tract include
aphonia, wet or breathy voice quality and inability to initiate a swallow (Pore and
Reed 1997).
Due to the high chemical reactivity of mustard gas, most of the acute damages
are limited to the upper respiratory tract (Iwaszkiewicz 1966). In the acute phase of
exposure mustard gas has direct contact with upper airway mucosa and irritates
them directly. Acute damage to the respiratory tract causes acute edema, inflamma-
tion, and destruction of the airway epithelial which its severity is different based on
the exposure dosage (Pechura and Rall 1993).
In high exposure doses, the clinical respiratory effects of SM after inhalation
include an immediate phase of coughing and choking. Upper and lower airways
edema with ulcerations and necrosis and tracheobronchitis may develop also in
severe exposure usually several hours after exposure (Kehe and Szinicz 2005).
In exposure to moderate SM doses, rhinorrhea, loss of smell and taste, nose and
throat discharge and lacrimation are the main observed symptoms (Kehe and Szinicz
2005).
In lower exposure doses, acute respiratory damage occur but the symptoms do not
appear immediately and usually there is a brief symptom free delayed period of few
hours which is followed by the development of a variety of acute respiratory symp-
toms such as rhinorrhea, pain, nose, sinus and pharynx discomfort, sinusitis, sinus
pain, sneezing, and sore throat as well as respiratory irritation symptoms including
dyspnea, tachypnea, coughing and choking and dysphonia. The early respiratory
symptoms usually develop 216 h after exposure. Rhinorrhea is common symptom
but bleeding from the nasal mucosa is rare (Borak and Sidell 1992; Tang and Loke
2012; Kehe and Szinicz 2005; Miller and Chang 2003). Hoarseness, dry cough and
sputum production are other symptoms that could develop following acute inhala-
tion of SM (Iwaszkiewicz 1996; Balali-Mood 1986; Taghadosi et al. 2002).
Early symptoms appear in chronological orders based on the dose and mode of
exposure, the environmental temperature, the extent of use of protective masks, and
the age. Rhinorrhea, sneezing, and sore throat usually develops within 26 h of
exposure. Aphonia, hoarseness, and non-productive cough appear after 624 h and
productive cough develops in 2448 h after exposure. Respiratory problems,
improve slowly, although some cough and weak hoarseness may remain for as long
as 6 weeks (Balali-Mood and Hefazi 2006; Papirmeister et al. 1991). However, it
may take longer time (12 months) to recover, particularly after secondary infec-
tions and necrotic bronchopneumonia (Papirmeister et al. 1991).
Based on the inhaled dosage, the damage can be mild to severe. Severe damage
induce epithelial destruction and sloughing and subsequent formation of pseudo-
membranes, which may progress to airway obstruction and result in death (Pechura
and Rall 1993). These pathologic changes in severe cases are manifested with pul-
monary edema, respiratory failure and death in less than 4 % of the patients (Borak
and Sidell 1992; Kehe and Szinicz 2005; Haber 1986). Inhalation of higher concen-
trations of vapor induce laryngeal damage with aphonia or husky voice and injury
to the upper medium-sized airways with tracheobronchitis, which usually occurs
several hours after exposure (Ghanei et al. 2006a) and is presented by a nonproduc-
tive hacking cough (Mx 2003).
In a study on acute effects of SM in chemical victims of Iraq-Iran war, 12 Iranian
victims were evaluated in Germany. These victims arrived in Munich 68 days
(17 days in one case) after exposure and were treated in 3 hospitals during 1984
1985 (Kehe et al. 2009). The patients distance from explosion was 530 m and
none of them had used protective equipment. In this study there was no relationship
between the age and the course of disease. The most common early clinical effects
of SM exposure in the upper respiratory tract were hoarseness, sore throat and pro-
ductive cough that were observed almost in all patients. Less common respiratory
symptoms were purulent sputum (8/12) and bloody sputum (5/12). Tracheal steno-
sis occurred in two patients with 10 and 60 % occlusion of the lumen. Twenty per-
cent of patients required tracheotomy (Kehe et al. 2009).
Pathologic changes detected in upper airways were edema (45 %), inflammation
(27 %) and obliterative necrosis (27 %). Also, it was found that the healing process
in the bronchial tract lesions is faster than that of the throat (Kehe et al. 2009).
Table 6.1 shows the manifestations of upper respiratory effects of SM reported in
different studies.
6.3.2 Late Clinical Features of the Upper Respiratory Tract

After Mustard Gas Exposure
Late effects of SM poisoning refer to all organ dysfunctions and abnormalities

that occur several years after the first exposure (Ghanei and Vosoghi 2002; Emad
and Rezaian 1997; Easton et al. 1988; Bijani and Moghadamnia 2002).
6
Table 6.1 Manifestations of Upper respiratory tract complications of Sulfur Mustard exposure in different studies
Upper respiratory
tract complications Large airway
Authors, year narrowing (%) Cough (%) Expectoration (%) Dysphonia (%) PND (%) Laryngitis (%) Sinuisitis (%)
a a a
Taghadosi et al. 82.7 64.4 4.6 28.7
(2002)
a a a a
Sohrabpour et al. 91 77 83
(1988)
a a a a a
Amini and 66 33
Oghabian (2013)
a a a
Akhavan et al. 90 74 82
(2009)
a
Balali-Mood et al. 97.7 88.4 79.1 41.9 14.8 55
(2010)
a a a a a a a
Ghanei et al.
(2006b)
a a a a
Namazi et al. 2.72 72.38 52.98
(2009)
a a a a a a
Emad and Rezaian 9.64
(1997)
a a a a a a
Hefazi et al. (2005) 15
Kehe et al. (2009) 0 96.1
Upper Respiratory Complications of Sulfur Mustard (SM) Poisoning
a a a a a
Ghanei et al. 24.2 100
(2004a, b)
a
Is not investigated in the study
145
Unlike early effects, there is big data in the literature about the delayed toxic
effects of SM in the respiratory tract which most of them are from the Iraq-Iran war.
However, little data is available about SM related late clinical effects in the upper
respiratory tract from Iraq-Iran conflict.
Evidences have shown that long-term respiratory effects may occur even in the
absence of early-phase symptoms. This suggest that late effects are not necessarily
dependent to the presence of acute-phase effects and they develop by independent
mechanisms (Pechura and Rall 1993).
Late respiratory complications are the major cause of long-term disability and
could occur from a few months to several years after exposure. Most available infor-
mation on late effects are related to the lungs and lower respiratory airway and there
is limited information about long-term effect of SM on upper airways.
Early symptoms in acute phase reduce and subside during a few weeks after
acute exposure to SM, but the damages persist and gradually progress into the
chronic forms. This condition is progressive and during several years, it will convert
to delayed complications.
In the first Iranian report on 236 veterans suffering from SM poisoning, the
most common complications were found in the respiratory tract (78 %) followed
by the central nervous system (45 %), the skin (41 %) and the eyes (36 %)
(Balali-Mood 1986). The patients with mild to severe toxicity were included in
the above mentioned study and were evaluated 228 months after SM exposure
(Balali-Mood 1986).
Khateri et al. (2003) study obtained results somewhat different from Balali-
Mood report. In their study on 34,000 Iranians veterans exposed to SM, the most
common complications were observed in the lung (42.5 %), eyes (39.5 %) and skin
(24.5 %) (Khateri et al. 2003). The difference between these two studies may be due
to the difference in the study population as the Balali-Mood patients had severe SM
exposure and were evaluated after 228 months while most of the patients in Khateri
et al. study had mild SM exposure and were evaluated 1823 years following expo-
sure (Khateri et al. 2003; Balali-Mood 1986). In a study on 43 male veterans by
Zojaji et al., the most common affected sites were the lung (95.5 %), peripheral
nerves (77 %), the skin (73 %), eyes (68 %), and head and neck (16.2 %), respec-
tively. The results of this study are similar to those of Balali-Mood et al. (Zojaji
et al. 2009; Balali-Mood 1986).
Delayed effects of SM in the upper airways are characterized by chronic inflam-
mation of the oral cavity, pharynx and larynx, inflammation and ulceration of the
palate, nasopharynx, oropharynx and laryngeal cancer with aphonia (Papirmeister
et al. 1991; Akhavan et al. 2009).
Laryngitis is one of the main delayed complications of upper respiratory tract among
Iranian chemical veterans (Razavi et al. 2013; WHO 1987). Other delayed complica-
tions of respiratory tract include chronic bronchitis, bronchiectasis, asthma, large air-
way narrowing, and pulmonary fibrosis (Balali-Mood 1986; Emad and Rezaian 1997).
Airway narrowing in the late phase is a sequel of acute damage to the trachea and
large airways and occurs due to the scarring or granulation tissue formation in the
acute phase. Airway stricture usually develops 2 years after exposure (Balali-Mood
et al. 2005; Ghanei et al. 2004a, b).
In the chronic phase, chronic cough and sputum production are the main symp-
toms of chronic bronchitis in the victims (Emad and Rezaian 1997; Ghanei et al.
2005). The most important causes for chronic cough in the late phase are broncho-
spasm, postnasal drip syndrome, gastroesophageal reflux disease, bronchiectasis,
tracheobronchial collapse and postnasal discharge due to chronic sinusitis (Ghanei
et al. 2006b).
In the first study in 1988, late respiratory effects of SM intoxication were inves-
tigated in 35 Iranian soldiers 6 weeks to 1 year after SM exposure. The most com-
mon upper respiratory tract symptoms were cough in 91 % and dysphonia in 83 %
of patients (Sohrabpour et al. 1988).
In a study that evaluated 39 patients with chronic cough exposed to a single
high dose of SM, paranasal sinus mucosal abnormalities was identified in 76.9 %
of the patients, in which 20.5 % had severe mucosal thickening (Ghanei et al.
2006b).
In another study carried out in Iran (Akhavan et al. 2009), late laryngeal effects
of SM was assessed in 50 victims after 20 years of SM exposure. That study found
hoarseness in 32 %, intermittent dysphonia in 74 %, and continuous dysphonia in
4 %, harshness in 14 % and chronic laryngitis in 82 % of patients. Unilateral vocal
cords paralysis was identified in three patients (6 %) and laryngeal nodules in 12 %
of victims. They reported vocal cord paralysis as a long-term neurotoxic effect of
SM and synechia and vocal cord nodules as a result of laryngeal and bronchial
infections. Also, they concluded that hypertrophy of false vocal cords is probably
due to dysfunction of the edematous true vocal cords and dysphonia. This study is
the only found report that focused on laryngeal effect of SM poisoning (Akhavan
et al. 2009).
Balali-Mood et al. in 2010 assessed delayed toxic effects of SM on respiratory
tract in 43 male victims of Iraq-Iran war 2025 years after poisoning. In their study,
dysphonia was found in 79.1 %, post-nasal discharge (PND) in 41.9 %, lower larynx
position in 30.2 %, vocal cords limitation in 25.6 % and mucosal inflammation of
larynx in 14.8 % of patients and therefore dysphonia and chronic sinusitis were the
most common delayed effects of SM in upper respiratory tract (Balali-Mood et al.
2010). Vocal cords paralysis and laryngeal nodules were not detected in their
patients. Mucosal inflammation of sinuses was found in 25.9 % of patients in Balali-
Mood et al. study while in 79 % in Akhavan et al. report. Balali-Mood and col-
leagues concluded that most of delayed toxic effects of SM in upper respiratory
tracts were inflammatory and infectious complications.
Namazi et al. (2009) studied long-term complications of SM intoxication in 134
chemical veterans about 20 years after exposure in Iraq-Iran battle. In their study, all
patients suffered from dyspnea, 72.38 % from coughing, and 52.98 % from expec-
toration (Namazi et al. 2009).
Table 6.2 demonstrates demographic and clinical feature of respiratory compli-
cations in different studies in the world.
Table 6.2 Demographic and clinical features of respiratory complications in several studies throughout the world
148
Duration Rate of
between respiratory
Acute/chronic exposure and complications Upper respiratory
Authors, year Country Number Population Mean age complications study (years) (%) tract complications
Taghadosi et al. Iran 87 Veterans 35.58 6.45 Chronic 12 1.5 90.8 *
(2002)
Sohrabpour et al. Iran 35 Veterans 28 10.4 Chronic 6 week-1 year 100 +
(1988)
Amini and Iran 62 Veterans 53 6.9 Chronic 20 2.4 100 *
Oghabian (2013)
Heydari and Ghanei Iran 19 Veterans + 41.32 4.63 Chronic >22 * +
(2011) civilian
Akhavan et al. Iran 50 Veterans 46.6 6.8 Chronic 20 100 +
(2009)
Balali-Mood et al. Iran 43 Veterans 50.6 8.9 Chronic 2025 * +
(2010)
Ghanei et al. Iran 39 Veterans 37.9 7.6 Chronic * 100 +
(2006b)
Namazi et al. Iran 134 Veterans 37.2 9 Chronic 1722 100 *
(2009)
Emad and Rezaian Iran 197 Veterans 34.39 5.95 Chronic 10 100 *
(1997)
Hefazi et al. (2005) Iran 40 Veterans 43.8 9.8 Chronic 1620 100 *
Khateri et al. (2003) Iran 34,000 Veterans 1730 Chronic 1320 42.5 *
Ghasemi Iran 600 Civilian 1980 Chronic 19 45.8 *
Boroumand et al. population
(2008)
R. Zojaji and M. Mazloum Farsi Baf
6
Ghasemi Broumand Iran 479 Militaries + 2160 Chronic * 32.1 *

et al. (2007) civilian
Etezad-Razavi et al. Iran 40 Veterans 43.8 9.8 Chronic 1620 95 *
(2006) (3276)
Kehe et al. (2009) Germany 12 Iranian 1846 Acute 417 days 100 +
Veterans
Bijani and Iran 220 Veterans <30 and >60 Chronic 613 100 *
Moghadamnia
(2002)
Ghanei et al. Iran 33 Veterans 43 8 Chronic 16 0.7 100 *
(2004a)
+
Presence of upper respiratory tract complications
*
This factor was not assessed in mentioned study
Upper Respiratory Complications of Sulfur Mustard (SM) Poisoning
149
6.3.3 Linkage of the Early and Late Toxic Effects of SM
In the respiratory tract, the early effects of SM usually progress to chronic

effects without disruption (Taghaddosinejad et al. 2011). Unlike the chronic
effects on the skin and eyes that recover during the time, respiratory complica-
tions usually progress and worsen over the years (Shirazi et al. 1988; Balali-
Mood and Hefazi 2006).
Chronic laryngitis, tracheobronchial stenosis, tracheobronchomalacia and
chronic bronchitis are the main delayed complications of SM exposure among
Iranian veterans (Akhavan et al. 2009; Ghanei et al. 2004a, 2006a; Emad and
Rezaian 1997; Khateri et al. 2003).
6.4 Upper Respiratory Tract Cancer
SM is a mutagenic and alkylating agent, which alkylates DNA. Experimental and

human studies have shown that SM is mutagenic and carcinogenic and could induce
mutation and chromosomal aberrations in animal model (Papirmeister et al. 1984;
Heston 1950; Takeshima et al. 1994).
Carcinogenicity of SM in human also, has been approved and the International
Agency for Research on Cancer (IARC) has confirmed SM as a human carcinogen
and has known it as a risk factor for occupational lung cancer (Ghanei and Vosoghi
2002; Nishimoto et al. 1998; Perchura and Rall 1993).
SM could induce malignant changes in various organs such as the hematopoietic
and respiratory systems.
However, most of available evidences about the mustard induced cancers of the
respiratory tract are related to the lung cancer and there is limited evidence about the
carcinogenic effects of mustard gas in the upper respiratory tract.
Most of primary data about the carcinogenicity of SM in human was about occu-
pational exposure obtained from workers of chemical factories with prolonged low
dose exposure to SM while there was no data on the carcinogenicity of single high-
or low- dose SM exposure (Easton et al. 1988; Manning et al. 1981; Wada et al.
1968; Dacre and Goldman 1996). Different studies also revealed increased risk of
respiratory tract cancers in the workers of chemical factories producing SM (Wada
et al. 1968; Manning et al. 1981; Easton et al. 1988).
Wada et al. (1968) study on 485 men showed a significant increase in death due
to the respiratory cancer including the lungs, pharyngeal and nasal cancer (33 cases
against 0.9 expected) among former workers of the Japanese poison gas factory
(Wada et al. 1968). The risk of the upper airway cancer in their study was 37 times
more than the normal population (Wada et al. 1968).
High incidence of cancer of the larynx, pharynx and other upper airways as well
as a moderately increased rate of mortality due lung cancer in the former workers of
a British SM manufacture was also reported (Manning et al. 1981).
In a cohort study by Easton et al. in 1988, the mortality due to cancer in 3530
men and women employed in the manufacture of mustard gas, highly significant
excesses of death was observed due to the cancer of gum and mouth, larynx and
pharynx compared to the national death rates of these cancers. Mortality due to the
lung cancer was even moderately excessive in comparison to the upper respiratory
tract cancers (Easton et al. 1988). The increased rate of death due to cancers of the
tongue, salivary gland, and nose was not significant. Also, it was found that the risks
for pharyngeal and lung cancer were significantly related to the duration of employ-
ment (Easton et al. 1988). They also compared the mortality of World War II Navy
veterans with low dose SM exposures to that of veterans without exposure and did
not find any increase in the risk of cause-specific mortality (Easton et al. 1988). In
their study relative risk for cancers of the pharynx, larynx, lung, and other upper
respiratory sites were associated with duration of exposure. Also, the risk of respira-
tory cancers among production workers was not considerably greater than that of
workers at other factory parts.
Nishimoto et al. (1988) in their study on 1632 workers of SM factory found a
fivefold increase in the risk of cancer in the workers employed in the production as
well as in other factory parts with direct contact with SM (Nishimoto et al. 1988).
They observed an excess of cancers in the nasal sinuses, pharynx, and larynx as
well. This study again confirmed the Easton et al. study (1988) and showed that the
risk of cancer was significantly associated with the duration of exposure. These
studies concluded that long time SM exposure is a risk factor for occupational can-
cers of upper respiratory tract and the lungs. They have also proved a causal rela-
tionship between occupational exposure to SM and respiratory cancers.
Evidence on the mutagenicity and carcinogenicity of mustard gas in human also
obtained from battlefield exposures and accidents as well (Hosseini-Khalili et al.
2009). Hosseini-Khalili et al. (2009) assessed p53 and KRAS (Kirsten rat sarcoma)
mutations in 18 SM victims with lung cancer. They found eight point mutations in
p53 but no mutation in KRAS. The frequent p53 mutation in these patients was
similar to that frequently observed in workers of factory with prolonged exposure to
SM (Hosseini-Khalili et al. 2009).
Although the carcinoma of the nasopharynx and bronchogenic carcinoma were
reported in Iranian veterans (Balali 1992) but a later study in 1997 on 197 chemical
veterans of Iraq-Iran war could not find any more cases of bronchial carcinoma or
other lung cancers in the victims after 10 years of exposure to SM (Emad and
Rezaian 1997). In agreement with this study, again another study on the chemical
veterans exposed to SM during the World War I failed to show any significant
increase in the observed deaths due to the cancer (2.5 % vs. 1.9 % in controls)
(Norman 1975).
However, few years later, results of British and American studies showed
increased incidence of lung cancer from the World War I battlefield SM exposures
(Somani et al. 2001). Gilasi et al., investigated the incidence of cancer among 500
Iranian victims after 18 years of exposure and 500 unexposed soldiers. They could
only detect three cases of cancer in exposed group (Gilasi et al. 2006). They found
no significant relationship between cancer and acute exposure to SM.
Again, another study in Iran performed on 43 chemical veterans after 2025

years of exposure to SM did not find any malignancy in upper or lower respiratory
tract as well as the lung (Balali-Mood et al. 2010). Easton et al. (1988) observed that
development of the lung, pharynx and larynx cancers in SM exposed patients is
dose dependent (Easton et al. 1988).
Zojaji and colleagues in their study, identified two cases of thyroid cancer and
one case of nasopharyngeal carcinoma in victims exposed to SM during Iraq-Iran
war (Zojaji et al. 2009). The other investigators based on their studies concluded
that toxic effects of SM may remain in the body, even after several years of expo-
sure, it may relapse or develop a new disease (Balali-Mood and Hefazi 2006; Bijani
and Moghadamnia 2002; Hefazi et al. 2005).
In the recent cohort of Zafarghandi et al., 7570 Iranian victims exposed to SM
during Iraq-Iran combat were compared with 7595 unexposed veterans (Zafarghandi
et al. 2013). They were followed up for about 2223 years for development of
cancer. During the follow-up period, 84 cases of cancer were identified in the
exposed group while 49 cases detected in unexposed group. The crude incidence
rate of cancer for SM exposure was 1.81 and its hazard ratio was 2.02.
Among these identified cancers 75 % were hematological and gastrointestinal
cancers, which were the most common types of cancers in both study groups. Two
patients were diagnosed with thyroid cancer in exposed group; one with undiffer-
entiated thyroid carcinoma and the second one with papillary carcinoma of a thy-
roglossal cyst that developed 12 and 14 years after SM exposure, respectively. In
addition, nasopharyngeal carcinoma was developed in a patient, 12 years follow-
ing SM exposure (Zojaji et al. 2009). The incidence of head and neck cancer
between the two groups was not significantly different but the incidence of lung
and bronchial cancer in exposed group was significantly higher than the unexposed
group (p < 0.001) (Zafarghandi et al. 2013; Zojaji et al. 2009). The cohort revealed
that SM exposure significantly increases cancer incidence in the victims
(Zafarghandi et al. 2013).
It must be stated that the mentioned delayed effects are of a single low dose
exposure to SM, and differ from those caused by chronic occupational exposure
(Easton et al. 1988).
6.5 Molecular Mechanisms Involved in Toxic Effects

of Mustard Gas in the Upper Respiratory Tract
Understanding of the mechanisms of SM effects could lead to development of new

ways for treatment. Mustard gas is an alkylating agent that exerts its deleterious
effects trough several mechanisms.
It degrades exposed tissues and causes severe chemical burns. SM is extremely
reactive bifunctional chemical and has antimitotic, mutagenic, carcinogenic, terato-
genic and cytotoxic properties (Prentiss 1937).
Fig. 6.4 Molecular mechanisms involved in pathogenesis of sulfur mustard toxicity
The skin, eyes and respiratory tract are the main target organs of SM while
DNA is the most important molecular target (Malhotra et al. 1999). It reacts with
membranes (phospholipids), DNA, RNA, and proteins (Somani and Babu 1989).
SM by disrupting DNA and proteins impairs cell homeostasis and induces cell
death (Balali-Mood and Navaeian 1986b; Dacre and Goldman 1996; Balali-Mood
et al. 1986a).
Mustard acts on affected tissues by addition of an alkyl group to the cell compo-
nents (Fig. 6.4). Alkylation, occurs extremely rapidly and it is very difficult to control
(Ivarsson et al. 1992). SM can attack DNA and break it at specific nucleotides. It usu-
ally alkylates DNA at the site of nitrogen residue of guanine (Wheeler 1962).
Alkylation of deoxyribonucleic acid, DNA depletion and inactivation of glutathione
by SM induce significant damage to the cell (Ball and Roberts 1972; Balali-Mood
1986).
Irreversible alkylation of protein and nucleic acids mediates mono adduct forma-
tion with components such as ring nitrogens or extracyclic oxygens of nucleotide
bases and induces acute toxic effects of mustard gas (Shulman 1993). This mecha-
nism by disrupting structural and functional integrity of cells and tissues, induces
blister formation with severe pain and burning (Watson and Griffin 1992). The most
important cellular effect of SM is inhibition of glycolysis.
After absorption, SM activates biological compounds and induces severe electro-
philic tissue reactions by forming carbonium ions and transient complexes with
large molecules. Various biochemical reactions and DNA changes contribute in
cytotoxic and mutagenic effects of mustard gas (Maynard et al. 1991).
Chemical reaction of SM with proteins induces massive damage to all tissues
(Dacre and Goldman 1996). In the lungs, it induces severe inflammatory reaction in
the tracheobronchial epithelium and cause severe leukocyte infiltration, alveolar
hemorrhage, thrombus formation and vacuolation of lung parenchymal cells (Pant

and Vijayaraghavan 1999).
Two possible important mechanisms can contribute in destructive effects of
SM. Formation of a reactive sulphonium ion is the first step of both pathways. The
first contributing mechanism acts by bonding and alkylation of the base compounds
in DNA. The bonding of SM may induce DNA strands breakages and the formation
of bridges between the two strands of the DNA molecule. Cell death due to DNA
bridge formation occurs when the DNA replicates and cell undergoes division
(Balali-Mood et al. 2008). Formation of these bridges interferes with normal DNA
functioning during cell division, which may cause severe cell injury and possibly
cell death (Ivarsson et al. 1992). However, at high dose exposures, other mecha-
nisms different from DNA cross-linking are important and induce more rapid cell
death (Fig. 6.4).
DNA damage may also cause mutations and disturbance of the natural DNA
repair mechanisms. The destructive effects of SM on DNA may interfere with cell
division leading to the increase of cancer incidence (Ivarsson et al. 1992).
Experimental studies have shown that DNA alkylation has an important role in
delayed toxic effects of mustard gas (Ball and Roberts 1972; Walker 1971).
Takeshima et al. in their study on the workers of mustard gas factory, identified
two double point mutations (G:C to A:T) in the p53 suppressor gene in the lung tis-
sues from 12 workers with lung cancer exposed to SM while such mutation was not
detected in 12 nonexposed workers. They suggested that this double mutation might
be characteristic of mustard gas exposure (Takeshima et al. 1994).
Hosseini-Kalili et al. could also identify the same mutation as well as seven fur-
ther mutations in p53 suppressor gene in 18 chemical veterans with lung cancer
exposed to SM during Iraq-Iran war. Before this study, mutation detection has not
been performed in chemical victims with single high or low dose exposure to mus-
tard gas during combats (Hosseini-Khalili et al. 2009).
These two studies show that p53 mutations are relatively similar in those with
small-prolonged exposure and those with single low or high dose SM exposure
(Takeshima et al. 1994; Hosseini-Khalili et al. 2009).
Interaction with intracellular glutathione is the second mechanism of SM action.
Glutathion is a small peptide molecule that protects the cell against destructive
effects of the free radicals formed during cellular oxidation. It has also a critical role
in reducing reactive oxygen species in the cell and preventing peroxidation and
preserving membrane integrity (Rankin et al. 1980; Eklow et al. 2004).
Bonding of SM to a large amount of glutathione interferes with its function and
by disturbing regulation of these free radicals leads to increase in free radicals in the
cell. As free radicals are very toxic, accumulation of these radicals in the cell may
lead to sever damages to various cellular mechanisms (Ivarsson et al. 1992).
The other mechanism that may have a role in acute effects of SM, is nicotin-
amide adenine dinucleotide (NAD) depletion, which inhibits glycolysis and impairs
energy generation in the cell (Kehe and Szinicz 2005; Brkle 2001).
In addition, mustard gas can exerts its destructive effects by binding to different
cell proteins. It binds to the functional groups such as the sulphydryl or amino
groups. If SM for example binds to the active site of enzymes, it could inhibit the
enzyme activity and could induce metabolic disorders. If it binds for example to the
membrane proteins, it could modify uptake of substances and disturb the inner cell
environment (Ivarsson et al. 1992).
Molecular mechanisms involved in pathogenesis of sulfur mustard toxicity have
been shown in Fig. 6.4.
6.6 Diagnostic Approaches to the SM Induced Injuries

in Upper Respiratory Tract
SM can induce damage to the different parts of the respiratory system. Various
diagnostic methods have been used to detect mustard induced complications in dif-
ferent parts of the respiratory apparatus.
6.6.1 High Resolution Computed Tomography (HRCT)
For assessment of respiratory complications of SM, the best method with optimal sen-
sitivity and specificity should be selected. Chest X-ray is not a proper method for detec-
tion of respiratory complications in the patients exposed to SM instead high resolution
computed tomography (HRCT) of the chest is the method of choice and the most accu-
rate modality for the assessment of the lung parenchyma and bronchi (Bagheri et al.
2003; Bakhtavar et al. 2008). It is the most sensitive imaging modality for evaluation of
respiratory complications including increased bronchial wall thickening, airway steno-
sis, bronchiectasis and bronchiolitisobliterans (Bagheri et al. 2003; Ghanei et al.
2004b). Although HRCT is a powerful modality for detecting SM induced complica-
tions of both airways and lung parenchyma, but it cannot estimate the severity of respi-
ratory damage or determine the overall patients condition (Hefazi et al. 2005).
In Emad and Rezaian study, chest HRCT was performed for 197 veterans with
the history of single heavy SM exposure and 86 non-exposed veterans. Upper air-
way narrowing was detected in seven patients (3.55 %) in the trachea (Emad and
Rezaian 1997).
In a case-control study by Ghanei et al., 39 patients with SM exposure and
chronic cough comparing 35 controls with chronic cough but without exposure
were evaluated. Paranasal sinus computed tomography (CT) scan was performed
for all patients. Among obtained CT scan (74 cases and controls) except one, all
showed some abnormalities. Mucosal thickening was identified in 30 patients in the
case group (76.9 %). They found no significant difference in sinus pathologic find-
ings between the two groups (p > 0.05).In their study, the prevalence of various
sinus abnormalities was the same in both groups with chronic chough and there was
no significant difference in this regard between the two groups (Ghanei et al. 2006b).
This study identified sinus abnormalities as a further cause of chronic cough in addi-
tion to the previously known conventional etiologies (Ghanei et al. 2006b).
In another study, we evaluated 43 male veterans after 2025 years of SM expo-
sure. Sinus CT scan and HRCT were performed for those with clinical indication.
In their study, chronic sinusitis (55 %), lower larynx position (30.2 %), and mucosal
inflammation of larynx (14.8 %) were the most common upper respiratory tract
findings. Mustard induced sinus complications in sphenoidal, posterior ethmoidal
and maxillary sinuses were observed more than the anterior and frontal sinuses
(p < 0.001). We found that in each patient at least one sinus was affected. No malig-
nancy was detected in the upper respiratory tract of the exposed patients. Also, lung
HRCT was normal in 23.5 % of patients (Balali-Mood et al. 2010).
In a study, Ghanei et al. examined 300 chemical veterans after 15 years of SM expo-
sure by chest HRCT as well as 20 healthy controls. They detected tracheal collapse in
13 patients (4.3 %) and air trapping in 137 cases (45.7 %) and in 5 (25 %) controls
(p < 0.001). They found a significant association between the presence and severity of
air retention and the severity of tracheomalacia in HRCT (Ghanei et al. 2006a).
They concluded that both bronchiolitis obliterans and tracheobronchomalacia
have the same underlying mechanism affecting small and large airways, respec-
tively (Ghanei et al. 2006a).
6.6.2 Laryngoscopy
Laryngoscopy is the other useful method for assessment of upper airways including
throat and larynx. It is a procedure for visualization of vocal cord as well as glottic
structures. Evidence about laryngoscopic findings of patients exposed to SM is lim-
ited and there are only two reports in this field.
Akhavan et al. in a case series evaluated laryngeal complications of 50 male
chemical war veterans by fiberoptic laryngobronchoscopy. In their study, various
degrees of inflammation were detected in 18 % of patients in supraglottic region and
in 6 % of the cases in infraglottic region. Inflammatory changes of true and false
vocal cords (TVC and FVC) were observed in 14 % and 44 % of chemical veterans,
respectively. Chronic laryngitis was diagnosed in 82 % of patients. True vocal cord
nodule was detected in 12 % of the cases, while synergy was observed in 12 % of
patients. They found tissue hypertrophy in the supraglottic region in 8 % of patients
and in FVC in 12 % (6) of cases as well. They diagnosed FVC hyperfunction in
48 % of the patients, hypofunction of TVC in 8 % and unilateral paralysis of TVC
in 6 % of patients (Akhavan et al. 2009).
In another study, we assessed late laryngeal effects of SM in 43 chemical victims
by direct laryngoscopy 2025 years after exposure (Balali-Mood et al. 2010). This
study found a lower larynx position in 30.2 %, vocal cords limitation in 25.6 % and
mucosal inflammation of larynx in 14.8 % of mustard exposed patients. Unilateral
paralysis of the vocal cords was not detected in our study while it was reported in
6 % of patients in the Akhavan et al. study. In addition, vocal cord nodule was
observed in six patients (12 %) in the Akhavan et al. survey while it was not observed
in our patients (Balali-Mood et al. 2010; Akhavan et al. 2009).
6.6.3 Video Laryngoscopy and Stroboscopy
Video Laryngoscopy is a useful tool for diagnosis of vocal cord lesions. Video
Laryngoscopy provides slow motion image of vocal cord vibration and offers valu-
able information about the motion of the vocal cord movements. This technique
makes it possible to detect vibratory asymmetries, structural changes, small masses,
sub-mucosal scars and other conditions. It is also useful for careful evaluation of
larynx during phonation and in detection of vocal cord paralysis.
There is no evidence about the functional damages of mustard agent on the
larynx and vocal cords in the literature. The only evidence in this regard is a uni-
versity thesis for a MD degree in our university. That study evaluated the laryngeal
damages by video laryngoscopy and stroboscopy in patients exposed to SM
(Hasanzadeh 2012).
In our study, 41 chemical veterans with dysphonia and a history of SM exposure
(25 years ago) were evaluated with video laryngoscopy. The most common com-
plaints of the patients were chronic cough in 88 %, post nasal discharge (PND) in
81 % and snoring in 63 % of patients. Sore throat was observed in 34 % of the cases.
Hoarseness was detected in all patients. Video laryngoscopic examination revealed
bilateral vocal cord injury in 12 % of patients while laryngitis and glottic abnormal-
ity were identified in all patients. Infraglottic and supraglottic abnormalities were
diagnosed 24 % and 19.5 % of patients, respectively. In 5 patients (12.2 %) simul-
taneous inflammation, hyperemia and stenosis of all three regions of supraglottic,
glottic and infraglottic were detected. Also in 5 patients (12.2 %) bilateral vocal
cords injury was identified. Supraglottic and infraglottic inflammation was identi-
fied in 24 % and 19 % of the patients, respectively (Hasanzadeh 2012).
In stroboscopy, complete glottic closure was seen in 171 % and incomplete clo-
sure in 9.8 % of patients. Hourglass configuration was observed in 48.8 % of cases
and posterior gap in 24.4 % as well (Hasanzadeh 2012).
Asymmetry of vocal fold vibration was detected in 95 % of the patients.
Perturbations of vocal folds were observed in right fold in 85 % of patients while in
80 % of the patients in the left fold. Non vibrating parts of vocal folds were present
in 80 % of patients in the right side as well as in 90 % of cases in the left side
(Hasanzadeh 2012).
It was concluded that sulfur mustard induces long term harmful effects on func-
tion of vocal folds and larynx and causes permanent damage to the vocal cords.
High rate of supraglottic and infraglottic inflammation and vocal fold asymmetry
shows that toxic effects of SM remain in the larynx for a long time after exposure
(Hasanzadeh 2012).
6.6.4 Speech Evaluation
There is only one study on changes in aerodynamics of speech in patients exposed

to SM.
Heydari and Ghanei for the first time in 2011 evaluated the effects of mustard
agent on speech aerodynamics. They assessed aerodynamics of speech in 19 chemi-
cal war veterans exposed to mustard gas and 20 healthy controls using by Glasgow
Airflow Measurement System (ST1) (Heydari and Ghanei 2011).
In their study, there were significant differences between the two groups in vital
capacity, maximum phonation time, phonation volume, vocal velocity index, total
expired volume, and phonation quotient as well. However, there was no significant
difference between the two groups regarding mean flow rate. The mean vital capac-
ity, maximum phonation time, phonation volume and total expired volume in the
patients with SM exposure were lower than the controls, but the vocal velocity index,
and phonation quotient in chemical veterans was higher than the controls. All differ-
ences between the two groups were statistically significant except for mean flow rate.
It was found that in patients exposed to SM respiratory capacity reduces and can-
not support breathing for speech. They concluded that mustard agent could impair
the values of speech aerodynamics (Heydari and Ghanei 2011).
In the Akhavan et al. study, a speech-language pathologist interviewed chemical
veterans. In objective speech evaluation, they found a normal speech pattern in 54 %
of patients as well as different degrees of dysphonia at 46 % of the cases. Hoarseness
was detected in 32 % of patients, and harshness 14 % of them (Akhavan et al. 2009).
6.7 Experimental Study of SM -Induced Upper Respiratory

Tract Diseases in the Animal Models
Experimental studies on animal models have provided valuable evidence about

damages induced by mustard gas in the upper respiratory tract as well as its molecu-
lar mechanisms of action.
Destructive effects of SM poisoning have been shown in experimental studies on
mice (Vijayaraghavan 1997; Pant and Vijayaraghavan 1999), rats (Weber et al.
2010; Capacio et al. 2008; Anderson et al. 1996) and guinea pigs (van Helden et al.
2004), pigs (Fairhall et al. 2008) and rabbit (Warthin and Weller 1919).
6.7.1 Acute Effects
Acute effects of mustard gas in the respiratory system were investigated in an early
study in 1919. That study reported severe damage of the nasal passages, pharynx,
larynx, trachea, and bronchi following mustard gas inhalation in rabbit model
(Warthin and Weller 1919). Such changes was also observed in upper respiratory
airways by Szarejkoin the rat model (Szarejko 1974).
Warthin and Weller found that mustard- induced respiratory damages are dose
dependent and severity of damages increases by elevation of the exposure doses. In
high exposure doses, SM induces epithelium necrosis, WBC (white blood cell)
infiltration and pseudomembranes formation in upper respiratory airways (Warthin
and Weller 1919). This finding was confirmed also in the study of respiratory effects
of SM in guinea pigs (Allon et al. 2009) as well.
Exposure of guinea pigs (head only) to various doses of SM vapor in Allon et al.
study, caused nasal erythema and swelling with extensive mucous secretion (with or
without bleeding) as early as 3 h after exposure (Allon et al. 2009).
Weber et al. examined the respiratory effects of various forms of SM (vapor vs.
aerosol) as well as different concentrations and exposure routes of SM in female
rats. In their study, animals that exposed to SM by nose-only inhalation progres-
sively lost their body weight by the time after exposure and developed respiratory
distress manifested by difficult, open-mouthed breathing and even cynicism. They
showed that exposure to SM vapor (3000 mg min/m3) by nose-only route induces
severe nasal injury and epithelial degeneration while minimal or no lung injury.
They concluded that SM droplet might penetrate and deposit more deeply into the
lung than the vapor form. Also, they observed that respiratory tract pathology
induced by the nebulized SM was similar to that of the vaporized form. Finally, they
concluded that respiratory distress induced following a nose-only exposure to SM
vapor oraerosolis not a direct consequence of the lung injury instead it is the result
of the nasal injury (Weber et al. 2010).
In van Helden et al. study exposure of guinea pigs to intratrachealaerosolized SM
induced severe epithelial injury and inflammation in the upper airways (van Helden
et al. 2004). Such exposure in rat and guinea pig models, induced acute inflamma-
tory response characterized by an obvious increase in the neutrophil count in bron-
chioalveolar lavage fluid, cellular enzymes release and changes of LDH, protein and
glutathione metabolism (Kim et al. 1996; Allon et al. 2009). Pathologic effects
induced by SM in the airways were partially controlled by pretreatment with
N-acetylcysteine but did not change significantly by niacinamide (Anderson et al.
2000). Experimental studies have shown efficacy of cysteine and other thiol agents,
and thiosulfate, in reduction of both SM and nitrogen mustards toxicity (Callaway
and Pearce 1958; Zhang et al. 1995; Paromov et al. 2008; Gross et al. 1993).
Additionally, it has been shown that protease inhibitors reduce general inflammation
of airways and its related tissue injury and necrosis as well (Anderson et al. 2009).
6.7.2 Chronic Effects
Chronic effects of SM in upper respiratory tract have not been studied in animal
models and most of available evidence is related to the lungs.
Studies on rat model, found no evidence of teratogenicity or mutagenicity following

different doses of SM (McNamara et al. 1975; Sasser et al. 1996). McNamara et al.
exposed rats to 0.001 mgm-3 (continuously) or 0.1 mgm-3 (6.5 h per day, 5 days per
week) SM for 152 weeks. They did not show teratogenicity or mutagenicity but they
found that SM induced skin malignancies at the sites of exposure, but not other tumors
at other sites (McNamara et al. 1975). It has been shown that mustard gas exposure
causes lung cancer by inhalation or intravenous injection and mammary tumors and
local sarcomas by injection in mice (International Agency for Research on Cancer
1975). In addition, long-term administration of SM in monkeys, caused toxic effects on
pulmonary function and histology (International Agency for Research on Cancer 1992).
Abell et al. showed that uracil mustards cause lung cancers in mice. The carcino-
genicity of uracil mustards was more than typical mustard gas (Abell et al. 1965).
Heston et al. studies also confirmed pulmonary carcinogenesis of SM in rodents
(Heston 1949; Heston et al. 1953). In the study by Shimkin et al., intravenous injec-
tion of SM into the highly susceptible strain A mice, significantly increased pulmo-
nary tumors (Shimkin et al. 1966).
6.8 Clinical Management of SM-Induced Damages

in the Upper Respiratory Tract
Up to now, various medications have been used for prevention and or treatment of
respiratory complications of mustard gas but none of them has been effective defi-
nite treatment. Understanding of the mechanisms of mustard induced injuries may
help to develop new treatment. A new approach is required to obtain optimal and
rapid healing, and to return the optimal appearance and function of damaged tissue
in the shortest period.
As the first step of intoxication (alkylation) occurs very quickly, it is very diffi-
cult to prevent or control its effects. Therefore, treatment are focused on suppressing
and reducing the development of symptoms and improving the opportunities for
early recovery (Ivarsson et al. 1992).
Yet, there is no curative treatment or antidote to control and reduce the basic
cause of SM induced damages. Instead, most of current treatments are focused on
the symptomatic treatment and decontamination of the patient to prevent further
exposure. The other advantage of decontamination is reducing the risk of exposure
to the medical and nursing staff.
6.8.1 Management of Acute Phase of Poisoning
One of the important points in the management of the acute phase is protection of
first aid workers, nursing and medical staff. They should be physically protected by
using protective masks, suits and gloves to avoid contamination while taking care of
the patients with acute SM poisoning.
The first step of patient care includes decontamination of the patient. However,
decontamination of inhalation poisoning is limited or not possible and it is more
applied for skin contamination. Most of therapeutic interventions for respiratory
signs and symptoms in the acute phase are supportive such as providing humidified
air or oxygen for mild to moderate symptoms. Also, it is important to maintain patient
hydration in acute phase to prevent dehydration and thickening of mucous and secre-
tions. In case of oral airways injury that limits the use of oral fluids, parenteral fluid
therapy may be required for maintaining patient hydration. In severe cases, endotra-
cheal intubation and mechanical ventilation may be required (Borak and Sidell 1992;
Kehe and Szinicz 2005). In case of upper airway obstruction due to laryngospasm
(stridor, hoarseness), early tracheostomy is crucial (Kehe and Szinicz 2005).
Bronchial lavage with isotonic saline may be beneficial in the management of early
respiratory effects of SM-inhalation to remove tracheobronchial debris that accumu-
lates from necrosis and sloughing of membranous tissue (Freitag et al. 1991; Zilker
and Felgenhauer 2002). Treatment with mucolytics such as N-acetyl-cysteine may be
also beneficial when it is difficult to remove secretions because of decreased ciliary
function or increased thickness of secretions (Borak and Sidell 1992; Shohrati et al.
2008),although the efficacy is lacking (Balali-Mood and Hefazi 2006). Antibiotic
therapy is indicated in the case of infection that is usually diagnosed clinically and
shall be confirmed by positive culture if at all possible (Kehe and Szinicz 2005).
Ina addition, immediate inhalation of high doses of corticosteroids such as fluti-
casone or beclomethasone has been recommended for prevention of lung oedema
after contamination. Taking five deep breaths of the medication, every 10 min is the
preferred recommended method (Dacre and Goldman 1996). Severe SM intoxi-
cated patients should be treated in an ICU with special care for chemical burns in
case of severe skin damages.
6.8.2 Management of Chronic Phase of Poisoning
The main goal of long-term management of the SM induced effects is to reduce

scarring that can lead to stenosis and restrictions, and to return reversible airway
obstruction by the use of inhaled corticosteroids and long-acting b2-agonists
(Ghanei et al. 2007). Bronchodilators are beneficial in patients with increased air-
way hyper-reactivity. It has been found that combination of a b-agonist (e.g., salbu-
tamol) and an anticholinergic (e.g., ipratropium bromide) is more effective than any
of the other drugs used alone (Aslani 2000).
Also, immunosuppressive therapy may be beneficial by reducing chronic
inflammation and improvement of the lung function (Ghanei et al. 2006c).
However, despite treatment, inflammation and light sensitivity may persist for the
lifetime.
Annual examination and follow-up of mustard gas victims by chest imaging,
tracheobronchoscopic exams, and tuberculin skin testing has been recommended
for early detection and treatment of long-term SM-poisoning effects (Graham and
Schoneboom 2013).
6.8.3 Treatments
Treatment of mustard poisoning includes two parts of antidotal therapy and organ
specific care.
6.8.3.1 Antidotal Therapy
Although there is no effective antidote or specific treatment for sulfur mustard poi-
soning but an antidotal therapy has been proposed. Treatment includes immediate
administration of 500 mg sodium thiosulphate per kilogram body weight to the
patient soon after SM exposure. Chemical reaction of sodium thiosulphate with
mustards in cyclic form makes it ineffective and protects against systemic intoxica-
tion especially when taken more than 1 h after exposure. Also, it can be used in
combination with a number of other drugs such as cysteine, sodium citrate, dexa-
methasone, promethazine, heparin and vitamin E, to increase its protective activity
against SM (Callaway and Pearce 1958; Foster et al. 1962). It has been shown that
sodium thiosulphate would be more effective if it given before or immediately after
SM exposure (Foster et al. 1962; Connors 1966).
Hexamethylenetetramine (HMT) is another chemical protection in addition to
the thiosulphate. HMT protects lung cells against toxic effects of mustard gas. The
efficacy of HMT in protection of the lungs against the mustard toxicity was exam-
ined and confirmed in the cell lines of human upper respiratory tract. Studies showed
that HMT does not exert its protective effects when is applied after SM exposure.
Therefore, it seems that HMT can be used as an effective prophylactic agent for
exposure to SM by inhalation (Andrew and Lindsay 1998).
6.8.3.2 Organ Specific Treatments
Although there is no definite curative treatment for mustard induced respiratory

complications, but supportive general and organ specific treatments are available.
These therapies include oxygen therapy, vaporized moist air, respiratory physio-
therapy (Balali-Mood and Hefazi 2005a, b), mucolytic agents, bronchodilators, oral
corticosteroids, inhaled corticosteroids, interferon, and long-acting beta-2 agonists
(Ghanei and Harandi 2007; Attaran et al. 2007), antioxidants (Elsayed and Omaye
2004; McClintock et al. 2006), surfactant (van Helden et al. 2004), magnesium ions
(Agin 2005), therapeutic bronchoscopy, laser therapy, respiratory stents (Freitag
et al. 1991) ICU care (Balali-Mood and Hefazi 2005a, b), early tracheostomy
(Wattana and Bey 2009) and ultimately lung transplantation (Freitag et al. 1991).
However, most of these treatments are specified for treatment of lower respiratory
tract and there is few therapies specified for the treatment of SM induced upper
respiratory tract damages. For example in the patients with tracheobronchial steno-
sis (TBS) placement of stents can be life-saving (Freitag et al. 1991).
The studies about early and late effects of SM in upper respiratory tract in the litera-
ture are scars and these effects are less known. This study reviewed the evidences
about acute and chronic effects and complications induced by SM in the upper respi-
ratory tract mostly from Iraq-Iran war. The most clinical symptoms of SM in upper
respiratory tract in acute phase are irritative and inflammatory symptoms while in
the chronic phase most symptoms are due to functional damages and cancers. The
most important parts affected by SM in the late phase in upper respiratory tract are
larynx and vocal cords as well as trachea and bronchioles. DNA alkylation and dam-
age is the most important molecular mechanism of these effects. Yet there is a gap in
knowledge regarding the different aspects of early and late effects of SM in upper
respiratory tract. Animal models are suitable candidate for investigating such effects
in the lab.
Therefore, we recommend future experimental studies using animal models for
assessment of the early effects of SM in upper respiratory tract as well as case-
control studies on chemical veterans exposed to SM previously (for example, during
Iraq-Iran war) for assessment of late respiratory effects of SM with focus on differ-
ent parts of upper respiratory tract.
Glossary
Aphonia The inability to produce voice. It is more severe than dysphonia

Bronchiectasis A disease in which there is permanent enlargement of parts of the
airways of the lung
Bronchospasm Sudden constriction of the muscles in the walls of the bronchi
Chronic bronchitis A chronic inflammatory condition in the lungs that causes
the respiratory passages to be swollen and irritated
CWA Chemical warfare agents: a chemical substance whose toxic properties
are used to kill, injure or incapacitate human beings
Cytotoxic The quality of being toxic to cells
Dysphonia An impairment in the ability to produce voice sounds using the vocal
organs
Dyspnea Shortness of breath or breathlessness is the feeling or feelings associ-
ated with impaired breathing
effect.
Hoarseness A harsh, raspy, or strained voice caused by a variety of conditions
Hypopharynx The area where the larynx and esophagus meet
Inhalation The flow of air into an organism. In humans, it is the movement of air
from the external environment, through the airways, and into the alveoli.
Inspiratory Relating to the act of breathing in

Intoxication An abnormal state that is essentially a poisoning
Laryngitis An inflammation of the larynx
Laryngoscopy An exam that gives doctors a close-up view of the larynx and the
throat
Lipophilic The ability of a chemical compound to dissolve in fats, oils, lipids, and
non-polar solvents
Mutagenic Capable of inducing mutation or increasing its rate
Nasopharynx A part of the pharynx lies in the upper part of the throat behind the
nose
Oropharynx This space lies behind the oral cavity, extending from the uvula to
the level of the hyoid bone
Pulmonary Alveol An anatomical structure at the terminal ends of the respiratory
tree in the lug parenchyma that has the form of a hollow cavity and is the site of
gas exchange with the blood.
Stroboscopy Direct examination of the vocal cords and surrounding structures
with the use of a stroboscope
Teratogenic A drug or other substance capable of interfering with the develop-
ment of a fetus, causing birth defects
Toxicity The degree to which a substance can damage an organism
Tracheobronchial stenosis Abnormal narrowing of the central air passage ways
Tracheobronchitis A condition involving inflammation of the windpipe orbronchi
Tracheobronchomalacia A condition characterized by flaccidity of the tracheal
support cartilage which leads to tracheal collapse with condition extends further
to the bronchi
Tracheotomy A surgical procedure, which consists of making an incision on the
anterior aspect of the neck and opening a direct airway through an incision in
the trachea
Turbinate or conchae A long, narrow and curled bone shelf that protrudes into
the breathing passage of the nose
Video laryngoscopy A form of indirect laryngoscopy in which the clinician does
not directly view the larynx. Instead, visualization of the larynx is performed
with a fiberoptic or digital laryngoscope inserted transnasally or transorally
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Chapter 7
Lower Airway Complications of Sulfur
Mustard Exposure
Mostafa Ghanei and Amin Saburi
Contents
7.1 Introduction .................................................................................................................. 172
7.2 Pulmonary Toxicity of SM........................................................................................... 173
7.3 Mechanism of Long-Term Respiratory Complications ............................................... 175
7.3.1 Chronic Inflammation ...................................................................................... 176
7.3.2 Increase of Proteolysis ..................................................................................... 177
7.3.3 Oxidative Stress................................................................................................ 178
7.3.4 Apoptosis ......................................................................................................... 178
7.4 Clinical and Pathological Findings and the Relationships with the
Bronchiolitis Obliterans Mechanisms ......................................................................... 180
7.5 Changes of Respiratory Ducts Due to SM ................................................................... 182
7.6 Tracheobronchomalacia and Air Trapping After SM Exposure .................................. 183
7.7 Sign and Symptoms ..................................................................................................... 183
7.7.1 Symptoms in Acute Phase................................................................................ 183
7.7.2 Late Symptoms ................................................................................................ 184
7.7.3 Chronic Coughing ............................................................................................ 184
7.8 Incidence and Diagnosis of Emphysema in SM Victims ............................................. 185
7.9 Other Damages to Airways and Lung Parenchyma ..................................................... 186
7.9.1 Ventilation and Perfusion Disorder .................................................................. 187
7.9.2 Hemoptysis....................................................................................................... 187
7.9.3 Biochemical Disorders of Lung ....................................................................... 187
7.10 Para-clinical Findings .................................................................................................. 187
7.10.1 Radiologic Data.............................................................................................. 187
7.10.2 Respiratory Function ...................................................................................... 191
7.11 Differential Diagnosis and Other Diagnosis Along with BO ...................................... 195
7.11.1 Asthma and COPD ......................................................................................... 195
7.11.2 Bronchiectasis ................................................................................................ 196
7.11.3 Pulmonary Fibrosis ........................................................................................ 196
7.11.4 SM and Lung Cancer ..................................................................................... 196
7.11.5 Cardiopulmonary Involvement of SM Exposed Patients ............................... 197
M. Ghanei (*) A. Saburi

Chemical Injuries Research Center and Faculty of Medicine,
e-mail: mghaneister@gmail.com; mghanei@hbi.ir; aminsaburi@yahoo.com

172 M. Ghanei and A. Saburi
7.12 Treatments .................................................................................................................... 197

7.12.1 Acute Phase .................................................................................................... 197
7.12.2 Chronic Phase................................................................................................. 198
7.13 Conclusion and Recommendations .............................................................................. 202
Glossary................................................................................................................................... 202
References ............................................................................................................................... 203
Abstract The pulmonary complications of sulfur mustard (SM) exposure is the

most mortal and morbid complication in exposed cases. In acute phase after expo-
sure, all symptoms can because of airways and alveolar injuries and cell necrosis.
Thus, treatment in this phase should be supportive and sometimes critical cares may
be mandatory. In the chronic phase, productive cough, thick sputum and dyspnea
are more frequent symptoms. Although chronic pulmonary sequels are common in
exposed patients, the mechanism of these complications was not clearly described.
Although corticosteroids extensively prescribed for treatment in chronic phase, its
efficacy is short-term and its complications are serious. Imbalance in apoptosis and
repair is seems to justify these symptoms. Therefore, treatments include antioxi-
dant, bronchodilators, and mucolytics in this phase. Lung carcinogenicity of a sin-
gle exposure to SM was not confirmed. Future studies may be helpful for assessing
the genome mutation to resolve prolong symptoms.
Keywords Sulfur mustard Lung Bronchiolitis obliterans Antioxidant
7.1 Introduction
Although the chemical and biological properties of sulfur mustard (SM) were dis-
cussed in the other chapters, our present scope is to discuss assessing the SM toxic-
ity and complications on the lower respiratory tract (LRT). SM is a famous
biochemical toxic warfare, known as a powerful alkylating agent (Balali-Mood and
Hefazi 2005; Steinritz et al. 2013). This property can determine mutations in the cell
genome, finally leading to apoptosis (Steinritz et al. 2013). Alkylation may occur at
the level of the membrane and also takes place on intra or extracellular proteins
(Everley and Dillman 2010). Therefore, cell signaling and multiple other cell func-
tions, on which depend the normal functioning of intracellular and membranous
proteins, become corrupted (Ham et al. 2012; Sagar et al. 2014). Also, the process
can induce hydrolysis and produce free radicals (Brimfield et al. 2012; Tewari-
Singh et al. 2014). As a result, necrosis commences, leading to apoptosis. For the
other cells, which are exposed only to a nonlethal dose of SM, genomic mutations
and other extracellular conditions, such as inflammation and persistence of toxic
materials, could be important (Ghabili et al. 2011; Ghanei and Harandi 2011).
Therefore, there are two biological pathways, each corresponding to the acute or
chronic phase of SM toxicity. Although it is not a water soluble compound, it is
however freely soluble in fat, which is abundant in the cell membrane (Balali-Mood
7 Lower Airway Complications of Sulfur Mustard Exposure 173
and Hefazi 2005; Mostafa Ghanei and Amin Saburi 2011). Its half time is long
enough to allow the SM to penetrate into the deeper tissues and even blood circula-
tion (Elsayed and Omaye 2004). Therefore, when a victim is exposed to SM locally,
e.g. via skin, other systemic effect should be expected (Ghabili et al. 2010, 2011).
Lungs are more sensitive to complications of SM exposure compared to other
organs because of their biological properties (Mansour Razavi et al. 2012). This
explains why the most common long-term complications encountered in the Iranian
victims of the Iran-Iraq conflict, are related to the respiratory system (RS). The
exposed cases are exhibiting chronic lung lesions, clinically translated in airway
hyper-responsiveness (AHR) or chronic obstructive pulmonary disease (COPD),
with manifestations ranging from mild to severe (Ghasemi et al. 2013; Balali-Mood
et al. 2011; Boskabady et al. 2008).
Since the RS is one of the structures most frequently affected in SM victims,
there are multiple reports of cases which confirm that different parts of the RS are
affected, ranging from large caliber airways, like the trachea and bronchus, to the
level of the bronchioles (Akhlaghpoor et al. 2011; Ghanei and Harandi 2007).
Therefore, before discussing the pathogenesis and clinical aspects of SM injuries,
we should take a brief look into the anatomy of the airways.
In its sequential divisions, the bronchus loses the cartilage of its wall. These divisions
without cartilage are called bronchiole. Each bronchiole enters a lobule and is further
divided into smaller branches, ending with terminal bronchioles. In addition to goblet
cells, the bronchioles contain Clara cells, alongside other types of intermediate and
undifferentiated cells. The mucous membrane on the surface of the epithelium is cov-
ered by actively mobile cilia, which remove additional particles similarly to a conveyor
belt. Velocity of this moving removal system composed of cilia and mucus is of 1 mm/
min in the bronchioles and 1030 mm/min in the bronchi. The matrix of the epithelium
of the bronchi and bronchioles, mainly composed of elastic fibers, is gradually converted
into smooth muscle, adventitial tissue, and also in irregular cartilaginous components.
At the level of the bronchi and bronchioles, the basal membranes become thicker and
their elastic tissue content increases (Guyton and Hall 1996; Mostafa Ghanei and Amin
2011). The mucous serous glands, located along the respiratory tract from the bronchi-
oles to the larynx, respond to neural stimulations and other local irritants such as SM.
7.2 Pulmonary Toxicity of SM
A wide range of respiratory complications were reported in patients exposed to

SM. However, the key point in the evaluation of toxicity is the time of exposure.
There are multiple differences in the pathogenesis and toxicity of SM between the
acute and chronic phases. After inhalation of the SM, the airways including the
upper and lower respiratory tract, are exposed to SM. During the first 24 h, symp-
toms, including chest tightness, mild dyspnea, coughing and increased secretion of
tears, saliva and sputum, are recorded (Mostafa Ghanei and Amin 2011; Ghabili
et al. 2010). The severity of these symptoms arising from the upper airway tracts
increases during the first 3 days. In the lower respiratory tract and alveoli, SM can
induce epithelial and subepithelial cell injury and the degree of the damage depends
on the amount of SM which reaches the cells. Although a great amount of SM is
taken by mucus and cilia of the upper airways, a small amount of SM is sufficient to
cause injury to the respiratory airways (Marrs and Al 2007).
After exposure, the cell injuries depend on the amount and duration of exposure.
For greater amounts and longer exposure due to the higher degree of proteolysis and
production of free radicals, cell necrosis occurs (Weinberger et al. 2011; Ghanei and
Harandi 2011). Cell membrane integrity is corrupted and proteolysis and denatur-
ation of the essential proteins and enzymes in the cells disrupt the cellular signaling
pathways and the necrosis becomes inevitable (Sayer et al. 2010; Karacsonyi et al.
2012). Consequently, inflammation is activated and acute phase inflammatory cells,
including neutrophils, gather locally. On the other hand, SM can impair the inflam-
matory cells migration (Steinritz et al. 2014). They release inflammatory molecules,
such as cytokines and interleukins, which induce the activation of a variety of
inflammatory pathways (Pohl et al. 2009; Choi and Levy 2011). Several interleukins
cause vasodilatation and permeate water via the capillary wall, ensuing an extracel-
lular edema and sputum secretion increase (during first days) (Choi and Levy 2011).
Capillary walls are also injured and red blood cells (RBC) reach to the extracellular
and alveolar spaces (Kadar et al. 2013). Several cascades, such as those of the cas-
pase group, promote injured cells apoptosis, which elevates the severity of tissue
damage (Choi and Levy 2011; Keyser et al. 2013; Ray et al. 2008). Protein-rich
edema fluid, containing cell remnants, proteins, cytokines and toxicants, leads to
diminished aeration and atelectasis (Malaviya et al. 2010). Therefore, the respira-
tory effort increases and the succession of tachypnea, dyspnea and then hypoxia
installs (27 days after exposure) (Choi and Levy 2011). The alveolar surfactant
becomes inactivate and a thick and streaky membrane forms from cellular debris
and proteins, covering the small lower respiratory tract and alveoli. This phenome-
non leads to increases of respiratory dead spaces, intrapulmonary shunting and
hypoxemia. Respiratory distress syndrome (RDS) develops (during the first or sec-
ond week) (Tang and Loke 2012; Mostafa Ghanei and Amin 2011; Choi and Levy
2011). After this exudative phase, the proliferative and then fibrotic phase begin.
Fibroblasts and other inflammatory cells, and also platelets, play a significant role
(Mirzamani et al. 2013; Ghane Zadeh et al. 2014). All this set of chronologic cel-
lular events following the acute injury in SM exposure being summarized in Fig. 7.1.
The processes mentioned above occur in patients who are exposed to a great
amount of SM. However, in slightly injured patients, the process is milder and the
healing begins more rapidly. In these patients, symptoms become evident a few
weeks after exposure. Nevertheless, in severely injured victims, mechanical
ventilation and other respiratory supports may be required and, likewise, the treat-
ment takes more time to produce its effects (Muskat 2008).
The late respiratory complications of SM exposure are more important, as they
have a greater morbidity and mortality. In several cases, chronic symptoms may
persist or reoccur after years. The most probable cause is a chronic process that this
chapter will focus upon.
Fig. 7.1 Flowchart of the cellular events in acute injury following sulfur mustard exposure.
(Red arrow: inhibitory pathway, Dotted line: under study or possible pathway, Boxes with lock
symbol; Steps needs more investigation)
7.3 Mechanism of Long-Term Respiratory Complications
Bronchiolitis, bronchitis, and COPD, asthma, pulmonary fibrosis, emphysema,

pneumonitis, pneumonia and interstitial lung diseases were suggested for justifying
the main lower respiratory complications of the SM poisoning (Mostafa Ghanei and
Amin Saburi 2011; Ghanei et al. 2008a; Ghanei and Harandi 2007; Razavi et al.
2013a; Hefazi et al. 2005). The lacks of a suitable response to specific treatments,
such as corticosteroids, and also further studies have clarified the specific character-
istics of these patients. Due to different and sometimes contradictory pathogenesis,
the term of Mustard Lung (ML) has been suggested by Ghanei et al. to be included
in the literature although there is concern about accuracy of the use of such term
(Beheshti et al. 2006). Also, it seems that the patients clinical and paraclinical find-
ings can be justified by diseases of the small caliber airways, such as constrictive
bronchiolitis or bronchiolitis obliterans (BO) (Ghanei et al. 2008a, 2011a, b; Ghanei
and Harandi 2007, 2011; Saber et al. 2012).
According to our experience and the literature, in these cases, thick sputum,
chronic cough, irritable airways, recurrent upper and lower respiratory tract infec-
tions, chest tightness and dyspnea are the most common clinical features. These
manifestations relate to an insufficient repair process and therefore, a chronic dam-
age and incomplete healing in the small airways, lead to chronic secretion and
injury of the epithelial layer.
Among the mentioned mechanisms of BO in the exposed cases, four mecha-
nisms are more important, as followings:
1. Chronic inflammation
2. Proteolysis
3. Lack of balance between oxidative stress and antioxidant mechanisms
4. Apoptosis
The following section of this chapter will discuss the above mentioned mecha-
nisms, to have an insight on the cellular and molecular processes, which would
enhance the understanding of the clinical and paraclinical presentations.
7.3.1 Chronic Inflammation
Chronic inflammation is the first mechanism that takes place in the pathogenesis
of SM exposure (Emad and Rezaian 1997). Different inflammatory mediators,
particularly interleukin (IL) 8 and 6 play important roles in pathology of these
patients (Pourfarzam et al. 2009). Emad and Rezaian (1999)., in one of the first
studies on these patients, considered pulmonary fibrosis (PF) as the main mor-
phopathological alteration of exposed lungs, making chronic inflammation as the
main responsible for the PF (Emad and Rezaian 1999). They described neutro-
philic alveolitis as the main feature in bronchoscopic biopsy, noting that neutro-
phils and eosinophil were the most frequent inflammatory cells in the
bronchoalveolar lavage (BAL) specimens (Emad and Emad 2007c). These media-
tors (inflammatory markers such as cell count and level of ILs) are also regarded
as biomarker with a strong correlation with the severity of the disease (Emad and
Emad 2007c). On the other hand, these findings are similar to biological findings
in COPD patients (Larsson 2008). When COPD is aggravated, IL-6 and IL-8
increase in BAL and serum samples. All inflammatory cells, which include mac-
rophages, B and T lymphocytes and neutrophils, are increased in the alveoli and
airways of patients with COPD (Ji et al. 2014). Inflammatory indices are also
considered as predicting factors of disease intensity and mortality in COPD (Celli
et al. 2012; Higashimoto et al. 2009).
To our experience and in congruence with previous studies, in the pathogenesis
of BO, as the main sequel of SM exposure, proliferation of fibroblasts and tissue
regeneration play important roles and peribronchial fibrosis can ensue (Myong et al.
2001). Different growth factors, among which transforming growth factor (TGF-
) is the most studied, can increase the reactivity of fibroblasts and increase collagen
accumulation. The excessive expression of TGF- in macrophages and endothelial

cells can be an indicator of the changes resulting from BO. The target cells of TGF-
are present in great numbers BAL samples and also target tissues of the patients
exposed to SM (Aghanouri et al. 2004; Mostafa Ghanei and Amin 2011).
A research conducted on 50 chemical war victims, compared levels of high-
sensitivity C-reactive protein (hs-CRP) with the control group. The pooled results
showed increased levels of this protein in exposed patients and a direct relationship
with the intensity of the disease (Attaran et al. 2009).
Levels of cytokines, such as IL-12, tumor necrosis factor-alpha (TNF-a), IL-6,
IL-1 beta, were higher in the study group compared to their peered controls. In addi-
tion, the high level of cytokines in this studys population was strongly correlated
with fibrosis intensity (Emad and Emad 2007a; Shohrati et al. 2014a). It is neces-
sary to note that PF was increased in the chemical victims in the pilot studies, an
issue which was later debated for years.
Further studies and evidence showed that fibrosis was not an evident finding in
the pulmonary pathology of these patients. In addition, although inflammation and
inflammatory processes, along with the oxidative stress phenomenon play impor-
tant roles in the pathology of the initial stages of exposure to SM, the interaction
between these two pathologies were more studied to declare the main
pathogenesis.
More recent trials have revealed that that the level of inflammatory mediators is
not high in these victims, and also, for some of them, such as CRP, IL-8, IL-1 and
rheumatoid factor (RF), the levels were even lower in comparison to the control
groups. No correlation between IL-8 level and pulmonary symptoms was found
(Pourfarzam et al. 2009). It should be noted that, although the main pathology in
chemical victims is bronchiolitis, this type of bronchiolitis has major differences
from the obstructive bronchiolitis, which results from pulmonary transplantation.
These differences, which were found between lung of the chemical victims and
other pulmonary patients, were responsible for different and unique appearances
and responses to treatment in the injured patients. The lack of a satisfactory response
to corticosteroid treatment in more than 50 % of these patients is an argument for a
decreased presence of active inflammation in them (Mostafa Ghanei and Amin
2011). The study, which was conducted on the samples obtained from open pulmo-
nary biopsy of these patients, revealed only mild to moderate lymphocytic infiltra-
tion, even for the cases with a severe pathology.
7.3.2 Increase of Proteolysis
Disruption of the balance between proteolytic and anti-proteolytic molecules causes

metabolic hyperactivity. The result of this phenomenon is a proteolytic destruction
of the healthy cells in patients with COPD. Since emphysema was not observed in
lungs of the chemical victims who did not smoke, the presence of proteolytic activ-
ity is not possible in the patients without emphysema (Ghanei et al. 2008c).
7.3.3 Oxidative Stress
There are multiple evidences on the presence of oxidative stress and oxidative interme-
diaries in patients with COPD. The markers relating to oxidative stress in these patients
are 4-hydroxynonenal (4-HNE), hydrogen peroxide (H2O2) and isoprostane, which are
end products of lipid peroxidation. The role of oxidative factors is evident when their
activity and effect overcome antioxidant factors. The result of this imbalance is damage
of lipids, proteins and DNA. This cellular damage process induces apoptosis and alter-
ations of pulmonary matrix, including elastin and collagenfibrillar structures (Sarsour
et al. 2009). Oxidative stress exerts its effect by inactivating antiproteases, such as
alpha-1 antitrypsin (AAT) or leukoprotease secretion inhibitors, or activating metallo-
proteinase oxidants. Oxidants play a major role in the inflammatory damage of lungs,
inducing the translation of proinflammatory genes (Demedts et al. 2006).
To study oxidative stress in chemical victims, the levels of glutathione (GSH)
and malondialdehyde (MDA) have been measured. Results have shown that the
victims with moderate to severe pulmonary damage had lower levels of GSH and a
higher rate of MDA. An increase of MDA indicates the increase of lipid peroxida-
tion, which is a consequence of the production of free radicals after exposure to
SM. Nevertheless, the reduction of GSH levels is not only limited to pulmonary
patients exposed to MS, as its levels decrease when they are exposed to other air-
borne toxins, such as ozone and tobacco (Fidan et al. 2005; Shohrati et al. 2010a).
Several studies have been conducted on the effect of apolipoprotein A1 (APOA1)
and S100 calcium binding protein family. High levels of these proteins indicate a
lack of balance between oxidant and antioxidant substances in chemical victims. Dr.
Mehrani et al., using a proteomic method, tried to identify different proteins
expressed in these victims, compared with healthy people. Results showed that
there was APOA1 in all BAL samples of patients exposed to SM, while none of the
healthy volunteers showed such protein. A direct relationship between the intensity
of pulmonary disease and APOA1 and isoform haptoglobin was also noted. The
S100 protein was also found in all patients who had moderate to severe pulmonary
damage (Mehrani et al. 2009; Mostafa Ghanei and Amin 2011).
7.3.4 Apoptosis
New information indicates the important role of apoptosis in the pulmonary pathol-
ogy of SM victims. It is necessary to note that two main pathways play a crucial role
in apoptosis, and they are termed the intrinsic apoptotic pathway and extrinsic apop-
totic pathway (Saburi et al. 2012a; Mostafa Ghanei and Amin 2011). It is necessary
to note that apoptosis is not an isolated process, also occurring in COPD pathogen-
esis, while other pathways, such as those of the oxidative stress, increase the com-
plexity of this process. Apoptosis is recognized as a method of cleaning performed
by neutrophils, classically evident in the process of inflammation. Wherever there is
oxidative stress in lungs, apoptosis will ensue. This indicates a positive relationship
between these two phenomena (Tse and Tseng 2014).
On the other hand, efferocytosis is a process in which the cells that have apoptosis
are cleaned by phagocytes (Simpson et al. 2013). If this is not done, the apoptosed
neutrophils will be a new inductive factor for oxidative stress. The efficiency of effero-
cytosis has been suppressed by oxidants and while antioxidants increases it (Simpson
et al. 2013; Lee and Surh 2013; McPhillips et al. 2007). Tumoral growth factor-beta
(TGF-) is one of the substances that have a relationship with efferocytosis. Zarin et al.
in their study stated that TGF-beta1 and TGF-beta3, but not TGF-beta2, secretion is a
result of efficient efferocytosis in chemically injured patients, playing a protective role
by improving airway remodeling and lung homeostasis in this group (Zarin et al.
2010). Deficiency in efferocytosis is encountered in multiple pulmonary diseases, such
as asthma and COPD. In vivo and in vitro studies have proved the presence of apoptosis
as one of the main causes involved in pulmonary damage in chemical victims. It was
shown that both the intrinsic and extrinsic apoptotic pathways are active in lungs of
chemical victims (Tang and Loke 2012; Saburi and Ghanei 2013). It has been shown
that different types of translations relating to TGF- and high levels of the TGF- pro-
tein are present in the BAL of the chemical victims, being measurable with ELISA
method. It has been concluded that TGF- may be responsible for the regeneration of
airways, hemostasis and slow progress of disease in chemical victims. As a result, it has
been suggested that TGF-1 and TGF-3 may improve efferocytosis and play impor-
tant roles in the regeneration of the airways of these patients. These capabilities of
TGF- are promoters of prolonged life in these patients, compared with the patients
with BO resulting from lung transplantation (Jonigk et al. 2010; Zarin et al. 2010).
As mentioned above, low levels of GSH is also an important factor for induction
of apoptosis in the chemical victims. Rosenthal et al. have shown the role of caspase
activity in the apoptosis of these patients and the early light-inducible protein
(ELIP), which is a protein similar to caspase-8, is affected after exposure of pulmo-
nary cells to low concentration of SM (Rosenthal et al. 2003; Saburi and Ghanei
2013; Saburi et al. 2012b).
Complementary studies indicated that the phenomenon of apoptosis in chemical
victims is not performed completely. For example, in the injured patients and con-
trol group, the caspase-3 level did not record considerable differences although
there are some contradictions (Pohanka et al. 2013; Pirzad et al. 2010). More analy-
sis of lung lavage fluid with annexinV-fluorescein isothiocyanate (FITC) kits proved
that the majority of the cells had necrosis and only few of them had completed the
phenomenon of apoptosis (Keyser et al. 2013). The homeostasis of calcium and
S100 protein are reduced in these patients, while the two play pivotal roles in the
regulation of apoptosis and regeneration of tissues (Mehrani et al. 2009). Figure 7.2
shows the suggested mechanisms of the long-term lung complications of SM.
Recently, accumulation of IL-17(+) cells in the injured areas of the lungs has
been suggested as the responsible reason of the lung squeal in chronic phase (Mishra
et al. 2012). This cell can affect all four aspects which were discussed above. If a
mutation would be found in this cell genome, it can be a new horizon for research
to find the mechanism of the Mustard Lung.
Cell & Tissue Injury with SM in Acute Phase
Insufficient Cell Anti-Oxidant storage
Membrane potential Inflammatory Apoptosis & Gene mutation regulating

imbalance modulator release Necrosis dysfunction
Thick Sputum
Impaired Cell & Tissue
Inflammatory cell TGF- Defect in

activation Adherin&Laminin
Gamma-glutamyl cysteine Defect in

synthetase inhibition Epithelialization Symptoms;
Infection
Thick mucus
GSH Dyspnea
Defect in Cough
Scavenging
Fig. 7.2 Flow chart of the suggested mechanisms of the long-term lung complications of SM
(Published in Critical Review in Toxicology, Informa Healthcare, with permission)
7.4 Clinical and Pathological Findings and the Relationships

with the Bronchiolitis Obliterans Mechanisms
Inhalation of SM can create different degrees of pulmonary diseases. Although the

previous studies have reported affliction with pulmonary fibrosis (PF) after expo-
sure to SM, our studies on the clinical and radiological manifestations of patients
who were permanently injured by SM exposure specified that PF was not the domi-
nant pathological pattern. Based on complementary reports, PF was the least impor-
tant pulmonary change in patients (Taghaddosinejad et al. 2011; Saber et al. 2012;
Hefazi et al. 2005). It is not clear if this disease presents with interstitial pneumonia,
similar to other interstitial pneumonia diseases, or has unclear clinical signs. The
pooled results are general and include parenchymal injuries and airways mucous
damages (Veress et al. 2010).
In novel studies based on High Resolution Computed Tomography (HRCT), air
trapping has been reported as the prevalent finding, which indicates the presence
of BO as the underlying disease (Ghanei et al. 2004b; Idani et al. 2012). Ghanei
et al. at their research demonstrated that the most frequent findings were; air trap-
ping 38 (76 %), bronchiectasis 37 (74 %), mosaic parenchymal attenuation (MPA)
36 (72 %), irregular and dilated major airways 33 (66 %) bronchial wall thicken-
ing (BWT) 45 (90 %), and interlobular septal wall thickening (SWT) 13 (26 %)
(Ghanei et al. 2004b) (Figs. 7.3 and 7.4).
To study BO and pulmonary air trapping in patients, HRCT should be performed
at the end of the exhalation phase, because obstruction in bronchioles is a cause of
air trapping and this state is observed during exhalation. Pulmonary air trapping of
more than 25 % on HRCT strongly indicates BO (Ghanei et al. 2004b). Mosaic
perfusion in BO results from hyperaeration of terminal alveoli to bronchioles due to
obstruction in proximal. On the other hand, the presence of areas with low blood
perfusion and non-engaged areas with normal perfusion or increased perfusion
Figs. 7.3 and 7.4 HRCT

findings in chemical
injured cases. Air trapping,
mosaic pattern and centri
lobular emphysema are the
most common findings
(Authors captured,
published in Resaneh
Takhasosi Publication,
Iran, with permission,
Mostafa Ghanei (2011))
create the mosaic pattern due to contraction of vessels, as visible on HRCT. Although
patients were not in a hyper aeration stage, the mosaic pattern was found on
HRCT. In the spirometry of these patients, obstructive, restrictive or sometimes
normal pattern was reported (Leung et al. 1998; Ghanei et al. 2010a).
In study of these patients, it was specified that there were not provided suffi-
cient tissue samples to differentiate the type of bronchiolitis after sampling by
needle biopsy or in the samples prepared with transbronchial method. The reported
pathology in these cases was organizing pneumonia and in cases where opening
sampling of lung tissues was performed or tissue sampling was prepared by Video
Assisted Trachoscopy (VAT), BO diagnosis was confirmed. The bronchoscopic
biopsy is an accepted technique for the diagnosis and follow-up of patients with
organizing pneumonia (Beheshti et al. 2006; Mostafa Ghanei and Amin 2011). In
our study on the histology of patients exposed to SM with bronchoscopic biopsy,
damage to the tracheobronchial tree, base membrane, edema, infiltration of mono-
nuclear cells into the lamina propria, fibrosis of lamina propria and muscular
mucous hyperplasia were found. These structural changes may indicate a chronic
reaction following the inhalation of SM (Beheshti et al. 2006; Ghanei et al. 2011a;
Ghanei and Harandi 2011).
7.5 Changes of Respiratory Ducts Due to SM
The extent of the changes in the respiratory ducts following contact with SM
depends on the duration of contact and the concentration of the chemical in the
inhaled air. In hot climatic conditions, the effects of hot SM on the respiratory sys-
tem are intensified. The findings obtained during the First World War are hardly
interpretable, due to the high incidence of secondary infections in the studied sam-
ples after death (Lewisite 1993).
In case of intensive contact and during the acute phase, the epithelial layers of the
larynx, lung and bronchi are necrosed and even pseudomembrane formation occurs.
In cases of contact with lower intensity, petechiae are prevalent on surface layers of
respiratory ducts. Similar injuries were also found in respiratory ducts of tested
animals, several months after contact with SM (Balali-Mood and Hefazi 2006;
Karacsonyi et al. 2009). During the First World War, severe conditions, such as
gangrenous changes, were found in lungs. In a study with the optical microscope,
the epithelial secretions of fibrin and mucous cells were intense (Lewisite 1993). In
this report, the base membrane was altered due to swelling and occurrence of edema
in epithelial tissues, along with infiltration of inflammatory cells and dilation of
blood vessels was a usual finding (Mostafa Ghanei and Amin 2011).
In more intensive cases of contact, injuries are expanded to the connective tissues
and smooth muscles of the wall of respiratory duct. During the healing stage, the
extensive metaplasia of the squamous epithelial cells and the first changes were also
visible in mucous glands ducts (Ghanei et al. 2006b). The laminated metaplastic squa-
mous epithelial cells completely cover surfaces of the injured regions (Lewisite 1993).
In these studies, the growth stages of these epithelial cells were not well described,
while the settlement of the pseudostratified columnar cells was not evident in the
epithelial layer.
7.6 Tracheobronchomalacia and Air Trapping After SM

Exposure
One of the side effects of SM in chemical victims is Tracheobronchomalacia.

Tracheobronchomalacia and air trapping are found in the thoracic HRCT of nearly
all patients exposed to SM (Ghanei et al. 2006a).
The findings of our studies first showed that air trapping and Tracheobronchomalacia
were related to each other, as long-term side effects of SM exposure. The accompani-
ment of air trapping and BO is induced by a main single process, which generates air
trapping in large and small airways in these patients. The intensive stricture of tra-
cheobronchial tree has been reported in several of these cases. Tracheobronchomalacia
is usually diagnosed by HRCT, while at present its standard global diagnostic method
is bronchoscopy (Ghanei et al. 2006a).
Given that air trapping is a more prevalent finding than tracheobronchomalacia
in the chemical victims, it can be concluded that air trapping is not the outcome of
Tracheobronchomalacia in this group, while similar pathological mechanisms,
which leads to small airways disease, i.e. BO, may lead to disruption of large air-
ways such as in the case of tracheobronchomalacia. In other words, SM may affect
epithelium of both small and large airways.
7.7 Sign and Symptoms
7.7.1 Symptoms in Acute Phase
The major characteristic of contact with SM is the occurrence of a period without

clinical signs and symptoms, several hours after contact. Duration of this period
depends on contact, ambient temperature and individual characteristics. Some peo-
ple show a superior sensitivity to SM compared with others. At time of chemical
injury, airways, eyes and skin are directly exposed to SM and clinical symptoms
appear at their level. Nevertheless, in case it is absorbed in a high amount by lung or
skin tissues, it can induce complications in the circulatory, digestive and central
nerve system (Razavi et al. 2013a).
At initial minutes (2060 min) after exposure, coughing, shortness of breath,
nausea, vomiting, eye pain (shooting pain) is sometimes found but symptoms may
often be absent. After some hours, fatigue, periorbital edema, intensive eye pain,
tears, skin erythema, and minor respiratory symptoms (e.g. coughing, shortness of
breath, rhinitis, sneezing, epistaxis, and hoarseness of voice) are clear. After 24 h,
vesicles become more evident and more considerable symptoms of respiratory

appear (Mostafa Ghanei and Amin 2011; Marrs and Al 2007).
7.7.2 Late Symptoms
The eyes, skin and respiratory system are the organs that are affected primarily and
secondarily by the poisonous action of this substance. The skin and eye lesions may
persist on the long-term or can be reduced. However, pulmonary complications are
the most prevalent delayed conditions of these patients, and they can progress over
time (Khateri et al. 2003). The effect of SM is affected by different factors. Exposure
intensity is so high that symptoms of the person at time of exposure reflect it.
Nonetheless, it should be noted that environmental factors and genetic factors can
change the organic response to SM (Hosseini-Khalili et al. 2008; Taravati et al. 2013).
Different studies indicate that the intensity of primary symptoms resulting from
SM exposure relates to the risk of pulmonary obstructive diseases. Patients with
primary weak symptomatology have a lower risk of obstructive diseases compared
to the patients with moderate to severe primary symptoms (Mostafa Ghanei 2011).
The pulmonary obstructive pattern is the most prevalent in the pulmonary function
test (PFT) after exposure to SM and does not have a relationship with moderate to
severe primary symptoms of the patients. In the patients who had mild to moderate
pulmonary symptoms after exposure, pulmonary function was normal and they
have had less pulmonary secondary pattern over time. It seems that, in case the
intensity of SM exposure induces primary symptoms and the hospitalization of the
person at time of exposure, the incidence of the late pulmonary complications will
be augmented (Ghanei et al. 2008a). However, changes in symptom intensity from
moderate to severe or periodical hospitalization in the hospital after exposure do not
lead to changes of the symptoms. Therefore, it seems that other factors, such as
personal susceptibility to the intensity of the primary symptoms and hospitalization
probability are more valuable (Mostafa Ghanei and Amin 2011).
Findings in patients presenting with the chronic phase have no relationship with
premature pulmonary symptoms. Our findings indicate that the increase of prema-
ture pulmonary symptoms intensity does not correlated with air trapping or mosaic
diffusion. These two findings have been found in radiographic images of symptom-
atic and asymptomatic exposed people. As mentioned before, histopathological
studies and radiological findings (HRCT) support the diagnosis of BO in SM
exposed victims (Ghanei et al. 2011a; Kehe et al. 2008).
7.7.3 Chronic Coughing
Regarding the SM victims who suffered from BO, our study showed that the afore-
mentioned causes influenced the incidence of coughing and intensity of chronic
coughing in patients exposed to SM. The conducted studies show that the principal
reason for chronic coughing in SM exposed victims was the contraction of bron-
chus, and therefore, it imposes the necessity to study them and prescribe adequate
treatment when available (Ghanei et al. 2005a, 2006c).
The importance of this issue, particularly in patients exposed to SM, increases
when we compare the results of our study with the unexposed people. More than
90 % of the studied patients had a combination of chronic coughing causes.
Therefore, it can be concluded that, despite the known causes of chronic coughing
in patients exposed to SM, each patient with chronic bronchitis induced by SM
should be evaluated more thoroughly for other causes of chronic coughing, particu-
larly in uncontrolled chronic coughing or recently intensified coughing. In addition,
since the number of related causes in SM exposed patients is considerably higher
than in the non-injured population, it is suggested to study potential exposure to
mustard gas when the patients have chronic coughing induced by different factors.
It should be emphasized that chronic coughing in chemical victims should not be
attributed only to SM exposure, and other causes of coughing such as gastro esopha-
geal reflux should also be considered, because coughing cannot be cured properly
in the absence of a clear diagnosis (Karbasi et al. 2013).
7.8 Incidence and Diagnosis of Emphysema in SM Victims
Emphysema is morphologically defined as resistant enlargement of distal airways

up to the level of the terminal bronchiole and destruction of its walls without evi-
dent fibrosis. Age and smoking are several of its main causes. Deficiency of AAT
enzyme is a genetic factor, which can lead to premature installation of emphysema.
The main methods used for identification of emphysema are PFT and chest radiol-
ogy. Although these methods are not sensitive enough for early identification of
functional and apparent abnormalities of airways, they are used in practice for
apparent evaluation of the extent and intensity of HRCT diagnosed emphysema
changes (Shohrati et al. 2010b).
In a casecontrol study, which was conducted on 20 smokers with low exposure to
SM and 20 smokers without exposure to SM, PFT and chest HRCT were conducted
on all participants for identification of emphysema. Sensitivity, specificity, and nega-
tive predictive value were calculated for PFT. In the group with low exposure to SM,
spirometry was not able to diagnose emphysema, while chest HRCT identified paren-
chyma changes in five patients. In the smoking group, 11 out of 20 persons (55 %) had
emphysema, as compared to the low exposure SM group, where only 5 out of 20 cases
(20 %) were diagnosed with emphysema (Ghanei et al. 2007a, b). There was no case
of deficiency of AAT enzyme in all 40 people (Shohrati et al. 2010b). It can be con-
cluded that, in the people who had a record of exposure to SM, cigar can induce
emphysema in younger ages. In this group, HRCT is more useful than the PFT for
early diagnosis, as the PFTs were reported normal in this group.
In our studies, there was no relationship found between air trapping in HRCT and
results of PFT. Pulmonary function test may not accurately identify longitudinal
changes in acinar structures, which are induced by specific factors, such as age and
smoking. When there is an additional pulmonary risk factor, as in the case of poisonous
gas exposure, symptoms of the disease, incidence and diagnostic approach may differ
from other patients. Considering the high incidence of such exposure in cities and
industries, it seems necessary to study emphysema in the patients with more than one
risk factor (Ghanei et al. 2007a, b).
In another study, the results of PFT were compared with HRCT findings of chest
in symptomatic smokers, with or without SM exposure, after studying the incidence
of emphysema and accuracy of PFT in smokers with a record of SM exposure.
Results showed that the Tiffeneau index forced expiratory volume in 1 s/forced
vital capacity (FEV1/FVC), was not a good criterion for the diagnosis of emphy-
sema in the SM exposed group, while chest HRCT confirmed the diagnosis in five
patients. In smoking patients, FEV1/FVC showed 100 % sensitivity for COPD. The
FEV1/FVC results show values that are more abnormal in the smoking group than
in the SM exposed group. On the other hand, chest HRCT results indicate a higher
rate of emphysema in the smoking group (Ghanei et al. 2007a, b, 2008b).
In summary, smokers with additional risk factors, such as exposure to respiratory
poisons, present the occurrence of emphysema at younger ages and, in this stage,
spirometry is also normal. Chest HRCT should be considered as a suitable tool for
early diagnosis of emphysema in smokers with exposure to poisonous gases. This
additional risk factor can intensify the symptoms during the initial stages.
Considering the mentioned evidence, it seems that an early diagnosis of emphy-
sema, before installation of symptoms, is possible (Ghanei 2011).
7.9 Other Damages to Airways and Lung Parenchyma
The SM damages the mucous layer of airways, depending on the inhalation exposure
rate. This damage starts from the upper airways and reaches smaller and terminal
airways in case of increasing inhalation dose. The intensity of the inflammatory reac-
tion varies from mild to severe, and induces respiratory epithelium necrosis.
Pulmonary damage is present under different forms, such as acute inflammation of
upper and lower air ways, secretion from upper parts of respiratory system, inflamma-
tory exudates and formation of pseudo membrane in the tracheobronchial tree. These
damages are slowly intensified within several days. Primary bronchitis is usually non-
infectious. The increase of leukocytes, fever and pulmonary infiltrations are found on
radiographic a imagery. This process occurs in the first 34 days and secondary infec-
tion ensues after 46 days. A daily study of the mucosa with gram staining and culture
should be conducted (Anderson et al. 1996; Fatal Exposure to Mustard Gas).
In a study investigating the chronic pulmonary complication induced by SM
exposure on 50 soldiers, 80 % of them had obvious abnormalities on chest radio-
graphs and while an increase in the thickness of bronchial wall was present in all
(100 %), and changes suggesting interstitial pulmonary disease (80 %) and bron-
chiectasis (26 %) were visible on HRCT lung scan (Bagheri et al. 2003). The afore-
mentioned study was compared with a separate study, in which 220 persons were
exposed to SM. Almost all people who had obstructive phenomena had symptoms
such as coughing, asthma and degrees of pulmonary obstruction, wheezing and
dyspnea (67 %), Hemoptysis (2.7 %) and respiratory distress with respiratory
accessory muscles (1.8 %) (Bagheri et al. 2003).
7.9.1 Ventilation and Perfusion Disorder
As mentioned above, accumulation of secretions in airways leads to obstruction.

Obstruction causes oxygen not to enter lung alveoli and carbon dioxide (CO2)
induced by cell metabolism not to exit from the lung. As a result, a type of hypoven-
tilation occurs during which PaCO2 increases and PaO2 decreases. At the same time,
it has been found in animal studies that the respiratory rate decreases following inha-
lation of a high dose of SM. This bradypnea can intensify the hypoventilation disor-
der resulting from bronchus obstruction (Vijayaraghavan 1997; Shohrati et al. 2012).
7.9.2 Hemoptysis
In the acute stage of SM poisoning, mucous bleeding is present, alongside inflam-

mation and severe injury of epithelium. In the case of excessive bleeding, it can lead
to airways obstruction in conjunction with the lung lesions inducing choking of the
patient. In chronic phase, hemoptysis can be due to new vascular proliferation and
chronic inflammation in these cases and it is not a reliable index for lung malig-
nancy (Ghanei et al. 2006b; Karami et al. 2011).
7.9.3 Biochemical Disorders of Lung
Increases of gamma-glutamyl-transpeptidase (GGT) activity indicate bronchus epi-

thelium damage, while LDH activity and increased concentration of proteins indi-
cate cytotoxic processes in lungs, resulting in damage of the epithelium of bronchi
(Foy and Schatz 2004).
The evolution of these destructive processes can be stopped by excluding the
induction factor. However, in particular cases, the lysis continues, accounting for
tissue destruction on the long-term (Biljak et al. 2013).
7.10 Para-clinical Findings
7.10.1 Radiologic Data
In diagnosis, treatment, follow-up and evaluation of the response to treatment in

chemical victims, radiological findings are very useful. For radiological study, chest
X-ray (CXR) and or high resolution computed tomography (HRCT) can be used.
7.10.1.1 Chest X-Ray
Radiological findings of the thorax in chemical victims are different between the
symptomatic acute condition and asymptomatic condition. Therefore, it is very
important to diagnose and interpret radiographical imagery in chemical victims in
different conditions. In this instance, we describe radiographical findings in pulmo-
nary symptomatic and asymptomatic patients.
In studies conducted on CXR among these patients, there was no mass or nodule,
while the X-ray was considered normal in 70 % of the cases. The most prevalent
abnormal radiological pattern was the infiltration surrounding bronchus in 34 % of
the patients and the increase in thickness of the bronchial wall and increase in vas-
cular marking. In 5 % of the cases, there was an increase in the thickness of pleura.
These particular findings indicate that the patients suffer from respiratory problems
despite normality of CXR. Therefore, radiography is not a reliable tool for the diag-
nosis and evaluation of lesions among these patients (Mansoor Ghanaei and
Alizadeh 1999; Razavi et al. 2013a).
Although the study of chemical victims in the secondary stage showed that most
of these patients (70 %) did not had abnormal findings on CXR despite respiratory
problems, however, changes of chronic bronchitis (15 %), reticular marking (9 %)
and bronchiectasis (6 %) were visible (Ghanei and Harandi 2010a).
In another study, CXR findings among the chemical victims reported emphy-
sema (26 %), increase in pleural thickness (40 %), bronchiectasis (15 %), and pneu-
matic infiltration (83 %). Although BO is the most clinicopatholic complication, it
could not be diagnosed by CXR (Amini and Oghabian 2013).
Considering the mentioned facts, it can be concluded that the most prevalent
finding of radiography is normality of lungs, and therefore, a normal X-ray does not
exclude pulmonary damage in chemical victims and more investigations should be
conducted, especially with HRCT, in case of suspicious diagnosis of pulmonary
complication in these patients.
7.10.1.2 Lung HRCT in Patients Without Symptoms
In a study on the people who attended the SM contaminated areas for a week
during which they did not had any early symptom of SM exposure, it was shown
that these individuals will suffer from complications induced by SM exposure
in future. The start of clinical symptoms varied from 23 years after attendance
in the SM contaminated areas. In the HRCT of 38 % of these patients, no posi-
tive finding was found. In other 38 % of them, there was only air trapping on
HRCT In addition, there was an increase of the thickness of the bronchus wall
in 14.7 % bronchiectasis in 8.8 % of them and mosaic pattern in one patient. It
should be noted that the presence of air trapping in more than 25 % of the
patients was considered as the most sensitive and accurate finding indicating
BO (Ghanei 2011).
In study by Dr. Bagheri et al., HRCT was used on 50 patients to study pulmonary
changes induced after SM exposure, and the results were compared with clinical
and chest radiography findings in the same patients. In all of these cases, HRCT was
abnormal, while CXR was abnormal only in 80 % of them. Increases of bronchus
wall thickness were reported as the most prevalent positive finding of HRCT
(100 %). Other positive radiography characteristics were interstitial lung disease
(ILD) (80 %), bronchiectasis (26 %) and emphysema (24 %). There was no statisti-
cally significant relationship between CXR findings and intensity of pulmonary
lesions intensity. In addition, the people who had normal radiography (20 %)
showed increased thickness of bronchus wall and interstitial lung disease (with
lower rate), which were reported in HRCT. Based on the results of this study, the
researchers concluded that an increase of thickness of bronchus wall, ILD and
emphysema were the most prevalent radiological findings in these patients. For this
reason, HRCT was suggested as the best radiological diagnostic tools for these
patients (Bagheri et al. 2003).
In the study by Dr. Hosseini and Balali-Mood (1998), bronchography was per-
formed in 11 individuals while HRCT was performed in 50 individuals to study a total
of 61 SM exposed patients, in the secondary stage, in which most of them (81 %) had
abnormal parameters in spirometry and pulmonary disease symptoms. Bronchiectasis
pattern on HRCT is found. The diagnosis of bronchiectasis had been performed by
bronchography in three patients and by HRCT in 13 patients. The involvement of the
lower lobes was higher, compared to the upper lobes (ten versus four cases). There
was no right middle lobes involvement. Although most patients did not presented
bronchiectasis in CXR, there were 12 cases of bronchiectasis (24 %) and they showed
a particularly bilateral involvement (Hosseini and Balali-Mood 1998).
In our study in 2004, we found that The most frequent findings were; air trap-
ping 38 (76 %), bronchiectasis 37 (74 %), mosaic parenchymal attenuation (MPA)
36 (72 %), irregular and dilated major airways 33 (66 %) bronchial wall thickening
(BWT) 45 (90 %), and interlobular septal wall thickening (SWT) 13 (26 %)
(Ghanei et al. 2004b).
Since about 25 % of the random population had lung air trapping, the presence
of more than 25 % of air trapping in a section of lung was considered as diagnostic
criterion of BO in this study. The characteristics for HRCT positive slides were
studied based on the type and number of lesions in all pulmonary lobes of all 50
patients. Afterwards, the numbers of each type of lesions were counted in the
engaged lobes. Air trapping and abnormal pattern were evident in 44 % of patients
(Mostafa Ghanei 2011; Ghanei et al. 2011b).
Exhaled HRCT is one of the diagnostic tests that should be performed in the
patients who may have small airways involvement, because multiple symptoms of
the involvement of small airways, such as air trapping, can be diagnosed only dur-
ing the exhalation phase. Mosaic parenchyma attenuation is one of the findings that
can be diagnosed easily during exhalation. These two signs are the most prevalent
abnormal signs during the exhalation state (Bakhtavar et al. 2008). There are some
sample HRCT cuts for example in Fig. 7.5ad.
Fig. 7.5 (ad) HRCT of

SM exposed patients after a
more than 20 years;
emphysema, septal
thickening, air trapping are
seen (Author captured)
d
7.10.2 Respiratory Function
7.10.2.1 Polysomnography
In a study that was conducted on 30 SM victims, the relationship between disease

intensity and sleep pattern was evaluated based on the GOLD (Global initiative for
chronic Obstructive Lung Disease) criteria and polysomnography. Results of this
research indicated that patients with a lower intensity of pulmonary symptoms had
more hypopnea and more frequent episodes of rapid eye movement. The first stage
of sleep in the patients who had higher FEV1 and the fourth stage of sleep in the
patients who had higher Diffusing capacity for carbon monoxide (DLCO) was the
dominant stages of sleep (Vahedi et al. 2012). These findings can indicate a greater
symptomatic response of these patients in more intensive stages of pulmonary dis-
ease and asthma.
In chemical victims, in acute phase, Arterial blood oxygenation (PaO2) and
saturation levels were significantly decreased at 12 h. Arterial blood carbon dioxide
(PaCO2) significantly increased, and arterial blood pH and bicarbonate (HCO3)
significantly decreased at 12 h. Shunt fraction was significantly increased at 12 h
(Jugg et al. 2013). Moreover, in chronic phase, there are evidence of imbalance in
po2, pco2 and other blood gas parameter (Balali-Mood et al. 2011).
7.10.2.2 Spirometry
Pulmonary Function Tests in Clinical Contact with SM
Pulmonary function tests (PFT) studies are useful for four indices of pulmonary
functions: airflow (spirometry), pulmonary volumes, gas exchange (diffusion coef-
ficient) and lung mechanics. In a study which was conducted on 77 persons who
attended the SM contaminated areas for at least 1 week, during which they did not
have any symptom of SM exposure, it was shown that, although these individuals
did not had any initial clinical symptom of the acute phase, they have now late SM
toxic effects. Specifically, the PFT pattern was restrictive in 5 % of these patients,
obstructive in 5 %, restrictive and obstructive in 8.82 % and normal in 85.3 %
Pulmonary Function Tests
In study of the pulmonary function, the chemical victims exposed to SM in acute

phase, who did not use suitable protective tools at the time of exposure to the chemi-
cal agent, showed an obstructive pattern (53 %), while a restrictive pattern was
evident in only 1.5 %, a mixed pattern in 18.7 % and a normal pattern in 21.8 %,
based on PFT findings (Sohrabpour 1987).
In another study, which was published in 1997, the pulmonary function was
studied in 130 SM victims hospitalized in Isfahan hospitals with acute injury. Of
these patients, 11.5 % had restrictive lesions, 32.3 % of them had obstructive
lesions, 21.5 % had FEV1 and forced mid-expiratory flow (FMF) reduction
(obstruction of small and large ducts) and 10.7 % had only reduction of maximum
mid-expiratory flow (MMEF) (small and peripheral respiratory duct obstruction).
Only 14 % of the patients had obstructive and restrictive lesion altogether, while
41 % of these people had normal spirometry. Considering the above results, it can
be concluded that the most prevalent finding of the abnormal spirometry in the
acute phase after SM exposure, is pulmonary obstructive lesion. Spirometry with
normal results also played considerable role. It is necessary to note that two thirds
of the patients who were above 41 years showed mixed obstructive and restrictive
disorders, indicating that the increase of age enhances the intensity of pulmonary
complication (M and AR 1997).
In the results of PFT, 35 chemical victims exposed to SM with intensive pulmo-
nary disease for 6 weeks to 1 year showed an obstructive pattern (55 %), restrictive
pattern (24 %), small airways involvement (13 %) and normal pattern (6 %)
(Sohrabpour et al. 1988).
The most prevalent spirometric changes and clinical symptoms of the flow-
volume loop among the people exposed to chemical bombing with mustard included
change in the flow-volume loop and smoothness of the middle part of the loop in 18
people (30 %). The obstructions with moderate and mild intensity or changes of
flow-volume loop manifested earlier than the spirometric evidence, or these changes
were only limited to the flow-volume loop. The maximum changes of the flow-
volume loop occurred in the fourth month after injury and the maximum changes in
spirometry occurred in the fourteenth month after injury. It was also reported that a
distance to the chemical bombing place of up to 500 m did not affect induction and
incidence of complication in this group. Nevertheless, it affected the intensity of the
complication (Ansarin and Rezvanyeh 1987).
At the end, it was concluded that all obstructive disorders evidenced on the flow-
volume loop included lower airways obstruction and no important flow limitation
related to upper airway obstruction was found until the end of the 2-year study.
In a cross-sectional study, long-term complications of pulmonary function were
studied in 197 chemical victims after 10 years. Findings of these victims were com-
pared with 86 soldiers who were not injured with mustard as control group. In this
study, patients were divided into three groups of asthmatic, chronic bronchitis and
pulmonary fibrosis patients. Results of PFT were evaluated in all three groups. In
this study, the relationship between fibrosis degree and DLCO and PFT test was
studied in patients with idiopathic pulmonary fibrosis (IPF), the results revealing
that there is only a direct relationship between the intensity of fibrosis and the per-
centage of DLCO (Emad and Rezaian 1999).
Since the following complementary studies rejected PF and ILD, therefore, the
results of the mentioned study cannot be used. Results of a report on 43 patients
exposed to SM, among whom the interval between time of injury and their study
time was 47 years and who had long-term symptoms of injury with SM, showed
that RFT had and obstructive pattern in 53 %, 43 % had a restrictive pattern and 5 %
had normal pattern. In fact, the most common pattern of the PFT of these patients
was obstructive. On the other hand, many of these patients had abnormal clinical
and spirometry findings, despite CXR normality. Considering that the patients who
were studied at shorter term after injury had a less abnormal pattern, it can be con-
cluded that the progression of pulmonary injury in these patients may be prevent-
able over time (Emad and Rezaian 1999; Attaran et al. 2006).
In another trial, which was conducted on 407 SM victims, changes of spirometric
tests after 10 years were compared with the levels at study enrolment. According to
the results, all indices of PFT were considerably reduced compared with the initial
test (Ghanei et al. 2007).
In a longitudinal prospective study in 1988 by Heidar Nejad et al., spirometry
parameters like FVC, FEV1, peak expiratory flow (PEF) and clinical symptoms of
1872 chemical victims who referred to the pulmonary clinic were studied
(Heidarnejad et al. 1988). The PEF and FEV1 had statistically considerable changes.
Considering the role of age increase in the reduction of pulmonary volumes, FEV1
was reduced in these patients averagely by 50 mm per year, which has also been
statistically considerable, especially when adding that 24 % of the patients were
new smokers. In the first year of study, there was no difference in FEV1 and FVC
between smokers and nonsmokers. On the other hand, because these people gave up
smoking in the following years, it was mentioned that the reduction of FEV1 values
related to effects of mustard. Parameters of PEF during these 10 years showed an
ascending trend, and, considering that the age of most patients was above 29 years,
this problem was regarded as normal (Heidarnejad et al. 1988). It is necessary to
note that inclusion and exclusion criteria of the participants were not specified and
no information was given for proving their contact with SM.
Dr. Motamedi et al., in other similar reports, assessed pulmonary capacities of
the chemical victims. The study of different pulmonary parameters included 480
chemical victims and the alterations of pulmonary spirometric parameters varied
largely from 1 to 150 days after exposure and even later, despite treatment. It
seems that the intervention of other factors, such as distance of the person to the
place of explosion of the chemical bomb was also important in the changes of
pulmonary volumes and capacities. Studies showed that there was a correlation
between the distances of the person to the chemical bomb explosion place, respon-
sible for a reduction of the spirometric parameters. However, there was no signifi-
cant relationship between the time interval of injury and the use of mask, or
smoking before injury and spirometric factors. If smoking after injury continues,
parameters like FVC, FEV1 and FEV1/FVC, will be significantly reduced
(Eftekharhosseini et al. 1987).
There was a significant correlation between the time interval when the injured
person left the chemically contaminated region and variables like functional resid-
ual capacity (FRC), total volume (TV), FVC and FEV1. In addition, there was a
correlation between the duration injury time and start of treatment, and also between
the time interval from clothes change and simple shower and the spirometric param-
eters of FVC, FEV1 and FEV1/FVC. With increasing time between injury and
clothes change, shower and treatment, the aforementioned parameters are reduced,
while the anorak and mask before chemical contamination is associated with a less
important reduction. At the end, it was specified that an allergy positive record in
the injured person reduced pulmonary capacities, particularly FEV/FVC, FRC and
FMF (Eftekharhosseini et al. 1987). As mentioned above, there is a direct correla-
tion between pulmonary changes and weight and there is a reverse relationship
between age and the parameters of FEV1 and FMF (S et al. 1987).
The existing evidence and experiences show that although the rate and intensity
of pulmonary involvement is considerably related to SM inhalation, this is not
always the rule. For example, there are individuals with a record of symptomatic
injury who showed normal PFT. On the contrary, other patients, who had low
intensity symptoms, or minimal clinical injury, had severe disorders in the next
years. This problem may underline the role of genetic differences between individu-
als on what concerns the intensity of pulmonary and dermal disease in future
(Mostafa Ghanei and Amin Saburi 2011).
Validity of the PFT in recent years was higher than in the previous years, consid-
ering the increase of the awareness level of the physicians and technicians, and
organizing training courses. When comparing PFT nowadays with the past years, it
becomes evident that the incidence of the obstructive pattern has increased in the
patients and a vice versa effect was recorded for the restrictive pattern. The reason
may be the low awareness level of medical personnel in the past, with the character-
istics of a standard spirometry. Pulmonary restrictive lesions of the injured people
may gradually transform into the restrictive type. However, this issue should be
analyzed more thoroughly in order to have a fundamented opinion. This obstructive
pattern is the most prevalent abnormal spirometric pattern in the injured people who
had asymptomatic exposure during war and showed pulmonary symptoms in the
next years (Mostafa Ghanei and Amin 2011).
In general, the abnormal PFT pattern (mostly obstructive pattern) of these
patients indicates that injury with SM causes the involvement of airways, rather
than lung parenchyma. Since pathological and radiological evidence indicates the
involvement of small airways, the diagnosis of a specific pulmonary pathological
alteration should take into account the changes and indices of changes at the level
of the small airways (Idani et al. 2012).
In summary, if we would want to clarify which is the highest spirometric pat-
tern in all chemical victims of Iran, based on the study on 34,000 chemical vic-
tims of the country, it can be said that the near predicted range pattern (obstructive)
is the most frequent pattern of PFT. From the studies conducted in acute and
chronic stages after injury, it can be concluded that the most frequent abnormal
pattern is obstructive pattern. Mixed states and restrictive states have been men-
tioned with different statistic results as the other patterns encountered
inspirometry.
7.11 Differential Diagnosis and Other Diagnosis

Along with BO
7.11.1 Asthma and COPD
In the early years, asthma, chronic bronchitis and emphysema had been introduced
as prevalent pulmonary diseases of the chemical victims of SM. Imaging findings
and spirometry studies have shown that the chemical victims are afflicted with
degrees of chronic lung obstruction, while emphysema was observed as the domi-
nant finding in pathological samples. On the other hand, considering that the revers-
ibility of airway obstruction is one of the diagnostic criteria of asthma, the
reversibility following intake of bronchodilator was low in chemical victims and, in
fact, it can be mentioned that airways obstruction is not reversible in chemical vic-
tims (Mirsadraee et al. 2005; Emad and Emad 2007b).
Sandall (1922) is one of the authors who have conducted studies in this field.
He studied 83 soldiers injured with SM in the First World War and there were
symptoms of emphysema in 26 % of them, while chronic bronchitis was present
in 20 % of them (Sandall 1922). In 1922, Hankins published another report in the
same year, which had similar results (Hankins and Klotz 1922). Berghoff (1919)
also studied clinical symptoms of 2000 American soldiers poisoned with SM
(Berghoff 1919). After 34 months following SM exposure, symptoms of chronic
bronchitis were reported in 30 % of these people and emphysema in 22 % of them.
In 1933, Gilchrist et al. evaluated long-term complications of injury with SM in
89 persons, and after 10 years, the findings of their study showed that 27 cases had
pulmonary complications, such as chronic bronchitis and emphysema (Institute of
Medicine (US) Committee on the Survey of the Health Effects of Mustard Gas
and Lewisite 1933). A study which was conducted in 1955 on 1267 British veter-
ans from the First World War showed that about 80 % of these soldiers suffered
from chronic bronchitis. Based on the conducted studies on the chemical victims
in 1989, iterative bronchitis and pneumonia have been introduced as the major
pulmonary complications of injury with SM, 2 years after exposure (Mostafa
Ghanei and Amin 2011).
A 2-year follow-up of more than 200 Iranian soldiers exposed to SM in the
Iran and Iraq war has shown that one third of these patients were afflicted with
long-term pulmonary complications (chronic bronchitis and asthma). The
United Nation (UN) has published a report on the effects of using chemical
gases by Iraq against Iran, which has studied the complications of SM poison-
ing in some of the injured soldiers. In 78 % of these injured soldiers, pulmo-
nary complications of chronic bronchitis and asthma were found (Report of the
mission dispatched by the Secretary-General to investigate allegations of the
use of chemical weapons in the conflict between the Islamic Republic of Iran
and Iraq).
7.11.2 Bronchiectasis
In reports of UN mission in 1986, bronchiectasis has been mentioned as one of the

long-term pulmonary complications induced by mustard in Iranian injured soldiers.
In addition, different studies, which have been conducted on 200 chemical victims
of SM, concluded that bronchiectasis is one of the main pulmonary complications,
and about 8.6 % of the chemical victims exposed to SM had findings supporting
bronchiectasis (Report of the mission dispatched by the Secretary-General to inves-
tigate allegations of the use of chemical weapons in the conflict between the Islamic
Republic of Iran and Iraq).
7.11.3 Pulmonary Fibrosis
Pulmonary fibrosis is one of the causes of the restrictive pattern in the pulmonary
function of the chemical victims. In a study that was conducted on 197 chemical
victims, PF was regarded as one of the causes of the restrictive pattern of PFT in
12.1 % of the patients. However, lung fibrosis was not regarded as the dominant
pathology finding in SM exposed patients. Complementary studies on carbon mon-
oxide diffusing capacity (DLCO) and HRCT did not confirm these findings based
on PFT (Emad and Rezaian 1997).
7.11.4 SM and Lung Cancer
As an alkylating agent, it was proved that SM can lead to genome mutation. First
reports confirmed this hypothesis that emphasis on the lung carcinogenicity of SM
but there is doubt in profile and properties of exposure. In 2006, Beheshti et al. con-
ducted an investigation on the lung specimen of SM victims who had a diagnosis of
lung cancer. They found p53 mutations in 5 of 16 cases but there is no mutation in
KRAS gene. They finally concluded that the distinguishing characteristics of lung
carcinogenesis in these mustard gas victims suggest that a single exposure may
increase the risk of lung cancer development in some individuals (Hosseini-Khalili
et al. 2009; Beheshti et al. 2006). Later reports confirm that a chronic, long-term,
high-dose SM exposure especially in cases with low storage of anti-oxidant may
promote lung cancer. Ghanei and Amini 2010 in a literature review demonstrated
that It is well documented that SM can cause human lung cancer after long-term
exposure, but there has not been strong and definitive evidence for only short-term
and acute, single, high-dose exposure until now (Ghanei and Harandi 2010b). In
spite of these evidence, after a long term cohort research, Zafarghandi et al. found
that the rate of some cancer increased in SM exposed cases but the investigated
cases had a single high dose exposure. They found a high incidence rate ratio and
hazard ratio of cancer as 1.81 and 2.02 compared to non-exposed controls. Although
the most exposed organs were skin, lungs and eyes, 75 % of all neoplasm was hema-
tological and related to gastrointestinal tract whereas lung cancer was found in 5
cases of 7570 exposed cases in comparison with 2 cases of 7595 unexposed cases
(Zafarghandi et al. 2013). By the way, lung cancer in these patients still is a chal-
lenging issue (Karbasi-Afshar et al. 2013a).
7.11.5 Cardiopulmonary Involvement of SM Exposed Patients
Cardiovascular systems involvements such as accelerated atherosclerosis, coronary

artery ectasia, pulmonary hypertension, and changes in myocardial perfusion were
reported in these patients (Shabestari et al. 2011; Karbasi-Afshar et al. 2013b).
There is no a constant relationship between the above disorders and pure toxicity
SM but when lungs are affected by SM and chronic inflammatory situation is estab-
lished, some inflammatory products and toxicants such as CRP and interleukins
may affects cardiovascular health (Attaran et al. 2009; Saburi et al. 2012b).
7.12 Treatments
7.12.1 Acute Phase
Before discuss about acute phase treatment, we should talk about prevention.
The environmental sustainability of sulfur mustard is high. Hence the agent is
able to remain in soil for at least 10 years and it can persist in the clothes and be
active in soil even for months at low temperatures (Ghasemi Broumand et al.
2007; Razavi et al. 2013b). Using mask and other personal protective equipment
are suggested. Antioxidant is the most studied pharmacological prevention (Jugg
et al. 2013). Das et al. in a lab study on rats, concluded that Pretreatment with
NAC for 3 and 30 days protected against 6976 % of the acute lung injury but
they could not suggest a dose for human study (Das et al. 2003). pre-exposure
therapy with protinin, ilomastat, vitamin E, dexamethasone and their combina-
tion, and antioxidant liposomes were successfully used for prevention and
decrease lung consequence of SM (Mukhopadhyay et al. 2010; Boskabady et al.
2011a; Anderson et al. 2009).
After exposure antidotal treatment with up to 500 mg sodium thiosulphate per
Kg body weight should be infused within 30 min post-exposure (Balali-Mood and
Hefazi 2005). Also, if anticholinergic symptoms occur, the treatments should be
considered. Some researchers approved the efficacy of the detoxification procedure
such as haemoperfusion and haemodialysis (Willems 1989). Pain killer or sedative
may be necessary. At the early phase, supportive treatments such as oxygen therapy,
treatments of lung edema or even ARDS, and anti-congestive agents were recom-
mended (Poursaleh et al. 2012).
To prevent progressing towards chronic and persistent sequel, some supportive
treatments including NAC, sodium citrate, promethazine, heparin and vitamin E
were recommended. All mentioned treatments are considered to provide cell anti-
oxidant supply (Balali-Mood and Hefazi 2005; Zhang et al. 1995; Wigenstam et al.
2009; Laskin et al. 2010; Poursaleh et al. 2012). Corticosteroids also may be useful
when lung edema and severe alveoli and airways inflammation are suspected
(Wigenstam et al. 2009; Poursaleh et al. 2012).
Tissue plasminogen activator (t-PA) was Intratracheally used by Veress et al. at
2015 for treating the acute impact of SM analouge in rats. They demonstrated that
Intratracheal t-PA treatment eliminated mortality (0 % at 48 h) and greatly
improved morbidity after lethal SM inhalation (100 % death in controls). tPA nor-
malized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved
respiratory distress (Veress et al. 2015). Therefore, Intra-airway tPA (optimal dose
: 0.7 mg/kg) may be prescribed for patients with ventilation and oxygenation failure
(Veress et al. 2013).
Moreover, there are some reports about some protective and therapeutic agents
which were used for acute toxicity of SM. Sawyer in 1999 reported that synergistic
protective effects were also achieved when L-nitroarginine methyl ester (L-NAME) was
added up to 8 h after HD exposure, if they were pretreated with L-thiocitrulline (L-TC)
(Sawyer 1999). In another interesting study, Pohanka et al. in 2011, used Melatonin for
treating the acute symptoms of SM toxicity in animal model. They found that 25 and
50 mg/kg, subcutaneously, melatonin can decrease toxicity of SM exposure as consider-
ably changes serum level of ferric-reducing antioxidant power (FRAP), thiobarbitu-
ric-acid-reactive substances (TBARS), and plasma protein carbonyls (Pohanka et al.
2011). Also, these findings support antioxidant-oxidant imbalance hypothesis (Korkmaz
et al. 2008). Although the mentioned treatments were successfully used in vitro, these
medications have not ever been used on humankind to approve its efficacy.
7.12.2 Chronic Phase
There are many clinical trials in which various treatment protocols were used for
healing the clinical and pathological disorders of long terms SM injuries. Initially,
corticosteroids were solely used for these cases. Ghanei et al. 2005 at a RCT evalu-
ate two form of corticosteroid (IV and Oral) and they finally concluded that intra-
venous methylprednisolone acetate 500 mg daily for 6 months and oral treatment
with prednisolone 1 mg/kg daily for 6 months improve respiratory indices signifi-
cantly (Ghanei et al. 2005b). Corticosteroids were administrated because it was
believed that one of the main pathogenesis of mustard lung is chronic inflammation.
Ghanei et al. used these medications for exacerbation in patients with SM induced
bronchitis. They concluded that short-term bolus steroid treatment to triage the
patients into responders and non-responders for subsequent treatment.
It seems that additional medications along with corticosteroids increase its effi-
cacy and it has opportunity to decrease the dose of corticosteroid to prevent its
adverse effects. Ghanei et al. at 2007 after a clinical trial study, recommend two
combination therapy for SM induced chronic bronchiolitis; first combination form
of fluticasone propionate and salmetrol, 500/100 g daily and second 1000 g daily
beclomethasone in addition to 800 g daily inhaler salbutamol for 12 weeks. They
declared that both protocols improve PFT and had similar efficacy (Ghanei et al.
2007a, b). Boskabady et al. in 2011 after adding Nigella sativa to corticosteroids for
pulmonary exposed guinea pigs claimed that inflammatory cells (neutrophils) were
significantly lower rather than corticosteroids alone (Boskabady et al. 2011b).
As well, Wigenstam et al. at 2009 conducted a study on mouse exposed to alkyl-
ating nitrogen mustard melphalan to evaluate the efficacy of Vitamin E in addition
to corticosteroids on airways inflammation. They interestingly demonstrated that
early single-dose treatment with dexamethasone protects against long-term effects
observed 24 weeks after melphalan exposure. This findings shows that dexameth-
asone can reduce lymphocytic response in airways and decrease collagen deposi-
tion. Eke, their findings showed that vitamin E (50 mg/kg) reduces acute
inflammatory cell influx, and suppresses collagen formation in lung tissue, indicat-
ing that this drug could be used in combination with corticosteroids for protection
against chemical-induced lung injury (Wigenstam et al. 2009).
Also, some antibiotics such as Macrolids (Azythromycine and rotrixomycine)
have been proven that may lead to clearance improvement of apoptotic material in
the airway and ultimately cause to reduce airway inflammation due to SM inhala-
tion (Poursaleh et al. 2012; Gao et al. 2010). Previously, it was shown that
Macrolides can module the immune response in airways. In diffuse panbronchiol-
itis, leukocytes and neutrophils are the main targets for modulatory effects of
Macrolides particularly erythromycin on host defense responses (Culic et al. 2001).
This antibiotic can also accelerate the efferocytosis process and also prevent fre-
quent and persistent pneumonia. Therefore, macrolids are still recommends as the
first line for these cases. Gao et al. after serial studies on interaction of Macrolids
and SM demonstrated that roxithromycin has inhibitory effects on the cytotoxicity
and inflammation provoked by SM in human respiratory epithelial cells. The
decreased cytotoxicity in roxithromycin-treated cells likely depends on the ability
of the macrolide to down-regulate the production of proinflammatory cytokines
and/or mediators (Gao et al. 2007). They Also found that Macrolids can reduce
iNOS expression and nitric oxide production (Gao et al. 2008) improve the degener-
ated chemotactic and phagocytotic functions of monocytes (Gao et al. 2010) and
protects against some damages associated with SM injury in the lung, particularly
in the upper respiratory tract (Gao et al. 2011).
After revealing the role of oxidant-antioxidants imbalance in pathogenesis of
mustard lung, antioxidant supply such as NAC was used. N acetyl-cysteine can be
used as a protective agent that enhance glutathione-S-transferase (GSH) synthesis,
as well as prevent oxidative activation of NF-B inducing endothelial cell death and
generate a local inflammatory reaction associated with the release of endothelial-
derived cytokines (Poursaleh et al. 2012; Dekhuijzen 2006; Soltan-Sharifi et al.
2007). NAC may be ordered between 600 and 1800 mg per days according to the
severity of the diseases. It can effectively improve lung indexes in addition to heal-
ing the symptoms such as thick sputum, dyspnea and cough (Shohrati et al. 2014b).
There are some limitations in the case of NAC prescription; first, a very high dose
should be prescribed to be effective in this patients, second, its first pass effect is
considerable, third, it should be used every days. It seems that nebulized NAC can
resolve all mentioned queries. Jugg et al. at 2013, multiple inhaled doses of NAC
(1 ml of 200 mg.ml1 Mucomyst at + 30 min, 2, 4, 6, 8, and 10 h post-exposure
administered for pigs exposed with SM and they concluded that Pigs which were
treated with nebulized NAC had significantly improved arterial blood oxygen satu-
ration, HCO3 levels, and shunt fraction compared to those of the sulfur mustard
controls. Also, they had significantly fewer neutrophils and lower concentrations of
protein in lavage compared to controls (Jugg et al. 2013). They did not add a group
with oral administration of NAC and it is a limitation for final conclusion.
As mentioned above, bronchodilators such as long-acting beta 2-agonists (e.g.
Salmetrol) and anticholinergic (e.g., ipratropium bromide) could be recommended
for cases with airways hyper reactivity symptoms (Boskabady et al. 2008). The
effectiveness of these medications increases when used in combination with corti-
costeroids (e.g. inhaled corticosteroids) (Ghanei et al. 2007a, b).
Although other medications such as Protease inhibitors were effective, they
should try in human model to approve it efficacy (Weinberger et al. 2011). Anderson
et al. in 2009 design a rat model study to evaluate the efficacy of protease inhibitors
aprotinin and ilomastat and the antioxidant trolox on lung complications of
SM. They prescribed intravenous aprotinin, 4.4 mg/kg; intraperitoneal (ip) ilomas-
tat, 25 mg/kg; or ip trolox, 500 g/kg. They finally concluded that Histopathologic
examination of lung tissue 24 h post-exposure showed minimal alveolar effects
caused by SM, while damage to bronchiolar regions was much more severe due to
the highly reactive nature of SM. While aprotinin and ilomastat both alleviated the
PF perturbations, surprisingly only aprotinin reduced the observed pathology, both
grossly and histologically. These early results indicate that treatment with aprotinin
and to a lesser extent ilomastat reduces some of the direct inflammatory response
and damage associated with SM-induced lung injury (Anderson et al. 2009).
Morphine also has been prescribed in these patients. Shohrati et al. in 2012 pre-
scribed 1 mg morphine sulfate diluted by 4 cc normal saline 0.5 % using nebulizer
once daily for 5 days in SM lung injured patients. They found that scores of VAS
for dyspnea, cough and quality of life and also respiratory rate, heart rate, and night
time awaking due to dyspnea and night time awaking due to cough improved sig-
nificantly after morphine nebulization without any major adverse events. Also pick
expiratory flow has been improved significantly after nebulization in each day
(Shohrati et al. 2012).
Interferon gamma-1b (INF -1b) plus a low-dose of prednisolone can improve
the lung function of these patients, Panahi et al. demonstrated at 2005 (Panahi
et al. 2005). Also, Ghanei et al. in 2006 evaluated 36 bronchiolitis patients were
receiving their conventional treatment (inhaled Felixotide and Servent) in addition
to 6 months combination of 200 g of interferon gamma-1b (given three times per
week subcutaneously) plus 7.5 mg of prednisolone (given once a day) (case group)
compared to alone conventional treatment (controls). They found that FEV1 and
FVC showed a significant increase in the case group compared control group
(Ghanei et al. 2006d).
Surfactant therapy (Malaviya et al. 2010) are also on the list of treatment that has
not proven their effectiveness as well as the above therapeutics. Although, based on
pathogenesis of SM, surfactant should be used at the acute phase, exogenous lung
surfactant curosurf and salbutamol were used for the treatment of Guinea pigs with
asthma like symptoms following intratracheal exposure of to SM aerosol. Van
Helden et al. at 2004 showed the efficacy of administration of the natural surfactant
Curosurf and the broncholytic Salbutamol on asthma like symptoms reflected by an
early bronchoconstriction and late asthmatic responses (LAR), and ARDS-like
symptoms secondary to SM exposure in large animal models. They concluded that
Intratracheal nebulization of a Salbutamol solution (10 g/kg), or I.T. bolus admin-
istration of Curosurf (62.5 or 125 mg/kg), tended to reduce mortality, although
Salbutamol appeared to be more effective than Curosurf in this respect (Das et al.
2003; van Helden et al. 2004).
Among herbal medications, Curcumin as a famous herbal antioxidant was suc-
cessfully used for this patients (Moghaddam et al. 2009; Biswas et al. 2013; Panahi
et al. 2014b). In the only conducted study, Panahi et al. in 2014, use Curcuminoids
supplements in 45 cases versus 44 controls in a RCT for 4 weeks and they finally
found that immunological parameters including IL-6 and 8, TNF, TGF, hs-CRP,
calcitonin gene related peptide (CGRP), substance P and monocyte chemotactic
protein-1 (MCP-1) and spirometric indices were changed significantly (Panahi et al.
2014a). Moreover, they evaluated serum antioxidant level and respiratory quality
index and quality of life in these groups and they demonstrated that significant
improvements in the total as well as subscale (symptoms, activity and impact)
SGRQ and CAT scores in both groups. Also, their study showed that curcumin
supplements significantly elevate GSH and reduce MDA. They prescribe curcumin
as 1500 mg/daily (500 mg every 8 h) in addition to piperine (15 mg/day). This result
supports the antioxidant-oxidant imbalance in these patients (Panahi et al. 2014b).
Regarding to the mentioned mechanism of long term consequences of SM expo-
sure, some potential treatments can be effective on these cases as recent studies have
proven their effectiveness (Saburi et al. 2012a). Hypertonic saline (Hom and
Fernandes 2011), and mannitol (de Nijs et al. 2011) were successfully examined in
non-mustard lung injuries and their efficacies in bronchiolitis and bronchitis were
confirmed.
It seems that Gastroesophageal reflux (GERD) may have an important role in
worsening the pulmonary symptoms in SM injured cases. Karbasi et al. in 2013
declared that Higher pepsin concentrations in sputum of SM exposed patients
compared with healthy control subjects indicate the occurrence of significantly
more gastric micro-aspiration in SM exposed patients (Karbasi et al. 2013).
However, Roushan et al. in 2014 showed that although the prevalence of GERD
in SM exposed cases is higher than controls, this difference was not statistically
significant (Roushan et al. 2014). Moreover, it was shown treatment of gastro
esophageal reflux (using proton pump inhibitors) is effective in the prevention of

disease progress (Saber et al. 2012). Emami in a double bind randomized cross
over clinical trial on 45 cases of SM induced pulmonary complications claimed
that 4 months treatment with omeprazol (20 mg twice per day) can improve
GERD symptoms as well as cough, and QOL, but not changed respiratory func-
tion indices (Emami et al. 2014).
Therefore, an effective therapeutic protocol should involve some of the above
medication relating to the patients situation.
Many questions remain in the pathogenesis of SM pulmonary consequences. Therefore,

there is no curative modality for the treatment. It seems that system biology approach
to lung injuries will be a key point in the future researches. On the other hand, although
acute phase and symptoms in this phase is so severe, mortality and morbidity of chronic
phase is higher. Therefore, study on impaired repair mechanism is important as study
on acute tissue and cells injuries. Other co-morbidity and other organ complications
such as GERD should be mentioned in addition to lung treatments.
Although population of victims of SM lung exposure is a small group compared
to patients groups of some respiratory disorders such as COPD, SM lung disorder
can be a good model for inhaled injured patients in addition to lung transplant
patients with BO.
Acknowledgment We would like to thanks chief and personnels of chemical injuries research
center, Baqiyatallah University of medical sciences, Tehran, Iran. Also, we thank Informa
Healthcare for their permission to use their journal content in this chapter. Moreover, we acknowl-
edge Dr. Amini-Harandi and Dr. Amin Abbasi in addition to Resaneh Takhassosi publication for
permission to use their book contents for this manuscript.
Glossary
Alveoli Cystic structures at the end of respiratory tree that have a thin layer of cell
which charged with the task of gas exchange.
Antioxidant A molecule which can prevent oxidation and cell injuries.
Apoptosis Is a gradual process of cell death due to programmed inter and intra
cellular signaling.
ARDS Or acute respiratory distress syndrome which is a complex syndrome due
to severe injury to the terminal components of airways and alveoli and pulmo-
nary edema and respiratory failure are its two important sequels.
Bronchiolitis obliterans The inflammatory involvement of terminal respiratory
airways.
Bronchitis The inflammation in the bronchi that can present with dyspnea and
productive cough.
Chemical Warfare agents Every chemical material which use for military and
terroristic propose.
Chest tightness A sense of heavy on the chest during the respiration
COPD Or Chronic Obstructive Pulmonary Diseases which includes two main ter-
ritories; emphysema and chronic bronchitis.
Corticosteroids Synthetic hormone-like medications using for inflammatory
suppression.
Dyspnea Sensing the breathing that leads to increase the respiration attempts.
Fibrosis A process in a damaged tissue that is characterized by gathering fibro-
blasts and collagen deposition.
Hemoptysis Coughing up the blood that can because of a lesion in upper or lower
respiratory tract.
Inflammation Is a biological interaction between immune cells, threatened cells
and pathogens that can present as an interaction complex containing warmness,
pain, and swelling.
Interleukin A class of cytokine family that usually in related to the immune sys-
tems cells.
Mutation A change in normal sequence of genome.
N-Acetyl cysteine Is a medication with mucolytics, antioxidative and immuno-
molulating properties.
Necrosis Is a form of cell injury that leads to the premature and unprogrammed
cells death.
Respiratory Airways Include Upper respiratory tract and Lower respiratory tract
which are responsible for providing a pathway to reach oxygen to the alveoli.
Sputum Liquid secret from mucus cells of airways
Sulfur Mustard An Alkylating chemical component which is used as a blistering
chemical warfare agent that affect lung, eye and skin more than other organs.
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Chapter 8
Dermatologic Aspects of Sulfur Mustard
Exposure
Masoud Maleki and Pouran Layegh
Contents
8.1 Introduction .................................................................................................................... 214
8.2 Pathophysiology of Mustard Poisoning ......................................................................... 215
8.2.1 SM Induced Cytotoxicity ................................................................................... 215
8.2.2 Inflammation ...................................................................................................... 218
8.2.3 Epithelial Damage/Protease Activation ............................................................. 220
8.3 Clinical Features ............................................................................................................ 220
8.3.1 Cutaneous Effects of Sulfur Mustard (SM) Exposure in the Acute Phase ........ 220
8.3.2 Delayed and Chronic Skin Complications of SM Exposure.............................. 223
8.4 SM Carcinogenesis ........................................................................................................ 226
8.5 Histopathology of Mustard Skin Injuries....................................................................... 227
8.6 Management of Vesicant Injury ..................................................................................... 229
8.6.1 Prophylaxis ........................................................................................................ 229
8.6.2 Decontamination ................................................................................................ 229
8.6.3 Conventional Therapy ........................................................................................ 231
8.6.4 Management of Acute Skin Lesions .................................................................. 231
8.6.5 Management of Chronic Skin Complications .................................................... 232
8.6.6 New Therapies ................................................................................................... 234
8.7 Conclusion ..................................................................................................................... 239
Glossary .................................................................................................................................. 239
References ............................................................................................................................... 242
M. Maleki
Dermatology Department, Cutaneous Leishmaniasis Research Center,
Emam Reza Hospital, Emam Reza Square, Ebne Sina Avenue, Mashhad, Iran
e-mail: malekim@mums.ac.ir
P. Layegh (*)
Dermatology Department, Cutaneous Leishmaniasis Research Center,
Ghaem Hospital, Dr. Shariati Square, Ahmadabad Avenue, Mashhad, Iran
e-mail: layeghpo@mums.ac.ir

214 M. Maleki and P. Layegh
Abstract The skin is one of the important affected target organs by sulfur mustard
(SM) as a chemical weapon, besides the eyes and lungs. Skin exposure with sulfur
mustard results in the onset of a multiple series of events including a full set of der-
mal responses for normal wound healing and their mutual influence on each other,
eventually leading to skin toxicity. In this process, various mediators that have a
regulating role in inflammation, apoptosis, immune responses and some signaling
pathways are involved. In this chapter we try to describe the current knowledge on
the potential mechanisms which mediate the SM actions on skin, the clinical mani-
festations in the acute phase of exposure and years later (delayed or chronic ones),
histopathology of SM-exposed skin and the potential therapeutic countermeasures.
Keywords Chemical weapons Alkylating agents Sulfur mustard Cutaneous

toxicity Skin Adjunct therapy
8.1 Introduction
Sulfur mustard (SM; 2, 2- dichloroethyl sulfide) is a strong alkylating agent that

reacts with all constituents of the cell. Skin is one of the first organs which is exposed
to SM and because of its extension, is the most vulnerable to damage. Moreover, the
lipophilic nature of SM and the skin affinity for lipophilic substances make the skin
an efficient transporting system for this agent. The extent of damage depends on SM
exposure duration, absorbed dose, type as liquid or vapor, temperature, humidity,
skin moisture and its anatomical site. Moisture and heat increase skin absorption, as
warm and moist areas of thin skin like the axilla, antecubital fossa, perineum and
external genitalia are much more sensitive than other areas (Naraghi et al. 2005;
Poursaleh et al. 2012). Some reports have indicated that even cumulative low doses
of SM could lead to serious side effects (Ghabili et al. 2011).
Small quantities of SM evaporate in 23 min whereas the amounts over several
hundred milligrams remain on the skin for hours. In general, 80 % of the SM com-
ing into contact with the skin will evaporate, and the remaining 20 % will penetrate
the skin. Of this amount, 12 % reacts with components in the skin and approxi-
mately 8 % is absorbed systemically (Balali-Mood and Hefazi 2005).
SM easily penetrates the skin within 35 min of contact. In human skin, the pen-
etration rate of saturated vapor or liquid mustard is 14 mg/cm2/min in 21 C (Balali-
Mood et al. 2005). After penetration of SM into the skin, it combines with tissue
components and could no longer be isolated or extracted. The estimated LD50 of
mustard liquid on human skin is 100 mg/kg. Erythema can be frequently observed
48 h after SM exposure at a threshold dose (vapor 100300 mg/min/m3; liquid
1020 g/cm2) while blister formation occurs at higher doses (vapor 10002000 mg/
min/m3; liquid 40100 g/cm2) and eventually higher doses of SM result in ulcer
(Kehe et al. 2009). It is of interest that some degree of difference in skin sensitivity
to SM were noted by other investigators (Nagy et al. 1946; Jenner and Graham 2013).
8 Dermatologic Aspects of Sulfur Mustard Exposure 215
8.2 Pathophysiology of Mustard Poisoning
Sulfur mustard, as a vesicant, whether in the gas or liquid form binds to tissues and
reacts irreversibly with a variety of cell constituents including DNA, RNA, nucleic
acid, proteins, lipids as well as small molecular weight metabolites such as glutathi-
one (Papirmeister et al. 1991; Noort et al. 2002; Laskin et al. 2010). The effects of
SM on the skin are complex which in this process various mediators having a role
in inflammation, Immune response and cell death aside with a number of signaling
pathways have been implicated. Despite some valuable knowledge on the skin tox-
icity of SM, the exact molecular mechanisms of SM toxicology are not completely
understood. But during the recent decades, both in vitro and in vivo investigations
have been conducted to describe the bio-mechanical and molecular mechanisms
related to SM injuries; their results have led to identification of several systems and
pathways playing a major role in signaling the cytotoxic effects of SM. The follow-
ing sections attempt to explain the most current proposed mechanisms of SM action.
8.2.1 SM Induced Cytotoxicity
8.2.1.1 DNA Damage and Activation of Poly (ADP-Ribose)

Polymerase (PARP)
Formation of a cyclic ethylene sulfonium ion intermediate is the initial reaction

which is then followed by an electrophilic attack on the target molecule. Target
molecules have reactive groups such as phosphates, sulfhydryls, carboxy and ring
nitrogens groups. Therefore, macromolecules including DNA, RNA, proteins, car-
bohydrates and lipids are the principle targets for SM alkylation (Noort et al. 2002;
Debouzy et al. 2002; Mol et al. 2008). In this series, DNA alkylation is believed to
be the most critical event in SM toxicity (Lodhi et al. 2001). After SM exposure, the
DNA double strand breaks resulting in the formation of monofunctional adducts
and bifunctional (intra- and intermolecular) cross-links. Using 35S-labeled SM has
led to the identification of DNA alkylation sites. About 65 % of DNA alkylation
products are monofunctional adducts on the N7 position of guanine, 17 % in N3 of
adenine, 0.1 % in 06 of guanine and approximately 17 % are bifunctional cross-
links on N7 guanine (Ludlum et al. 1994).
Although in comparison with others, the 06 position of guanine is rare but is
regarded as a critical position, because removing the SM adduct at this position by
the human DNA repair mechanism is not successful (Ludlum et al. 1986). The
extent of SM cytotoxicity due to DNA alkylation and cross-linking is influenced not
only by cell capacity in DNA repairing, but also by the activation of specific repair
mechanisms. Inter strand cross-links are believed to interfere with replication and
finally lead to double strand breaks (Andreassen et al. 2006). This process appears
to result in the up regulation of a family of repair enzymes of DNA called Poly
(ADP-ribose) polymerases (PARPs) (Papirmeister et al. 1985; Shall and de Murcia

2000). High SM concentrations strongly activate PARP-1 which is the authorizing
member of the PARP family with the highest enzymatic activity, also a capsase-3
substrate in the early phase of apoptosis (Kaufmann et al. 1993) and lysosomal
proteases in necrosis (Gobeil et al. 2001). Its activation leads to subsequent deple-
tion of its substrate, the intercellular cellular NAD+ (nicotin amide adenine dinucle-
otide) that are normally used to produce ATP and also inhibition of glycolysis
(Bennion and David-Bajar 1994; Martens and Smith 2008; Poursaleh et al. 2012).
The depletion of ATP in epidermal cells may lead to apoptosis or necrotic cell death,
depending on the level of depletion or cell type (Rosenthal et al. 2001; Kehe et al.
2009). It can also induce a hypoxemic state that may result in oxygen radical forma-
tion and the subsequent reaction of free radicals with cell-structures and damage it
(Somani and Babu 1989). Moreover, ultrastrutural analysis of mouse models and
skin samples have shown mitochondrial swelling after SM exposure (Brown and
Rice 1997). Although the alkylation of mitochondrial DNA due to SM is less promi-
nent than nuclear DNA, it should be considered that the mitochondria are a central
regulator of the intrinsic apoptotic pathway and the most prominent feature in this
pathway is the mitochondrial outer membrane permeabilization (Green and Kroemer
2004). Moreover, opening a pore in the inner mitochondrial membrane has been
shown to be mostly involved in necrotic cell death (Nakagawa et al. 2005). Cell
damage is highly dependent on the amount of alkylated DNA due to SM. Cellular
responses including cell cycle arrest, terminal differentiation, and apoptosis are due
to limited DNA damage, while necrosis is the result of excessive DNA alkylation
(Rosenthal et al. 1998; Martens and Smith 2008).
8.2.1.2 SM Induced Apoptosis in Keratinocytes
Studying primary cultures of human keratinocytes has led to useful information

about the underlying mechanisms of SM-induced apoptosis. Decrease in anti-
apoptotic protein Bcl-2 and increase in pro-apoptotic protein p53 has been observed
in keratinocytes after SM exposure (Rosenthal et al. 1998, 2000). Exposure of
human keratinocytes to 100300 M SM leads to the auto activation of the caspase-
8, which induces the Fas-dependent death receptor pathway and at the same time
caspase 9, that initiates the mitochondrial apoptotic pathway (Rosenthal et al. 2003).
Active caspase 8 leads to subsequent activation of downstream effector cas-
pases (caspases 3; 6; 7). These two pathways come together to activate caspase-
3 that is the principle killer protease (Zimmermann et al. 2001). Such death receptors
function as cell sensors that detect the presence of specific extracellular death sig-
nals and rapidly trigger cellular destruction by apoptosis.
Another form of programmed cellular death which is common in epithelial cells
is referred to as anoikis or detachment-initiated apoptosis (Chiarugi and Giannoni
2008). Cells usually stay close to the tissue to which they belong, since the com-
munication between proximal cells as well as between cells and the extra cellular
matrix (ECM) provide essential signals for growth or survival. When there is a loss
of normal cellmatrix interactions, they may undergo anoikis. For example, essen-
tial components of hemidesmosome such as integrins 64 and 31 interact

directly with laminin-332. These are matrix proteins which are found on the baso-
lateral keratinocyte surface (Schneider et al. 2007). Some molecules such as caveo-
lin, paxillin, integrin signaling kinase and various growth factor receptors have
associations with integrin and seem to transduce anchorage-dependent survival sig-
nals (Frisch and Screaton 2001; Chiarugi and Giannoni 2008). If these signals are
absent, epidermal cells undergo anoikis either by mitochondrial pathways of apop-
tosis or Fas-dependent ones. SM can modify the dynamic of cytosolic proteins such
as intracellular actin microfilaments and keratin intermediate filaments (keratin 5
and 14) that have important roles in the attachment of keratinocytes to the basement
membrane (Hinshaw et al. 1999). Moreover, SM can alkylate ECM proteins of the
skin and also reduce the ability of keratinocytes to deposit laminin at the dermo-
epidermal junction, which in turn can affect the maintenance of the basal keratino-
cyte connection with the basement membrane (Gentilhomme et al. 1998).
8.2.1.3 Altering Metabolism
Reaction with Glutathion/Lipid Peroxidation
Another suggested mechanism of cell death due to the alkylation effects of SM is

based on changing of metabolisms which could lead to oxidative stress in
SM-exposed skin. SM directly reacts with gluthatione (GSH) that is a free radical
scavenger forming various metabolites which lead to intracellular GSH depletion
and finally enhancement of reactive oxygen species (ROS) production (Kumar et al.
2001). ROS induced damage to macromolecules is well recognized and includes
DNA base oxidation, which could interfere with the DNA replication and repair
processes; lipid peroxidation which could produce highly reactive electrophilic
lipid peroxidation end products; and protein oxidation that could modify the struc-
tural proteins and enzymes activity such as increasing the activity of antioxidant
enzymes including catalase, glutathione-s-transferase and superoxide dismutase. It
could also inhibit antioxidant enzymes such as thioredoxin reductase, so shifting the
intracellular environment toward a more oxidized state and disrupt cellular redox
homeostasis (Laskin et al. 2010). This condition unprotects cells against oxidative
free radicals and activates certain inflammatory responses (Miccadei et al. 1988;
Gentilhomme et al. 1992; Gross et al. 1993; Atkins et al. 2000).
Calcium Homeostasis
Intracellular free calcium is a well-known marker of cell stress (Ruff and Dillman
2007). Some studies have shown that toxicants like SM with a non clarified mecha-
nism induce a rise in intracellular levels of free Ca2+ in keratinocytes which could
result in cell death (Berridge et al. 2000; Rosenthal et al. 2003). Treatment of primary
human epidermal keratinocytes with buthionine sulfoxamine decreased the level of
reduced glutathione but increased intracellular Ca+2 (Ray et al. 1993). It seems that
the alkylation of sulfhydryl groups in the Ca2+ adenosine triphosphatase (ATPase) in

the cell membrane leads to an increase in cytosolic calcium and cell death (Orrenius
et al. 1985). Changes in intracellular calcium are accepted to activate the mitochon-
drial pathway of apoptosis. A pivotal director of calcium-dependent proteins is
calmodulin. Recently, a study demonstrated the key role of calmodulin 1 (CAM1) in
SM apoptosis (Simbulan-Rosenthal et al. 2006). CAM dependent apoptosis is medi-
ated by Protein kinase II or calcineurin (Canning et al. 2006). There is some evidence
regarding the role of Ca2+ CAM calcineurin pathway in SM-induced apoptosis.
Bad which is a pro-apoptotic Bcl-2 family member can be activated by SM. It pres-
ents in the inactive phosphorylated form in viable cells. Calcium dependent activation
of Bad may be one of the mechanisms by which SM promotes apoptosis in keratino-
cytes. There is evidence that cyclosporine as a selective inhibitor of calcineurin and a
Bad phosphatase, can inhibit SM induced apoptosis (Donald et al. 2009).
Oxidative Stress and Nitric Oxide Signaling
Calmodulin and Ca2+ have an essential role in the formation of nitric oxide. Reactive
nitrogen species (RNS) and peroxynitrite have been proposed as key mediators of
SM cytotoxicity (Korkmaz et al. 2006; Yaren et al. 2007). In a two-step reaction,
nitric oxide is synthesized from arginine and oxygen by nitrogen oxide synthases
(NOSs). Three forms of this enzyme have been recognized, including the endothelial
(eNOS) and neuronal type (nNOS) which are low output isoforms and inducible
(iNOS) or macrophage which could be expressed in both epithelial cells as well as
activated macrophages and neutrophils and is the high output form of the enzyme.
Nitric oxide as a molecule containing a single unpaired electron can react with many
constituents of the cell and could lead to toxicity. One particular importance is the
reaction of ROS superoxide anion with nitric oxide and the generation of a more
long-lived RNS, peroxynitrite formation which is a strong oxidant and nitrating
agent with well-known capacity to trigger oxidative injury (Virag et al. 2002; Laskin
et al. 2010). Low intracellular free Ca2+ can restrict NOS activity while its rise
librates eNOS. There are evidence that represent the SM ability to activate eNOS
and upregulate iNOS (Bloch et al. 2007). SM probably raises free intracellular Ca2+
where the subsequent association of Ca2+- CAM can lead to NOS liberation and
activation. Nevertheless, further studies are necessary to detect the actual role of
RNS in SM skin injury.
8.2.2 Inflammation
8.2.2.1 Inflammatory Mediators
Based on years of experience, it seems that the inflammatory response to vesicant

injured skin has to be biphasic; as it has a minor role in the initial events of SM
cutaneous injury but much greater importance at the later stages (Papirmeister et al.
1991). However, in more recent studies some evidence has been presented that
inflammation through inflammatory cells and mediators may indeed contribute
directly to early vesication (Cowan and Broomfield 1993). Leukocyte infiltration of
the papillary dermis and epidermis has been reported both in human skin explants
and in mice after SM exposure (Lindsay and Rice 1996; Wormser et al. 2005). In
addition, in the rabbit model, within 2 h of SM exposure, an increases in mononu-
clear cells and granulocytes has been reported that persists for 24 h (Dannenberg
et al. 1985). Results of multiple in vitro and in vivo studies have documented that
SM exposure induces the expression of pro inflammmatory cytokines and chemo-
attractants including interleukin (IL)-1, IL-1, IL-6, IL 8, interferon gamma
(IFN), tumor-necrosis factor alpha (TNF) and some others. These chemokines
have strong chemotactic activity for macrophages and neutrophils. Furthermore,
some inflammatory mediators such as free arachidonic acid and its cyclooxygenase
and lipooxygenase products have been detected in the skin after SM exposure
(Tanaka et al. 1997; Blaha et al. 2000; Lefkowitz and Smith 2002; Dachir et al.
2004). The increased capillary permeability due to some of these mediators would
allow the influx of additional inflammatory substances such as complement compo-
nents, kininogens and fibrin into the dermal interestitium (Rikimaru et al. 1991).
The effective results of using non-steroidal anti- inflammatory agents (NSAIDs) in
SM injury suggests the important role of these mediators in SM toxicity (Casillas
et al. 2000).
8.2.2.2 Signal Transduction Pathway
SM has been reported to activate many molecular signaling pathways (Ruff and
Dillman 2007) which control cytokines expression and mediate many responses
such as cell proliferation, differentiation, apoptosis and inflammation. Some that
have a role in inflammation include the transcription factor nuclear factor- kappa B
(Nf-B), activator protein-1 (AP-1) and p38 mitogen-activated protein kinases (p38
MAP kinase) (Zenz et al. 2008). The role of NF-B and MAPK activation in the
regulation of the genes coding for inflammatory cytokines after SM exposure has
recently been considered (Rebholz et al. 2008; Kehe et al. 2009; Mishra et al. 2010).
Nf-B is a pivotal mediator of inflammatory responses which is involved in cellular
responses due to cellular stress (Karin and Greten 2005). There is much evidence
that Nf-B induced by SM within 24 h after exposure (Atkins et al. 2000;
Minsavage and Dillman 2007) may enhance the synthesis of the aforementioned
cytokines (Donald et al. 2009).
Also p38 MAP kinase could be activated in response to harmful stimuli such as
ultraviolet radiation, heat and pro-inflammatory stimuli. The p38 MAPK signaling
is one of the common members of the MAPK cascade which its important role in
SM-exposed normal human epidermal keratinocytes has been newly demonstrated.
SM induces phosphorylation of p38 and activation of upstream kinase MKK3/
MMK6 in a dose dependent manner (Dillman et al. 2004). Inhibition of these pro-
cesses results in the decreased production of inflammatory cytokines due to SM
exposure in vitro (Kehe et al. 2008).
8.2.3 Epithelial Damage/Protease Activation
Although the exact series of events of blister formation after exposure of skin with
SM have not been demonstrated, recently, an important role for proteases has been
considered in this process. The evidences show that some attachment proteins which
regulate cell adhesion, migration and morphogenesis are degraded by SM. These
are laminin-5 and integrin 64 that facilitate the assembly of basement mem-
branes. Degradation of these proteins by SM prompted the search for proteases
involved in SM induced skin blisters. Increased protease activity has been reported
after SM exposure in vitro in human peripheral lymphocytes (Cowan et al. 1993)
and human skin explants (Lindsay and Rice 1996), and in vivo in hairless guinea pig
skin (Cowan et al. 1993; Kam et al. 1997). Matrix metalloproteinase (MMP) activ-
ity has been detected in culture fluids of rabbit skin after SM exposure (Woessner
et al. 1990). An increase in the elastase, calpain, tryptase and gelatinase (MMP-2
and MMP-9) activity within 24 h of SM treatment of mice ears has been reported
(Powers et al. 2000). The source of gelatinase is likely to be infiltrating neutrophils,
epidermal keratinocytes and dermal fibroblasts. Latent gelatinase activity is
increased after SM injury and remains elevated for at least 7 days in the mouse ear
model (Shakarjian et al. 2006). Furthermore, some studies on mouse model have
indicated that MMP-9 is the most upregulated MMP in SM exposed skin (Shakarjian
et al. 2006; Ries et al. 2009). Gelatinases and elastase have the ability to cleave the
basement membrane components and disrupt the dermo-epidermal junction
(Malemud 2006). Early elevation of these proteases after SM exposure suggests that
they are potential effectors of SM vesication. Inhibiton of these proteases could
reduce the extent of injury and be a useful therapeutic strategy (Cowan and
Broomfield 1993).
8.3 Clinical Features
8.3.1 Cutaneous Effects of Sulfur Mustard (SM) Exposure

in the Acute Phase
These effects depend on different variables including the SM concentration, vapor

or liquid SM contact, anatomical site of skin exposure, individual characteristics
mainly immunologic status, use of protective equipment, measures taken to elimi-
nate contamination, environmental conditions (e.g. temperature and humidity, rain
and wind) and the duration of exposure (McNamara et al. 1975; Sidell 1990;
Papirmeister et al. 1991; Bennion and David-Bajar 1994).
The onset of symptoms and their severity largely depend on the SM concentra-
tion and exposure time. In cases of low contamination (vapor 100300 mg/min/m3,
liquid 1020 g/cm2), mild and delayed signs and symptoms occur. They are most
commonly limited to erythema and itching. In moderate to severe exposure (vapor
10002000 mg/min/m3, liquid 40100 g/cm2), the signs and symptoms present
earlier, even in a few minutes and are more severe, including necrosis and scar for-
mation (Willems 1989; Bennion and David-Bajar 1994).
The clinical course of skin signs and symptoms of mustard poisoning can be divided into
five phases: the latent phase, erythema phase, blistering phase, necrosis phase, and healing
phase (Bennion and David-Bajar 1994).
In the first 6 h, itching is usually the prominent symptom. At 648 h post SM

edema and cyanosis succeed the itching and burning sensation in the skin. If the
dose of exposure is too high, blisters also appear (Willems 1989; Smith and Dunn
1991; Momeni et al. 1992).
Erythema usually manifests 224 h after exposure which is mostly accompanied
by intense itching (Willems 1989). After 18 h, small vesicles form within the area of
erythema which gradually coalesce to form subepidermal blisters containing a clear
yellow fluid; they become more apparent after third day. The size of bullae may be
variable from small vesicle to large blister up to 15 cm (Momeni et al. 1992) (Fig. 8.1a).
A positive Nikolskys sign usually present in these patients (Momeni et al. 1992;
Kehe and Szinicz 2005).
Because all chemical reactions are complete within a few minutes of the agent
penetrating the skin, the fluid in the blister caused by SM does not contain active the
vesicant (Mellor et al. 1991; Kehe and Szinicz 2005).
Late-onset bullae have seen in 6 % of patients over normal appearing skin after a
few days or weeks of injury (Momeni et al. 1992). The anatomical location of the
exposed skin is highly important. Bullous lesions are more likely to occur on warm,
moist areas such as genitalia, axilla, and areas where tight clothing is worn (Smith
et al. 1919; Smith and Dunn 1991) as shown in Fig. 8.1b. Because of their special
condition like warmth, moisture and less thickness these area have a lower dermal
barrier function (Kehe and Szinicz 2005). Also abundance of hair follicles (Smith
et al. 1919; Smith and Dunn 1991), and high density of sweat glands (Mellor et al.
1991) may prepare a suitable condition for skin absorption of SM which makes
them as a common site of SM skin injuries.
With blister formation the itching normally diminishes (Sohrabpour 1984;
Willems 1989). However, pain and itching are still the main clinical symptoms at
this stage. Large blisters usually rupture resulting in erosions, this can also lead to
ulceration and full-thickness skin loss, in which secondary infection may follow.
During the first and second days, skin necrosis may occur, leading to worsening
of the pain. At the start of the 4th day, eschar formation becomes apparent at the site
of necrosis which begins to slough by 46 days, leaving a hyperpigmented scar
(Mellor et al. 1991).
Superficial blisters and ulcers usually heal within 2 weeks while deep ulcers
mostly heal during 48 weeks, by leaving a scar. SM-induced ulcers heal more
slowly than thermal burns (Mellor et al. 1991), that maybe due to DNA alkylation
which results in the reduced proliferation of epidermal cells, particularly in the
basal layer and systemic immunosuppression via immune cell damage and death
(Bennion and David-Bajar 1994). Moreover, the healing rate differs based on body
Fig. 8.1 (a) Vesicle and bullae formation within the area of erythema (Unpublished slide of a SM
veteran under Prof. Balali-Moods medical care, taken with permission of the patient). (b) Erythema,
erosion and bulla formation of buttocks, intergluteal and thighs after sulfur mustard exposure.
(Unpublished slide of a SM veteran under Prof. Balali-Moods medical care, taken with permission
of the patient)
sites. Blisters which form in the face heal faster usually during a week, whereas
blisters of other body sites may require 26 weeks and occasionally up to 12 weeks
for healing; this is mostly true for the lesions located on the feet. This difference is
partly due to abundance of adnexa such as hair follicles in the face (McNamara
1960). Also the healed mustard burns are hypersensitive to mechanical trauma
(Chiesman 1944; Mellor et al. 1991).
Regions with severe exposure usually lose their pigments whereas the surround-
ing areas of lesions having milder injuries, become hyperpigmented (Klehr 1984;
Requena et al. 1988; Mellor et al. 1991; Smith et al. 1995). The resulting poikilo-
derma is a characteristic cutaneous late complication of SM poisoning that may
persist for decades (Kehe and Szinicz 2005).
The majority of SM victims are adults, although, in cases that civilians were
targeted resulting in child injuries in which the rate of involvement was higher than
adults. One of the main reason may be their thinner skin compared to adults
(Momeni and Aminjavaheri 1994).
There are some discrepancies in the common signs and symptoms of acute SM
skin injuries in different studies which demonstrated in Table 8.1 (Balali-Mood
1984; Moradi et al. 1986; Balali-Mood et al. 1991; Momeni et al. 1992; Naraghi
et al. 2005).
8.3.2 Delayed and Chronic Skin Complications of SM

Exposure
The studies performed in this respect can be categorized into two groups:
8.3.2.1 Delayed Complications in War Veterans
That includes studies on individuals who have been exposed to sulfur mustard usu-
ally once in battlefields; which is better to use the term delayed or late for their
complications.
In this group similar to the acute phase, complications may vary depending on
the severity of poisoning and other aforementioned factors.
Based on some studies, while the respiratory tract complications of sulfur mus-
tard often intensify and the eye symptoms remain unchanged, the cutaneous com-
plications alleviate over time (Shirazi and Balali-Mood 1988).
Table 8.1 Prevalence of acute and chronic complications of sulfur mustard skin injuries based on
the results of different studies
Acute Chronic
Prevalence Prevalence
Complication (range %) Complication (range %)
Erythema 2184 Pruritius 2595
Pruritus 4097 Burning sensation 1052
Burning sensation 4097 Dry skin 244
Vesicle and/or Blister 1581 Cherry angioma 1238
Ulcer Up to 71 Erythematous papules 042
Hyper pigmentation 2084 Hyper pigmentation 655
Pain 2577 Hypo pigmentation 040
Depigmentation 04
Scar (Atrophic & Hypertrophic) 231
Hair loss 039
The long-term cutaneous complications has been reported in 23 % (Ghassemi-

Broumand et al. 2008), 24.5 % (Khateri et al. 2003), 41 % (Balali-Mood 1986) and
90 % (Emadi et al. 2008b) of victims. However, in another study conducted on Iraq-
Iran war veterans with severe injuries, skin involvement has been reported in 75 %
of the cases (Balali-Mood et al. 2005).
Mild injuries in the acute phase causing limited signs such as eryethma and
edema result in complete healing and do not leave any foot prints in the delayed
phase (Warthin et al. 1918; Chiesman 1944), while in cases which blisters and
ulcers develop; the healing process may lead to pigmentary changes or scar forma-
tion (Fig. 8.2a).
The most common symptom in such patients is itching followed by a burning
sensation and dryness of skin which is more common on the extremities, particu-
larly the arms and legs (Fig. 8.2b).
The main cause of itching seems to be skin dryness which is intensified in cold
and dry climates and with repeated bathing with hot water and the use of detergent.
The mechanism of long-term dryness of skin has been evaluated in several studies.
In a study the level of skin hydration (SH) and trans-epidermal water loss
(TEWL) was measured at four different body locations: forehead, suprasternal,
palm and dorsum of hand compared in SM-exposed veterans with the healthy sub-
jects and patients with eczema. The interval between their last hygiene toilets before
the measurements was at least 3 h.
Accordingly, although the prevalence of dry skin (xerosis) was significantly
higher in the SM-exposed group and in patients with eczema compared to the nor-
mal population, interestingly skin hydration in the dorsal and palmar sides of hands
and the forehead areas was higher in the SM-exposed group than the non-exposed
subjects. In addition, TEWL in SM-exposed subjects was significantly higher than
the control group only in the suprasternal region and dorsal side of the hands
(Davoudi et al. 2009).
Moreover, regarding the measurement of skin sebum content and elasticity in
four areas: forehead, suprasternal, palm and back of the hands, skin sebum was
higher in participants who presented with dermatitis and had history of contact with
SM than other control groups; the difference was only statistically significant on the
forehead (Davoudi et al. 2010).
In another study (Layegh et al. 2015), the skin hydration and sebum content of
SM veterans in the flexor and extensor aspects of the forearm and medial and lateral
sides of the legs was less than the control group; the difference being significant only
for the lateral side of the legs. In the mentioned study, the common sites of dry skin
were studied and the interval from the last hygiene toilet was determined as 24 h.
The differences between results of these studies may be due to selection of body
location or time interval between the last toilet of the patients and the skin sebum
and hydration measurement. The common signs and symptoms seem to vary in dif-
ferent studies as demonstrated in Table 8.1 (Fekri and Janghorbani 1995; Heidari
et al. 2000; Moosavi et al. 2001; Toosi et al. 2002; Hefazi et al. 2006; Rezvani et al.
2006; Emadi et al. 2008b; Moin et al. 2009).
They mainly include itching, skin dryness, hyper- and hypopigmentation, poiki-
loderma and scar formation. Furthermore, multiple erythematous papules may be
seen mainly on the trunk and arms of patients that does not match any skin disease
Fig. 8.2 (a) Skin dryness with fine scaling of the forearm (Unpublished slide of a SM veteran
under Prof. Balali-Moods medical care, taken with permission of the patient). (b) Scaring with
hypo and hyperpigmentation of skin on back of the thigh (Unpublished slide of a SM veteran under
Dr. Layegh and Dr. Malekis medical care, taken with permission of the patient). (c) Multiple and
eruptive cherry angioma on the trunk. (Unpublished slide of a SM veteran under Prof. Balali-
Moods medical care, taken with permission of the patient)
(Balali-Mood et al. 2005; Rezvani et al. 2006). Previously injured sites were
reported to be sensitive to subsequent mechanical injury and showed recurrent blis-
tering after mild injury (Chiesman 1944; Mellor et al. 1991).
Several studies have reported a higher prevalence of eczema (Mellor et al. 1991;
Momeni et al. 1992; Fekri and Janghorbani 1995; Balali-Mood et al. 2005; Emadi
et al. 2008b; Moin et al. 2009), hair loss (Fekri and Janghorbani 1995; Toosi et al.
2002; Hefazi et al. 2006), urticaria and angioedem (Fekri and Janghorbani 1992;
Rezvani et al. 2006; Emadi et al. 2008b), vitiligo (Fekri and Janghorbani 1992;
Emadi et al. 2008b), psoriasis (Emadi et al. 2008b) in SM-exposed patients in com-
parison to the healthy population.
There are certain differences in various studies about some skin diseases such as
acne vulgaris and pityriasis versicolor. Some researchers have reported an increase
(Moosavi et al. 2001; Emadi et al. 2008b; Moin et al. 2009) and others a decrease in
their incidence (Fekri and Janghorbani 1995) while they have not been addressed in
other similar studies. It seems the differences in these results may be due to the
severity of injury and the time interval between exposure and the time of study.
Overtime, additional lesions may develop in such patients. Multiple and eruptive
cherry angioma has been reported in several studies over 10 years from the exposure,
whereas it was not mentioned in the early reports (Firooz et al. 1999; Moosavi et al.
2001; Moradi and Aghaei 2004; Balali-Mood et al. 2005; Maleki et al. 2006; Moin
et al. 2009) (Fig. 8.2c). Also there is evidence that veterans with severe itching have a
significant less quality of life than patients with milder symptoms (Panahi et al. 2008).
8.3.2.2 Chronic Complications Due to Occupational Exposure
This part includes studies on subjects who have been exposed to sulfur mustard
while working in mustard gas factories and have often been under chronic and pro-
longed contact with small amounts of this material; that is better to use the term
chronic for their long-term side effects.
Occupational exposure to SM could induce pigmentary changes, skin ulcers and
increase the probability of skin cancers (Klehr 1984).
In a study from Japan on 488 former workers of a SM factory, 155 cases were
reported with pigmentary disorders in the form of hyperpigmentation and depig-
mentation as a rain drop even on the covered areas of the body (Sidell 1998).
In another study 22 cases with Bowens disease, BCC and hyperkeratotic papular
eruptions were described (Wada et al. 1962; Inada et al. 1978).
8.4 SM Carcinogenesis
Based on laboratory studies, SM is a carcinogenic agent and several clinical studies

have reported increased rates of lung and skin cancer among workers of SM facto-
ries (Wada et al. 1968; Inada et al. 1978; Nishimoto et al. 1983; Easton et al. 1988).
Regarding war veterans, despite several reports indicating a rise in lung and
skin cancers, yet the subject is still controversial and requires long-term cohort
studies. Carcinoma of the nasopharynx, bronchogenic carcinoma, adenocarci-
noma of the stomach, as well as acute myeloblastic and lymphoblastic leukae-
mia, have been reported in Iranian veterans (Balali-Mood 1992; Ghanei and
Vosoghi 2002).
In one study cancer incidence was significantly increased in Iranian war vetre-
rans exposed to SM. The incidence rate ratio of cancer was 1.81 (95 % CI 1.27
2.56) with hazard ratio of 2.02 (95 % CI 1.412.88). Finally, the authors concluded
carcinogenesis of SM following acute exposure during war and recommended
improvement care programs such as routine screening schemes for exposed veter-
ans (Zafarghandi et al. 2013).
Regarding skin cancer, a study performed on 800 war veterans, skin cancer was
diagnosed in 1.1 % which in comparison to the normal population (0.01 %), showed
a statistically significant difference. These cancers included BCC, SCC, Bowens
disease, dermatofibrosarcoma protuberans and Mycosis fungoides which mostly
developed at the site of SM-induced scar lesions (Emadi et al. 2008b).
Also a case of Merkel cell carcinoma at the site of SM-induced scar has been
reported (Maleki et al. 2008).
Although there are some evidences about carcinogenicity of SM in war veterans
till now, but this relationship is uncertain and considering prolonged time needed to
elapse for development of skin cancers, a long-term follow-up is recommended.
8.5 Histopathology of Mustard Skin Injuries
The majority of data on the histopathology of mustard skin injuries has been gath-
ered from experimental animal models. Although within several minutes after expo-
sure, mustard fixes to the tissue, histopathological changes are not evident until
3060 min later and do not complete till 23 days after exposure (Bennion and
David-Bajar 1994).
The histopathology of SM exposed skin including light microscopy findings and
ultrastructural pathology by electron microscopy could be classified in 3 stages;
prevesication (46 h after exposure); vesication (624 h post exposure) and scar
(after 1 year). During prevesication, the earliest changes limited to individual basal
keratinocytes occur by becoming dyskeratotic and pyknotic. Nuclear chromatin
margination, nuclear envelope dilatation, mitochondrial swelling of basal cells,
intercellular spaces widening, disqualifying of desmosomes and hemidesmosomes
also succeed.
In the vesication stage suprabasal and stratum spinosum cells show nuclear
pyknosis, vacuolation in cytoplasm, mitochondrial density and endoplasmic retic-
ulum swelling. These changes lead to microvesicle formation within the lamina
lucida of the basement membrane which primarily appears at 12 h post exposure.
Then, the microvesicles in lamina lucida are infiltrated with inflammatory cells
especially leukocytes, phagocytic cells, cellular debri, degenerating cells and tis-
sue fluid to form blisters and bullae. The presence of large amounts of melanin in
all epidermal layers even the horny layer and numerous malanophages filled with
coarse melanin granules in the upper dermis have also been described in this stage
(Fig. 8.3ac).
Finally in SM-induced scars, marked epidermal atrophy, acanthosis with flat-
tened rete ridges, alteration in basal layer pigmentation, perivascular mononuclear
infiltrate scattered through the papillary dermis, melanophages in the upper dermis,
nonspecific dermal fibrosis, and atrophy of dermal appendages such as sebaceous
glands, hair follicles and sweat glands have been reported (Balali-Mood and Hefazi
2006; Emadi et al. 2008a; Emadi et al. 2011; Poursaleh et al. 2012). Hypodermis
has been described as normal with no changes in nearly all available reports
(Coppens and Roels 1986).
a b
Fig. 8.3 (a) Subepidermal and intraepidermal hemorrhagic blister and also regenerative changes
after several days (H&M 400). (b) Apoptosis/necrosis and mitotic activity in basal layer and lay-
ers above it (H&M 400). (c) Basal hyperpigmentation, increased basilar melanin along with
upward transmigration of the melanin (Fontana, 400) (Reprinted with permission from Naraghi
et al., and John Libbey Eurotext publishing)
Naraghi et al. have described the histopathologic features of acute cutaneous

lesions of 32 Iranian veterans at 5th day of SM exposure, as four distinct patterns:
interface dermatitis, vacuolar type and lichenoid type; spongiotic dermatitis and
bullous dermatitis with or without acantholysis; pigmentary disorders pattern,
increase in epidermal melanization; alteration of dermis/hypodermis, vasculopathy
and appendageal inflammatory response; sclerodermoid pattern. Despite descrip-
tion of some specifications related to SM skin injury, they concluded that these
findings were compatible with the histopathologic changes in any chemical burns
(Naraghi et al. 2005).
8.6 Management of Vesicant Injury
In spite of considerable investigation regarding the treatment of SM damages during

the past 20 years, no effective treatment or specific antidote has yet been developed
for skin injuries due to SM exposure. As yet, the mainstay of treatment has been
symptomatic therapy. The management of SM casualties can be divided into several
phases: prophylaxis, decontamination and treatment of lesions (Bennion and David-
Bajar 1994).
8.6.1 Prophylaxis
Prophylaxis consists of using protective equipment, avoidance of contaminated

areas and destruction of the chemical capability of the enemy (Bennion and David-
Bajar 1994). Personal protective clothing includes gas mask, gloves, suit and foot
protection. Also for skin areas located at the junction of protective clothing like the
wrist, waist, neck and ankle, some topical skin protectants like butyl rubber or poly-
vinyl chloride gloves and boots can be used (Poursaleh et al. 2012).
8.6.2 Decontamination
SM is infamous for its persistence and ability to adhere to fomites. Mustard casual-
ties and fomites could be decontaminated passively by absorption to inert sub-
stances or deactivation and detoxification by chemicals (active decontamination)
(Jenner and Graham 2013). The standard way to inactivate mustard compounds
includes the US Armys M13 decontamination kit which contains a dusting pad of
fullers earth that absorbs liquid SM and the newer one, M258 containing a solution
of chloramide and a mixture of phenol, ethanol and sodium hydroxide (Bennion and
David-Bajar 1994).
One of the best examples for chemical neutralization is reactive skin decontami-
nation lotion (RSDL), a product that has been approved by the FDA in 2003 and
undergone military use by the American forces. It has an efficacy equal to fullers
earth (Taysse et al. 2007). The solvent portion of this lotion solubilizes chemical
weapons away from the skin whereas its oxime component readily reacts with mus-
tards and even nerve agents to produce less toxic products (Sawyer et al. 1991a, b).
Treatment of domestic pigs with RSDL, 5 min after exposure to SM eventuated
significantly less injury after 3 days (Taysse et al. 2007).
Other chemical agents from this group include sodium thiosulfate, as a reducing
agent (Owens and Hatiboglu 1961; Bonadonna and Karnofsky 1965) and 2,
3-dimercapto-propen sulfonic acid (DMPS) which has shown protective effects in
mice exposed to SM vapor (Pant et al. 2000).
In the absence of standard kits, washing skin repeatedly with soap and/or sham-
poo and large amounts of warm water could inactivate large quantities of mustard
(Aasted et al. 1987). Beside water, washing with other substances such as oil, gaso-
line, kerosene and surgical spirits have also been proposed (Jelenko 1974; Gold
et al. 1993; Wormser et al. 2002).
If water is not easily available, application of absorbent powders such as grain
flours, talcum powder (van Hooidonk et al. 1983), fullers earth that is clay-rich soil
which has an almost irreversible bond to SM (Chilcott et al. 2001), and activated
charcoal or even mechanical scraping could be used. Strong basic solutions like
ammonia and lye or chlorinated acids such as sodium hypochlorite especially when
used in a proper ratio to SM like 1000:1, and in appropriate concentrations of 0.5 and
4 % solution could effectively hydrolyze SM and may be used to decontaminate fomi-
tes (Papirmeister et al. 1985; Bennion and David-Bajar 1994; Wormser et al. 2004).
Vaporized hydrogen peroxide which generally used in industry as a gaseous ster-
ilant has been shown to be an effective SM decontaminant in the presence of ammo-
nia (Wagner et al. 2007).
Creams containing fluorinated cross-linker monomers could also be applied as a
decontamination agent (Liu et al. 1999). The rate of skin absorption has reduced by
18-fold after using perfluorinated creams in some cases (Chilcott et al. 2002).
Active ingredients within the cream by actively reacting with SM, can decon-
taminate it. Because chemical agents could snare in these creams and prevent natu-
ral off-gassing, they should be administered with caution. It is to be noted that in
cases of vapor exposure to SM, decontamination is not effective (McNamara 1960).
In a study by Vijayaraghavan et al, 20 % of CC2 (N,N-dichloro-bis
[2,4,6-trichlorophenyl] urea) in hyroxypropyl cellulose was reported as a safe
chemical substance and a personal SM decontaminant (Vijayaraghavan et al. 2002).
Oral administration of amifostine and ethyl phenyl sulfoxide which is a newly
synthetized compound has been reported to be effective as a prophylactic agent
against SM toxicity (Kumar et al. 2002). Also the combination therapy of acet-
aminophen and N-acetylcysteine on human skin fibroblast cells before or concomi-
tant with SM exposure, could reduce its toxicity (Saberi and Zaree Mahmodabady
2009).
8.6.3 Conventional Therapy
Supportive care similar to that performed for severe thermal burns has the principle
role in the treatment of SM skin lesions and their related symptoms. The patients
must be carefully monitored for limiting associated complications. Extensive dam-
age to the epidermal barrier results in increased fluid loss which could lead to hypo-
volemia, electrolyte imbalance, renal insufficiency and sepsis.
8.6.4 Management of Acute Skin Lesions
Skin injuries with less than 20 % involvement of body surface area are unlikely to
lead to significant complications, essentially due to electrolyte and fluid imbalances
(Chan 1987). They could be managed in a non acute care setting such as a dermatol-
ogy ward.
Topical care is initiated after precisely cleansing and decontaminating the
involved areas. Careful daily wound care is essential. Erosions and denuded areas
should be monitored for early signs of bacterial infection. Cleansing the wounds
and if necessary their debridement is essential to avoid developing infections. Anti-
infectious creams such as silver sulfadiazine or mafenide acetate (sulfamylon)
should be used to inhibit bacterial colonization and infection of erosions and
denuded skins. Topical antibacterial ointments or creams such as bacitracin, neomy-
cin and polymyxin B (Neosporin) could be protective against erosions and bullas
and accelerate re-epithelialization (Winton and Salasche 1985). Using biosynthetic
dressings such as hydrocolloids and hydrogels could lead to absorption of wound
fluids and could induce faster wound healing, accelerate reepithelialization and
reduce pain (Eaglstein 1985).
Aspiration and deroofing are the main actions for larger blisters, in order to
accelerate the healing process (Graham et al. 2005). Several recent studies have
focused on the use of physical debridement of injured tissue via surgical removal
followed by skin grafting or application of Xeroform petrolatum and scarlet red
ointment dressing (Graham et al. 2000, 2006), by CO2 laser (Graham et al. 1997,
2000) or even dermabrasion (Rice et al. 2000), to enhance the rate of wound
healing.
The most common symptoms related to skin damage are pain and itching. Itching
could be controlled by antihistamines such as hydroxyzine and doxepin. In areas
with severe itching and resistant to antihistamines, topical corticosteroids may be
helpful. However, it should be considered that these drugs may slow the healing
process. Beside standard analgesics such as codeine, non-steroidal anti-inflammatory
drugs such as naproxen can be used for relieving pain and reducing inflammation
(Bennion and David-Bajar 1994).
The management of acute skin lesions are summarized in Diagram 8.1.
Management of SM- skin Injury
Decontamination Therapy
Prophylaxis RDSL
Protective M13, M258 kits
equipments Stabling the
Fullers earth
patient
Gas Mask Sodium
Gloves thiosulfate
Suit & Foot Washing with
Treatment of Skin injury
protection soap or shampoo
and plenty of
Water
Acute skin injury

Chronic complications
Potential new
Conventional therapy
therapies*
Control of
Wound care
symptoms Reduction of deleterious
effect Inactivation of SM
Debridement Cooling the

Anti - skin
Cleansing
histamine
Avoid Trichloro
Topical & acetic acid
infection
systemic
Antibiotic steroids Vitamine E
cream Niacin
Analgesic
Bio-synthetic Methenamine
NSAIDs
dressing mandelate
Protease
Anti- Antioxidants &
Inhibitors* Calmodulin
inflammatory scavengers*
MMP-9 inhibitors*
Bifunctional Pre treatment
PARP inhibitors Trifluoperazine
compound with
inhibitors* GM 1489 Glutathion
Thioridazine
Capsaicin Niacinamide Ilomastat Pentamidine NAC
Vanilloids Doxycycline Anesthetics Oral or topical
Anti-TNF Povidone Aloe vera
iodine
Diagram 8.1 Algorithmic approach to management of Sulfur mustard skin injury (* Some of
these therapies have just been shown effective in in-vitro which has been mentioned in the text
while their cutaneous administration is not recommended. Non- steroidal anti-inflammatory
drugs. Matrix metalloproteinase 9. N-acetyl cysteine)
8.6.5 Management of Chronic Skin Complications
One of the most common late skin complications of SM-exposed individuals in

almost all studies is pruritus and/or burning sensation.
Like many other skin disease with pruritis, oral antihistamines, the well- known
anti pruritus drugs, have been used for SM-induced pruritus. Previous studies
showed efficacy of hydroxyzine 25 mg/day, cetrizine 10 mg/day and doxepin 10 mg/
day for 4 weeks in decreasing the severity of such complaints in SM-injured patients
(Shohrati et al. 2007b, c). In these studies doxepine had the same efficacy as
hydroxyzine taken once a day, but had greater efficacy than cetrizine (Shohrati et al.
2007b, c).
Equal efficacy of doxepin cream to betamethasone was observed in a recent clin-

ical trial which suggests topical doxepin as a potential alternative for controlling the
pruritus caused by sulfur mustard in exposed veterans (Panahi et al. 2011).
Based on several clinical trials of Iranian researchers on SM veterans of the Iraq-
Iran war (19801988), mild to moderate topical corticosteroids are the first line
treatment for Pruritus (Vogt et al. 1984; Shohrati et al. 2007a; Panahi et al. 2007,
2008, 2009). Furthermore, in some of these studies, the efficacy of betamethasone
cream in controlling SM induced pruritus was compared with other preparations
such as capsaicin (Panahi et al. 2008), pimecrolimus (Panahi et al. 2009) doxepin
cream (Panahi et al. 2011) and Aloe vera/olive oil cream (Panahi et al. 2012a) which
among all, betamethasone cream 0.1 % was superior or as effective as others in
reducing chronic skin signs and symptoms caused by sulfur mustard exposure. It is
to be considered that although these are effective drugs, their long-term side effects
particularly in extensive areas, limit their application.
Calcineurin inhibitors such as tacrolimus and pimecrolimus are available as topi-
cal formulations which were first developed for the treatment of atopic dermatitis.
They are non-steroidal anti-inflammatory drugs that have been also applied in man-
aging pruritus, burning sensation and skin dryness of SM veterans. The results of a
clinical trial showed that they require a longer period of time to achieve the same
effects (Panahi et al. 2009), while being free of the many adverse effects of topical
corticosteroids. However, other issues such as higher price, age limitations and prob-
ability of burning sensation should be considered in the application of these drugs.
In another study, Unnas boot which is a compression dressing usually made of
cotton and impregnated with glycerine, zinc oxide paste and calamine was used and
compared with betamethasone ointment for managing SM related pruritus; it
showed promising results (Shohrati et al. 2007a). The zinc oxide paste in the Unnas
boot helps ease skin irritation and keeps the area moist.
Capsaicin or Trans 8-methyl-N-vanillyl-6- nonenamide, is a natural alkaloid
and the active agent causing spicy taste in hot chili peppers. It is formulated today as
a topical cream or lotion which may be used as an adjunct topical analgesic in con-
trolling pain (Lin 2007) and occasionally in intractable pruritus (Lysy et al. 2003).
An investigation revealed significant effects of capsaicin cream 0.025 % (twice a
day for 6 weeks) in reducing SM-induced pruritus and skin dryness (Panahi et al.
2008). However, in comparison to the betamethasone cream, it was less effective
and less well tolerated.
Until recently, it was thought that capsaicin decreases pain via selective excita-
tion of peripheral un-myelinated afferent C-fibers by releasing of substance-P and
finally depleting it; but experimental and clinical studies have shown that depletion
of substance P from nociceptors has little, if any, causative role in pain relief. Rather,
it acts in the skin through a process best described as defunctionalization of noci-
ceptor fibres (Anand and Bley 2011).
Panahi et al in a randomized control trial investigated the anti-inflammatory
effects of curcumin in 96 male Iranian veterans who were suffering from chronic
SM-induced pruritic skin lesions and concluded that curcumin supplementation
effectively alleviate pruritus and improve their QoL (Panahi et al. 2012b).
Aloe vera/olive oil cream was as effective as betamethasone 0.1 % in the treat-
ment of sulfur mustard-induced chronic skin complications and might serve as a
promising therapeutic option for the alleviation of symptoms in mustard gas-
exposed patients (Panahi et al. 2012a).
Another topical preparation that has been investigated for controlling
SM-pruritus is the combination of menthol 1 % and phenol 1 %; it is used twice
a day for 6 weeks and has shown significant effects in decreasing pruritus (Panahi
et al. 2007). Menthol is an old medicine which contains major monoterpene in
the essential oils of some menthe species (Lamiaceae). It is a widely used over-
the-counter topical drug for the treatment of pain and its antipruritic effects have
been described in several studies (Bromm et al. 1995; Panahi et al. 2007; Haught
et al. 2008).
Menthol can relieve itch in some patients by activating the cold-sensitive recep-
tors in the skin that transmit a cool sensation, thereby reducing the perception of itch
(Kibbi et al. 1992). In this sense it is similar to capsaicin (Anand 2003).
Another common delayed skin complications in SM-injured patients are skin
dryness (xerosis) and eczema. Its treatment is very similar to other causes of skin
dryness or eczema. Use of emollients especially the thicker and greasier ones like
petrolatum and eucerin that are occlusive and prevent trans-epidermal water loss are
preferable. Only mild soap or soapless cleansers such as some pains and body
creamy shampoo are recommended while prolonged bathing or excessively warm
baths and showers should be avoided (Firooz et al. 2011).
Regarding high concentration of chlorine in some swimming pools which could
aggravate skin dryness and itching, we recommend moisturizing the skin after
swimming or if available using chlorine free ones.
To date, for abnormal skin pigmentation (hyper or hypopigmentation) due to
SM, no effective treatment has been identified (Poursaleh et al. 2012).
The management of chronic skin lesions are summarized in Diagram 8.2.
8.6.6 New Therapies
Recent researches have focused on two main strategies as the principles to finding
new treatments for SM injury: (a) Deactivation of SM before causing significant
damage to tissues and (b) Reducing SMs harmful effects.
8.6.6.1 Inactivation of Mustard Compounds
Antioxidants and Scavengers
Skin absorption could continue from free, non-fixed SM compounds or from con-
taminated fomites, so treatments specifically focusing on inactivating SM may be
useful even after the initial exposure. The aim of using these chemical scavengers is
No effective treatment till

Pigmentation
now
Oral or topical
antihistamine
Hydroxyzine
Cetrizine
Doxepine
Doxepine cream
Oral or topical steroids
Calcineurin Inhibitors
Pimecrolimus
Tacrolimus
Capsaicin cream 0.025 %
Chronic skin
Pruritus & Burning
complications
Menthol & Phenol 1%
Aloe vera & olive oil cream
Curcumin supplement
Unna s boot
Emollients
Skin dryness Mild cleansers
Avoiding hot bathing
Diagram 8.2 Algorithmic approach to management of chronic skin complications due to sulfur
mustard
to inactivate the free radical forms of SM or the oxygen or nitrogen radicals which
result from SM activation (Donald et al. 2009). This type of treatment should be
used within minutes to deliver maximum effects, because SM reacts with body tis-
sue within the first minutes of exposure. Most studies that have focused on the
therapeutic effects of these scavengers are related to pulmonary exposure or have

investigated their ability to reduce leukopenia (Papirmeister et al. 1991) and there is
limited research on the skin injury.
There is evidence that thiols or compound containing sulfhydryl groups can
decrease the toxic effects of mustard (Walker and Smith 1969; McKinley et al.
1982). Sodium thiosulfate, a potent antioxidant and scavenger, is a thiol containing
compound that is currently used to treat cyanide poisoning. It has been used sys-
temically for reducing leukopenia and thrombocytopenia in the treatment with
nitrogen mustard prior to exposure (Bonadonna and Karnofsky 1965; McKinley
et al. 1982). Nevertheless, it has limited effects on SM cutaneous injuries (Vojvodic
et al. 1985; Zhang et al. 1995).
Considering the pivotal role of glutathione in maintaining the intracellular reduc-
ing state, pretreatment with glutathione may protect cells against SM toxicity. There
are several in vitro studies that have shown pretreatment of various cell lines with
glutathione itself or the cysteine precursor; 10 mM L-oxothiazolidine-4-carboylate
provides resistance against SM toxicity (Andrew and Lindsay 1998; Amir et al.
1998; Simpson and Lindsay 2005). However, the use of reduced glutathione, once
before and twice after SM exposure did not keep mice from toxicity (Kumar et al.
2001). Since cutaneous application of glutathione is difficult, it has not been consid-
ered as an appropriate therapeutic agent for this purpose.
N-acetyl cysteine (NAC) is an antioxidant, inducer of glutathione synthesis and
redox-active agent. As SM could reduce glutathione in the cell, its recovery may
provide increased tissue survival.
In an in vitro study, pre treatment with NAC elevated intracellular glutathione
levels and protected the cells against SM exposure (Atkins et al. 2000). It has also
been reported to prevent apoptosis in different cell lines such as lymphocytes, neu-
rons and vascular endothelial cells (Dabrowska et al. 1996; Atkins et al. 2000).
In a mouse model, the effect of oral and topical Aloe vera gel on toxicity and skin
lesions caused by SM was evaluated. It showed protective results on SM -induced
oxidative stress. The results were more prominent for topical administration but
were partial for the oral type (Anshoo et al. 2005).
Protease Inhibitors
One of the mechanisms of dermo-epidermal separation in SM-exposed skin with strong

evidences in the literature is basement membrane damage by MMPs. Up-regulation of
MMP expression especially MMP-9 has been shown in some studies following expo-
sure to SM (Danne et al. 2001; Sabourin et al. 2002; Shakarjian et al. 2006).
There is some evidences supporting the use of protease inhibitors like MMP-9
inhibitors GM 1489 (Gerecke et al. 2005), Ilomastat (Schultz et al. 2004) and doxy-
cycline (Schultz et al. 2004; Guignabert et al. 2005; Lindsay et al. 2008) on human
or animal skin cells in vitro.
The beneficial effects of post SM- exposure treatment with iodine have been
shown in several studies on rodents. Povidone-iodine ointment is an efficient pro-
tective agent against chemical injuries and heat stimuli (Wormser et al. 1997, 2000).
Treatment with iodine significantly increases epidermal hyperplasia and reduces

inflammation and necrosis.
Both human and animal studies showed that the ointment should be used imme-
diately after SM exposure (Brodsky and Wormser 2007). The proper time for
achievement to protective effect of topical iodine application varied in different
studies from 15 to 30 min or even up to 60 min (Wormser et al. 2004). The shorter
interval between exposure and treatment, the better was the protection achieved
(Wormser et al. 2000).
The mechanisms by which iodine take care of the skin against chemical injury
such as SM exposure is reduction of collagenolytic activity (Wormser et al. 2002)
or may be due to reduced inducible nitric oxide synthase expression (Nyska et al.
2001). Combination of povidone-iodine preparation with anti-inflammatory agents
could improve their efficacies and preserve them as a potent antidote against SM
skin lesions (Vijayaraghavan et al. 2009).
Although in the majority of these research, reduction of MMP-9 or 2 or decreas-
ing cellular detachment have been reported, their results showed various degrees of
impact on SM injury. Therefore, the effect seems to be related to time of administra-
tion (before, post or co-exposure to SM) and the method of application as topical or
in media.
PARP Inhibitors
It has been supposed that activation of PARP due to SM-induced cellular damage
could result in the depletion of cellular NAD+ which finally led to blister formation.
So, PARP inhibitors seem to have ability as a useful pretreatment compound to
reduce SM induced injuries.
Niacinamide, a precursor for NAD+ synthesis and an inhibitor of PARP, has been
demonstrated as an appropriate pretreatment compound to reduce SM-induced skin
injury. Pretreatment and post-treatment application of niacinamide alone (Yourick
et al. 1992) or in combination with promethazine and indomethacin in a hairless
guinea pig skin exposed to SM reduced erythema and microvesicle formation
(Yourick et al. 1995).
Calmodulin Antagonists
Calmodulin antagonists and anesthetics were investigated in hairless mice and

observed that they may be considered as a choice for treatment of SM-induced skin
injuries. Topical pluronic base ointments including lidocaine or pentamide showed
beneficial effects when administered immediately after SM exposure on the skin of
pig (Kadar et al. 2000). Potent calmodulin antagonist such as trifluoperazine
(0.51 %) and thioridazine 2 % significantly prevented the development of
SM-induced skin lesions. Also pentamidine 10 % showed the similar effect.
Anesthetics drugs such as lidocaine and pentobarbital with concentration more than
5 % demonstrated some protective effect (Kim et al. 1996).
Anti-Inflammatory Drugs
Protection effects of steroidal and non steroidal anti inflammatory drugs (NSAIDs)
against SM toxicity, given systemically or topically, have been demonstrated the
key role of inflammation in SM-skin injury. Although glucocorticoids are effec-
tive in reducing edema in the early phase of injury, they do not seem to affect the
overall rate of healing. In animal models, using either systemic preparations such
as hydrocortisone or dexamethasone or topical steroids like clobetasol prior or
after SM exposure resulted in reduction of inflammation (Babin et al. 2000;
Casillas et al. 2000; Dachir et al. 2004; Reid et al. 2008). Also the findings on the
administration of NSAIDs like indomethacin given from hours before till minutes
or hours after SM exposure has been effective in reducing early edema but not the
late effects (Babin et al. 2000; Casillas et al. 2000; Kiser et al. 2001). Co- treat-
ment of steroids with NSAIDs has shown more significant results such as less
erythema, reduced damage area and occurrence of fewer lesions (Dachir et al.
2004). More recently, bifunctional compounds including NSAIDs (Diclofenac or
Ibuprofen) which have the ability to bond with pyridostigmine, were to some
extent effective against SM toxicity (Amitai et al. 2005). In addition, there are
some other preparations such as capsaicin and its structural analogs known as
vanilloids that their anti inflammatory effects including inhibition of edema, leu-
kocyte migration and mast cell degranulation have been previously demonstrated
(Brand et al. 1990; Bunker et al. 1991).
These compounds interfere with the release of neuropeptides from sensory fibers
and produce desensitization (Campbell et al. 1993). Some studies have shown that
pretreatment of skin with vanilloids like olvanil before SM exposure, significantly
reduces edema as well as cytokine and chemokine mRNA induction (Casillas et al.
2000; Babin et al. 2000, 2003; Sabourin et al. 2003). Other analogs of capsaicin
such as heptyl isovanillamide and homovanillamide have shown similar protective
effects against SM (Casbohm et al. 2004).
Also anti-tumor necrosis factor- antibodies may be a new treatment approach
in SM injuries. Because in a mouse ear model study, SM- induced ear edema
reduced following anti TNF- administration (Wormser et al. 2005).
8.6.6.2 Reduction of Deleterious Effects
Cooling the skin and the application of trichloro acetic acid crystals could signifi-
cantly reduce skin vesication or detachment (Papirmeister et al. 1991). Anesthetized
swine skin that was first exposed to 15 C for 24 h and then exposed to SM showed
significantly less damage after 7 days (Sawyer et al. 2002). Also human skin kerati-
nocytes exposed to SM and cultured at 25 C had less injury after 24 h than that
grown at 37 C (Sawyer and Risk 1999).
Nevertheless, subsequent studies showed that this effect was transient and just
slowed the rate of damage rather than reducing overall injury. Interestingly,
temperature-mediated inhibition of tissue damage was reversible as soon as return-
ing the tissue to normal body temperature (Risk et al. 2001).
The mechanism by which cooling with ice bags could inhibit mustard toxicity is
unknown and little work on the effect of cooling in reducing or preventing of injury
has been performed. Yet, it may be due to a decrease in mustard reaction with tissue
substrate or reduction of skin penetration at lower temperatures.
Some researchers have considered cooling as a temporary measure that prepares
a therapeutic window for performing other medical interventions (Nelson and
Sawyer 2006; Sawyer and Nelson 2008). Vitamin E, niacin and methenamine man-
delate are other drugs that have been reported to relieve the toxic effects of SM on
the skin (Papirmeister et al. 1991).
8.7 Conclusion
It seems that for the efficient treatment of sulfur mustard injuries more research is
required in order to better understand the basic mechanisms of SM injuries.
Although several studies have been done in the past two decades regarding this
issue, the present information is still not enough and the search is ongoing.
Despite the large number of biochemical pathways that have been identified
regarding SM injury, the main difficulty in finding the most effective preventive or
countermeasure is to find which one of these pathways has higher pharmaceutical
significance in this respect. Indeed, among the mentioned therapeutic modalities,
only anti-inflammatory drugs appear to have moderate effectiveness in SM injury
treatment. Currently, the most effective way to enhance overall efficacy in the man-
agement of patients exposed to SM is combination therapy in order to influence
different stages of SM injury. Also, in case of the possibility of using some topical
preparations such as antioxidants, protease inhibitors or scavengers, while consider-
ing the problems related to drug penetration in the skin, focusing on the drugs phar-
maceutics or delivery systems such as encapsulation of certain drugs in nanoparticles
or liposomes could be highly recommended.
Glossary
AP-1 Activator Proein-1: a transcription factor which regulates gene expression

in response to a variety of stimuli, including cytokines, growth factors, stress,
and bacterial and viral infections.
Blister (bullae): Elevated, circumscribed lesion, >1 cm in diameter primarily
filled with clear fluid.
CAM Calcium Modulated Protein: a calcium-binding messenger protein, a
multifunctional intermediate messenger protein that transduces calcium signals
by binding calcium ions and then modifying its interactions with various target
proteins.
Desmosome: A type of junction that attaches one cell to its neighbor. One of a
number of differentiated regions which occur, for example, where the cytoplasmic
membranes of adjacent epithelial cells are closely apposed. It consists of a circu-

lar region of each membrane together with associated intracellular microfilaments
and an intercellular material.
Dyskeratosis: Abnormal keratinization occurring prematurely within individual
cells or groups of cells below the stratum granulosum.
e NOS: endothelial Nitrogen Oxide Synthases.
ECM Extra Cellular Matrix: a collection of extracellular molecules secreted by
cells that provides structural and biochemical support to the surrounding cells.
Eczema: A pruritic papulovesicular dermatitis occurring as a reaction to many
endogenous and exogenous agents.
Epidermis: The external, nonvascular layer of the skin. It is made up, from within
outward, of five layers of epithelium: (1) basal layer (stratum basale epidermi-
dis); (2) spinous layer (stratum spinosum epidermidis); (3) granular layer (stra-
tum granulosum epidermidis); (4) clear layer (stratum lucidum epidermidis); and
(5) horny layer (stratum corneum epidermidis).
Erosion: Partial loss of epidermis (epithelium).
Erythema: Redness of the skin produced by congestion of the capillaries. This
condition may result from a variety of causes.
Eschar: A slough or piece of dead tissue that is cast off from the surface of the
skin, particularly after a burn injury, but also seen in gangrene, ulcer, fungal
infection.
Hemidesmosome: An anchoring junction of the cell to a non-cellular substrate,
similar in morphology to halves of desmosomes. They are composed of special-
ized areas of the plasma membrane where intermediate filaments bind on the
cytoplasmic face to the transmembrane linkers, integrins, via intracellular attach-
ment proteins, while the extracellular domain of the integrins binds to extracel-
lular matrix proteins.
Hypodense: Abnormality which is less dense than the reference structure.
IL Interlukin: a group of cytokines (secreted proteins and signaling molecules)
that were first seen to be expressed by white blood cells.
INF Interferon: a dimerized soluble cytokine that is the only member of the
type II class of interferons.
iNOS: inducible Nitrogen Oxide Synthases.
LD50 Lethal Dose, 50 %: The dose amount of poisonous or toxic substance or
dose of ionizing radiation required to kill 50 % of the tested population.
MAPK Mitogen-activated protein kinases (MAPK): A superfamily of protein-
serine-threonine kinases that are activated by diverse stimuli via protein kinase
cascades. They are the final components of the cascades, activated by phosphory-
lation by mitogen-activated protein kinase kinases, which in turn are activated by
mitogen-activated protein kinase kinase kinases.
Melanophage: A histiocyte that contains phagocytized melanin.
MMP Matrix Metalloproteinases (MMPs): zinc-dependent endopeptidases
that are capable of degrading all kinds of extracellular matrix proteins, but also
can process a number of bioactive molecules.
n NOS: neural Nitrogen Oxide Synthases.
NAD+ Nicotinamide adenine dinucleotide: A coenzyme composed of ribosyl-

nicotinamide 5-diphosphate coupled to adenosine 5-phosphate by pyrophos-
phate linkage. It is found widely in nature and is involved in numerous enzymatic
reactions in which it serves as an electron carrier by being alternately oxidized
(NAD+) and reduced (NADH).
NF-KB Nuclear factor kappa-light-chain-enhancer of activated B cells:
Ubiquitous, inducible, nuclear transcriptional activator that binds to enhancer
elements in many different cell types and is activated by pathogenic stimuli.
Nikolsky sign: A skin finding in which the top layers of the skin slip away from
the lower layers when slightly rubbed.
NOSs Nitrogen Oxide Synthases: a family of enzymes catalyzing the produc-
tion of nitric oxide (NO) from L-arginine. NO is an important cellular signaling
molecule.
NSAIDs Non-steroidal Anti-inflammatory Drugs: a class of drugs that provides
analgesic and antipyretic effects, and, in higher doses, anti-inflammatory effects.
Papillary dermis: Is the uppermost layer of the dermis. It intertwines with the
rete ridges of the epidermis and is composed of fine and loosely arranged col-
lagen fibers.
Papule: Elevated, circumscribed lesion, <1 cm in diameter which its elevation is
due to increased thickness of the epidermis and/or cells or deposition within the
dermis.
PARPs Poly (ADP-ribose) polymerase: a family of proteins involved in a number
of cellular processes involving mainly DNA repair and programmed cell death.
Poikiloderma: A skin condition that consists of areas of hypopigmentation,
hyperpigmentation, telangiectasias and atrophy.
Pyknosis or karyopyknosis: The irreversible condensation of chromatin in the
nucleus of a cell undergoing necrosis or apoptosis.
RNS Reactive Nitrogen Species: a family of antimicrobial molecules derived
from nitric oxide (NO) and superoxide (O2 ) produced via the enzymatic activ-
ity of inducible nitric oxide synthase 2 (NOS2) and NADPH oxidase respectively.
ROS Reactive Oxygen Species: Molecules or ions formed by the incomplete
one-electron reduction of oxygen. They contribute to the microbicidal activity
of phagocytes, regulation of signal transduction and gene expression, and the
oxidative damage to nucleic acids; proteins; and lipids.
RSDL Reactive Skin Decontamination Lotion: a proposed replacement for the
existing skin and equipment decontamination kit.
Scar: Areas of fibrous tissue (fibrosis) that replace normal skin after injury.
agents with the ability to form large blisters on the exposed skin and in the lungs.
Spinousum cell: One of the layers of the epidermis, composed of several layers
of polygonal cells. It lies on top of the stratum basale and beneath the stratum
granulosum.
TEWL Trans Epidermal Water Loss: The quantity of water that passes from
inside a body (animal or plant) through the epidermal layer to the surrounding
atmosphere via diffusion and evaporation processes.
TNF Tumor Necrosis Factor , cachexin, or cachectin): a cell signaling pro-

tein involved in systemic inflammation and is one of the cytokines that make up
the acute phase reaction.
Ulcer: Full-thickness loss of epithelium (epidermis).
Vesicle: Elevated, circumscribed lesion, <1 cm in diameter primarily filled with
clear fluid.
Xerosis: The medical term for abnormally dry skin. This name comes from the
Greek word xero, which means dry.
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Pharmacol Ther 92:5770
Chapter 9
Ocular Injury by Mustard Gas;
Early and Late Complications
Nasser Shoeibi, Mojtaba Abrishami, and Alireza Eslampoor
Contents
9.1 Introduction .................................................................................................................... 254
9.2 Ocular Pathophysiology ................................................................................................. 255
9.2.1 Anatomy............................................................................................................. 255
9.2.2 Tissue Toxic Effect of Mustard Gas ................................................................... 258
9.3 Clinical Manifestations .................................................................................................. 260
9.3.1 Anterior Segment ............................................................................................... 260
9.3.2 Posterior Segment .............................................................................................. 266
9.4 Treatment ....................................................................................................................... 267
9.4.1 Acute Phase........................................................................................................ 267
9.4.2 Chronic Phase .................................................................................................... 268
9.5 Conclusion and Recommendation ................................................................................. 269
References ............................................................................................................................... 270
Abstract Sulfur mustard (SM) is an oily liquid. It rapidly reacts with ocular tissues.
Eyes are the most sensitive organs to SM. In acute phase, it results in conjunctivitis,
corneal swelling and edema. Gradual spontaneous recovery usually occurs within
few days of severe pain and blepharospasm, with regeneration of the corneal epithe-
lium appearing within 57 days. In late stages, severe dry eye, limbal stem cell
deficiency, corneal vascularization and corneal ulcers and probably retinochoroidal
changes may complicate the course of disease several years after exposure. Copious
irrigation, topical steroids, artificial tears, cycloplegics and corneal protection are
the mainstay of treatment in acute phase. Late complications occur in a small per-
cent of those initially severely wounded. In chronic phase, current approaches are
chiefly conservative and symptomatic by nature: wearing contact lenses to improve
visual acuity, managing tearing problems as well as ocular surface instability via
N. Shoeibi, MD (*) A. Eslampoor, MD, FICO

Eye Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
e-mail: shoeibin@mums.ac.ir; Eslampoura@mums.ac.ir
M. Abrishami, MD
Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences,
Tehran, Iran
e-mail: mojtaba_abrishami@yahoo.com

254 N. Shoeibi et al.
administering artificial tears, occluding the puncta on a temporary basis, tarsorrha-

phy and blepharorrhapy. Inflammation can be partially reduced using topical as well
as systemic steroids. More drastic action, i.e., corneal transplantation, is needed in
cases of scar formation, abnormal deposition, vascularization loss of eye sight and
severe stromal thinning eventually leading to loss of global integrity.
Keywords Sulfur mustard Mustard gas Eye Cornea Ocular surface Retina
9.1 Introduction
Sulfur mustard (SM) [bis-(2-chloroethyl) sulfide] is an oily liquid. SM and similar

bifunctional agents have been produced as chemical weapons since nineteenth century.
High dose exposure of SM may result in death within hours or weeks. But low-dose
exposure may cause injury to the eyes, skin, and respiratory tract without any mortality
(Balali-Mood and Hefazi 2005). SM is regarded as one of the most important agents
of chemical warfare because of its simple and cheap chemical synthesis that makes it
readily available for both terrorist and military use. SM acts as an alkylating agent that
induces disruption of nucleic acids and proteins, impairing cell homeostasis and even-
tually causing cell death. It rapidly reacts with ocular, respiratory and cutaneous tis-
sues, as well as bone marrow and the mucosal cells of the gastrointestinal tract,
resulting in several devastating long-term effects on human health, many of which are
not clinically or pathologically well defined (Ghabili et al. 2010). Chemical warfare
agents (CWA) such as SM were widely used against Iranian troops and even civilians
by Iraqi forces between 1983 and 1988 (United Nations Security Council 1984).
Sulphur mustard was first synthesized at around 1822 by Despretz. Its vesicant
properties were noted by Guthrie in 1860 and it was first prepared in pure form by
Meyer in 1886. Fritz Haber was responsible for developing SM for use as a chemi-
cal warfare agent in WWI. Its first use was on 12 July 1917, in a field near Ypres,
Belgium, where during 10 days of attack more than one million mustard shells were
fired at Allied troops by Germans. Thereafter, it was employed extensively by both
sides and was responsible for more than 80 % of all documented chemical casual-
ties. It is estimated that over 1,200,000 soldiers were exposed to mustard gas during
the war and about 400,000 of them needed prolonged medical observation (Balali-
Mood and Hefazi 2005).
Briefly, SM use has been reported in several conflicts since the First World War:
the UK against the Red Army (1919), Spain against Rif insurgents in Morocco
(19211927), Italy in Libya (1930), the Soviet Union against Japan in Xinjiang
(1930s), Italy against Abyssinia (19351940), Poland against Germany, Germany
against Poland and the Soviet Union and Japan against China during the Second
World War, Egypt against North Yemen (19631967), Iraq against Iran (1983
1988), Armenians against the Azerbaijanis in the Nakhchivan Autonomous Republic
(1992), and Sudan against insurgents (19951997) (Ghabili et al. 2010). Regardless
9 Ocular Injury by Mustard Gas; Early and Late Complications 255
of WW I, the greatest use of SM, however, has been by the Iraqi army against
Iranian and even against its own Kurdish population. In one particularly distressing
event, some 5000 Kurdish civilians were killed in the Iranian-occupied village of
Halabja in 1988. Several CWAs, including SM and sarin, were identified in this
massacre (United Nations Security Council 1984). According to data reported by
Veterans and Martyrs Affair Foundation (VMAF) which is responsible for taking
care of the war victims in Iran, the Iranian people were 387 times attacked with
chemical bombs, rockets and artillery shells during the 8-years war by Iraq (1980
1988) (Kehe et al. 2009). Sardasht is a city in north-west of Iran. Iraqis released four
250 kg bombs containing mustard gas on this city at 4 pm, July 27, 1987 injuring
4,500 civilians (Ghanei et al. 2003). Use of mustard gas against Iranian soldiers by
the Iraqis was reported to the international commissions by Iran in 1984 and in 1986
application of this agent by Iraqis was documented by the United Nations observa-
tory team led by M. Dominguez. There were 398,587 veterans who needed long-
term follow-up during the war. 52,195 of them (13 %) were chemically injured
(Zargar et al. 2007).
9.2 Ocular Pathophysiology
9.2.1 Anatomy (Britannica 1987)

9.2.1.1 Eyelid
Eyelid is a thin fold of skin that covers and protects the eye. The levatorpalpebrae
superioris muscle retracts the eyelid to open the eye. This can be either volun-
tarily or involuntarily. The human eyelid features a row of eyelashes along the eye-
lid margin, which serve to heighten the protection of the eye from dust and foreign
debris, as well as from perspiration. Palpebral (and blepharal) means relating to
the eyelids. Its key function is to regularly spread the tears and other secretions on
the eye surface to keep it moist, since the cornea must be continuously moist. They
keep the eyes from drying out when asleep. Moreover, the blink reflex protects the
eye from foreign bodies.
The eyelid is made up of several layers; from superficial to deep, these are: skin,
subcutaneous tissue, orbicularis oculi, orbital septum and tarsal plates, and palpe-
bral conjunctiva. The meibomian glands lie within the eyelid and secrete the lipid
part of the tear film.
9.2.1.2 Conjunctiva
The conjunctiva lines the inside of the eyelids and covers the sclera (white part of
the eye). It is composed of non-keratinized, stratified squamous epithelium with
goblet cells, and also stratified columnar epithelium. The conjunctiva helps
lubricate the eye by producing mucus and tears, although a smaller volume of tears
than the lacrimal gland. It also contributes to immune surveillance and helps to
prevent the entrance of microbes into the eye. The conjunctiva is typically divided
into three parts:
Palpebral or tarsal conjunctiva: Lines the eyelids.
Bulbar or ocular conjunctiva: Covers the eyeball, over the anterior sclera. This
region of the conjunctiva is tightly bound to the underlying sclera by Tenons
capsule and moves with the eyeball movements.
Fornix conjunctiva: Forms the junction between the bulbar and palpebral conjuncti-
vas. It is loose and flexible, allowing the free movement of the lids and eyeball.
9.2.1.3 Cornea
Cornea is the transparent anterior part of the human eye. Because transparency is of
prime importance, the cornea does not have blood vessels; it receives nutrients via
diffusion from the tear fluid through the outside surface and the aqueous humour
through the inside surface, and also from neurotrophins supplied by nerve fibres that
innervate it. Transparency, avascularity, the presence of immature resident immune
cells, and immunologic privilege makes the cornea a very special tissue.
The cornea has unmyelinated nerve endings sensitive to touch, temperature and
chemicals; a touch of the cornea causes an involuntary reflex to close the eyelid. It
borders with the sclera by the corneal limbus.
The human cornea has five (possibly six) layers. From the anterior to posterior
the layers of the human cornea are: corneal epithelium, Bowmans layer (also
known as the anterior limiting membrane), corneal stroma (also substantia propria),
descemets membrane (also posterior limiting membrane), and corneal endothe-
lium: a simple squamous or low cuboidal monolayer.
9.2.1.4 Lens
The crystalline lens is a transparent, biconvex structure in the eye that, along with
the cornea, helps to refract light to be focused on the retina. The lens, by changing
shape, functions to change the focal distance of the eye so that it can focus on
objects at various distances, thus allowing a sharp real image of the object of interest
to be formed on the retina. This adjustment of the lens is known as accommodation
(see also below). Accommodation is similar to the focusing of a photographic cam-
era via movement of its lenses. The lens is more flat on its anterior side than on its
posterior side.
The lens has three main parts: the lens capsule, the lens epithelium, and the lens
fibers. The lens capsule forms the outermost layer of the lens and the lens fibers
form the bulk of the interior of the lens. The cells of the lens epithelium, located
between the lens capsule and the outermost layer of lens fibers, are found only on
the anterior side of the lens. The lens itself lacks nerves, blood vessels, or connec-
tive tissue.
9.2.1.5 Sclera
The sclera (from the Greek skleros, meaning hard), also known as the white of the
eye, is the opaque, fibrous, protective, outer layer of the eye containing collagen and
elastic fiber. The sclera forms the posterior five-sixths of the connective tissue coat
of the globe. It is continuous with the dura mater and the cornea, and maintains the
shape of the globe, offering resistance to internal and external forces, and provides
an attachment for the extraocular muscle insertions. In humans the whole sclera is
white, contrasting with the colored iris. In the elderly, fatty deposits on the sclera
can make it appear slightly yellow. The scleras blood vessels are mainly on the
surface. Along with the vessels of the conjunctiva (which is a thin layer covering the
sclera), those in the episclera render the inflamed eye bright red.
9.2.1.6 Choroid
The choroid, also known as the choroidea or choroid coat, is the vascular layer of
the eye, containing connective tissue, and lying between the retina and the sclera.
The choroid provides oxygen and nourishment to the outer layers of the retina.
Along with the ciliary body and iris, the choroid forms the uveal tract. The structure
of the choroid is generally divided into four layers (classified in order of furthest
away from the retina to closest):
Hallers layer outermost layer of the choroid consisting of larger diameter blood
vessels;
Sattlers layer layer of medium diameter blood vessels;
Choriocapillaris layer of capillaries; and
Bruchs membrane (synonyms: Lamina basalis, Complexusbasalis, Lamina vitra)
innermost layer of the choroid.
9.2.1.7 Retina
The retina is the light-sensitive layer of tissue, lining the inner surface of the eye.
The vertebrate retina has ten distinct layers. From closest to farthest from the vitre-
ous body that is, from closest to the front exterior of the head towards the interior
and back of the head:
Inner limiting membrane basement membrane elaborated by Mller cells
Nerve fibre layer axons of the ganglion cell nuclei (note that a thin layer of Mller
cell footplates exists between this layer and the inner limiting membrane)
Ganglion cell layer contains nuclei of ganglion cells, the axons of which become
the optic nerve fibres for messages and some displaced amacrinecells.
Inner plexiform layer contains the synapse between the bipolar cell axons and the
dendrites of the ganglion and amacrine cells.
Inner nuclear layer contains the nuclei and surrounding cell bodies (perikarya) of
the amacrine cells, bipolar cells and horizontal cells.
Outer plexiform layer projections of rods and cones ending in the rod spherule and
cone pedicle, respectively. These make synapses with dendrites of bipolar cells.
In the macular region, this is known as the Fiber layer of Henle.
Outer nuclear layer cell bodies of rods and cones
External limiting membrane layer that separates the inner segment portions of the
photoreceptors from their cell nucleus
Photoreceptor layer rods/cones
Retinal pigment epithelium single layer of cuboidal cells (with extrusions not
shown in diagram). This is closest to the choroid.
The optics of the eye create an image of the visual world on the retina (through
the cornea and lens), which serves much the same function as the film in a camera.
Light striking the retina initiates a cascade of chemical and electrical events that
ultimately trigger nerve impulses. These are sent to various visual centers of the
brain through the fibres of the optic nerve. These can be simplified into four main
processing stages: photoreception, transmission to bipolar cells, transmission to
ganglion cells which also contain photoreceptors, the photosensitive ganglion cells,
and transmission along the optic nerve. At each synaptic stage there are also later-
ally connecting horizontal and amacrine cells.
9.2.1.8 Optic Nerve
The optic nerve is a central tract of many axons of ganglion cells connecting primar-
ily to the lateral geniculate body, a visual relay station in the diencephalon (the rear
of the forebrain). It also projects to the superior colliculus, the suprachiasmatic
nucleus, and the nucleus of the optic tract. It passes through the other layers creating
the optic disc in primates.
9.2.2 Tissue Toxic Effect of Mustard Gas
Shortly after SM exposure, cell DNA alkylation has been identified as a major trig-
ger of apoptosis, which includes mono-functional SM-DNA adducts as well as
DNA crosslinks. As a consequence, DNA replication is blocked, which leads to
cell-cycle arrest and DNA single- and doublestrand breaks. The SM-induced DNA
damage results in poly (ADPribose) polymerase (PARP) activation. High SM con-
centrations induce PARP over activation, thus depleting cellular nicotinamide
adenine dinucleotide (NAD) and ATP levels, which in turn results in necrotic cell
death (Ghasemi et al. 2013). SM-induced apoptosis has been linked both to the
extrinsic cell surface death receptor (Fas) or intrinsic (mitochondrial) pathway
(Ghasemi et al. 2013; Keyser et al. 2013).
SM-DNA adducts (DNA binding covalently to SM) have been found in internal
organs such as brain, kidney and spleen in animal models (Batal et al. 2014). This
may explain the late findings of deep ocular findings in these patients.
In addition, in acute phase SM upregulates many pro-inflammatory mediators
including interleukin (IL)-1a, IL-1b, IL-6, IL-8, tumor necrosis factor-alpha
(TNF-a) and nuclear factor kappa-B (NF-B) cells activation 18. Likewise, levels of
four pro-inflammatory mediators (IL-1b, TNF-a, IL-6, and IL-8) increases in the
aqueous humor and the level of matrix metalloproteinase (MMP) 2 and 9 in the
cornea in chronic phase (McNutt et al. 2012). Furthermore, SM inhibits the effect of
collagenase on corneal collagen. This impaired collagenase activity due to the
remodeled collagen structure induced by SM may result in an insufficient corneal
repair process. Epithelial migration and endothelial redistribution, which depend on
the collagenase activity, become challenged, because collagenase cannot make
them move over the corneal collagen, which seems to make cornea susceptible to
ulceration and opacity (Naderi et al. 2010).
Oxidative stress involves inducible nitric oxide synthase (iNOS) which also
leads to peroxynitrite (ONOO) production that in turn activates NF-B and activator
protein-1 (AP-1), and expression of pro-inflammatory genes lead to promoting of
inflammation. In addition, ONOO could directly damage all biomolecules including
lipids, proteins and DNA within the cells. Individuals with moderate-to-severe
SM-induced injuries showed a decreased serum level of glutathione (GSH) and an
increased serum level of malondialdehyde (MDA) activities compared with the mild
injuries 20 years after exposure, which represents their tendency to oxidative stress
19. Superoxide dismutase, catalase and GSH peroxidase activities in bronchoalveo-
lar lavage (BAL) fluid, plasma and erythrocytes are significantly higher in exposed
patients. The increased glutathione-S-transferase activity in BAL fluid was associ-
ated with a depletion of GSH and an increase of MDA levels 20. Overall decrease
in the activity of superoxide dismutase by SM exposure is probably mediated by
direct inactivation of the extracellular type of this gene or enzyme itself, which may
be due to CysCys disulfide bonding cleavage 21.
It has been shown that total protein and Calcitonin Gene-Related Peptide (CGRP)
decreases, and vascular Endothelial Growth Factor (VEGF) concentration increases
in tears of SM-intoxicated patients who suffer from chronic ocular complications
(Panahi et al. 2012). This may explain the increased risk of corneal vascularization
in these patients.
In chronic SM toxicity, significant reduction in serum albumin and paraoxonase-1
activity may lead to long-term accumulation of reactive oxygen metabolites which
in turn subsequently lead to oxidative stress state. These changes might contribute
to morbidity and occurrence of other complications, such as atherosclerosis and
rapid or premature aging in these patients which may explain the possible findings
in choroid of these patients 22. In chronic toxicity, the level of IL-8 in tear as an
inflammatory cytokine does not decrease in SM-exposed cases with ocular surface
abnormalities as occurs in controls with ocular surface abnormality (Ghasemi et al.
2012).
Using human mononuclear leukocytes, post-exposure cell death process induced
by HD did not initiate before 4 h, but continued in a dose dependent manner after-
ward. Antioxidants (niacinamide and 3-aminobenzamide, PARP inhibitors) are
effective in preventing cell death partially if administered as early in the first 12 h
23 (Ghasemi et al. 2013). In rabbits, at 24 h, all corneal epithelial cells presented
degenerative changes, with the epithelium eventually detaching from the underlying
basement membrane at the level of the lamina lucida. Microblisters, a characteristic
SM-induced skin pathology of the basement membrane zone of animals, were
absent in this corneal study. Edema, degenerating fibroblasts and inflammatory cel-
lular infiltrates were persistent stromal responses. Immunopathological effects
included changes in antigenicity of bullous pemphigoid protein, laminin, desmo-
somal protein, Ki67 and p53 (Petrali et al. 2000).
9.3 Clinical Manifestations
9.3.1 Anterior Segment

9.3.1.1 Acute Phase
As aforementioned and owing to the excess lipophilicity as well as high reactive-

ness of the gas, corneal epithelial cells, with their considerable turnover and high
metabolism, are shown to be largely affected when exposed to the agent detected in
large quantities in the oily tear layer of the eye (Graham and Schoneboom 2013).
Other epithelial cells undergo apoptosis when exposed to lower doses and apoptosis
as well as necrosis in high concentrations.
This agent seems less capable of infiltrating the cornea. Nevertheless, anterior and
middle layer involvements have been observed on confocal microscopy in cases of
chronic and delayed MGK. The degree of involvement was reported as high as 7590 %.
On exposure, the affected individual may present with both early and late signs
and symptoms, with the delayed ones more debilitating regarding visual morbidity.
The latter may ensue either following immediate injury as persistent smoldering
inflammation (chronicity) or with latency of varying length. Presentations also vary
in severity and time of occurrence based on parameters namely quantity of the agent
used as well as duration of exposure (Blodi 1971).
At the incipient stage, there are complaints of foreign body sensation as well as
mounting ocular soreness. The blood-shot appearance, which is initially no more
than local hyperemia, will later progress to all-out edema an, in the end, acute con-
junctivitis. Excessive lacrimation, blepharospasm, diminished sight and even trans
lent loss of vision will develop few hours following exposure, with them all reach-
ing their zenith a day or two afterwards. Thence, symptoms commence to regress
spontaneously (after day 2) Though the patient may remain symptomatic in the
following 6 weeks, (s)he will regain full eye sight along with complete corneal epi-
thelial healing in no later than 5 days.
As for early lesions, there are three distinct classes in term of the severity of
symptoms: When exposed to 70 mg/m3/min, mild eyelid erythema as well as mild-
to-moderate congestion of the conjunctive occur, with vessels on site minimally
engorged minus any substantial chamois (Fig. 9.1). In such cases, cornea often
remains intact, hereinafter full repair is accomplished. Majority of the victims in
Iran-Iraq war presented with this class of symptoms (burning and painful eye after
SM exposure). A plausible explanation is the possible impurities of the agent used
by Iraqis or SM mixing with dust in the battle field.
A dose of 100200 mg/m3/min can lead to moderate complaints involving eye-
lids, conjunctiva and cornea. Vesication often occurs in the corneal epithelium, most
notably in the palpebral fissure. Almost similar symptoms mentioned in the mild
category can be seen but with higher intensity. Besides, punctate erosions, chiefly
on the interpalpebral fissure, arise on the corneal abrasion, infiltration, ulcer and
eventually perforation 96, more often than not seen in middle and inferior cornea to
the mild lesions, regression after 48 h and regeneration in 45 days, with full recov-
ery procrastinated to 6 weeks comprise the concluding scenario in such cases
(Fig. 9.2).
Lesions tend to intensify on exposure to in excess of 200 mg/m3/min, along with
systemic toxicity involving skin, respiratory and GI systems (Solberg et al. 1997).
Ocular damage is admittedly more profound in the palpebral fissure and tempro-
nasal zones owing to high exposure. Naso temporal limbi often show necrosis and
whitening due to vascular involvement.
Eyelids may rarely adhere to the eye ball as toxicity often involves the exposed
palpebral fissure. Low-grade iridiocyclitis minus synechia, cataract and full-
thickness corneal injury and more often not, a temporary increase in intraocular
pressure can be detected in exposure to doses greater than 400 mg/min/m3.
Fig. 9.1 Mild eyelid

erythema and conjunctival
congestion in acute
keratopathy with mild SM
exposure
Fig. 9.2 Diffuse punctate

keratopathy at the acute
phase of keratopathy with
moderate SM exposure
An orange-peel is a schematic description word phrase stromal edema as well

major corneal epithelial defects, which remain stain-free on fluorescein applica-
tion. In addition, possible nerve ending degeneration diminishes ocular protection,
breaching corneal privilege in such condition. Epithelial erosions and ulcerations
create an environment that makes the cornea prone to infections including
Pseudomonas aeruginosa. Pseudomonas aeruginosa is the notorious super-infec-
tion culprit, urging therapeutic PK, or worse, evisceration. Deeper layers of cornea
may also be affected in severe SM exposure when eyelids also ulcerate. This is
seen in the form of severe ischemia and necrosis following early congestion and
chemosis. Corneal edema resolution and as well as improving uveitis heralds
recovery in 12 week (s). Intraocular bleeding may occur as a result of corneal
neovascularization during recovery, leaving white opacities following degenera-
tion. Superinfection may complicate the course of keratitis (Javadi et al. 2005;
Solberg et al. 1997).
In severe toxicity, three scenarios can be described: the initial thorough resolu-
tion minus more inflammation, persistent complaints indicative of chronicity,
late-onset lesions emerging years after. The first course often takes 26 weeks, with
severe lesions disappearing but photophobia persisting. The patient will ultimately
become symptom/sign-free. Throughout the chronic course, acute lesions recover,
yet dry eye, photophobia and foreign body sensation perpetuates. There will also be
sequelae namely limbal ischemia, corneal erosions and peripheral corneal neovas-
cularization (Ashkenazi et al. 1991). Intrastromal exudation of plasma lipids and
amyloid deposition can be seen in the latter scenario (Fig. 9.3). Deeper layers of the
cornea are involved, creating descemetoceles, occasionally eventuating in perfora-
tion. What has to be borne in mind is that a quiet eye is not synonymous with
resolution as inflammation may go on. The misleading on-going improvement in
ocular conditions for weeks. Only to resurge even years (1440) needs constant
vigilance and attention.
9.3.1.2 Chronic Phase
Irreversible, idiopathic corneal inflammation tends to occur in below 1 % of cases

of acute intoxication with SM, often after a period of latency. This type of chronicity
is often dubbed MGK. Having been initially detected in WWI veterans, the patho-
physiology of MGK is not thoroughly recognized yet. Ever since, both experimental
and human studies have been conducted to unearth the unknown pertaining to
chronic ocular injury in such cases (Solberg et al. 1997).
Having investigated Iranian cases with delayed keratitis following a being
asymptomatic for long revealed that chronicity is essentially distinct from acute
circumstances, with MGK eventuating in permanent vision loss, either partial or
total (United Nations Security Council 1984).
A number of theories have been put forward to account for the deterioration. An
auto-immune process whereby corneal antigen attired by the offensive agent is
destroyed, or a degenerative reaction which results in damage to cornea and limbus,
toxic by-products and subsequent necrosis (confirmed by the presence of
spindle-shaped keratocytes on confocal microscopy) are to name but a few (Saladi
et al. 2006; Solberg et al. 1997; Javadi et al. 2005).
Delays in presentation can vary from 0.5 to 40 years in MGK, with slowly devel-
oping ocular discomfort as well as multisystem involvement. Ocular complaints
resemble those seen in delayed form, yet they persist and progress to a full-blown
inflammatory catastrophe. Limbus, conjunctiva and delayed involvements. The for-
mer is the most commonly affected while eyes may be involved asymmetrically
(Balali-Mood et al. 2005).
Limbal lesions, identical to Moorens ulcer, present with infiltration and melting.
Unless rendered avascular previously, vascular engorgement could also be seen,
with tortuosity, segmental narrowing and sporadic dilated areas clear on observa-
tion. Narrowing in their extreme forms may make dilations appearing as blood
pools (Etezad-Razavi et al. 2006).
Fig. 9.3 Limbal ischemia,

corneal abrasion and
peripheral
neovascularization as well
as lipid keratopathy are
sequels of acute phase
mustard gas keratopathy
Though seldom commencing intra-stromally, corneal problems more often than

not presents with peripheral infiltration plus/minus thinning of the limb. As the
condition recurs, the damage extends deeper into underlying layers, leaving crystal-
line deposits and giving cornea a silky appearance owing to progressive
degeneration.
Neovascularization, causing irregularity and thinning on cornea, is an ominous
premonition of later degenerative changes. Beside inflammation, melting recurs
during relapse, forming descemetocele and causing perforation. Corneal sensation
diminishes, intercellular epithelial attachments are impaired and basal tear secretion
decreases, predisposing the eye to infections. Eye lid changes, similar to chronic
blepharitis, include lid margin thickening and meibomian gland dysfunction.
In contrast, corneal lesions can be most detected in naso-temporal regions owing
to higher exposure, likewise limbal vascularity are more affected in these areas, yet
the account differs by nature as it is speculated that fewer number of stem cells
makes the limbus prone.
Despite the report by Javadi et al. investigating the link between the course of the
condition and climate dryness, the fact still remains to be unraveled via further
investigation. What still perplexes researchers is the capricious nature of the dis-
ease, with erratic periodicity regarding exacerbation and relapse. Symptomaticity
and ocular surface instability coincide with inflammation on an alternate basis
(Javadi et al. 2005).
Grading of SM-delayed keratitis are as follows (Solberg et al. 1997).
Mild: deformed vessels on conjunctiva in the form of segmentation, tortuosity
and telangiectasia minus corneal involvement (Fig. 9.4).
Moderate: conjunctivalisation: limbal ischemia and peripheral vascular invasion
plus/minus corneal opacity (Fig. 9.5).
Severe: conjunctival ischemia, corneal neovascularization, corneal thinning and
melting and secondary degenerative changes (Fig. 9.6).
Three mechanisms were proposed to account for delayed-keratitis presentations
(Javadi et al. 2011):
1. The on/going loss of stem-cells, initially incomplete and irregular but further
progressing to conjunctivalisation, neovascularization and sustained epithelial
erosions, limbal ischemia is also claimed to bring about neurotrophic and trophic
changes namely thinning, descemetocele formation and perforation
2. SM-induced vasculitis in the limbal vessels leads to sustained gradual ischemia
mainly on the limb of the palpebral fissure.
3. Lipoid and amyloid concentration, causes degenerative changes possibly in rela-
tion to chronic melting stromal inflammation and thinning.
There is no solid explanation to account for the exact effect of SM on limbal
stem cells; nevertheless, higher grades of keratitis are known to be in a positive
correlation with the severity of stem cell deficiency. Histopathological findings
corroborate the assumption stressing the resilience of stem cells against the direct
impact of the mustard gas owing to their low mitotic activity. Atypical conjunctival
Fig. 9.4 Mild stage of

delayed keratopathy
following SM exposure
Fig. 9.5 Moderate stage

of delayed keratopathy
after SM exposure
Fig. 9.6 Severe stage of

delayed keratopathy due to
SM exposure
presentations namely telangiectatic, leaking, tortuous vascularity in peripheral

cornea rather than completely vascularized pannus formation defy the account of
goblet cells presence in relation with delayed MGK. Similarly, LSCD findings do
not match corneal manifestations. A possibility is DNA alkylation in the stem
cells, impairing epithelial regeneration. A secondary process, prolonged impaired
innovation as a result of ischemia and diminished growth factors, is also claimed
to have caused LSCD in the vicinity of stem cells. It is hypothesized that matrix
metalloproteinase creates pathological conditions for stem cells in this respect.
Stem cells deficiency may also explain cells pleomorphism as well as irregularity
in boundaries on confocal microscopy. Another finding in the latter investigation
regards the lack of sub basal nerve plexus, possibly owing to Bowman layer
destruction and concurrence of sub epithelial fibrosis. Yet dry eye effect cannot be
categorically denied. Typical wallerian degeneration as well as a severe process of
the same nature inflicting nerves could be observed in animal experimentation on
MGK. In line with this observation, sensation problems in the form of hypoesthe-
sia/anesthesia were reported in cases where SM had led to neurotrophic keratitis
(Javadi et al. 2007, 2011; Baradaran-Rafii et al. 2010).
SM exposure allegedly causes eye and skin inflammation in animal models. Yet,
neither acute and delayed corneal injuries nor neovascularization did not seem to
respond to anti-inflammatory agents as expected. As far as autoimmune process are
concerned whereby altered corneal proteins (collagen-mustard compound) eventu-
ates in keratitis, Naderi et al. failed to prove the efficacy of collagenase when applied
to inhibit stromal collagen degradation. A plausible explanation in this respect will
be that over-expression of collagenase can degrade normal stromal collagen, giving
rise to pro-inflammatory cytokines. Hematoxylin-eosin staining on light micros-
copy reveals severe inflammation, stromal scarring and epithelial changes. A case in
point is the disappearance of goblet cells, similar to what can be seen in dry eye
condition. In a histopathological examination off 22 cases of chronic and delayed
MGK, Javadi et al. encountered ulceration, thinning, diminishing epithelial cells,
inflammation, vascularization and degeneration. Corneal buttons showed the
deterioration of Bowmans layer, epithelium, pannus formation neovascularization
and stromal scarring. Necrosis was detected together with lipid and amyloid con-
centrations. Scrape cytology revealed conjunctival epithelial cell dysplasia in com-
batants formerly exposed to mustard gas. Aberrant pleomorphic keratocytes were
reported on confocal microscopy of MGK cases, which is not normally seen in
unoperated cases, perhaps owing to the mutagenicity as well as teratogenicity of the
offensive agent (Javadi and Baradaran-Rafii 2009).
9.3.2 Posterior Segment
Electroretinographic (ERG) findings in an animal model showed no abnormality

67 weeks after exposure (Banin et al. 2003).
The study performed by authors (unpublished data) in survivors of Iran-Iraq war

(19801988) about 20 years after exposure showed a general reduction of retinal
photoreceptor function in chronic SM exposure. This effect involves both cone and
rod photoreceptors in terms of amplitude and implicit time. There was also a gen-
eral thinning of retina in macular area as evaluated by optical coherence tomogra-
phy (OCT). These findings in ERG traces and OCT records of SM veterans show
that SM intoxication may have late complications on neurologic tissues such as
retina.
9.4 Treatment
9.4.1 Acute Phase
There is no specific therapy for sulphur mustard poisoning; the sole aim of clinical
management in such cases is to maintain vital organ systems and alleviate symp-
toms. However, experience in the clinical management of several Iranian casualties
from the IranIraq War (19841987) demonstrated that those with severe burns will
require weeks of hospital care followed by lengthy convalescence and that, despite
the superficial nature of the burn, it is all too easy to underestimate the period of care
for such patients (Solberg et al. 1997). Historically, in Munich experience of treat-
ment of Iranian veterans of mustard toxicity, it is mentioned that in Tehran centers
eyes were washed with Ringers lactate solution. Mydriatics (e.g., cyclopentolate)
and sulfonamide antibiotics (e.g., sulfacetamide) were topically applied. The
patients were advised to keep the eyes closed for 2448 h. In Munich centers, myd-
riatics (e.g., atropine) and antibiotic (neomycin, gentamicin, acidamphenicol,
polymyxin-B-sulfate) eye drops were used (Kehe et al. 2009).
Recommendations from CDC (Centers for Disease Control and Prevention)
declare that immediately the patient should be removed from the source of exposure.
Eyes should be washed with large amounts of tepid water for at least 15 min.
Moreover, eyes should not be covered with bandages.
In sum, based on available records and reviews we recommend the following
steps:
First of all, as soon as possible ocular injuries should be washed by vigorous and
copious irrigation with water, facilitated by the use of topical anesthetic drops. While
isotonic fluids such as saline might have theoretical advantage, as they are available
rarely in the scene, it is recommended to use large quantities of clean water. The lids
should be held away from the globe. Irrigation should last at least copious fluid should
be irrigated into the eye and allowed to drain for at least 15 min to leach out all pos-
sible traces. If needed, any visible matter should be removed straight away. Reassurance
and pain management with systemic analgesics and dark glasses are needed. To pre-
vent lid adhesion, petroleum jelly can be used to. Bandages should be avoided.
Early assessment is vital to determine the extent and severity of the injuries. The
extent of the corneal chemical injury can be graded. Admission to hospital is advised
if there are grade 3 or 4 changes based on Hughes classification of ocular burn
(Brodovsky et al. 2000). Like other chemical burns in acute phase, some or all of the
following may be used by ophthalmologists. Ideally, preservative free topical drops
should be used where available. It is obvious that lubrication with topical artificial
tear, especially preservative free, is the mainstay of the treatment (Amir et al. 2000;
Brodovsky et al. 2000; Singh et al. 2013; Logothetis et al. 2014).
1. Steroids: Should be used very frequently up to hourly for the first week and then
tapered off. In the early phase, the anti-inflammatory leucocytic inhibitory action
of steroids is valuable to prevent secondary tissue damage by invading poly-
morphs. However, adverse effects as impairing epithelial regeneration and col-
lagen repair, may induce corneal melt. Therefore their use ought to be reduced
after the first week.
2. Vitamin C or potassium ascorbate drop: Vitamin C acts as a cofactor in collagen
synthesis, and as an antioxidant, and may prevent damage by chemically active
free radicals, released at the time of injury. These drugs consumption is better to
stop when the epithelium has healed.
3. Acetylcysteine: systemic or topical drop four times a day for up to 2 weeks is
used to induce cross linkage and maturation of new collagen and inhibits colla-
genase enzymes in tissue damages.
4. Antibiotics. If there is loss of corneal epithelium, their use prevents secondary
infection. Chloramphenicol drops or fluoroquinolones 4 times a day.
5. Cycloplegia with atropine 1 % or cyclopentolate 1 % drops twice daily, for the
acute phase for pain management and prevent synechiae formation.
6. Corneal Protection: If there is significant damage to the lid or corneal exposure,
the ocular surface is vulnerable to severe damage. Blink reflex and tear spread
over the ocular surface is essential in preserving the eye. In any form, treatment
by moist protective chamber, constant lubrication with preservative free drops,
or ointment is mandatory.
9.4.2 Chronic Phase
Chronic and delayed mustard keratitis have not been totally treated so far. Current
approaches are chiefly conservative and symptomatic by nature: wearing contact
lenses to improve visual acuity, managing tearing problems as well as ocular surface
instability via administering artificial tears, occluding the puncta on a temporary
basis, tarsorrhaphy and blepharorrhapy (Baradaran-Rafii et al. 2013). Inflammation
can be partially reduced using topical as well as systemic steroids. If failed, immu-
nosuppressive agents (azathioprine, cyclosporine) are indicated.
More drastic action, i.e., corneal transplantation, is needed in cases of scar for-
mation, abnormal deposition, vascularization loss of eye sight and severe stromal
thinning eventually leading to loss of global integrity. This will be particularly indi-
cated when central corneal opacity occurs without severe limbal involvement.
Under special circumstances, both lamellar keratoplasty and PK are warranted. If
severe dry eye, limbal ischemia or peripheral corneal involvement exist, PK graft
outcome will be affected as reported by Javadi (a rate of 21.7 % following 41months
and greater during longer follow-up).
Lamellar graft can be most effective in the management of the disease as anterior
stroma harbors most corneal lesions, let alone the fact that the course is inherently
recurrent and/or chronic. LK is merely an alternative to full-thickness PK, which is
warranted in cases of profound lesions or perforation (Feizi et al. 2013). One distinct
advantage of LK is that it can be re-conducted without difficulty in case graft opacity
diminishes vision or causes irritation. PK-treated patient are prone to cataract and
glaucoma as well. LK seems less invasive owing to its extraocular nature, reducing
the need for topical and systemic corticosteroid use in such patients (Feizi et al. 2013).
More frequent graft rejection episodes have been reported in eyes undergoing
simultaneous stem cell transplantation than in sequential cases. Whether concomi-
tant stem cell transplantation is an independent risk factor for graft failure in PKP or
if these eyes are already more predisposed to rejection and failure remains an unre-
solved issue (Javadi et al. 2011).
In conclusion, corneal involvements in MGK can be best managed with conven-
tional LK in the majority of cases. However, PK is still inevitable in certain condi-
tions and can provide acceptable outcomes despite a higher rate of graft rejection
reactions and failure relative to LK, especially with regular follow-up examinations,
adjunctive measures such as punctal occlusion and tarsorrhaphy, and use of immu-
nosuppressive agents.
9.5 Conclusion and Recommendation
Mustard gas has a high lipophilic activity and tropism to tear film, so eye surface
disorders are among the most common clinical features of mustard gas exposure.
After SM exposure, ocular lesions present within a spectrum of severity. Mild
and moderate cases usually resolve uneventfully; however, severe cases may follow
three courses: complete resolution, chronic smoldering inflammation, and reappear-
ance after a latent period of varying length (delayed form). Late complications
occur in a small percent of those initially severely wounded. In contrast to immedi-
ate damage, chronic and delayed mustard gas lesions usually cause progressive and
permanent reduction in visual acuity and even blindness. Delayed manifestations
have been reported up to 40 years after exposure.
Symptoms and signs of mustard gas exposure are:
Immediate damage:
1. Mild exposure (1270 mg/m3/min mustard gas exposure): red eye and conjunc-
tival hyperemia without significant chemosis; no corneal involvement.
2. Moderate exposure (100200 mg/m3/min mustard gas exposure): red eye, eye
pain and photophobia are among common symptoms. We can find chemosis,
hyperemia and corneal edema and punctuate epithelial erosion in ophthalmic
exam especially in pulpebral fissure area.
3. Severe exposure (>200 mg/m3/min mustard gas exposure): involvement of deep
corneal layers and limbal vessels is obvious. Eyelids are edematous, erythema-
tous and sometimes ulcerated. Corneal epithelial defects may occur.
Chronic smoldering inflammation
Limbal ischemia, recurrent microscopic erosions and impaired corneal sensation
result in corneal thinning and descematocele.
Reappearance after a latent period.
Following SM exposure, lesions heal in a few weeks. After a latent period of
varying length, lesions reappear. Conjunctival, corneal and limbal involvements
are key features. Symptoms start with tearing, photophobia and decreased visual
acuity. Limbal hyperemia, vascular tortuosity, narrowing and intervening dila-
tion of limbal vessels occurs. Infiltration and melting of limbus imitating
Moorens ulcer may occur. Corneal neovascularization and crystalline deposi-
tions are the end results.
Copious irrigation, topical steroids, artificial tears, cycloplegics and corneal pro-
tection are the mainstay of treatment in acute phase. In chronic phase, current
approaches are mainly conservative and symptomatic by nature: wearing contact
basis, tarsorrhaphy and blepharorrhapy. Inflammation can be partially reduced
using topical as well as systemic steroids. In case of corneal opacity, keratoplasty
may be needed.
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Chapter 10
Immunological and Hematological
Complications of Sulfur Mustard Poisoning
Bamdad Riahi-Zanjani and Mahmoud Mahmoudi
Contents
10.1 Introduction .................................................................................................................. 274
10.1.1 Brief on Physiology of Immunology .............................................................. 274
10.1.2 Brief on Biological Effects and Mechanisms of Action of SM ...................... 275
10.2 Experimental Studies ................................................................................................... 276
10.3 Human Studies ............................................................................................................. 278
10.3.1 Hematological Outcomes of Sulfur Mustard .................................................. 278
10.3.2 Immunological Outcomes of Sulfur Mustard ................................................. 280
10.4 Conclusions .................................................................................................................. 286
References ............................................................................................................................... 286
Abstract Sulfur mustard (SM) is an incapacitating chemical warfare agent, which

has been widely employed in particular regions. The short and long term biological
effects of SM have been clinically and basically studied. Sulfur mustard has been
demonstrated to induce a broad continuum of pathological effects in affected indi-
viduals. In addition to skin, lung, eyes and gastrointestinal manifestations, SM has
been shown to induce hematological and immunological complications. The acute
and chronic immune-hematological outcomes of individuals exposed to SM are
reviewed here. It seems that the dysfunctions of the immune system in these patients
may contribute to the increased incidence of a myriad of diseases that have been
documented in SM exposed veterans, including cancers. As the toxic effects of SM
are progressive and the clinical outcome of veterans can worsen over time,
developing additional therapeutic strategies is needed. Some of these strategies
might be based on immunopotentiating interventions.
B. Riahi-Zanjani
Medical Toxicology Research Center, School of Medicine,
e-mail: riahib@mums.ac.ir
M. Mahmoudi (*)
Department of Immunology and Allergy, Immunology Research Center, School of Medicine,
e-mail: mahmoudim@mums.ac.ir

274 B. Riahi-Zanjani and M. Mahmoudi
Keywords Sulfur mustard Immuno-hematological complications Veterans
10.1 Introduction
10.1.1 Brief on Physiology of Immunology
The immune system protects the human body against infection and conditions that
result in altered/depressed host immune functions consequently increase their risk
for infections and/or development of certain cancers or other pathologies. The
evolution of the immune system starts late in the embryonic phase and attains the
maximum capacity about the time of puberty. The immune response contains spe-
cific responses of lymphocytes (B- and T-cells) and general actions of other leu-
kocytes such as polymorphonuclears, monocytes, macrophages, and natural killer
cells. The leukocytes are produced in the bone marrow and lymph tissues and then
as the mobile cells of the immune system circulate throughout the body and are
transported to different parts of the body via the lymphatic and blood circulation
systems where they are going to be acted. The immune system function includes
a complex continuum of cellular and biochemical phenomena. Once a foreign
antigen enters the body, the antigen is phagocitized by macrophages and pro-
cessed via intracellular enzymatic hydrolysis. Afterwards, the segments of the
antigen are taken to the cell membrane of the macrophage to be presented to
T-helper cells. T lymphocytes are activated through the interaction of their spe-
cific receptors with antigen segments and the major histocompatibility complex
(MHC). The activation of B cells and cytotoxic T cells needs existing of a series
of lymphokines and cytokines secreted by T cells and other cells. The number of
activated T and B cells is increased through clonal expansion and then they are
converted to memory T and B cells, respectively. As a result, a strong and rapid,
specific immune response is induced following the next exposure to the same
antigen. The specific antibody produced by B cells neutralizes and inactivate the
foreign antigen while effector T cells destroy the cells containing antigens. The
immune system can recognize and destroy foreign agents efficiently if these
mechanisms work properly. Dysfunction of the immune system can happen at
anywhere along the pathway of cellular and biochemical processes, resulting in a
variety of immunological disorders from hypersensitivity to immunosuppression.
For instance, exposure to immunotoxic agents can lead to immunosuppression,
resulting in altered host resistance. The consequence of immune suppression is
affected by the amount and mode of action of the immunotoxins. In suppressed
condition, the immune system is not sufficiently able to respond to foreign and
dangerous antigens. Some of adverse outcomes include severe disseminated infec-
tious diseases caused by a number of agents that are usually not pathogenic
(Delves et al. 2011; Pechura and Rall 1993).
10 Immunological and Hematological Complications of Sulfur Mustard Poisoning 275
Chemical agents that suppress the function of bone marrow can influence reser-
voirs of stem cells that are required to replace cells and consequently affect on blood
line cells. because of the rapid proliferation of stem cells, they seem to be vulnera-
ble targets for toxic agents. Xenobiotics or various drugs that are toxic to the bone
marrow can lead to deep immunodeficiency due to lack of stem cells (Pechura and
Rall 1993).
10.1.2 Brief on Biological Effects and Mechanisms of Action of SM
Sulfur mustard (SM) is a vesicant chemical warfare agent that causes skin and
mucous membrane blisters on contact. Despite extensive worldwide research, there
is not any effective therapy for the treatment of patients exposed to SM yet. The
main acute pathologic findings due to SM exposure in humans include dermal, ocu-
lar and respiratory injury; other effects that become apparent soon thereafter are
reproductive, developmental, gastrointestinal, and hematological effects, and some
types of cancers (Namazi et al. 2009; Heinrich et al. 2009; Rowell et al. 2009). After
20 years, chronic consequences of these types of SM exposure are becoming
increasingly apparent for exposed individuals (Hassan et al. 2006). Several papers
reporting on the delayed toxic effects of SM in veterans have been published
(Ghanei 2004; Balali-Mood and Hefazi 2005, 2006; Balali-Mood et al. 2005, 2008,
2011; Hefazi et al. 2006; Shabestari et al. 2011). In spite of decades research, the
mechanism of SM chronic effects is unknown. considering the short half-life of SM
(1924 min in normal saline and 3060 min in the blood), the development of
delayed pulmonary effects after a single SM exposure is unexpected (Hambrook
et al. 1993).
In parallel to skin, lung, eyes and gastrointestinal problems, SM has been dem-
onstrated to cause hematological and immunological complications (Hassan et al.
2006). However, among various systems, the one that has been not studied mecha-
nistically is the immune system. Immune system dysfunction is considered as the
main cause of opportunistic infections and death in SM-exposed veterans (Ghotbi
and Hassan 2002).
It has been well known that sulfur mustard is able to induce toxicity through
production of reactive electrophilic intermediates, which can covalently modify
nucleophilic groups in macromolecules including DNA, RNA, and protein (Somani
1992). Intrastrand DNA cross-linking consequently lead to the toxic effects on rap-
idly proliferating tissues, such as lymphoid organs. These injuries may be tempo-
rary or permanent. A decrease in lymphocyte count in veterans is likely due to the
toxic effect of this alkylating agent on precursors of lymphocyte cells of bone mar-
row (Hefazi et al. 2006).
On the other hand, SM toxicity appears to be partially mediated by production of reac-
tive oxygen species (Naghii 2002; Han et al. 2004), and oxidative stress plays an essential
role in the pathogenesis and propagation of damages (Naghii 2002). For instance, the
major findings of a study conducted by Shohrati et al. were the decreased and increased
levels of serum glutathione (GSH) and malondialdehyde (MDA), respectively, in
SM-exposed patients compared to controls (Shohrati et al. 2010). In another study in
mice models, it was shown that exposure to SM lead to a significant decrease of serum
GSH (Pant et al. 2000). Therefore, mechanisms such as NAD and glutathione depletion
are involved in inducing cell death from SM toxicity (Hefazi et al. 2006). Among mecha-
nisms which have been considered to describe pathogenesis of delayed SM effects, the
failure in the cell repair mechanisms is the most important (Ghanei and Harandi 2011).
Oxidative stress due to the lack of cellular glutathione (GSH) plays a pivotal role in this
pathway (Shohrati et al. 2010). Also, previous studies showed a direct relation between
lack of harmony in cellular oxidative-antioxidative system and ineffective cell repair
which had been approved by raise in tissue growth factor beta (Mehrani et al. 2011;
Adelipour et al. 2011; Ghanei and Harandi 2011). The higher risk of cancers and the
higher level of TGF- among SM-exposed veterans, signalize the hypothesis that a
genetic mutation might have occurred in one of the components of repair pathways
(Hassan et al. 2006).
The short and long term hematological/immunological (both cellular and
humoral) consequences of individuals exposed to SM are reviewed here. For a bet-
ter understanding of SM effects on immune system a systematic search was per-
formed based on information available in known international medical databases
such as ISI, Medline, Scopus and Iranian databases such as Iranmedex and Irandoc.
The main criterion for qualification and selection of the manuscripts was their pub-
lication in approved medical journals.
10.2 Experimental Studies
In a review of the literature, it has been demonstrated that SM cause immunosup-

pression in animals. In a study conducted by Hektoen and Corper, antibody produc-
tion in dogs and rabbits were decreased after SM administration IV and IP. The SM
showed a depressed effect on neutralizing of invading foreign agents by antibodies
and on the leukocyte count of the blood in experimental animals (Hektoen and
Corper 1920). These studies categorized SM in a group which has frequently been
related to leukopenia, pancytopenia, anemia, and aplastic or hypoplastic bone mar-
row (Klaassen 2013).
Later studies on rabbits showed that leukocytes count elevated instantly after
exposure to SM (inhalation) but later reduced morphologically. The basophiles
showed unusual developments of the nucleus and dissolution of the granules.
The lymphocytes also showed degenerative changes (Hektoen and Corper
1920). In parallel to above results, in a study performed on euthymic hair less
guinea pig, it was shown that, after a leukocyte elevation on the day 1, leukocyte
count reduction occurs on days 46 after the exposure to SM, (Gold and Scharf
1995). Similar results were attained in experiments in which SM was injected
intravenously. Basophils increased following injection and then decreased rap-

idly. Zimmerman reported that lymphocyte parsing started within 5 h after
intravenous injection of SM and most of the lymphocytes disappeared during
first 24 h (Zimmerman 1942).
Quantitative histopathological studies of the effects of intravenously injected SM
on albino rats showed leukopenia, lymphopenia, and neutropenia, as well as hypo-
plasia and hyperemia of bone marrow. The volume of lymphoid organs decreased
because of the destruction of lymphocytes (Kindred 1947). In this study, the bone
marrow reacted to the SM more slowly than the lymphoid organs, but it got hyper-
plastic. In addition, some destruction of the mature granulocytes were observed
(Kindred 1949).
The aforementioned results on the albino rat have been joined by researches
demonstrating that SM-exposed dogs experience lymphoid organs and bone mar-
row toxicity as well as a decrease in peripheral blood cells. In this study a decrease
in peripheral blood cells of dogs were seen. The level of cytotoxicity was directly
associated with the dose of the SM administered (Kindred 1949). Furthermore,
Spurr clarified the effect of mustard compounds on immune function of rabbits by
intramuscular injection of typhoid vaccine and nitrogen mustard, simultaneously.
The results showed that nitrogen mustard suppresses antibody-forming processes
by lymphocytes (Spurr 1947).
More recent studies have generally confirmed earlier evidence that laboratory
animals exposed to mustards experience immunosuppression, alteration of host
defense mechanisms against pathogens and neoplasia. In a study performed by
Coutelier and colleagues the effects of SM on mice splenocytes were analyzed one
week after administration. A significant reduce in spleen cellularity was observed in
mice which had received high dose of SM. B-cells were relatively more vulnerable
than T-cells by this toxicant. However, B-cell function, measured by thymidine
incorporation and antibody secretion in the presence of lipopolysaccharide, was not
significantly affected. Also, SM did not show any changes in T-cells function
because their proliferation response to concanavalin A or to an anti-CD3 antibody
was not depressed (Coutelier et al. 1991). Studies conducted on mouse models indi-
cated that sulfur mustard caused a significant suppression of DTH responses to
Sheep Red Blood Cell (SRBC) and a significant decrease in the antibody titers to
SRBCs (Hassan and Ebtekar 2001).
Blank and colleagues (1991) investigated and compared the immunotoxic effects
of SM and nitrogen mustard on acquired immunity of mice. The effects on thymus
and spleen weight, spleen cellularity, and the production of antibody were similar to
earlier laboratory results. Splenic and thymic weight reduction was observed with
both compounds. When cellularity of spleen was depressed, the total number of B
cells was reduced. Only when SM reached lethal doses were the total spleen cells
producing antibody response at a level equivalent to that observed following
nitrogen mustard administration. Nitrogen mustard had an extra immunotoxicity in
favour of decreasing host resistance to tumor cells that was not seen with sulfur
mustard (Blank et al. 1991).
10.3 Human Studies
10.3.1 Hematological Outcomes of Sulfur Mustard

10.3.1.1 Short-Term Outcomes
As mentioned earlier, SM is considered as an alkylating agent which is particularly

toxic to rapidly proliferating cells such as bone marrow cells. The most common
hematologic finding during the first few days after intoxication is leukocytosis. The
number of white blood cell, then begins to reduce on days 3 and 4 after exposure
and reach their minimum level on day 9. This condition is followed by a decrease in
megakaryocytic series and finally in the erythropoietic precursors (Willems 1989;
Balali-Mood et al. 1991; Tabarestani et al. 1990). Thrombocytopenia and anemia
are observed later among the surviving patients (Sohrabpour 1984). The bone mar-
row studies disclose a severe decrease in total cell count and fatty change, and
nuclear changes in erythrocyte precursors. If cytopenia is not present and the
remaining stem cells stay viable, recovery will occur during upcoming days (Balali-
Mood et al. 1991; Tabarestani et al. 1988, 1990; Krumbhaar and Krumbhaar 1919).
The hematotoxicity of SM is dose dependent and it is concluded that it might lead
to aplastic or ineffective hematopoiesis. Exposure to SM at high doses was demon-
strated to induce a cytotoxicity on hematopoietic precursors and pancytopenia was
reported in exposed soldiers (Tabarestani et al. 1988). A significant decrease in the
number of peripheral blood leukocytes, however, is a factor that leads to secondary
infections and higher mortality rates in these patients. Patients with leukocytes
count of <200 cells/ml died during their initial admissions (Willems 1989). In case
of blood lymphocytes a published study showed initial marked lymphopenia in
36 % of the veterans, while during the recovery phase, the number of blood lympho-
cytes increased to >40 % in 18 % of the patients (Tabarestani et al. 1990).
10.3.1.2 Delayed Outcomes
Most of studies performed on individuals exposed to sulfur mustard focused on the

WBC, RBC, Hb, Hct, and platelets statuses. Data from a 5 year follow-up study
showed that the mean values of leukocytes, lymphocytes and neutrophils of exposed
veterans were decreased, but monocytes were increased in comparison with the first
evaluation at the beginning of the study (Ghanei 2004). In addition, in some exposed
patients, reactive or atypical lymphocytes included more than 20 % of the total lym-
phocytes. In Keramati et al study performed on 42 Iranian veterans poisoned by SM
23 years after exposure, hematological parameters of veterans (except the reticulo-
cyte count) did not show any changes in comparison with control. However, a
marked increase in the percentage of the patients reticulocytes was seen. The sig-
nificant increase in the reticulocyte percentage is likely secondary to the hypoxemic
status of the veterans as a result of their respiratory problems. Both increase in

reticulocyte percentage and unchanged RBC counts show that the RBC lifespan
of the patients might be less than normal. This condition is similar to hemolytic
anemia (Keramati et al. 2013) (Table 10.1). In another study, the analysis of com-
plete blood count (CBC) of all poisoned veterans that had been exposed to SM
1620 years before performing this study and had severe clinical complications
showed that total counts for WBC and RBC, as well as Hct percentage were signifi-
cantly higher in the patients than in the controls. The percentages of blood neutro-
phils and lymphocytes of the veterans were within the normal range and that of their
monocytes were significantly higher than in the controls. The authors of aforemen-
tioned study described that leukocytosis might relate to the high frequency of respi-
ratory infections in the veterans rather than a direct bone marrow toxicity. Of course,
in the case of the mentioned studies, all the changes were within the normal hema-
tological parameters ranges (Mahmoudi et al. 2005). Ten year follow up studies on
the clinical conditions of the SM-exposed veterans of the Iraq-Iran war showed the
long term consequences of sulfur mustard contamination. Studies revealed that sur-
viving Iranian victims are still suffering from high incidences of malignancies and
recurrent infections. Epidemiological studies have shown that the incidence of
AML is 18 fold and the incidence of ALL is 12 fold in comparison to normal popu-
lation (Zakery Neia 1995).
Table 10.1 Comparison SM-exposed

of hematological indices Parameter Controls veterans
of veterans and control
WBC (cell/l) 6380 230 6540 220
subjects 23 years after SM
exposure (Keramati et al. RBC (cell/l) 5700 90 5620 130
2013) Hematocrit (%) 46.56 0.56 46.12 0.61
Hemoglobin (g/dl) 16.23 0.18 15.96 023
Platelet (103 cell/l) 243 9 236 7
Neutrophil (cell/l) 3490 170 3740 150
Lymphocyte (cell/l) 2370 100 2300 100
Monocyte (cell/l) 400 30 390 30
Eosinophil (cell/l) 88 10 78 10
Reticulocyte count (%) 0.67 0.03 0.82 0.04*
MCH (pg) 28.58 0.35 28.82 0.29
MCHC (g/dl) 34.88 0.18 34.61 0.17
MCV (fl) 81.91 0.79 83.26 0.70
MPV (fl) 9.95 0.16 10.05 0.13
Data shown are mean SE. Value is significantly different
vs. control at *p < 0.05
WBC white blood cell, RBC red blood cell, MCH mean
corpuscular hemoglobin, MCHC mean corpuscular hemo-
globin concentration, MCV mean corpuscular volume,
MPV mean platelet volume
10.3.2 Immunological Outcomes of Sulfur Mustard
10.3.2.1 Short-Term Outcomes
Cellular Components
Publications on the status of blood immune cells of the veterans exposed to SM

within the day 1 up to the 7th week of exposure indicated a significant decrease in
T cell count in 54 % of the patients and a marked decrease in the number of mono-
cytes in 95 % of the patients. Also, the number of eosinophil reduced in 35 % of the
veterans in the first week and in 65 % of them in the 7th week after exposure. The
number of neutrophil r in 89 % and 60 % of the patients in the first week and the 7th
week, respectively. B lymphocyte counts were within the normal range from day 1
to the 7th week after exposure (Razavimanesh 1988).
In case of toxicity of SM on function of innate immunity, we found a few papers
on phagocytic cell function as the most important cells involved in innate immunity.
In a study, neutrophil function tests of the SM exposed veterans, during 1 month
after exposure showed a marked decrease, so that, in some cases the phagocytic
index diminished to one fifth of normal range. These changes were returned back to
normal condition after 3 months (Bahar et al. 1988). Keyhani and colleagues per-
formed a functional test for neutrophill called NBT on 121 Iranian veterans, and
they found the function of neutrophils to be decreased in a period up to 51 days after
exposure in all cases. This finding might indicate lack of neutrophil function
(Keyhani 1988). In contrary, Zandiyeh et al determined cell movement, chemotactic
factors, and plasma and cell opsonins among the SM exposed patients and the
results were within the normal range (Zandiyeh 1991). One mechanism by which
the innate immune system protects human body is by the phagocytic uptake and
subsequent destroying of pathogens, partly, via an oxidative bactericidal pathway
called the respiratory burst. The act of ingesting a foreign particle by a phagocytic
cell such as macrophage or neutrophil activates NADP oxidase that, in turn, start up
the process of generation of a significant amount of highly potent bactericidal ROS
from molecular oxygen. (Meydani et al. 1995; Chew and Park 2004). Thus, low
levels of ROS are essential for daily survival (Boxer et al. 1979; Victor et al. 2004).
On the other hand, if ROS is over-produced, or the antioxidant content is low, the
cells damage. Phagocytic cells are under oxidative stress when there is an disbal-
ance between pro-oxidants and antioxidants (Victor et al. 2004). Phagocytic cells
are especially vulnerable to oxidative injury because of the high amount of polyun-
saturated fatty acids in their surfaces and their high generation of ROS, which
contribute to damage. As the level of these fatty acids in the surfaces is elevated, the
potential for membrane lipid peroxidation mediated by ROS is also elevated. Lipid
peroxidation reduces membrane fluidity, which adversely influence immune
responses. Thus, the equilibrium between pro-oxidant generation and antioxidant
protection is critical for an accurate cell function, whereas a disturbance in this
equilibrium towards the oxidants indicates to an oxidative stress (Hughes 1999;
Victor et al. 2003). Thus, oxidative stress caused by SM is probably an important

mechanism of SM-induced immunotoxicity as measured/reflected by decreased
phagocyte (i.e., phagocytic) functionality.
Molecular Components
It has been reported that SM possesses a toxic effect on animal B-lymphocytes and
therefore, hypogammaglobulinemia is a significant finding. In case of short term
effects of SM on antibody status of veterans, Keyhani et al study is absolutely the
best. In this study, the serum concentrations of IgG, IgA and IgM of SM-exposed
veterans were determined from day 3 up to 1 month after exposure. The concentra-
tions of serum IgG in veterans showed a significant decrease on day 3 after exposure
to SM. On the other hand, the levels of serum IgG of the patients non-significantly
increased during 418 days after exposure. The increase in the levels of serum IgG
of veterans during 1931 days after exposure was found to be significant as com-
pared to controls. The concentrations of IgA in the sera of the veterans during 1
month after exposure showed fluctuations similar to those of IgG, but the variation
of the patients serum IgA, were not significant in comparison to controls. The serum
concentrations of patients IgM did not show any significant differences during 1
month after exposure to SM as compared to controls (Table 10.2). The justification
of Keyhani and colleagues for the initial decrease in serum concentration of IgG in
veterans was a possible leakage of IgG into the skin blisters and into other severely
influenced parts of the body such as lungs, whereas the subsequent elevation in
serum IgG was interpreted as a result of an auto-antigenic stimulation of the veter-
ans immune systems (Keyhani et al. 2007).
Table 10.2 The SM-exposed

concentrations of Immunoglobulin (mg/dl) Controls veterans
immunoglobulins isotypes in
IgG (3 days after exposure) 1110 220 950 200*
serum veterans during 1
month after exposure to SM IgG (418 days after exposure) 1170 250 1060 270
IgG (1931 days after exposure) 1110 220 1520 420**
IgM (3 days after exposure) 150 140 190 200
IgM (418 days after exposure) 150 110 170 150
IgM (1931 days after 150 140 140 76
exposure)
IgA (3 days after exposure) 220 91 200 180
The sera were collected from the veterans on day 3, during 418
days and during 1931 days after exposure (Keyhani et al. 2007)
Data shown are mean SE. Value is significantly different vs.
control at *p < 0.05 or **p < 0.001
In our search we found one study regarding short-term effects of SM on comple-

ment system showing normal level of C3, C4 and CH50 during the first week and
up to the 6 months after SM exposure.
10.3.2.2 Delayed Outcomes
Cellular Components
Sulfur mustard is a debilitating agent with long-term adverse effects on the immune
system. Several studies have tried to investigate the potential of sulfur mustard to
induce delayed outcomes of SM on cellular components of the immune system. In
this field, there are numbers of contradictory studies related to delayed effects of
SM on immune cells that some of them will be discussed.
In a study, all poisoned patients that had been exposed to SM 1620 years prior
to this study and had severe clinical complications were studied (Mahmoudi et al.
2005). The analysis of blood immune cells for the patients and 35 healthy age-
matched controls showed that the percentages of monocytes and T-cells were sig-
nificantly higher and the percentage of natural killer cells was significantly lower in
patients. According to Mahmoudi et al the impaired innate immunity due to a
decrease in the number of blood NK cells is probably responsible for the increased
risk of infections in these patients (Table 10.3).
Similar study was performed by Ghotbi and Hassan investigating a total of 75
veterans with an average age of 40. They all had been exposed to SM about 10 years
before the study begins. They classified the patients into mild, moderate and severe
groups. It was shown that the percentage of NK cells was significantly lower in
severe patients. The results also showed that the function of NK cells in severe
group is appreciably higher in comparison to the control (Ghotbi and Hassan 2002).
They proposed that a marked reduction in the absolute counts of NK cells in severe
patients is likely due to the harmful effect of SM on NK cell precursors of bone
marrow. Their other conclusion was that the function of NK cells has increased to
compensate the reduction in the number of these cells. Finally, they concluded that
Table 10.3 Flow cytometric SM-exposed

analysis of blood SM exposed Parameter Controls veterans
veterans and control groups
Lymphocyte (%) 30.5 8.0 31.5 8.4
1620 after exposure
(Mahmoudi et al. 2005) Monocyte (%) 3.9 1.1 4.8 1.6*
Neutrophil (%) 65.4 8.7 63.8 8.7
CD3+ lymphocyte (%) 65.6 10.7 71.1 8.6*
CD4+ lymphocyte (%) 57.8 8.1 57.7 5.3
CD8+ lymphocyte (%) 34.1 7.8 37.1 8.3
CD19+ lymphocyte (%) 13.6 6.2 11.9 5.9
Data shown are in terms of mean SD. Value is signifi-
cantly different vs. control at *p < 0.05
their results indicate that higher risk of cancer and also recurrent infections in
SM-exposed veterans might be due to the reduction of NK cell number.
According to a comprehensive study (Sardasht-Iran Cohort Study) on the
immune and pulmonary system of SM exposed civilian cases 20 years after expo-
sure, performed by Ghazanfari and colleagues, results indicated a significant
decrease in T lymphocytes and T helpers percentages. It was also shown that periph-
eral blood NK cell counts were highly elevated in exposed patients. The data dem-
onstrated a marked negative correlation between T lymphocyte counts and FVC
percentage and positive correlation with FEV1/FVC%. It was also showed that
blood monocytes counts had a negative correlation with FVC%. They concluded
that NK and T cells are likely to be involved in the pathogenesis or immune reac-
tions to the long term pulmonary problems induced by SM. (Ghazanfari et al. 2013).
In another study conducted by Akbari et al., the potential delayed toxic effects
of sulfur mustard on white blood cells was investigated on 113 Iranian veterans,
nearly 25 years after exposure. Total leukocyte counts and percentage of polymor-
phonuclear cells were significantly higher in exposed patients. The analysis showed
that the percentages of T helpers were significantly lower in exposed veterans
whereas, T cytotoxic lymphocyte percentage and CD4+/CD8+ ratio statistically
remained unchanged (Mohammadhoseiniakbari et al. 2008). They concluded that
blood diathesis is still present in sulfur-mustardexposed veterans 25 years after
exposure.
Shaker and colleagues in another study determined the total leukocyte and the
status of T helper and T cytotoxic cells on 75 exposed veterans 10 years after expo-
sure to SM. Their results showed that leukocyte percentages were normal in all
patients while the percentage of T helper and T cytotoxic cells showed a significant
decrease in the severely affected patients as compared to mild contaminated group.
Also the CD4+/CD25+ cells in the most severely affected patients were statistically
increased in comparison with the mildly and moderately affected groups (Shaker
et al. 2003). Therefore, in their opinion, 10 years after exposure to SM, the immune
system of the veterans is still impaired.
Molecular Components
The longterm effects of SM on molecular components of immune system of veter-

ans 1620 years after exposure was completely studied by Mahmoudi and col-
leagues. Serum levels of IgA, IgG, IgM, IgE, and complement components C3 and
C4 were measured. Other factor included plasma protein electrophoresis. In this
study, serum IgA, IgE, and C4 did not show any significant changes in comparison
to control, whereas IgM and C3 concentrations were significantly higher in veter-
ans. Regarding plasma protein analysis, the serum absolute levels of 1-globulin,
2-globulin and -globulin in veterans were higher than control. The albumin/globu-
lin ratio of patients was also elevated. They concluded that these protein changes
might be related to the acute phase response to frequent infections (Table 10.4)
(Mahmoudi et al. 2005).
Table 10.4 Serum SM-exposed

immunoglobulins, proteins Parameter Controls veterans
and complement levels of the
Total protein (g/l) 76.7 3.82 79.1 5.08*
veterans and control subjects
1620 after exposure Albumin (%) 55.2 2.74 52.4 4.96*
(Mahmoudi et al. 2005) Albumin (g/l) 41.9 1.94 40.4 6.75
IgA (mg/dl) 233.1 59.3 302.6 142.1
IgG (mg/dl) 1140.0 244.2 1438.6 486.1
IgM (mg/dl) 136.8 58.3 235.3 84.4***
IgE (IU) 86.5 146.3 92.4 112.1
C3 (mg/dl) 90.9 14.8 109.8 30.1*
C4 (mg/dl) 35.5 15.4 31.1 11.6
1 Globulin (%) 2.96 1.87 2.99 1.48
1 Globulin (g/l) 2.24 1.43 2.36 1.19*
2 Globulin (%) 8.95 1.04 11.1 2.1***
2 Globulin (g/l) 6.82 0.86 8.75 1.75***
Globulin (%) 14.0 2.07 15.4 2.80*
Globulin (g/l) 10.7 1.17 12.1 2.50**
Globulin (%) 18.9 2.40 17.9 3.37
Globulin (g/l) 14.5 2.60 14.3 3.31
Albumin/ 1.23 0.14 1.12 0.22*
Globulin (%)
Data shown are in terms of mean SD. Value is signifi-
cantly different vs. control at *p < 0.05, **p < 0.01 or
***p < 0.001
Through the immune system, cytokines are key regulators of immune cell func-
tion and differentiation; thus, disturbances in concentrations of these regulators are
probably related to various human diseases/pathologies (Rothlein et al. 1986). In a
cohort study performed by Ghasemi et al on 370 veterans who had been exposed to
SM 20 years before, serum and tear IL-8 levels in all exposed people showed a sig-
nificant decrease (Ghasemi et al. 2012). Pourfarzam and colleagues in another study
showed that the serum levels of IL-8 and IL-6 in SM exposed veterans significantly
decreased compared to the control group (Pourfarzam et al. 2009). On the contrary,
Attaran et al determined serum levels of IL-6 of veterans with pulmonary complica-
tions of SM poisoning and found it to be higher than that of control groups (Attaran
et al. 2010). Finally, they proposed that elevation of IL-6 in patients might be associ-
ated with airflow limitation. In a cohort study in Sardasht, Iran, Yaraee et al mea-
sured the serum level of pro-inflammatory cytokines and observed that these
mediators were significantly lower in the SM-exposed group. There was also a sig-
nificant positive correlation between level of all measured cytokines (TNF, IL-1,
IL-1 and IL-1Ra). In this study it was suggested that the decrease of these pro-
inflammatory cytokines had better be considered in diagnosis and therapeutic mea-
sures chosen to improve clinical complications (Yaraee et al. 2009).
In a study serum cytokines profiles (including IL-1, -2, -4, -5, -6, -8, -10, and 12,
IFN, TNF, TNF, ICAM-1, and sVCAM-1) of Forty-four male Iranian veterans
who had been exposed to SM >23 years earlier have been comprehensively investi-
gated (Riahi-Zanjani et al. 2014). The results of this study indicated that serum lev-
els of ICAM-1 were significantly higher in the samples from SM-exposed veterans
versus control. On the other hand, serum IL-1, IL-8 levels and TNF, were signifi-
cantly lower for the veterans than the controls. Levels of other assayed cytokines,
e.g., IL-2, -4, -5, -6, -10, and -12, IFN, TNF, and sVCAM-1 did not significantly
differ as compared to control (Table 10.5).
In the case of the SM veterans in Riahi-Zanjani et al. study, since the patients had
a sedentary lifestyle due to low levels of daily physical activity and the fact that they
were often suffering from ongoing medical complications present, it was expected
to be observed higher level of all the determined inflammatory cytokines. However,
surprisingly, it was seen that instead of being elevated, serum levels of IL-1, IL-8
and TNF in the veterans were significantly lower than those in the control subjects
and all the other measured proteins were not significantly different from the con-
trols. As mentioned earlier, inflammatory cells are particularly sensitive to oxidative
damage because of the high level of polyunsaturated fatty acids in their surfaces and
their high generation of reactive oxygen species (ROS), which contribute to injury
(Riahi et al. 2011). As a result, SM might affect immune responses such as
inflammatory cytokine secretion. Finally, they proposed that the sulfonium ion
formed from SM can alkylate the cellular DNA in cytokine producing cells resulting
in cellular oxidative stress and that this gives rise to the observed reductions in
IL-1, IL-8 levels and TNF. However, this kind of very selective targeting of cyto-
kine genes would be highly unusual; thus, further studies are required to examine
this hypothesis. irrespective to how the decreases in IL-1, IL-8 and TNF concen-
trations evolve, each of these cytokines plays an essential role in innate immunity
and can act on cells (such as lymphocytes) to give improvement to acquired immune
responses (Sims and Smith 2010). These low levels of IL-1, IL-8 and TNF could
Table 10.5 Comparison of SM-exposed

serum cytokine profiles of Parameters Controls veterans
SM-exposed patients vs
sICAM-1 (ng/ml) 710.15 20.00 772.81 15.14*
control subjects (Riahi-
Zanjani et al. 2014) sVCAM-1 (ng/ml) 653.00 227.75 856.72 181.10
IFN- (pg/ml) 23.44 10.41 21.72 8.06
TNF- (pg/ml) 5.53 0.12 4.48 0.09*
TNF- (pg/ml) 14.76 2.90 10.95 1.70
IL-1 (pg/ml) 4.30 0.21 3.78 0.10*
IL-2 (pg/ml) 7.01 0.32 6.35 0.24
IL-4 (pg/ml) 10.52 1.78 11.50 1.89
IL-5 (pg/ml) 6.92 1.91 4.67 0.31
IL-6 (pg/ml) 3.00 0.94 2.38 0.24
IL-8 (pg/ml) 84.59 20.29 21.04 6.08*
IL-10 (pg/ml) 118.43 35.45 117.24 29.74
IL-12 (pg/ml) 1.77 0.29 2.71 0.79
Data shown are in terms of mean SE. Value is significantly
different vs. control at *p < 0.05
likely reflect upon dysfunctions in the innate immune systems and disrupted
acquired immunity in the SM-exposed veterans. Such disruptions would, in turn,
result in an elevated risk of infection/development of certain cancers; epidemiologic
studies of what has become to an increasing extent apparent among SM-exposed
veterans support this concept (Balali-Mood and Hefazi 2006; Balali-Mood et al.
2008, 2011; Hefazi et al. 2006).
10.4 Conclusions
Evidence from experimental (animal) studies confirms that SM induces adverse

effects on immune system functions. Animal models are most precious in the
investigation of the physiological and molecular mechanisms involved in SM
hematological and immunological effects. However, results obtained from experi-
mental studies cannot be used alone to confirm or to refuse association between
SM exposure and delayed disorders, nor can they be used to assess exactly the size
of the effects in humans. On the other hand, clinical observations in humans pro-
vide the most direct evidence of the immunologic effects of mustard agents. Of
course, the data presented here shows that clinical studies as a whole support a
close synchrony between animal and clinical observations regarding the immuno-
suppressive properties of SM.
Finally, the immunological complications of the patients discussed here could
likely reflect the dysfunctions in the immune systems (both cellular and molecular
components) and disrupted innate and acquired immunity in the SM-exposed veter-
ans. Such disruptions would, in turn, result in an increased risk of infection/develop-
ment of certain cancers. As the toxic effects of SM are progressive and the clinical
outcome of veterans can worsen over time, developing additional therapeutic strate-
gies is needed. Some of these strategies might be based on immunopotentiating
interventions.
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Prepared for the Office of Scientific Research and Development
Chapter 11
Psychiatric Complications of Sulfur
Mustard (SM) Poisoning
Mohammad Reza Fayyazi Bordbar, Farhad Faridhosseini, and Ali Saghebi
Contents
11.1 CNS and Peripheral Complications of Patients with Delayed
Complications of Sulphur Mustard Poisoning ............................................................. 292
11.1.1 Headache ........................................................................................................ 293
11.1.2 Neuropathy ..................................................................................................... 293
11.1.3 Fatigue ............................................................................................................ 294
11.1.4 Impaired Memory and Concentration............................................................. 294
11.1.5 Seizure ............................................................................................................ 295
11.1.6 Other Neurological Symptoms ....................................................................... 295
11.2 Posttraumatic Stress Disorder ...................................................................................... 296
11.2.1 Treatment ........................................................................................................ 298
11.3 Depression and Anxiety ............................................................................................... 300
11.3.1 Depression (Definition and Symptoms) ......................................................... 300
11.3.2 Relationship between Life Events, Trauma, and Depression ......................... 301
11.3.3 Anxiety (Definition and Typology) ................................................................ 302
11.3.4 Relationship between Traumatic Stress, Anxiety, and Depression ................. 302
11.3.5 War, Depression and Anxiety ......................................................................... 303
11.3.6 Treatment ........................................................................................................ 305
11.4 Sexual Dysfunction ...................................................................................................... 307
11.5 Sleep Disorders ............................................................................................................ 308
11.5.1 Sleep-Wake Disturbance ................................................................................. 308
11.6 The Impact of SM Injury on the Quality of Life and Mental Health ........................... 309
References ............................................................................................................................... 311
Abstract Study of Sulfur Mustard (SM) effects on the central nervous system and
its neuro-psychiatric complications have been proved difficult to deal with. In the
First World War, and the Iran-Iraq war SM was extensively used and its medical and
psychological complications can still be observed in the veterans years later. We
M.R. Fayyazi Bordbar (*) F. Faridhosseini A. Saghebi

Department of Psychiatry, Psychiatry and Behavioral Sciences Research Center,
e-mail: fayyazimr@mums.ac.ir; drfayyazibordbar@gmail.com; faridhoseinif@mums.ac.ir;
farhad.faridh@gmail.com; saghebial@mums.ac.ir; ali.saghebi@gmail.com

292 M.R. Fayyazi Bordbar et al.
have tried to include the findings of all available research literature regarding the
neuro-psychiatric complications of SM.
The popular neurological complications attributed to SM exposure include:
headache, fatigue, chronic neuropathy, impaired memory and concentration, and
seizure. Post-traumatic stress disorder (PTSD) is one of the most common psychi-
atric disorders due to combat experience, especially chemical warfare agents (CWA)
including SM. Its clinical features, and pharmacological and psychological treat-
ments are covered in this chapter.
Other psychiatric complications commonly observed in the victims of SM expo-
sure are: depression, anxiety, sleep disorders and sexual dysfunctions. Clinical fea-
tures and treatment options of each disorder, in addition to their incidence and
prevalence rates are discussed.
The last but not the least are the changes in quality of life of these patients due to
chronic medical and psychological complications of SM exposure.
All in all, its safe to say that the neuro-psychiatric complications are serious and
quite common outcome of SM exposure and need specific clinical attention.
Keywords PTSD Anxiety Neuropathy Headache Depression Sulfur mus-

tard Poisoning Psychological complications
11.1 CNS and Peripheral Complications of Patients

with Delayed Complications of Sulphur Mustard
Poisoning
Delayed neurotoxic complications of chemical warfare agents (CWA), such as sul-

phur mustard (SM), in human beings have not been investigated in detail, due to
methodological limitations for confirmation and attribution of potential neurologi-
cal complications years after the exposure to SM. Even some researches that have
studied and reported SM-induced psychological disorders put that they did not
observe neurological complications in chronic poisonings (Balali-Mood and
Navaeian 1986). However, neurological manifestations such as cholinergic syn-
dromes and CNS depression are prominent following nerve agent exposure (Balali-
Mood 1992; Balali-Mood 2008). Nerve agents have a much higher mortality rate
than blistering agents and SM. Animal studies have shown that severe exposure to
SM could affect CNS and cause seizure in the animal (Anslow and Houk 1946). Yet,
examining the effects of SM on CNS in human beings is associated with certain
difficulties. For example, SM exposure dose cannot be measured exactly in combat
environments, since this factor is influential in causing neurological complications.
Although there are some definitions such as severe chronic complications including
skin blisters for assessing severity, they are not always applicable in battle condi-
tions as the exposure dose is not always measurable; especially, when the soldiers
have to stay in the exposed area for a long time. There are several other factors
affecting the severity of complications, such as temperature, humidity, wind
11 Psychiatric Complications of Sulfur Mustard (SM) Poisoning 293
direction, personal protective equipment, and activity level of the soldier (Balali-
Mood and Hefazi 2006; Perrotta 1996). Since, all veterans of Iran-Iraq war were
poisoned with SM via inhalation; influence of the other routes of entry (Newmark
2007; Lotti and Moretto 1999) on neurological system of the soldiers cannot be
assessed.
Chronic effects of SM exposure have been investigated in several studies. However,
a number of reports have mentioned different complications, the most important and
prevalent neurological complications include: headache, fatigue, chronic neuropathy,
paramnesia, impaired concentration, dizziness, tremor, and seizures.
11.1.1 Headache
In a study on SM-exposed patients in Iran, headache was reported as the most preva-
lent neuropsychiatric manifestation (71 %) (Parchami 1994). Most headaches pres-
ent with heaviness and pressure in the temporal, vertex, and sometimes frontal
areas.
These headaches are precipitated and aggravated by physical and psychological
stresses, which can be another manifestation of their psychogenic origin. On aver-
age, headaches last 12 h, and even for several hours in severe cases, and dont
respond to common pain relievers and even more specific treatments. Sometimes,
frontal headaches were associated with severe ocular pains, which could be because
of paranasal sinuses complications secondary to nasal congestion caused by mucosa
inflammation in patients suffering from persistent rhinitis. Darchini reported 83.7 %
rate of headache in 43 SM-exposed patients (2227 years after the exposure)
(Darchini-Maragheh et al. 2012).
Some studies with a larger sample size have not reported a high prevalence of
headache in these patients; for example, Namazi et al. in a research on 134 exposed
patients reported it only as high as 26.86 % (Namazi et al. 2009).
Uncertainty in the rate of headache in SM-exposed veterans increases in studies,
where the SM-exposed and non-exposed veterans are compared. In an investigation,
Parchami compared 75 SM-exposed with 105 none-exposed soldiers, with similar
demographic information and comparable front line service. The rate of headache
about 45 years after the service at the front were 51.4 % and 58 %, respectively,
indicating no significant difference (Parchami 1994); thus, attribution of this symp-
tom to SM-exposure should be carefully interpreted.
11.1.2 Neuropathy
A highly investigated nervous system complication is neuropathy caused by expo-

sure to SM. In a study on 43 Iranian veterans (2227 years after exposure to SM or
tabun) impairments such as paresthesia (88.3 %), hyperesthesia (72.1 %) and hypo-
esthesia (11.6 %) were reported (Darchini-Maragheh et al. 2012).
Balali and Hefazi (2005) reported 77.5 % peripheral neuropathy in 44 SM-exposed

patients with more sensory than motor nerve dysfunctions. The patients underwent
electro-physiologic investigations through electromyography (EMG). In addition
Nerve Conduction Velocity (NCV) was done to provide a better explanation for
their neuropathy. EMG and NCV findings showed an abnormal pattern in 7 patients
(16.3 %) in Darchinis study (Darchini-Maragheh et al. 2012). All detected NCV-
disrupted patterns were of axonal type, observed in both upper and lower extremi-
ties. Holisaz reported that 5 out of 100 chemical warfare victims were suffering
from axonal neuropathy (Holisaz 2006).
Some neurologic reflexes of SM-exposed patients are impaired. In a study, pal-
momental reflex (25.5 %), Babinski reflex (18.6 %), and Glabella reflex (13.9 %)
were reported (Darchini-Maragheh et al. 2012).
In addition, cranial nerve disorders were reported in 50 % of the patients.
Olfactory (53.4 %) and auditory (41.9 %) nerves were the most involved ones. The
direct, significant relationship between the patients cranial nerves and age is worth
noting (Darchini-Maragheh et al. 2012).
11.1.3 Fatigue
Darchini, et al. reported chronic fatigue as the most common objective complication
(93 %). It is interesting that based on this study, fatigue was more prevalent in
patients of lower age at the time of exposure; in that, there was a significant inverse
correlation between fatigue and age at the time of exposure (p = 0.008) (Darchini-
Maragheh et al. 2012).
In a study by Parchami in 1994 on patients with a single exposure to SM (28
years after the accident), fatigue and lethargy were the second most prevalent neu-
ropsychiatric symptoms (69 %) (Parchami 1994). It seems that over time, conse-
quences of SM-exposure increase the feeling of fatigue.
Fatigue and lethargy in such patients generally do not disrupt their daily life, and
mostly include lassitude and easy fatigability. Although in some of the victims,
fatigue may be on of the patients chief complaints, or cause severe inability in per-
forming normal activities (Parchami 1994). Fatigue can also be a part of patients
psychiatric symptoms including depression or anxiety.
11.1.4 Impaired Memory and Concentration
Darchini, et al. reported the frequency of impaired memory and concentration to

be 65.1 % and 58.1 %, respectively, in 43 SM and tabun-exposed patients
(Darchini-Maragheh et al. 2012). Balali and Hafezi also reported the same rate of
impairment in memory and concentration (Balali-Mood and Hefazi 2005). Studies
with larger sample size, as Pages, reported two significant complications of

impaired concentration and sleep disturbances after CWA exposure in a telephone
survey of 4022 military volunteers. Page observed these complications in 30.2 %
of the subjects (Page 2003). Parchami reported general memory impairment in up
to 27.7 % of the patients 45 years after exposure to SM. According to him, all
types of memory problems ranging from learning and retention to short-term and
long-term memory impairments, simultaneously or individually, may be observed
based on the intensity of the exposure. He reported that the complaint about this
problem was more frequent in the victims who wanted to study or continue their
education (Parchami 1994). It is interesting that comparison of amnesia between
SM-exposed veterans and non-exposed ones showed that the second group was
suffering from memory problems at least two times the first group, which again
suggests that this symptom cannot necessarily be attributed to exposure to SM
(Parchami 1994).
11.1.5 Seizure
Although studies suggest that severe exposure to SM causes seizure in animal

(Parchami 1994), it happens rarely in human exposure. Darchini, et al. in a study on
43 patients observed a history of generalized tonic-clinic seizure only in two of
them (4.6 %). Both patients had normal EGGs at the time of the experiment (2227
years after the exposure to SM) (Darchini-Maragheh et al. 2012). Namazi, et al. in
a study on 134 patients reported the frequency of epilepsy to be 16.42 % (Namazi
et al. 2009). Parchami observed epilepsy in 7 % of the exposed patients, with only
one case of abnormal EGG. He found symptoms such as myoclonus, fibrillation,
and fasciculation in 12 % of the patients (Parchami 1994).
11.1.6 Other Neurological Symptoms
Vertigo is relatively a common complication. Namazi et al. reported 11.94 % preva-

lence of vertigo in 134 patients (Namazi et al. 2009). Parchami observed it in 20.8 %
of the SM-exposed patients (Parchami 1994). This difference in prevalence may be
due to the difference in the exposure-study time interval (Namazi et al. investigated
the subjects, on average, 20 years after exposure, whereas Parchami studied the
patients 45 years after the exposure). Differences in methodology, sample types,
and severity of exposure may also play a role in that regard.
Another important complication is tremor. Namazi, et al. (2009) and Parchami
(1994) also reported the incidence of this problem in 4.68 % and 22.2 % of the
patients, respectively. Similar to vertigo, it seems that the frequency of this problem
decreases over time.
11.2 Posttraumatic Stress Disorder
The most common psychiatric disorder attributed to exposure to severe traumatic

events such as war and combat is post-traumatic stress disorder (PTSD). It was first
described during the civil war and was named soldiers heart. Since then it has
been described under various names such as irritable heart, effort syndrome, combat
stress reaction, and Persian Gulf War syndrome (Sadock et al. 2014). It affects
approximately 9 % of the general population and up to 30 % of individuals who
have experienced combat. It seems to develop more frequently in women when
exposed to comparable traumatic events (Sadock et al. 2014).
There are some risk factors predisposing an individual to PTSD such as: being
female, neuroticism, past history of prior trauma (especially in childhood), past his-
tory of PTSD, depression, or anxiety disorders, comorbid Axis II disorders (espe-
cially borderline, paranoid, dependent, or antisocial personality disorder), family
history of mood, anxiety, or substance abuse disorders, disrupted parental attach-
ments, severity of exposure to trauma, lower social support, and lower intelligence
quotient (IQ). High premorbid intelligence may be protective against PTSD (Hales
et al. 2008; Sadock et al. 2014; McNally 2009).
The biological models proposed for PTSD include: limbic hyperactivity and cor-
tical hypo-responsivity to traumatic stimuli, dysfunction of the hypothalamic-
pituitary-adrenal axis, noradrenergic dysregulation, abnormality in endogenous the
opioid system, sensitized serotonergic system, and reduced hippocampal volume
(Hales et al. 2008; Sadock et al. 2014; McNally 2009; Ehlers 2003).
The clinical diagnostic features of PTSD are categorized in four domains accord-
ing to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5). The first domain is intrusive symptoms, which include recurrent, involun-
tary, and intrusive recollections, dreams, dissociative flashbacks, psychological dis-
tress, and physiological reactions related to the traumatic event. The second domain
is avoidance of stimuli associated with the traumatic event, whether internal (mem-
ories, thoughts, or feelings) or external (people, places, conversations, activities,
objects, situations). The third one is negative alterations in cognitions and mood.
This includes Inability to remember an important aspect of the traumatic event,
negative beliefs or expectations about oneself, others, or the world, or about the
cause or consequences of the traumatic event, persistent negative emotional state,
diminished interest in significant activities, feelings of detachment or estrangement
from others, and a persistent inability to experience positive emotions. The last
domain is marked alterations in arousal and reactivity, including irritable behavior
and angry outbursts, reckless or self-destructive behavior, hypervigilance, exagger-
ated startle response, problems with concentration, and sleep disturbance (American
Psychiatric Association 2013).
It can also be accompanied by dissociative symptoms such as derealization and
depersonalization, auditory pseudo-hallucinations (such as hearing ones thoughts
spoken), as well as paranoid ideation (American Psychiatric Association 2013).
Survivor guilt (guilt about having survived or not having prevented the trau-
matic experience, or about what one had to do in order to survive) is another feature
commonly seen in combat-related PTSD. Other probable accompanying symptoms
include: depression, anxiety, panic attacks, feelings of rejection, humiliation, shame,
and rage, and emotional numbness. Prolonged episodes of intense affect or explo-
sive, hostile and impulsive behavior may also happen (Hales et al. 2008; Sadock
et al. 2014; McNally 2009; Ehlers 2003).
Other conditions complicating PTSD may include substance abuse, self-injurious
behavior and suicide attempts, and impaired occupational or interpersonal function
(Hales et al. 2008).
PTSD has a varied range of clinical presentation. In some people re-experiencing,
emotional and behavioral symptoms predominate. In others anhedonia, dysphoric
mood and negative cognitions are the major source of distress. Still in others, symp-
toms of arousal or dissociative symptoms may be prominent (American Psychiatric
Association 2013). Comorbidity of PTSD with depression and anxiety disorders
(such as OCD, panic disorder and phobia) is rather the rule than the exception, and
it is associated with higher impairment in functioning. PTSD can have a delayed
expression, even years after the traumatic events, but may be overlooked or ignored.
The disorders, most commonly considered in the differential diagnosis of PTSD
include: acute stress disorder, major depression, adjustment disorder, panic disor-
der, generalized anxiety, disorders with intrusive thoughts and perceptual distur-
bances (e.g. obsessive compulsive disorder, schizophrenia), agoraphobia, specific
phobia, adjustment disorder, borderline personality disorder, dissociative disorders,
factitious disorders, and malingering (Hales et al. 2008; Sadock et al. 2014; McNally
2009; Ehlers 2003).
The majority of patients develop PTSD symptoms immediately after the trau-
matic event. Delayed onset is found in 11 % of the cases. Of those with initial
PTSD symptoms, about 50 % will recover during the first year. Almost one third
of those with PTSD have a chronic course. In 80 % of the cases, PTSD lasts lon-
ger than 3 months, in 75 % longer than 6 months, and in 50 % for 2 years dura-
tion. The average time for PTSD remission is 36 months for those who seek help
for any mental health problem (not necessarily for PTSD), and about 64 months
for those who never seek help. A minority can remain symptomatic for years or
decades. Predictors of worse outcome include: female sex, being very young or
very old, greater number or severity of PTSD symptoms, slow onset of the symp-
toms, longer duration of the symptoms (more than 6 months), higher numbing or
hyperarousal to stressors, history of childhood trauma, poor premorbid function-
ing, poor social supports, and comorbid psychiatric, medical, or substance-
related disorders (Hales et al. 2008; Sadock et al. 2014; McNally 2009; Ehlers
2003).
Toxic exposure to sulfur mustard almost always happens in combat and war
conditions and might significantly increase the likelihood of PTSD development in
the exposed individuals up to 4060 % (Falahati et al. 2010; Mohaghegh-Motlagh
et al. 2012; Hashemian et al. 2006; Schnurr et al. 1996, 2000). It leads to reduced
physical health, higher rates of chronic illness and disability (which is quite com-
mon in individuals exposed to sulfur mustard), greater functional impairment, and
higher likelihood of health care supports (Schnurr et al. 2000).
Individuals exposed to both high-intensity warfare and chemical weapons have
been shown to have higher rates of PTSD than those exposed to high-intensity war-
fare but not to chemical weapons (Hashemian et al. 2006).
Psychological reactions at the time of mustard gas exposure have a strong asso-
ciation with PTSD symptoms. Strong peritraumatic stress or dissociative reactions
may lead to psychobiological changes which may persist for decades in some
cases (Jankowski et al. 2004). Female gender, older age, non-volunteer status,
lower preparation for combat, witnessing trauma in others (especially loved ones),
prohibited disclosure of the experience, poor physical health, chronic illness and
disability, healthcare usage, and functional impairment are the main predisposing
factors for PTSD in individuals exposed to mustard gas (Schnurr et al. 2000;
Jankowski et al. 2004). The number of exposures to sulfur mustard can also predict
lifetime PTSD. Academic education in the individual or his/her spouse can be a
protective factor against PTSD in individuals exposed to mustard gas (Karami
et al. 2013).
11.2.1 Treatment
11.2.1.1 Pharmacotherapy
Selective Serotonin Reuptake Inhibitors (SSRIs) are recommended as first-line

medication treatment for PTSD, as they are well tolerated and have a favorable side
effect profile, require once daily dosing, have documented efficacy in all four PTSD
symptom clusters, are effective treatments for psychiatric disorders that are fre-
quently comorbid with PTSD, and may help with clinical symptoms (such as sui-
cidal, impulsive, and aggressive behaviors) that often complicate management of
PTSD. Sertraline and paroxetine have been approved by FDA for the treatment of
PTSD. Other SSRIs have also shown to have similar efficacy.
Venlafaxine, mirtazapine, and bupropion have also comparable efficacy to
SSRIs.
Tricyclic Antidepressants (TCAs) (especially imipramine (Tofranil) and amitrip-
tyline) have shown modest results. Mono Amino Oxidaze Inhibitors (MAOIs) may
be superior to TCAs, especially for intrusive symptoms. (Phenelzine seems to have
good effects on re-experiencing symptoms and insomnia.)
Other medications can be tried when adequate response is not achieved with the
first-line options or for the additional treatment of specific PTSD symptoms or
comorbid disorders. Benzodiazepines may be useful in reducing anxiety and
improving sleep. Anticonvulsant medications (divalproex, carbamazepine, topira-
mate, lamotrigine) may have benefit for treating symptoms related to re-experiencing
the trauma. Second-generation antipsychotic medications (olanzapine, quetiapine,
risperidone) may be helpful for chronic treatment-resistant PTSD with disorganized

behavior and for those with comorbid psychotic symptoms.
Anti-adrenergic agents (propranolol, clonidine, prazosin) can be used for imme-
diate treatment in the emergency medical setting as a secondary prevention for
chronic PTSD. Prazosin is also used for nightmares and daytime intrusions. Lithium
can lead to improvement in intrusive symptoms and irritability. Triiodothyronine
can also lead to improvements, possibly due to its antidepressant response (Hales
et al. 2008). Trazodone and diphenhydramine are used for the sleep disturbance,
which is a common distressing complaint in PTSD patients. Several studies have
shown some benefit for buspirone. Combinations of medications might be neces-
sary for patients with more severe symptoms and with complex patterns of comor-
bidity. Transcranial Magnetic Stimulation (TMS) might have marked but transient
efficacy in decreasing core PTSD symptoms (Ursano et al. 2004; Sadock et al. 2014;
Ehlers 2003; Gabbard 2007).
11.2.1.2 Psychosocial Interventions
Some form of psychotherapy is generally necessary in the treatment of

PTSD. Cognitive and Behavioral Therapies (CBT) are the mainstream of psycho-
logical treatments for PTSD patients. These therapies include: graded exposure
(imaginal and/or in vivo), imagery rehearsal, prolonged exposure techniques, vir-
tual reality exposure, self-monitoring of intrusive symptoms, cognitive reprocess-
ing, anxiety management (stress inoculation), affect management, eye movement
desensitization and reprocessing (EMDR), relaxation techniques, and progressive
muscle relaxation. Both exposure programs (exposure to traumatic memories,
exposure to avoided stimuli associated with those memories, or both) and anxiety/
stress management techniques have been shown to be effective in reducing PTSD
symptoms. However, some studies indicate that although anxiety management
techniques are effective more rapidly, the results of exposure programs are larger
and last longer.
Psychodynamic psychotherapy might also be helpful for some PTSD
patients. Psychological debriefing is very beneficial if delivered soon after the
accident. Psycho-education and support for the patient and his/her family has
also a major role in the management of PTSD. Hypnosis can be useful in elicit-
ing traumatic memories and managing associated painful affects. Anger man-
agement programs are effective in reducing anger in patients with severe anger
reactions.
Present-centered and trauma-focused group therapies and support groups have
also proved to be highly beneficial for PTSD patients. Family therapy can help sus-
tain a marriage through periods of exacerbation.
Finally, it is worth noting that some evidence seems to suggest that civilian PTSD
is more responsive to treatment (especially exposure techniques) than is PTSD
among chronically ill war veterans (Ursano et al. 2004; Hales et al. 2008; Sadock
et al. 2014; McNally 2009; Ehlers 2003; Gabbard 2007).
11.3 Depression and Anxiety
Exposure to chemical gases is a major traumatic incident that can risk the patients
mental health in short-term and long-term, and cause permanent psychological
problems. Anyone who has been severely exposed to chemical agents, experiences
feelings of helplessness, anxiety, and decreased perceived safety (Hashemian et al.
2006). The subsequent chronic physical complications pose more challenges to the
patient, make him prone to psychological symptoms and disorders, and affect his
quality of life. Apart from PTSD and neuropsychiatric impairments discussed
above, depression, anxiety, sexual, and sleep disorders, as well as long-term impact
of SM injuries on quality of life have been investigated in some studies. In the fol-
lowing sections, the symptoms, disorders, and therapeutic methods will be dis-
cussed in the summary.
11.3.1 Depression (Definition and Symptoms)
Depression, as a symptom, is a state defined by depressed mood and lack of interest

in usual activities. It can affect ones thoughts, behaviors, and sense of well-being.
Life looks terrible to anyone who suffers from depression, making it challenging,
overwhelming (Comer 2010). Such conditions can normally be transient, especially
in the face of adverse events of life. Depression becomes clinical, requiring attention
and treatment, when lasting persistently, or causing considerable disruption to the
patients function (Sadock et al. 2014). According to Diagnostic and Statistical
Manual of Mental Disorders, Fifth Edition (DSM 5), depression can be diagnosed
only if it inhibits ones ability to function nearly every day for a period of at least 2
weeks (American Psychiatric Association 2013). Furthermore, depression is regarded
as a clinical syndrome in need of treatment when in addition to feeling depressed, it
is associated with other symptoms in thinking, motivational, behavioral, and emo-
tional areas, as well as physiological signs, each of which can be hard to bear.
In term of thinking, patients have negative self-view, regarding themselves
incompetent, despicable, and lower than others. They may be preoccupied with, or
ruminate over, thoughts and feelings of death, worthlessness, and inappropriate
guilt (Comer 2010; Sadock et al. 2014; APA 2013). It is hard for them to focus on
tasks and they are incapable of solving everyday problems. They mostly have a
pessimistic world view and feel incapable of changing the situation (Comer 2010).
In terms of motivation, they are no longer interested in their everyday activities.
They lose their initiative and spontaneity so that they have to force themselves to
go to work, socialize with relatives and friends and engage in pleasurable activi-
ties such as going to parties, eating meals and having sex. Therefore, they become
behaviorally inactive, isolated, and inefficient (Comer 2010). Other unpleasant
emotions such as anxiety, anger, irritability and feelings of emptiness are preva-
lent in these patients (Sadock et al. 2014). Other painful symptoms of clinical
depression include changes in sleep patterns and appetite and unexplained medi-
cal symptoms (e.g., dyspepsia, headache, constipation, and pain). Patients usually
complain of anorexia and insomnia, but sometimes they have increased appetite
and hypersomnia (APA 2013).
Depression syndrome is considered as a main feature in a class of mental ill-
nesses known as mood disorders or affective disorders. According to DSM 5, major
depression is diagnosed only if the patient has one or more depressive episodes with
5 or more of the criteria mentioned earlier, for more than 2 weeks and does not have
a history of manic episodes (which in many cases is exactly the opposite of depres-
sive state). If a patient suffers for more than 2 years from some depressive symp-
toms which are usually less severe than major depression (less than 5 criteria), he/
she is diagnosed with dysthymic disorder (APA 2013). However, clinical depression
syndrome is seen along with other psychiatric disorders including anxiety disorders,
PTSD, and stress-related disorders (Vieweg et al. 2006). In addition, depression
occurs due to a reaction to losses, grief and stressful life events (either everyday
stressful events or traumatic events) (Kendler et al. 1999), and also because of phys-
ical illness (most of the chronic and incurable medical conditions such as neurologi-
cal disorders, cancer, and diabetes) (Rustad et al. 2011). It can worsen the underlying
disease process or delay recovery from grief and stressors.
11.3.2 Relationship between Life Events, Trauma,

and Depression
Traumatic and stressful accidents are associated with the onset (or recurrence) of
depression (Kendler et al. 1999). This association is relatively causal-comparative.
There is also a complex two-way interaction between an individuals biologic,
genetic, and personality traits with environmental stressful events. In fact, these
patients may be more vulnerable to the stresses of life, due to genetic predisposi-
tions and family history, and deficiencies in adaptive skills may further expose them
to high risk environments (Kendler et al. 1999). Yet, stress plays a major role in
triggering depression. About 6070 % of the patients with depression have experi-
enced stressful events in their life 6 months to 1 year before the onset of clinical
depressive symptoms; although, in chronic cases and future recurrence, the role of
negative events fades (Kessing and Bukh 2013). Yet, negative and stressful circum-
stances prolong the symptoms of depression, making them chronic (Hardy and
Gorwood 1993). Over the years, the role of negative life events in depression does
not disappear completely while there is a doseresponse relationship between stress
and depression (Horinouchi and Nagayama 2001). In long-term studies on chemi-
cally injured patients, depression has been reported in many cases. It seems that
exposure to chemical gases, as a traumatic incident, plays a role in the onset of the
symptoms of depression. Furthermore, long-term problems of these patients, such
as marital conflicts, health complications, different medical diseases, lack of social
support, and financial and occupational problems may prolong the symptoms of
depression. The majority of these studies includes long-term follow-up (20 years on
average) after the exposure to SM, and has assessed only the symptoms of depres-
sion. The onset of the symptoms of depression and the underlying psychiatric con-
dition (i.e. major depression, anxiety disorders, PTSD, or chronic diseases from
which the majority of patients suffer) are not determined.
11.3.3 Anxiety (Definition and Typology)
Anxiety as a state of mind is defined as a negative emotion in which a person feels

an uncertain danger in the future (in contrast to fear which is experienced in the
presence of danger). The anxious person feels tense and edgy (Comer 2010), living
in a state of inner turmoil. Like other negative emotions, anxiety may have cogni-
tive, behavioral, and physiological manifestations. Worry, rumination and lack of
concentration are common symptoms of anxiety. Nervous behaviors such as nail
biting, rocking backward and forward, and restlessness can be the signs of anxiety.
Among the physical symptoms of anxiety are rapid breathing, palpitations and mus-
cular tension. Anxiety can act as an adaptive response to prepare an individual
against threats, but if it grows out of portion, or become severe and persistent, it is
considered a clinical syndrome (Sadock et al. 2014). The most prevalent patho-
logic clinical anxiety is generalized anxiety disorder. In this disorder, the person
feels excessive worry about everyday affairs such as occupational and financial
issues, as well as safety of self and beloved ones, which is very difficult to control.
This concern interferes with the individuals work and performance, and hinders the
persons concentration (Bitran et al. 2012). In addition to these signs, patients suffer
from a wide range of physical symptoms: shaky hands, shoulder pain, tension head-
aches, chest tightness, irritability, dysphagia, nausea, diarrhea, sweating, dry mouth,
fatigue, and urinary frequency (Bitran et al. 2012). These symptoms are typically
observed in stress related disorders such as PTSD and depression (Henningsen et al.
2003). Physical symptoms affect the patients sense of well-being and quality of
life, and lead them to an excessive and improper use of healthcare system (Beard
et al. 2010).
11.3.4 Relationship between Traumatic Stress, Anxiety,

and Depression
Many patients with anxiety disorders simultaneously suffer from another type of
anxiety disorder as well. Comorbidity of PTSD with other anxiety disorders and
depression is very common (Vieweg et al. 2006; Ginzburg et al. 2010; Rojas et al.
2014). The symptoms of anxiety disorders and depression highly overlap,
especially in veterans. In Ginzburg et al.s study (2010), 7480 % of the veterans

had comorbidities of depression, anxiety, or both, respectively (Ginzburg et al.
2010). Anxiety disorders, like other psychiatric syndromes, have multi-factorial
reasons. Exposure of a person with genetic/personality predispositions to environ-
mental stresses may lead to anxiety disorders. Studies have shown that people who
live in threatening conditions (poverty, war, economic insecurity, and political sup-
pression) are more prone to symptoms such as tension, uneasiness, intensified
startle-reactions, and sleep disorders which are common signs of an anxiety disor-
der (Comer 2010). Extended exposure to stressors develops sensitivity and lowers
the threshold of physiological responses to stressful factors, making the person
prone to anxiety disorders. Disruption of diurnal rhythms of sleep and wakefulness
aggravates the course of secondary anxiety disorders (Greenwood et al. 2014).
According to ODonnell et al. (2004), post-traumatic psychopathology is a set of
mixed anxiety, depression, and PTSD symptoms, at least 3 months after the trau-
matic injury. He has conceptualized it as general traumatic stress including a
combination of PTSD, depression, and anxiety symptoms. Comorbidity of these
three types of symptoms signifies them as a single diagnostic class, rather than sepa-
rate disorders (ODonnell et al. 2004).
11.3.5 War, Depression and Anxiety
War is one of the most evident stressors that cause mortality and long-term physical
and psychological complications in the military and among civilians. Change in the
form of wars, as well as the use of new weapons leads to novel impacts and compli-
cations in human societies. Iran-Iraq war (19801988) is specifically important due
to the extended use of different weapons such as chemical agents against Iranian
armed forces and civilians (Hashemian et al. 2006). Thus, all studies have been car-
ried out on Iranian soldiers, as well as citizens of Iranian border towns and Halabja,
a city in Iraqi Kurdistan, which were attacked by Iraqi SM chemical bombs.
The study on 1428 Iranian veterans neuropsychiatric complications, 39 years
after being exposed to SM, showed that the prevalence of psychiatric symptoms,
among them was still high in long-term (Balali-Mood et al. 2008). In addition to
anxiety (15 %) and depression (46 %), disorders such as personality, conversion,
and psychosis (3 %) have also been reported (Razavi et al. 2012). General symp-
toms such as weakness, decreased energy, decreased concentration, loss of libido,
somatoform complaints, neurasthenia, and increased sensitivity to sensory inputs
were observed (Balali-Mood et al. 2008), some of which are justifiable with psychi-
atric disorders, especially PTSD.
In a cross-sectional randomized survey by Hashemian et al. (2006), in addition
to the prevalence of PTSD, severe anxiety (65 %) and depression (41 %) symptoms
were also reported. In this study, three cities, chemically attacked (Onaviya, Robat,
and Sardasht) were investigated. The results were assessed using the Beck
Depression Inventory and Hamiltons Anxiety Rating Scale. In this research, 134
chemically injured citizens were studied. Those exposed to chemical weapons suf-
fered from severe anxiety and depression symptoms more than those with non-
chemical war trauma; suggesting that being chemically exposed is an independent
risk factor for depression (OR = 7.2), and anxiety (OR = 14.6), excluding PTSD
(Hashemian et al. 2006).
A cross-sectional study was conducted on all the victims (1336 cases whose files
are available) of the border city Sardasht, which was attacked by Iraqi chemical
weapons (SM). 15 subjects along with 154 healthy residents of Sardasht were ran-
domly selected and compared. To assess the rate of stress, accompanied with
depression and anxiety symptoms, DASS-42 was employed. All three variables
were higher in SM exposed victims than the general population 20 years after the
war. A high rate of the victims (79.3 %) suffered from severe depression and anxi-
ety. Although, in this study the type of anxiety disorder was not investigated sepa-
rately, the symptoms are similar to those of generalized anxiety disorder. In addition,
there was no control group, including non-chemically injured veterans. However,
since the control group was selected from the local residents of Sardasht, they had
the history of war and air strike traumas. Finally, the authors concluded that the dif-
ference in the prevalence of common psychological disorders (depression and anxi-
ety) could be attributed to the impact of direct exposure to chemical agents.
Nevertheless, finding a typical causal relationship, and estimating dos-response
relationship is not possible, due to the nature of retrospective studies (Ahmadi et al.
2010). Furthermore, the effect of physical diseases in veterans, which may be asso-
ciated with psychological consequences, could not be excluded from the study,
despite the lack of correlation between the percentage of injury and scores of stress
and anxiety.
A retrospective cohort study was conducted in Direh, an Iranian border village,
on 460 villagers who had been exposed to chemical bombs, using Becks Depression
Inventory and Hamiltons Anxiety Rating Scale. Results suggested that 95.4 % of
the subjects (29.5 % in the control group) had anxiety symptoms, mostly with mod-
erate severity, indicating a correlation with the level of disability due to chemical
injuries. Moreover, 98.7 % of the subjects (59.1 % in the control group) exhibited
depressive symptoms. This difference was more significant at higher levels of
depression, and like anxiety had a direct correlation with the severity of depression
(Falahati et al. 2010). Although, these people were exposed to small doses of SM (in
comparison to the residents of Sardasht who were severely exposed, due to the
bombing attacks), they had more mental-health problems than the control group
who had also experienced the psychological trauma of war. Therefore, it seems that
exposure to chemical gases, including SM, even to a little degree, can have additive
impacts in endangering the mental health of the exposed victims. The more
SM-induced disabilities and medical problems increase, the more mental disabili-
ties and problems will develop.
In some studies, chemically and non-chemically injured veterans have been
examined. For example, Vafaee and Seidy in a study of 100 veterans out of which
31 were exposed to chemical agents (mostly MS), assessed their depression with
Zhung Depression scale, and concluded that its prevalence in chemical victims was
higher (92 %) than that of non-chemical victims (57 %) and normal population. In
addition, severity of depression was higher in chemical victims (Vafaee and Seidy
2004). This study investigates the incidence of depression among the Iran-Iraq war
victims, some with a history of chemical weapon exposure, drawing a post-hoc
comparison between the two groups. However, in a more recent study by Motlagh
et al., no difference was seen between the two groups in prevalence of depression
and anxiety (Mohaghegh-Motlagh et al. 2012). contrasting the majority of previous
studies. Most studies have shown that exposure to chemical attacks is considered as
a more severe trauma, posing further psychological risk to the victims. Moreover, it
seems that following the trauma caused by chemical attacks, primarily PTSD symp-
toms will develop in the victims; depression and anxiety are the secondary conse-
quence of long-term PTSD, and MS-induced disabilities and medical problems
(Mohaghegh-Motlagh et al. 2012).
11.3.6 Treatment
So far, no study has been conducted specifically on the treatment of psychiatric

disorders, including depression, anxiety and PTSD in people injured by chemical
agents, specifically sulfur mustard (MS). The treatment of these individuals seems
to pursue two objectives: First: improvement of mental health and physical-mental
security of the patients through treatment of psychiatric symptoms up to the point of
remission (symptom reduction of at least 50 %), along with the treatment and man-
agement of other morbidities associated with the trauma (including complication of
other organs such as the eyes, skin and lungs). Second: improvement of patients
coping strategies, quality of life and return to a psychological status that indicates a
sense of security and trust (Vieweg et al. 2006). Committing suicide is one of the
risks that threaten patients with depression. Veterans and soldiers returning from
war who suffer from PTSD are at a high risk of committing suicide (Pompili et al.
2013). In a retrospective study conducted by Tavallaie et al., on veterans causes of
deaths of (1463 cases), 70 cases (4.9 %) were due to suicide, more common in indi-
viduals younger than 40 years (Tavallaie et al. 2006). Frequent visits and psychiatric
monitoring, complete treatment of psychiatric disorders, resolving financial prob-
lems, increasing the level of social supports, and treatment of medical problems of
chemical victims can be effective in lowering the suicide risk (Tavallaie et al. 2006).
11.3.6.1 Pharmacotherapy
Depression and anxiety in these patients could be treated by medication, psycho-

therapy or a combination of both. Drug therapy can be used to reduce the symptoms
(the first objective), especially when the patient suffers from chronic depression,
and has severe symptoms, suicidal ideation, and psychiatric or medical comorbidi-
ties (which may undermine active participation in psychotherapy) (National Institute
for Health and Clinical Excellence 2009) As noted earlier in the literature review,
many veterans and patients injured by mustard gas meet some of the mentioned
conditions; therefore use of medication seems reasonable for them. Pharmacotherapy
of these patients should take into consideration the medical comorbidities and con-
current medications. Medications should not have complications that exacerbate the
medical conditions of patients or have adverse interactions with the patients other
medications. On the other hand, given the high comorbidity of anxiety, depression
and PTSD in these patients, medications that are effective in all three conditions can
be used.
Selective Serotonin Reuptake Inhibitor antidepressants (fluoxetine, sertraline,
paroxetine, citalopram and escitalopram) are the first-line treatments for depres-
sion and anxiety because of their safety and a favorable side effect profile (Lam
et al. 2009; Baldwin et al. 2012); and there is more evidence in favor of their
impacts on PTSD. In fact, they are now the first line treatment for this disorder
as well (Berger et al. 2009). Selective Serotonin-Norepinephrine Reuptake
Inhibitors (such as venlafaxine or duloxetine) are also appropriate medications
effective in controlling symptoms of both anxiety and depression (Dunlop and
Davis 2008). In this class of medications, withdrawal symptoms occurring due to
abrupt discontinuation or missed doses, hypertension at higher doses, and risk of
drug-drug interactions (Paroxetine and fluoxetine also share this problem.)
should be taken into account (National Institute for Health and Clinical
Excellence 2011). Other classes of antidepressants, including tricyclic antide-
pressants (e.g., amitriptyline, imipramine, and nortriptyline) can also be used.
But they are not considered as the first-line treatment because of their adverse
side effects (anticholinergic properties, orthostatic hypotension) and are usually
reserved for treatment-resistant cases or due to intolerance of the side effects of
other medications (Baldwin et al. 2014).
Benzodiazepines are effective in the acute treatment of anxiety disorders and
can be used in cases where depression is associated with overt symptoms of anxi-
ety (Dunlop and Davis 2008). But due to risk of tolerance, dependence, and abuse,
the adverse effects on cognitive processing, risk of paradoxical disinhibition, and
increased risk of aggression, particularly in patients with PTSD (which, as was
noted, it is more the rule than the exception in chemical agent victims), their use
should be limited and prescribed under the strict control of specialist (Vieweg
et al. 2006; Baldwin et al. 2014). In chemical agent victims suffering from psy-
chological problems and psychiatric comorbidities, failure in remission of symp-
toms and the presence of residual symptoms after remission with one of the
recommended medications is possible. In these cases, physicians should consider
augmenting treatment and the use of combination therapy. For the treatment of
refractory depression and PTSD patients, we can add atypical antipsychotics such
as olanzapine, quetiapine, and risperidone (Schaffer et al. 2012). However, in pre-
scribing these drugs, the risk of weight gain and metabolic syndrome should be
considered (Sadock et al. 2014). The use of anticonvulsants has been recom-
mended as well; lamotrigine for depression, gabapentin and pregabalin for gener-
alized anxiety, and carbamazepine and topiramate for PTSD (NICE 2011; Schaffer
et al. 2012; Baldwin et al. 2014.
11.3.6.2 Psychotherapy
The ultimate goal of psychotherapy is to increase adaptive ways of thinking and pat-
terns of behavior which can lead to a better adjustment with the stresses of life (physi-
cal, social or family) in victims of the chemical attacks. Psychotherapy and psycho-social
interventions are used alone (for mild cases) or in combination with pharmacotherapy.
Addition of psychotherapy to the treatments of patients with multiple psychiatric
comorbidities and those who have not responded to medical treatment adequately will
lead to improved outcome and will be effective in relapse prevention (Parikh et al.
2009). There is broad clinical and research consensus that among various psychothera-
peutic approaches Cognitive Behavioral Therapy (CBT) is effective in treating depres-
sion and anxiety disorders (Schaffer et al. 2012). An important point in Iranian victims
of chemical attacks is the key role of spirituality (especially patriotism and religious
beliefs) in better coping of these patients with mental-physical outcomes of mustard
gas poisoning (Ebadi et al. 2009; SiratiNir et al. 2013). This should be considered by
therapists who work with victims of chemical attacks in Islamic Iranian culture.
Applying and strengthening these spiritual values in the course of therapy, help these
patients adapt more effectively to the challenges of life (SiratiNir et al. 2013).
11.4 Sexual Dysfunction
Another condition reported in SM-exposed patients, which has been studied in a

small number of studies is sexual dysfunction. It should be noted that it is not clear
whether these disorders are primary and should be considered as a separate disorder
or they are secondary and related to PTSD, depression, anxiety, relational problems,
medical and psychiatric medications, and neuropathic complications of SM. The
result of the study by Ranjbar et al., on 185 SM-injured veterans showed that 65.4 %
of these patients suffer from sexual dysfunction. Erectile problems and decreased
libido or sexual desire were more prevalent (49.2 and 48.6 % respectively). Patients
were assessed based on a researcher- made questionnaire developed according to
the DSM-IV-TR criteria. Its face validity was confirmed by a psychiatrist and an
urologist (Balali-Mood et al. 2008). In a study conducted by Balali et al., decreased
sexual desire and impaired sexual function was reported in 52 % and 9 % of patients,
respectively (Tabatabaee 1988). Furthermore, SM exposure can cause reproductive
system complications such as oligospermia (Balali-Mood et al. 2005).
11.5 Sleep Disorders
One of the most common symptoms of psychiatric disorders, particularly depres-

sion, anxiety and stress-related disorders is the sleep complaints including primary
insomnia (difficulty in falling asleep), interrupted sleep or early morning awakening.
People with these symptoms may develop hypersomnia or their sleep-wake rhythm
may be disrupted. In the study by Parchami (1994) sleep disturbances in SM exposed
patients were studied separately, rather than in the context of other psychiatric disor-
ders. In this study, 93 SM injured individuals suffering from bronchiolitis obliterans
were assessed in terms of their sleep quality using the Pittsburgh Sleep Quality
Index. Results reflected the poor quality of sleep in these patients indicating the need
for special attention to this problem (Tavallaie et al. 2006). Impaired sleep quality
subscales included the time to fall asleep, subjective quality of sleep, usage of hyp-
notic drugs, and morning dysfunction. However, this study was cross-sectional-
descriptive with no comparison with the control group (Tavallaie et al. 2006).
11.5.1 Sleep-Wake Disturbance
Another condition afflicting victims of chemical warfare is sleep-wake Disturbance.

It might present as initial, middle or terminal insomnia, impaired quality of sleep,
breathing-related sleep disorders, or parasomnias (which are disorders character-
ized by abnormal behavioral, experiential, or physiological events occurring in
association with sleep, specific sleep stages, or sleep-wake transitions).
The factors contributing to this condition include:
The medical complications of the SM poisoning (including breathing-related
conditions, painful conditions, or any other condition which can cause a signifi-
cant distress for the person)
The psychiatric disorders caused by the incident can be accompanied by prob-
lems in the sleep.
Sleep disorders directly caused by the deleterious effects of the SM poisoning on
the central nervous system
It must be noted that the sleep disorder caused by the underlying medical or psychi-
atric condition can affect the prognosis of the causative condition negatively as well.
In a study by Parchami (1994), prevalence of insomnia was 61 %, compared to
51 % in the control group (which included victims of war, not exposed to chemical
agents). Madarshahian and Hassanabadi also found similar results in their study
(2009). This is comparable to results of the study by Parchami (1994) which reported
a prevalence of 64 % for insomnia in victims of SM poisoning.
The quality of sleep was impaired in 94 % of chemical warfare victims, com-
pared to 60 % of healthy controls (Tavallaie et al. 2006). Similar results have been
shown in other studies as well (Tavallaie et al. 2005). The impairment in the quality
of sleep is correlated with the general health level of chemical warfare victims
(Abbasi et al. 2012).
As for the treatment of the sleep disorders, the first line of treatment is adequate
sleep hygiene training and treatment of underlying medical/psychiatric conditions
contributing to the sleep disorder.
If these measures fail to work, one might consider the use o sedative-hypnotic
medications.
Extra care should be exercised to rule out breathing-related sleep disorders
before prescribing a hypnotic medication, because it can worsen the sleep problem
and in some cases it can lead to fatal results.
11.6 The Impact of SM Injury on the Quality of Life

and Mental Health
Quality of life (QOL) is the perceived quality of various aspects of daily living
(especially those aspects that are important from the individuals perspective) and
feelings of well being and satisfaction (The WHOQOL group 1998). Health-Related
Quality Of Life (HRQOL) is the individuals perception of the impact of their health
status on the quality of life and sense of well being. That is, from their own perspec-
tive, what are the impacts of their physical illness, mental disorders or disabilities
on their quality of life, What is their assessment of their health status; regardless of
whether or not they are ill and the kind of disease they have (Hennessy et al. 1994).
Assessment of HRQOL can elucidate how diseases affect the mental experience of
individuals of their health status and their function, and facilitates the calculation of
the burden of disease. It also provides the information necessary for policy making
in the health care system; as the ultimate goal of all treatments, medical interven-
tions and health care system policies is to make society members feel good, increase
their level of satisfaction and improve their function (Guyatt et al. 1989).
A number of studies on the victims of chemical gases in Iran-Iraq war (mostly
sulfur mustard gas) have studied the long-term impact of injury on quality of life,
and mental health (in general, not in specific psychiatric disorders). The long-term
effects of sulfur mustard injuries, including cutaneous, ocular, respiratory, endo-
crine, immunological and digestive effects can cause long-term negative impacts
on the quality of life, in terms of both physical, health-related and psychosocial
aspects (Biat Saeed et al. 2014). The negative impacts of physical disability in
social, family and occupational roles and performing daily tasks is significant in
these patients (Ghaedi et al. 2012). There are various tools for measuring HRQOL,
but SF-36 has been mostly used in the studies conducted on SM injured patients. It
is a 36-item self-report questionnaire that measures eight distinct areas of physical
function, physical role function, social function, pain, emotional role function,
vitality, general health and mental health (Deborah 2009). Another measurement
tool used in these studies to assess the mental health and prevalence of psychiatric
symptoms in this group of patients is the Symptom Check List 90-Revised (SCL-
90-R). This test has 90 questions to assess psychiatric symptoms that are reported
by the client. It was first designed to show the psychological aspects of the physi-
cal illness. This test measures a broad range of psychological problems and symp-
toms. It includes 9 primary symptom dimensions and three global distress indices.
The symptom dimensions include the following: somatization, obsession, interper-
sonal sensitivity, depression, anxiety, hostility, phobia, paranoia and psychoticism.
The three general indices include: Global Severity Index, Positive Symptom
Distress Index, and the Positive Symptom Total (Prinz et al. 2013).
In a descriptive study of 149 Iranian war veterans with severe SM induced eye
injuries, their mental health was examined by SCL-90-R and Global Severity Index
(GSI). The results showed that these patients have lower scores in psychological
health than the average of Iranian community. Their somatization, obsession, anxi-
ety and depression scores were above the cutoff point of Iranian average population
(Ghaedi et al. 2012). Mousavi et al. (2009) assessed quality of life in veterans with
eye problems using the SF-36 questionnaire. The results of this study, like the previ-
ous ones, indicated that the overall quality of life scores in these patients is lower
than the general population, although an average of 21.6 years had passed since
exposure to mustard (Mousavi et al. 2009).
In their historical cohort study in 2013, Roshan et al. compared 367 individuals
exposed to sulfur mustard in Sardasht with 128 people that were not exposed to this
gas from the city of Rabat. In this study, SCL-90-R was used to assess the mental
health of individuals. The group exposed to SM had higher scores in somatization,
obsession, depression, anxiety and hostility compared with the control group.
Therefore, exposure to SM seems to lead to many psychological symptoms and
reduced level of mental health, even 20 years after exposure; (Roshan et al. 2013)
indicating the detrimental effects of sulfur mustard gas on physical and psychoso-
cial functioning of people exposed to this agent. There appears to be a significant
correlation between the severity of physical illness and mental disorders and dis-
ability in chemical gas victims (Riddle et al. 2003). Sulfur mustards devastating
impact on the mental health and quality of life, several years after the Iran-Iraq war,
has been confirmed in other studies as well (Karami et al. 2013; Biat Saeed et al.
2014). In a recent study published in 2014, Ebadi et al., examined quality of life in
chemical warfare veterans. The study was conducted on 242 patients with respira-
tory symptoms and lung injuries. The measuring tool was SF-36 and patients had
low scores in all dimensions. The lowest scores in SF-36 subscales were related to
role-physical and general health. The results of this study indicated that physical
symptoms and the number of organs involved have a significant correlation with
quality of life; in that with the higher number of organs are involved, the scores of
the quality of life are lower (Ebadi et al. 2014). Therefore, exposure to sulfur mus-
tard is associated with physical complications. That is, with an increase in severity
of symptoms and the number of organs involved (to more than one), the quality of
life of patients and their mental health are adversely affected.
Many studies have discussed neurologic and psychological complications of SM

exposure.
Headache is one of the most common central and peripheral nervous complica-
tions of SM, which has been reported in 2783 % of victims in different studies.
Several probable causes have been speculated for this symptom. Chronic neuropa-
thies such as paresthesia, hyperesthesia, and hyposthesia are also common in
these patients. Chronic fatigue is seen in up to 93 % of the victims; much more
than what can be explained by psychological complications such as depression.
Impaired memory and concentration is seen in these patients, but when compared
with other war veterans, SM exposure doesnt seem to play a major role in this
regard.
PTSD is one of the most common psychological complications in the victims of
SM exposure. It is not clinically different from PTSD patients due to other causes,
and it has similar pharmacological and psychological treatments.
Depression and anxiety have been reported in up to 65 % of veterans exposed to
SM. Many studies have considered exposure to chemical agents as an independent risk
factor for depression and anxiety, after having controlled for the symptoms of PTSD.
Sexual dysfunction is present in up to 65.4 % of the victims of SM exposure, the
most common of which are erectile problems and decreased desire. Sleep disorders,
especially changes in the sleep-wake cycle, also need due attention and proper
treatment.
The chronic medical complications of SM injury can lead to adverse effects on
the quality of life of these patients in physical and health-related as well as psycho-
social aspects. This has been documented in several studies.
The study of psychological and neurologic complications of SM exposure is dif-
ficult, has not been adequately done, and needs further research. However, the cur-
rent evidence indicates a high prevalence for these complications, and warrants
special attention of clinicians to psychological issues of these patients in addition to
their medical problems.
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Chapter 12
Genotoxicity, Teratogenicity and Mutagenicity
of Sulfur Mustard Poisoning
Effat Behravan and Mitra Asgharian Rezaee
Contents
12.1 Introduction .................................................................................................................. 318
12.2 Genotoxicity................................................................................................................. 319
12.2.1 Genotoxicity Tests .......................................................................................... 319
12.3 Genotoxicity of Sulfur Mustard ................................................................................... 320
12.3.1 Mechanisms of SM Genotoxicity ................................................................... 321
12.4 Mutagenicity of Sulfur Mustard................................................................................... 322
12.5 Carcinogenicity of Sulfur Mustard .............................................................................. 323
12.6 Teratogenicity of Sulfur Mustard ................................................................................. 324
12.7 Application of Laboratory Tests in Evaluation of Genotoxicity of Sulfur Mustard .... 324
12.7.1 Measurement of DNA Damage Induced by Sulfur Mustard .......................... 324
12.7.2 Evaluation of Proteins Involved in DNA Damage
Signalling in Sulfur Mustard Toxicity ............................................................ 325
12.7.3 Evaluation of Proteins Involved in DNA Repair
Signalling in Sulfur Mustard Toxicity ............................................................ 326
12.7.4 Measurement of Oxidative Stress in Sulfur Mustard Toxicity ....................... 326
12.7.5 Evaluation of Chromosomal Aberration in Sulfur Mustard Toxicity ............. 327
12.8 Report of the Results and Discussion .......................................................................... 328
12.9 Conclusions and Recommendations ............................................................................ 340
Glossary................................................................................................................................... 340
References ............................................................................................................................... 342
Abstract Sulfur Mustard (SM) or mustard gas is the most widely used chemical
weapons throughout the history. It has been used in World War 1 and recently in
Iran-Iraq conflict. Disabilities produced by SM are continuing problems and vari-
ous cancers as a consequence of SM exposure were reported. Different in vitro and
E. Behravan (*)
Medical Toxicology Research Center, School of Medicine, Mashhad
University of Medical Sciences, Mashhad, Iran
e-mail: effatbehravan@gmail.com
M.A. Rezaee
Department of Toxicology and Pharmacology, Faculty of Pharmacy,
Kerman University of Medical Sciences, Kerman, Iran
e-mail: rezaeem77@gmail.com
318 E. Behravan and M.A. Rezaee
in vivo studies showed DNA damage and mutations following sulfur mustard
exposure. These findings along with the other reported delayed complications as
cancer following SM toxicity, suggest instability in the genetic system. The most
accepted theory of SM toxicity is alkylation reactions with DNA, RNA and pro-
teins in the cell. DNA is the main target for SM toxicity and DNA cross links and
adducts constitute 15 % and 85 % of DNA damages respectively. Several studies
have documented the mutagenic effects of SM in mammalian cells, in vivo and
in vitro test systems. Measurement of DNA damage, measurement of proteins
involved in DNA damage and repair signalling, measurement of markers of oxida-
tive stress and evaluation of chromosomal aberration are among the most impor-
tant tests for evaluating of SM genotoxicity. There is no treatment for SM toxicity
yet, therefore, increasing our knowledge about the mechanisms of SM genotoxic-
ity, would help us better understanding about prevention and treatment of SM tox-
icity in human. Few studies are available regarding the reproductive effects of SM
in animals and humans and the results are controversial.
Keywords Sulfur Mustard Genotoxicity DNA damage Telomere

Chromosomal aberration
12.1 Introduction
Sulfur Mustard (SM) or mustard gas is the most widely used chemical weapons
throughout the history. It has been used in the World War 1 and recently in the Iran-
Iraq conflict. More than 300,000 of Iranians are still suffering from SM complica-
tion and about 4,000,000 veterans of the First World War showed a late complication
(Balali-Mood et al. 2005). SM is a very reactive and stable oily liquid, and is catego-
rized as highly toxic chemical agent. Different studies have shown that SM has
mutagenic, carcinogenic and antimitotic effects (Malhotra et al. 2013). Mustard
compounds have cytotoxic properties and a mustard analogue, Nitrogen Mustard,
has been used as an anti proliferative and anti cancer treatment (Saladi and Persaud
2005). SM in the format of oily liquid may persevere in the environment for decades.
There are still remaining site of wars where may contaminate and release SM many
years after initial usage (Munro et al. 1999). The main site of SM injury in cells is
nucleus and DNA is the most important target for SM toxicity.
After initial exposure the first symptoms occur in the eyes, skin and respiratory
system. Acute symptoms may take minutes to weeks after exposure. Delayed toxic
effects may take months or years after a single exposure and the main effective
organs are the respiratory system, skin, ophthalmic, immune system and neurologic
system (Balali-Mood and Hefazi 2006). Almost 25 years after the exposure to SM,
there are still evidences of its long-term complication in Iranian veterans which
need treatment. These findings along with the other reported delayed complications
following SM toxicity, suggest instability in the genetic system. There have been a
12 Genotoxicity, Teratogenicity and Mutagenicity of Sulfur Mustard Poisoning 319
lot of studies on clinical manifestations of SM toxicity in human. Also, molecular

and cellular mechanisms of SM toxicity have been discussed in vitro and in vivo and
different hypotheses have been proposed through in vitro studies on blood and
respiratory cell lines. But there is not enough information about SM genotoxicity
and its mechanisms in human. As there is no treatment for this toxicity yet, increas-
ing our knowledge about the mechanisms of SM genetic toxicity, would help us
better understanding about prevention and treatment of SM toxicity in human.
12.2 Genotoxicity
Genetic toxicology is a recently developed branch of toxicology and is defined as the

ability to interact with DNA and genetic materials (Brusick 1980). Genotoxins are
compounds that damage DNA direct or indirectly and result in repair, cell death or
mutation. Genotoxicants should be monitored carefully, as they cause mutations and
sometimes directly inducing cancers (Shugart and Theodorakis 1996). Mutagens
cause hereditary and permanent changes in the deoxyribonucleic acid (DNA)
sequences that are reserved in somatic cell divisions and germ cells and passed onto
future generations. All mutagens are genotoxic, but not all genotoxins are mutagenic.
Mutations are prevented by cell defence mechanisms, DNA repair or apoptosis; how-
ever, those damages which could not be fixed lead to mutation (Auerbach et al. 1947).
The molecular basis for genes is DNA which is packaged and organized in chro-
mosome. Chromosomes are structures of macromolecules consisting of DNA, pro-
tein and RNA (Saenger 1984). DNA molecules contain highly reactive groups and
are thus targeted to numerous attacks by internal and external genotoxic compounds
such as reactive oxygen species (ROS), metabolites and electrophiles every day
(Gregus and Klaassen 2001). These chemical compounds may impact genome
either directly through interaction with nucleotides or indirectly through effect on
DNA replication and DNA transcription. Free radicals, carbonium ions and episul-
fonium ions are amongst the electrophilic compounds which react directly with the
nucleophilic parts of DNA molecules (Williams and Weisburger 1991).
12.2.1 Genotoxicity Tests
Genotoxicity tests are defined as in vitro and in vivo tests designed by researchers
to detect toxic substances which induce genetic damage. The basis of these tests is
the evaluation of DNA damage and repair in cells exposed to toxic agents. DNA
single and double strand breaks, cross links, point mutations and chromosomal
aberrations are amongst DNA damage tests (Kornberg and Baker 1980) (Table 12.1).
Following genetic damage, cells undergo DNA repair mechanisms in the form of
gene mutation, recombination or chromosomal damage. Aneuploidy and larger
scale numerical chromosomal damage are of vital genetic changes and might have
Table 12.1 In vitro and In vitro assays

in vivo genotoxicity tests
Bacterial reverse mutation test (AMES test)
In vitro mammalian cell gene mutation test
Escherichia coli, reverse assay
In vitro mammalian chromosome aberration test
Sex-linked recessive lethal test in Drosophila
melanogaster
In vitro sister chromatid exchange assay in mammalian
cells
Comet assay
DNA damage and repair, unscheduled DNA synthesis in
mammalian cells in vitro
In vitro mammalian cell micronucleus test
HPRT (Hypoxanthine phosphoribosyltransferase) assay
Mouse lymphoma assay
In vivo assays
Mammalian erythrocyte micronucleus test
Mammalian bone marrow chromosome aberration test
Mammalian spermatogonial chromosome aberration test
Comet assay
Unscheduled DNA synthesis
been associated with malignancy (Weaver et al. 2007). Compounds that detect such
kind of damage and are positive in genotoxicity tests are considered to be potential
carcinogens (Lichtfouse et al. 2012). There is a confirmed relationship between
exposure to particular chemicals in humans and carcinogenesis. Genotoxicity tests
have been almost used for cancer prediction. Therefore the outcome of genotoxicity
tests can be valuable for the interpretation of carcinogenicity studies. Mutations are
usually associated with human diseases.
Genotoxicity tests are usually performed in bacterial, yeast, and mammalian
cells and the findings would help us to control and improve the cellular defense
against genotoxic substances (Kolle Susanne 2012).
12.3 Genotoxicity of Sulfur Mustard
Sulfur Mustard is regulated under the Chemical Weapons Convention (CWC)

among the classes of chemicals which monitored under the highest risk class
(Ganesan et al. 2010). Although sulfur mustard may be lethal in higher doses, it
usually causes extensive acute and chronic injuries in different organs. LC t50 (lethal
concentration-time product) of SM for humans is 900 mg-min/m3 for 210-min
exposures (NRC 1997). Sulfur Mustard (SM) and its analogs are of the first chemi-
cal agents which their genotoxic and mutagenic effects has been confirmed (Fox
and Scott 1980). SM is responsible for over 80 % of all chemical injuries which
have been reported and the most recent use of SM was in Iran-Iraq war (Chilcott
et al. 2000).
In vitro studies in prokaryotic organisms (Salmonella typhimurium and
Escherichia coli) and eukaryotic organisms (HeLa cells, mouse lymphoma, and rat
lymphosarcoma) are among the first studies which propose the genotoxicity of sul-
fur mustard. DNA cross-links formation, DNA alkylation, inhibition of DNA syn-
thesis and repair, point mutation, and chromosome aberration formation were
suggested mechanisms. Increasing the frequencies of chromosomal aberration in a
dose dependant manner and mutation induction in HPRT (hypoxanthine guanine
phosphoribosyl transferase) test are of the first genotoxic studies (Jostes et al. 1989).
Low doses of SM induce DNA cross links and thus replications and repair errors
in DNA, which may cause mutation (Papirmeister et al. 1991). In a study on rat
epidermal keratinocytes cultures exposed to SM, a dose related interstrand crosslink
of DNA has been confirmed. These cross links effects on DNA synthesis and
induces cell cycle block (Lin et al. 1996). Another study showed a mismatch repair
in DNA bases of monkey kidney cells following exposure to SM (Fan and Bernstein
1991). SM causes DNA alkylation in a bacteriophage and the most common sites of
DNA alkylation were on 5-AA, 5-GG, and 5-GNC sequences on the DNA tem-
plate strand. SM at the doses of 0.50.1 mM produced single strand breaks
(Venkateswaran et al. 1994).
In-vivo studies in Drosophila showed that SM injection caused point mutation in
male flies (Auerbach et al. 1947). Positive micronucleus test in mouse bone marrow
exposed to sulfur mustard was also evidence of SM genotoxicity (Ashby et al. 1991).
Ludlum exposed human white blood cells to labelled SM in vitro, and he measured a
SM DNA adduct 7- (2-hydroxyethylthioethyl) guanine in cell culture media (Ludlum
et al. 1994). Fishermen who were exposed to sulfur mustard shells, showed sister
chromatid exchanges in their lymphocytes (Wulf et al. 1985). Emison observed DNA
damage in human epithelial cell culture after exposure of the cells to SM. A cell cycle
block was found at the G1-S and G2-M phases at the concentrations of below and
equivalent of vesicating concentration of SM (100 M) (Emison and Smith 1996).
12.3.1 Mechanisms of SM Genotoxicity
The most accepted theory of SM toxicity is alkylation reactions with DNA, RNA
and proteins in cells. After absorption, SM comes in the form of an ionic intermedi-
ate, ethylene episulfonium. Ethylene episulfonium cation undergoes intramolecular
cyclisation and transforms to a very active carbonium ion. Carbonium ion rapidly
reacts with nucleophiles such as DNA and a large number of electron- rich mole-
cules such as sulfhydryl and amine groups of proteins and nucleic acids (Wheeler
1962).
SM induces DNA adducts and cross links between and inside DNA strands and
causes DNA breaks and inhibition of protein synthesis (Walker 1971). Thus, the
results are creating abnormal chromatids and inhibition of DNA, RNA and protein
synthesis. The main DNA alkylation occurs on the N7-position of guanine (Kehe
and Szinicz 2005).
Cross links and adducts constitute 15 % and 85 % of DNA damages respectively,
but the cytotoxicity of SM is related to cross links which prevent DNA replication
(Matijasevic et al. 2001). DNA damage by SM exposure activates poly (ADP-
ribose) polymerase-1 (PARP-1) and stimulates several DNA repair pathways,
including base excision repair, nucleotide excision repair, and homologous recom-
bination. If this genotoxic stress cannot be repaired, the cell will start the apoptotic
program (Jowsey et al. 2012).
DNA strand breaks activate DNA repair enzymes; especially poly ADP ribose
polymerase (PARP) and this reduce nicotinamide adenine dinucleotide (NAD)
resources in cells. ATP is also used for the synthesis of NAD and this caused a
reduction in the cellular pools of ATP and disruption in the supply of cell energy
(Lindahl 1979). Alkylation and inactivation of sulfhydryl-containing proteins and
peptides such as glutathione is the other mechanism of cell death. These proteins are
crucial in stabilizing the oxidation redox position of cells (Maynard 1995).
Other mustard analogs such as Nitrogen mustard and 2-chloro-ethylethylsulfide
(CEES), have shown the mutagenic and lethal effects in a number of studies (Fox
and Scott 1980) (Povirk and Shuker 1994).
12.4 Mutagenicity of Sulfur Mustard
Several studies have documented the mutagenic effects of SM in mammalian cells,

in vivo and in vitro test systems (Papirmeister et al. 1991). An aims assay on salmo-
nella with tester strains TA97, TA98, TA 100 and TA102 at the concentrations of
0.01250 g per plate of SM, was not able to show the mutagenic response by any
of the strains (Stewart et al. 1989). However, The mutagenic properties of mustard
compounds have been confirmed in other organisms including Ecoli and Neurospora
by Horowitz and Tatum (Horowitz et al. 1946; Tatum 1947). SM is mutagens in
diverse assays, including ames tests for germ cell mutations in drosophila and domi-
nant lethal in mice and in salmonella TA97a and TA102 strains (Vijayan et al. 2014).
Two major reasons of mutation induced by SM are point mutations (mis-
matched) and mutations in repair enzymes (mis-repair). DNA repair enzymes enter
a base in the damaged area and in front of alkylating purines, but if the base is incor-
rectly inserted, may cause errors during DNA replication and mutations. Mutation
in tumor suppressor genes or oncogenes, causes uncontrolled cell proliferation. For
example, mutations in p53 in Japanese workers at factories produced mustard gas
have been reported (Yanagida et al. 1988). In a survey on lung tumors of workers
who had worked in SM factory, p53 mutations were found which were the similar
to mutations in lung tumors of tobacco smokers except with the prominence of
double mutations in workers of SM factory (Takeshima et al. 1994).
Lung cancer biopsies from Iranian patients, who had a single exposure to SM
during Iran-Iraq conflict, have been analyzed. DNA was extracted from the tumor
tissue, PCR amplified and sequenced to detect p53 mutation. Eight p53 mutations
with two double p53 mutations have been observed and the dominant site of muta-
tions was G to A (Hosseini-khalili et al. 2009).
12.5 Carcinogenicity of Sulfur Mustard
SM is categorized as a carcinogen (IARC 1994) and several epidemiologic studies

provide sufficient evidence that SM is carcinogenic in humans, particularly in the
upper respiratory tract. Although there is not a doseresponse relationship in carci-
nogenicity of SM in human studies, laboratory studies have shown a relationship
between SM and respiratory cancers, skin cancers and leukemia (Pechuta and Rall
1993).
In a study, male and female strain A mice exposed to SM through breathing for
every 15 min showed a significantly higher incidence of lung cancers than their
controls with no SM exposure (Heston 1953). Another study on guinea pigs, mice,
rabbits, and dogs that were exposed to sulfur mustard in the air for 312 months did
not reveal any cancers except for the squamous cell carcinoma (SCC) in rats skin
(McNamara et al. 1975).
Occupational studies of Japanese and British workers, who manufactured SM,
have shown higher incidence of respiratory cancers compared to normal populations.
British factory workers who had manufactured SM, had a significant rate of death
from larynx, pharynx, lung, mouth, esophagus and stomach cancer compare with
death in the normal population (Easton et al. 1988). In Japanese workers, the number
of deaths from cancers of the respiratory tract were higher compared to fatality
expected from such cancers (37 vs. 0.9 respectively) (Wada et al. 1968). Another fol-
low up study in Japanese factory workers was performed 1720 years after 79 years
of exposure to SM. Of all the reported deaths, 28 % were because of cancers com-
pared with 7.7 and 8.5 % in two groups of unexposed residents of the same area. The
most common types of cancers were squamous cell carcinoma and small cell carci-
noma (Yamada 1963). Nishimoto Investigated 2068 Japanese factory workers.
Among the workers, those who had the highest SM exposure had three times more
deaths of cancers compared to the area male population (Nishimoto et al. 1983). The
same study was performed on German factory workers who manufactured SM. In a
20 year follow up, malignant bronchial carcinoma, leukemia and bladder carcinoma
were significantly increased (Weiss and Weiss 1975). An epidemiologic study of
World War 1 veterans who were exposed to SM was done 15 years after their expo-
sure. This study revealed that the number of deaths due to lung cancer was doubled
compared to controls (Case and Lea 1955). In another study on American veterans of
World War 1, the incidence of cancers of upper respiratory tract was slightly higher
than control (Beebe 1960). Nasopharynx carcinoma, bronchogenic carcinoma, gas-
tric adenocarcinoma, ALL (Acute lymphoblastic leukemia) and AML (Acute
myeloid leukemia) have been reported in chemically injured Iranian veterans with
SM (Ghanei and Vosoghi 2002). However Emad and Rezanian in a study of 197
Iranian veterans 10 years after acute SM toxicity in the Iran-Iraq conflict couldnt
show any upper respiratory tract malignancies (Emad and Rezaian 1997).
12.6 Teratogenicity of Sulfur Mustard
Few studies are available regarding the reproductive effects of SM in animals and
humans and the results are controversial. Intravenous injection of SM in male mice
causes a transient damage to the testes and inhibition of spermatogenesis with a full
recovery 4 weeks after exposure (Graef et al. 1948). Another study in mice who
were receiving SM intraperitoneal during the gestation period, different types of
birth defects, including craniofacial and septal defects as well as the limb malforma-
tions was observed (Sanjarmoosavi et al. 2012). SM exposure in rats who gavaged
by different doses of SM did not reveal any significant damage on fertility and
reproductive activities in two generations study (Sasser et al. 1996). In a study in
male rats, exposure to 0.1 mg/m3 of SM 5 days/week for up to 52 weeks signifi-
cantly increased the rate of lethal mutations in somatic and germ cells (9.4 % in SM
compared to 3.9 % in controls) (Rozmiarek et al. 1973).
In Iranian veterans with exposure to SM, in the first 5 weeks after exposure, the level
of testosterone has been decreased with an increase in FSH and LH, however all hor-
mones had returned to normal after 12 months. Of those veterans, (29 %) had decreased
sperm count below 20 million. In a testicular biopsy performed on 50 % of men with
sperm count below two million cells per ml., complete or relative arrest of spermato-
genesis was confirmed (Azizi et al. 1995). Another study on Iranian SM veterans 39
years after exposure also showed significant reduction in the number of sperms and
motility of sperms compared to healthy controls (Balali-Mood and Hefazi 2005). On
the other hand, in a 12 month survey following SM exposure in a group of SM exposed
veterans, the incidence of infertility was almost close to this number for a worldwide
average (Ghanei et al. 2004). Another study in Iranian SM veterans reported a signifi-
cant increase in the rate of fetal deaths and congenital malformations in children who
were borned after single exposure to SM compared to control (Pour-Jafari et al. 2011).
12.7 Application of Laboratory Tests in Evaluation

of Genotoxicity of Sulfur Mustard
12.7.1 Measurement of DNA Damage Induced by Sulfur

Mustard
The most important mechanism of SM pathogenesis is the reaction of SM with

DNA which creates DNA mono adducts or cross links (Ashby et al. 1991). DNA
mono adducts are thought to be more related to delayed genotoxicity of SM and
result to mutations in cells, which survive from SM toxicity (Jowsey et al. 2012).
Cross links induce DNA double strand breaks during DNA replication, and activate
DNA repair enzymes such as poly (ADP-Ribose) polymerase-1 (PARP-1)
(Papirmeister et al. 1991). Thymocytes which exposed to different concentrations
of SM over a period of 24 h, showed an increased level of DNA fragmentations
followed by laddering pattern suggesting apoptosis (Michaelson 2000). Another
study showed a dose-dependent increase in DNA damage in TK6 lymphoblastoid
cells incubated with a SM analogue, CEES (2-chloroethyl ethyl sulfide) (Jowsey
et al. 2009).
Comet assay is a rapid and sensitive test to detect DNA damage in vitro. A modi-
fied comet assay technique using DNA repair enzymes formamido-pyrimidine-gly-
cosylase (FPG), endoglycosylase III (ENDO III) and 3-methyladenine-glycosylase
(AAG) was able to show SM induced DNA damage in pigs skin cells. Repair
enzymes increase the sensitivity of the comet assay and are able to detect DNA
damage at the SM concentration of 30 nmol/L (Kehe et al. 2009). A study on Iranian
veterans using comet assay 20 years after exposure to SM showed DNA damage in
DNA lymphocytes, which was significantly higher than non SM exposed controls.
Mutations in DNA repair genes of the hematopoietic cells at the time of the initial
exposure are a possible explanation for the delayed DNA damage. The other sug-
gested mechanism of such finding is DNA damage due to a general inflammatory/
oxidative stress mechanism (Behravan et al. 2013).
12.7.2 Evaluation of Proteins Involved in DNA Damage

Signalling in Sulfur Mustard Toxicity
DNA damage signalling cascades orchestrates through ATM (ataxia telangectasia

mutated) and ATR (ataxia telangectasia related) protein kinases. These kinases
respond to different types of DNA damage. For example, ATM, is activated fol-
lowing DNA double strand breaks (DSB) while ATR is activated by different
types of DNA damage, including DNA cross links and adducts (Hurley and Bunz
2007). Checkpoint kinase 1 (Chk1) and Checkpoint kinase 2 (Chk2) regulate cell
functions such as DNA replication and cell cycle progression or apoptosis. There
are common substrates for both Chk1 and Chk2 and combination of these 2 mol-
ecules has been documented (McGowan 2002). Some Chk1 and Chk2 effectors
can be categorized as tumor promoter or tumor suppressor genes (Bartek and
Lukas 2003). Activated ATR and ATM, phosphorylated many target proteins,
including checkpoint kinases (Chk1, Chk2) and p53. Chk2 mainly activated by
ATM in response to DNA double strand breaks (DSBs). Chk1 is believed to be
associated with the ATR, however cross connection with ATM has also been seen
(Gatei et al. 2003).
P53 is a tumor suppressor protein and is activated following cellular stress, such
as DNA damage and hypoxia. This protein causes cell cycle arrest or apoptosis, to
inhibit malignant transformation of cancer cells. The lack of a normal p53 protein,
allows the mutations to accumulate and create a tumor(Ghosh et al. 2004). These
proteins have a vital role in preventing genetic lesions by slowing down the cell
cycle, regulating the transcription and increasing the power of DNA repair in cells
(Ljungman 2005). An in vitro study on lymphoblastiod cell line exposed to sulfur
mustard demonstrated the dose- and time-dependent activation of DNA damage
signalling pathways, in particular the phosphorylation of CHK1, CHK2 and p53
(Jowsey et al. 2012).
12.7.3 Evaluation of Proteins Involved in DNA Repair

Signalling in Sulfur Mustard Toxicity
To have a better understanding of sulfur mustard toxicity and to provide a treatment, we

should increase our knowledge about the mechanism that cells utilize to protect against
sulfur mustard damage. Simple DNA adducts and lesions caused by oxidative stress,
such as methylation are repaired by base excision repair pathway (BER). A DNA
repair enzyme known as PARP-1 (Poly (ADP-ribose) poly merase-1) plays a critical
role in the BER pathway. Cells lacking PARP-1 protein are very sensitive to chemicals
which induce DNA alkylation (Dantzer et al. 2000). Bulky DNA adducts, such as DNA
cross linking are repaired by NER (Nucleotide excision repair) (Jowsey et al. 2009).
Severe DNA damage induced by SM decreases NAD and cell repair restoration and
cellular ATP (Burkle 2001). While the small DNA lesions activates DNA repair path-
ways and caused DNA repair, severe DNA injuries caused cell apoptosis and cell death.
Rad proteins are important DNA repair checkpoints which arrest cell cycle pro-
gression at early stage of DNA damage. These proteins ensure the transmission of
undamaged genetic material to daughter cells (Abraham 2001). The DNA repair
signalling pathway was studied in mouse liver percutaneously exposed to SM. DNA
repair proteins Rad23, Rad50, Rad51, Rad52, and Rad54l were decreased during a
week after exposure and results indicated that SM promotes DNA double strand
breaks (DSB) which caused cell death(Anand et al. 2009). Incubation of TK6 lym-
phocytes with SM and studying DNA repair pathways showed that homologous
recombination (HR) is the major repair cascade protecting against acute SM toxic-
ity while NER has also positive effects in this pathway (Jowsey et al. 2012). A host
cell repair assay in Chinese hamster ovary cells showed that nucleotide excision
repair (NER) involves in repairing DNA damage caused by SM and decreases SM
toxicity (Matijasevic et al. 2001).
12.7.4 Measurement of Oxidative Stress in Sulfur Mustard

Toxicity
The cytotoxicity of SM has been proposed to result from a series of alkylation reac-
tions and production of reactive oxygen substances (ROS). After absorption of SM
into the body, it forms the highly reactive carbonium ion which reacts with DNA,
proteins and other molecules such as glutathione. Glutathione depletion increases

the level of ROS production (Kehe and Szinicz 2005). Also, ROS are changed into
highly toxic oxidants that cause membrane phospholipids to form lipid peroxides,
leading to loss of membrane function. Over stimulation of poly (ADP ribose) poly-
merase (PARP) following SM induced DNA damage also leads to consumption of
cell energy and generation of reactive oxygen species (Korkmaz et al. 2008). In a
study, antioxidant enzyme activities were measured 24 h after dermal exposure of
rat with SM. As a result of glutathione and NAD depletion, glutathione peroxidise
activity decreased significantly in white blood cells, spleen and liver (Husain et al.
1996). Another study in mice after 12 weeks of chronic exposure to SM showed
increased lipid peroxidation and reduced levels of antioxidant enzymes, glutathione
reductase and glutathione peroxidise (Sharma et al. 2009). In human acute SM tox-
icity induces oxidative stress and decreases the glutathione reserves (Balali-Mood
and Hefazi 2006).
There is a direct relationship between SM toxicity and oxidative stress.
Antioxidant therapy in the protection and treatment of SM poisoning has been pro-
posed previously. Various studies in laboratory animals have been shown the protec-
tive effects of antioxidants in SM toxicity (Gautam et al. 2007; Pohanka et al. 2011,
2013). Also, some studies have shown oxidative stress in Iranian veterans who were
exposed to SM and a significant decrease in the activities of some antioxidant
enzymes has been found (Shohrati et al. 2010).
12.7.5 Evaluation of Chromosomal Aberration in Sulfur

Mustard Toxicity
The incidence of chromosomal abnormalities caused by SM depends on the amount

of primary alkylation, deletion prior to DNA replication and cell repair capacity
after DNA replication. Cross linking of DNA induced by SM causes chromosomal
abnormalities and it is suggested that inter-strand cross links are more responsible
for these abnormalities compared to intrastrand cross links. DNA cross- linking
due to SM cause chromosomal aberration and although it is not observable until
mitosis, the exact damage has been induced during DNA replication (Papirmeister
et al. 1991). It has been seen that chromosomal damage in SM is dose dependent
and end G1 and early S phases of the cell cycle are the most sensitive sites to SM
damage (Savage and Breckon 1981). P53 protein has an important regulatory role in
cell cycle and genetic stability and mutations in the p53 gene has already been dis-
cussed in SM toxicity. P53 mutation is considered to be an important cause of aneu-
ploidy (Takeshima et al. 1994; Schmitt et al. 2002; Karami et al. 2007).
A significant increase in the incidence of sister chromatid exchanges has been
reported in the peripheral lymphocytes of fishermen who were exposed to SM (Wulf
et al. 1985). Rat lymphocyte cell line incubated with SM was examined for the
evaluation of chromosomal damage. DNA and RNA alkylation and chromosomal
aberration were found and the amount of damage was the same as chromosomal
damage due to X-irradiation (Scott et al. 1974a, b).
Another study was performed on Iranian chemical veterans, 7 years after expo-
sure to SM and it showed aneuploidy in the type of hyperdiploidy (22 of 27). All
patients were classified as severe disability due to SM injury (Hassan and Ebtekar
2002). The results of the same study in a similar group of Iranian veterans revealed
hyperdiploidy and Philadelphia chromosomes in bone marrow aspiration (Ghanei
and Vosoghi 2002).
There have been many research studies on in vitro, in vivo and clinical impacts
of sulfur mustard toxicity. In Tables 12.2, 12.3, 12.4, and 12.5 we reviewed them.
12.8 Report of the Results and Discussion
In summary, DNA alkylation, the well-known toxic mechanism of mustard com-

pounds, have been shown in numerous in vitro and in vivo studies. Inter-strand and
Intra-strand crosslinks, also are reported on SM toxicity (Walker 1971; Shahin et al.
2001; Jost et al. 2010).
7-(2-hydroxy-ethylthioethyl) guanine was detected as the abundant adduct,
accounted for 61 % of the total SM-DNA alkylation (Fidder et al. 1994; Ludlum
et al. 1994). SM and its derivatives induce DNA damage in a time and dose depen-
dent manner (Meier and Millard 1998; Lakshmana Rao et al. 1999; Steinritz et al.
2007). SM-DNA adduct was detectible 21 days after dermal exposure in experimen-
tal model (Batal et al. 2013). Whereas the results of a clinical study (Behravan et al.
2013) showed DNA damage in Iranian veterans even 25 years post SM exposure.
The mutagenic effects of SM and its analogues have been reported in several
studies. DNA alkylation in position of O-6 guanine and formation of
O6-ethylthioethylguanine have a main role in mutation induced by mustard com-
pounds (Ludlum et al. 1986). There are several reports indicating significant muta-
tions in the workers of poison gas factories or war veterans (Wulf et al. 1985;
Yanagida et al. 1988). Several studies have shown the increased risk of cancers in
cases of exposed-mustard gas. The upper respiratory tract and lung cancers fre-
quently have been reported in workers of poison gas factories and war veterans.
Furthermore adenocarcinomas of the stomach, acute myeloblastic and lymphoblas-
tic leukemia have been detected in Iranian veterans (Balali-Mood 2009). Mustard
gas has been classified as class 1 carcinogen compound by the International Agency
for Research on Cancer (IARC) since 1987.
The teratogenic effects of mustard compounds have been investigated in several
animal models. Some data represented the teratoginicity of SM and CEES (a SM
analogue), such as limb malformation, cleft and craniofacial defects and fetal death
in mice and rats (Sasser et al. 1993; Hassanzadeh-Nazarabadi et al. 2012;
Sanjarmoosavi et al. 2012). However, in another study following parental mustard
toxicity, no teratogenic effects were observed. These controversial results may be
Table 12.2 In vitro studies on the genotoxicity of SM and its analogues
Compound
(SM, NM, Concentration/
2CEES) Assay duration Cell line Results Ref.
SMa Ames test (salmonella/ SM: 10 and Salmonella strains Aminofostine analogs (chemical Vijayan et al.
microsome assay) 50 g/plate (TA97a, TA98, TA100, radioprotectors) decreased SM-induced (2014)
TA102, TA104) in the mutagenicity
presence and absence of
S9 mix
NMa Comet assay, NM: JB6 (mouse epidermal NM induced inter-strand cross-link, DNA Inturi et al. (2014)
immunofluorescence, 0.75 M/472 h cells) double strand break, decreased cell growth
confocal microscopy, and S-phase arrest
Western blot Homologous recombination repair (HRR)
showed as a key pathway involved in repair
of NM-induced DNA Double strand break
SM, CEESa Western blot SM: 0.11 M TK6 lymphoblastoid Homologous recombination (HR) was the Jowsey et al. (2012)
CEES: cells, Fibroblast cells major repair pathway protecting against the
100500 M (GM04312 & GM15876) acute SM toxicity with nucleotide excision
Chinese hamster ovary repair (NER) and non-homologous end
(CHO) cells joining (NHEJ) also contributing to cell
(EM9, EM9-XH, V-C8 survival
and V-C8 + B2) Dose and time-dependent activation of DNA
damage signalling pathways was shown after
SM exposure, in particular phosphorylation
of Chk1, Chk2a and p53
SM Western blot, SM: 1, 5, 20 or Hela, Chinese hamster DNA double strand breaks after SM exposure Jowsey et al. (2010)
12 Genotoxicity, Teratogenicity and Mutagenicity of Sulfur Mustard Poisoning
Immunofluorescence 100 M for 24 h ovary cells (V-C8, Cells lacking the homologous recombination
assay V-C8 + B2) and DNA repair (HR) pathway were more
lymphoblastoid cells sensitive to the SM toxicity
(TK6) Chemical activation of the HR protein offer
cellular protection against SM
329
(continued)
330
Compound
SM UV/V is spectroscopy, SM: Rat liver active (S1 and Unfolding of the chromatin was shown in Jafari et al. (2010)
Gel electrophoreses 251000 M S2) and inactive (P2) concentration <500 M of SM, at higher
chromatin concentrations condensation of chromatin
due to forming cross-links between the
chromatin components was detected
The incidence of condensation was higher in
S2 phase
SM Neutral red uptake SM: HeLa, A549, HepG2, The LC50 (0.2 M of SM) was associated Jost et al. (2010)
assay, XTT, Comet 0.12250 M AA8 with the lowest concentration that
assay DNA cross-links were found
The higher concentration (10 M) of SM
resulted in inhibiting the basal metabolism
CEES Western blotting, CEES: Lymphoblastoid cell CEES induced dose-dependent increase in Jowsey et al. (2009)
comet assay, DNA 0.21 mM lines (TK6, DK0064, DNA damage via induction of P53 and Chk2
adduct immunoassay LB707, LB708) phosphorylation
Also, the role of base excision repair (BER)
and nucleotide excision repair (NER)
pathways were shown in CEES-DNA
damage repair
SM & Bacterial and cell SM: Bacteria: The presence of a functional NERa pathway Matijasevic and
CEES survival, 50200 mM MV1161, wild type; increased survival and reduced mutagenesis Volkert (2007)
Host cell reactivation CEES: MV1273, uvrA6; whereas the presence of a functional BERa
assay 2001000 mM MV1174, alkA1; pathway reduced survival, increased
MV1302, alkA1 uvrA6 mutagenesis and decreased repair
Mammalian cells: Mouse
embryonic fibroblasts
(MEF); wild type and
3-alkyladenine DNA
E. Behravan and M.A. Rezaee
glycosylase null mutant

Compound
SM TUNELa, Western blot SM: Pulmonary A549 cells SM induced DNA fragmentation and Steinritz et al.
301000 mM dose- dependent increase in PARPa after 24 h (2007)
for 30 min Also, increased AChEa activity was detected
in SM-exposed cells
SM RT-PCRa, Western blot, SM: 100, 200 Primary human The activation of calmodulin, calcineurin and Simbulan-Rosenthal
Spectrofluorometry, and 300 M keratinocyte Bad during SM-induced apoptosis in et al. (2006)
Hoechst staining keratinocytes
SM Microarray 200 M for 2 h Human epidermal The transcriptional profile of SM compared Platteborze (2005)
keratinocytes with lewisite as a vesicant agent also with a
genotoxic agent (Cisplatin)
Apoptotic transcripts were found in Lewisite
but not in SM
SM, CEES Luciferase activity test SM: 10100 M Chinese hamster ovary NER-competent cells were more resistant to Matijasevic et al.
CEEM: cells (wild type and the toxic effects of SM and CEES, indicating (2001)
1001000 M nucleotide excision the role of NER in repairing DNA damage
repair (NER) deficient) also in decreasing their toxicity
SM Quantitative SM: 50500 M Human epidermal SM produced significantly higher levels of Shahin et al. (2001)
polymerase chain keratinocytes both total adducts and crosslink in genomic
reaction (QPCR) and DNA than mitochondrial DNA
southern hybridization DNA inter-strand crosslink introduced as the
critical lesion induced by bi-functional
alkylating agents
SM Agarose gel SM: Human peripheral blood Exposure to SM caused a time-dependent Meier and Millard
electrophoresis 0.01 M1000 lymphocytes shift from apoptosis to necrosis (from an (1998)
M/024 h oligonucleosome-sized DNA ladder
incubation characteristic of apoptotic cell death to a
broadband pattern characteristic of necrotic

cell death)
DNA fragmentation decreased when poly
ADP-ribose polymerase (PARP) inhibitors
were applied within 8 h of SM exposure
331
(continued)
332
Compound
SM Gel mobility shift SM: 40200 M Lac UV5 promoter DNA alkylation by SM preferably occured Masta et al. (1996)
assay at; 5-AA, 5-GG and 5-GNC sequences on
the DNA
SM GC-Massa SM: 131 M Human blood (DNA Identification of 7-(2-hydroxy-ethylthioethyl) Ludlum et al.
extracted from WBC guanine as the most abundant adduct, (1994)
after exposure) accounted for 61 % of the total alkylation
SM HPLCa analysis of [35 s] labeled of Human blood and calf N7-[2-[(2-hydroxyethyl)thio]ethyl]guanine Fidder et al. (1994)
adduct SM thymus DNA was detected as the abundant adduct
SM DNA alkylation and SM: Yeast Saccharomyces SM induced DNA alkylation independent to Kircher and
quantitative purine (0.022 mM) cerevisiae cell sensitivity Brendel (1983)
derivatives assay
SM Giemsa staining of the SM: Primary Syrian hamster The sharp peak of chromatic aberrations was Savage and
sites replicating DNA 0.05 M/20 min fibroblast shown 1216 h after SM-exposure Breckon (1981)
incubation
CEES Ames test CEES: 02 mM Escherichia coli (repair Identification of mutation sites following Gilbert et al. (1975)
deficient variants K12, CEES; alkylation at the N3 position of
B/r, B) adenine and the N7 position of guanine and
spontaneous depurination of these alkylated
bases
Activation of endonuclease II-polymerase I
excision-repair system reduced mutagenicity
and lethality of CEES
Compound
SM Radioactive labeling, SM: Rat lymphosarcoma cell SM- induced Chromosomal damage in Scott et al.
X-irradiation, 101000 ng/ml line (Yoshida), Mouse sensitive and resistance cell lines but less in (1974a, b)
Cytogenetic assay by lymphoma cell line the resistant cell line. There was no
Orcein staining (L5178Y) difference in capacity of DNA repair between
both cell lines after SM or X-ray
SM [35 s] labeled of SM SM: 6 g/ml E-coli (B/r and Bs-1 Inter-strand crosslink (ICL) following SM Venitt (1968)
strain) and repair of ICL in resistant strain (B/r)
a
Abbreviations: SM sulfur mustard, NM nitrogen mustard, HRR homologous recombination repair, CEES 2-chloro-ethylethylsulfide, NER nucleotide excision
repair, NHEJ non-homologous end joining, CHK1,2 Checkpoint kinase1,2, TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling, PARP Poly
ADP-ribose polymerase, AChE Acetylcholineestrase, RT-PCR reverse transcription-polymerase chain reaction, GC-MS gas chromatographymass spectrom-
etry, HPLC high performance liquid chromatography
333
Table 12.3 In vivo studies on the genotoxicity of SM and its analogues
334
Compound
CEES) Assay duration Exposure and species Results Ref.
SM Apoptosis assay (TUNEL) 2, 6 and 60 mg/ Dermal exposure/SKH-1 Dose and time dependent formation of Batal et al. (2013)
HPLCMS/MS; analyses kg/4 h mice (skin biopsy after 6 DNA adduct which detectable 21 days
of DNA adduct and 21 days) post-exposure
DNA adduct was correlated with
apoptosis of skin cells
CEES Western blot 2.3 mol/cm2 Dermal exposure/Big SM reduced epidermal level of Abel et al. (2013)
Single Blue C57BL/6 mice glutathione
application Sulforaphane (5 mol) as a treatment
could increase level of glutathione
CEES Mutation frequency assay 200 mM, Dermal exposure/ CEES induced dose and time dependent Boulware et al.
(lacI mutant screening), 400 mM/Single C57BL6 mice increase in mutation frequencies. (2012)
immunohistochemistry for dose 2,6-Dithiopurine (DTP) as a carcinogen
detection of cytokeratin 6 scavenger could reduce mutation
frequencies
SM Comet assay 5, 20, and 80 mg/ Percutaneous exposure/ The amount of ICL(inter-strand Stetina et al.
kg rat (biopsy of bone cross-links) was similar in different (2010)
marrow, liver and tissues and correlated with peripheral
peripheral lymphocytes lymphocytes
24, 48 and 96 h post
exposure)
CEES Western blot, DNA 0.052 mg/ Female SKH-1 hairless CEES induced oxidative stress. Pal et al. (2009)
oxidation assay by HPLC, Dermal exposure mice Activation of transcription factors AP-1
Electrophoretic mobility and NF-B was shown via upstream
shift assay (AP1, NFB)a signaling pathways including MAPKsa
and Akta
Increase in the formation of 8-oxo-2-
deoxyguanosine indicated DNA
oxidation
SM Western blot; Low, medium Percutaneous injection/ Chronic exposure of SM induced Sharma et al.
Bcl2, Bax, Cytochrome c, and high dose of Male Swiss albino mice oxidative stress and apoptosis in a dose (2009)
Caspase 3, P53 SM dependent manner also increased p53
Daily for 12 expression in neuronal tissue
weeks (chronic P53 may target the mitochondrial
toxicity) pathway for inducing apoptosis in
response to SM-DNA damage
SM DNA agarose gel Dermal exposure: Dermal and inhalation During dermal exposure; dose- Lakshmana Rao
electrophoresis, 38.7, 77.4, exposures/female mice dependent DNA damage in all organs et al. (1999)
DNA fragmentation assay 154.7 mg/kg (Biopsy of liver, lung, except lung and in inhalation route;
(using DAPIa) Inhalation: 10.6, spleen and thymus) dose and time-dependent toxicity in all
21.2 and organs were reported. By both routs
42.3 mg/m3 for liver and spleen were most affected
1 h duration organs
a
Abbreviations: AP1 activator protein 1, NFB nuclear factor-kappaB, MAPKs mitogen-activated protein kinases, Akt protein kinase B (PKB), DAPI
4,6-diamidino-2-phenylindole (see Table 12.2 legend for additional abbreviations)
335
Table 12.4 Clinical studies on the genotoxicity of SM and its analogues
336
Compound
(SM, NM, Exposure/
CEES) Assay Individual evaluation time Results Ref.
SM Comet assay; on lymphocytes 25 men, veterans Iran-Iraq war The significant DNA damage was detected Behravan et al.
of blood samples veterans in by comparison with the control group (2013)
198388)/ 2327
years post exposure
SM Cohort study 117 veterans (90 Iran-Iraq war No correlation was found between SM Ghanei et al.
with at least one veterans exposure and infertility in comparison with (2004)
partner and 27 the control group
with both partners
exposed to SM)
SM DNA index (hypo diploid, 75 men veterans Iran-Iraq war Patients with severe SM-exposure had Hassan et al.
diploid & hyperploid) using veterans/8 years aneuploidy (2002)
flowcytometry after exposure
SM Immunochemical and mass 2 Iranian war 22 and 26 days Development of two methods for evaluating Benschop et al.
spectrometric detection of veterans after exposure SM toxicity. Immunochemical assay was (1997)
DNA adduct based on detection of N7-guanine adduct in
lymphocyte whereas N-terminal valine
adduct in globin was performed by GC-
mass spectrometric.
The valine adduct levels corresponded with
those found in human blood after in vitro
treatment
SM Laboratory evaluation 58 men Iran-Iraq war Exposure to SM resulted in very low Azizi et al.
(testosterone, veterans androgen levels and hypo-responsiveness to (1995)
dehydroepiandrosterone, GnRHa in the first 5 weeks and
follicle-stimulating hormone normalization by the 12 week post exposure
(FSH), luteinizing hormone
(LH) and prolactin) and
histopathology assay
Compound
(SM, NM, Exposure/
CEES) Assay Individual evaluation time Results Ref.
Mustard PCRa 12 workers with Occupational P53 mutation frequency in the MG-exposed Takeshima et al.
gas (MG) lung cancer exposure cases was similar to the non-exposed (1994)
(workers of a controls
poison gas factory) However double mutations (G:C to A:T
transition) observed in two cases may be
related to MG exposure
SM Interview 1000 men Iran-Iraq war Significant increase of abortion following Pour-Jafari
veterans SM exposure of parents (1992)
SM Epidemiologic study 21,138 live births Iran-Iraq war 79 cases of newborns cleft lip and palate Taher (1992)
between 1983 and veterans were recorded that 30 cases associated with
1988 parental SM exposure
SM Epidemiologic study 2498 men and Occupational Highly significant increased rate of mortality Easton et al.
1032 women exposure associated with upper respiratory track due (1988)
(workers of to SM exposure (Cancer of the Larynx,
mustard gas Pharynx, other upper respiratory sites and
manufacture) lung cancer)
SM Examination of T lymphocytes 28 men (workers Occupational Increased frequency of somatic mutation in Yanagida et al.
lacking the hypoxanthine of a poison gas exposure workers (1988)
guanine phosphoribosyl manufacture)
transferase (HGPRT) activity
SM Sister chromatid exchange 11 men Fisher men exposed Significant increase of mutations even after Wulf et al.
(SCE) test on lymphocyte test to SM gas shell 3 week of SM exposure (1985)
SM One-dimensional 456 children of Parental exposure Total 36 protein variants were detected, Yamakido et al.
electrophoretic examination workers of the family history was found for 32 of them (1985)
poison gas factory One protein variant was found in one child
that was not inherited. This child was born

with cleft palate
a
Abbreviations: GnRH Gonadotropin-releasing hormone, PCR polymerase chain reaction, MG mustard gas (see Table 12.2 legend for additional abbreviations)
337
Table 12.5 Teratogenicity of SM and its analogues (in vivo studies)
338
Compound
(SM, NM, Exposure and
CEES) Assay Concentration/duration species Results Ref.
SM External 0.75 and 1.5 mg/kg/In 11, 13 IPa/Mice Craniofacial and septal defects and Sanjarmoosavi et al.
examination and 14 weeks of gestational limb malformation (adactyly and (2012)
age syndactyly) were the most common
types of defects
SM Stereomicroscopy 0.75 and 1.5 mg/kg in different IP/Mice SM exposure on the 11th day of Hassanzadeh-
periods of gestation gestation significantly increased the Nazarabadi et al.
incidence of cleft defects. The (2012)
teratogenic effects of SM depended
on the threshold dose and time of
gestation
SM Imprinting and 0.0017 g/kg17.0 g/kg Injection/Chick SM elicited significant defects in the Wormser et al. (2005)
locomotor activity Days 2 and 7 of chicken egg model intermediate part of the
assay, incubation hyperstriatum ventrale (IMHV)
immunoblotting related imprinting behavior. Also
parallel decreases were shown in the
level of membrane PKCa in the
IMHV
SM Histopathology 0, 0.03, 0.1, or 0.4 mg/kg SM Gavages/Sprague No significant effects were found on Sasser et al. (1996)
evaluation (5 day/week for 13 weeks prior Dawley rats the reproductive function or
to mating and throughout pregnancy outcome in either
gestation, parturition, and generation except an altered sex ratio
lactation in a 42-week, in the 0.4 mg/kg group
2-generation study) A dose-related lesion of the
squamous epithelium of the
forestomach (acanthosis) was
observed in adults of both sexes and
both the F1 and F2 generations
SM External 0.08, 0.20 or 0.50 mg/kg Gavages/Sprague Early fetal resorptions, pre- Sasser et al. (1993)
examination 5 days a week for 10 weeks Dawley rat implantation losses and decrease in
SM-exposed male total live embryo implants were most
rats mated to observed at a dose of 0.50 mg/kg of
untreated females at SM
2 and 3 weeks
post-exposure
SM External and Rats: 0.5, 1.0, and 2.0 mg/kg/ Female Rat and In rats, the fetal toxicity observed in Hackett et al. (1987)
histopathology day on gestation days 615, Rabbit/Gastric dose levels that also caused maternal
examination, Fetal Rabbits: 0.4, 0.6, and 0.8 mg/ intubation toxicity
viscera and kg/day on gestation days 619 No evidences of teratogenicity were
skeleton observed in rabbit
evaluation
SM 0.1 mg/m3 Female rat/inhalation No significant malformation was Rozmiarek et al.
During gestation observed (1973)
SM Histopathology No available Mice/Precutaneous, Spermatogenesis impairment Graef et al. (1948)
NM examination subcutaneous, following mustard exposure and also
intravenous, oral and post-exposure recovery after 2 weeks
gassing
a
Abbreviations: IP intraperitoneal, PKC protein kinase C (gamma isotype) (see Table 12.2 legend for additional abbreviations)
339
due to different experimental protocols, including species, time of exposure and

dose of mustard compounds.
There are limited studies concerning the SM teratogenicity in human. Some
reports have indicated the significant increase in the newborns cleft lip and palate
and abortion associated with parental SM exposure during the Iran-Iraq war (Pour-
Jafari 1992; Taher 1992)
Moreover, reduction in spermatogenesis have been reported in clinical and
in vivo studies (Graef et al. 1948). Azizi et al. (1995) have shown significant reduc-
tion in the level of androgens and hypo-responsiveness to GnRH in the first 5 weeks
of SM exposure in Iranian veterans.
12.9 Conclusions and Recommendations
Genototoxic effects of SM have been confirmed in human and animal studies. SM

has a relevant impact on DNA, which induces DNA adducts or cross links. The cur-
rently available laboratory tests are useful tools for detecting genotoxicty of SM
depending on the species and duration of exposure. Mice, rats, Chinese hamster
ovary (CHO) and human lymphoblasts and keratinocytes are the most important
species and cells to serve as host in the genotoxicity studies in the evaluation of SM
toxicity. Comet assay, western blotting, chromosomal aberration, immunohisto-
chemistry, quantitative PCR and Ames test were found to be the main experimental
tools in the investigation of SM genotoxicity.
We recommend future studies, including evaluations of enzymes involved in
DNA methylation and quantitative miRNA expression pattern to have a better
understanding of the regulators of protein-coding gene expression and epigenetic
mechanisms in SM toxicity. We should consider that these findings might help for a
novel therapeutic agent for SM toxicity. Also, we can apply these findings for iden-
tification and validation of genetic biomarkers that persists beyond the presence of
SM in the body.
Glossary
Adduct An adduct is a product of a direct addition of two or more distinct mol-

ecules, resulting in a single reaction product.
Adenosine triphosphate (ATP) ATP is a nucleoside triphosphate used in cells as
a coenzyme and transports chemical energy within cells for metabolism.
Alkylation The attachment of an alkyl group to an organic compound, usually by
the addition or substitution of a hydrogen atom or halide group
Apoptosis A natural process of self-destruction by degradative enzymes in certain
cells. Also called programmed cell death.
Carcinogen A substance or agent that can cause cancer.
Cell cycle The cell cycle, or cell-division cycle, is the series of events that take
place in a cell leading to its division and duplication (replication).
Chemical weapon A chemical agent or toxin, such as mustard gas, lewisite, or
sarin, that has been prepared for release on the battlefield or within a civilian
population in sufficient concentration to cause widespread illness or death.
Chromosomal aberration A chromosomal aberration reflects on a typical num-
ber of chromosomes or a structural abnormality in one or more chromosomes.
Cross-link A cross-link is a bond that links one polymer chain to another. They
can be covalent bonds or ionic bonds.
DNA Deoxyribonucleic acid (DNA) is a molecule that encodes the genetic instruc-
tions used in the development and functioning of all known living organisms.
DNA fragmentation The breaking of a (DNA) into smaller parts.
DNA replication DNA replication is the process of producing two identical cop-
ies from one original DNA molecule.
Electrophile An electron-deficient chemical compound or group that is attracted
to electrons and tends to accept electrons.
Eukaryote A domain of organisms having cells, each with a distinct nucleus
within the genetic material is contained.
Genotoxicity Genotoxicity describes the property of chemical agents that dam-
ages the genetic information within a cell causing mutations, which may lead to
cancer.
Glutathione A molecule that acts as a co-enzyme in cellular oxidation-reduction
reactions.
Leukemia Leukemia is a type of cancer of the blood or bone marrow character-
ized by an abnormal increase of immature white blood cells called blasts.
Methylation The addition of a methyl group to a cytosine residue on double-
stranded DNA, a process which plays a major role in regulating gene expression.
Mutation A change in the nucleotide sequence of the genome of an organism or
virus, sometimes resulting in the appearance of a new character or trait not found
in the parental type.
Nicotinamide adenine dinucleotide (NAD) Nicotinamide adenine dinucleotide
is a coenzyme and signaling molecule, whose oxidized form is NAD+.
Oxidative stress Increased oxidant production in animal cells characterized by
the release of free radicals and resulting in cellular degeneration.
P53 P53 is a tumor suppressor protein that is crucial in multicellular organisms,
where it regulates the cell cycle and, thus, functions as a tumor suppressor that is
involved in preventing cancer.
Poly (ADP-ribose) polymerase (PARP) PARP is a family of proteins involved
in a number of cellular processes involving mainly DNA repair and programmed
cell death.
Prokaryote The prokaryotes are a group of organisms whose cells lack a mem-
brane-bound nucleus (karyon).
Recombination Several processes by which genetic material of different origins
becomes combined. It most commonly occurs between the two sets of parental
chromosomes during production of germ cells
RNA Ribonucleic acid (RNA) is a ubiquitous family of large biological mole-

cules that perform multiple vital roles in the coding, decoding, regulation, and
expression of genes.
Sister chromatid exchange Sister chromatid exchange (SCE) is the exchange of
genetic material between two identical sister chromatids. Used as a mutagenic
testing of many products.
Spermatogenesis The process by which sperm develop to become mature sperm,
capable of fertilizing an ovum.
Sulfur mustard A class of related cytotoxic and vesicant chemical warfare agents
with the ability to form large blisters on the exposed skin and in the lungs
Telomere A telomere is a region of repetitive nucleotide sequences at each end
of a chromatid, which protects the end of the chromosome from deterioration or
from fusion with neighboring chromosomes.
Teratogen Any agent or factor that induces or increases the incidence of abnor-
mal prenatal development.
Transcription Transcription is the first step of gene expression, in which a par-
ticular segment of DNA is copied into RNA by the enzymes, RNA polymerase.
Tumor promoter A substance that has no intrinsic carcinogenic potential, but
which, when applied repeatedly, amplifies cancer-inducing effects of other (ini-
tiator) substances.
Tumor suppressor gene A gene that its function is to limit cell proliferation
and loss of function leads to cell transformation and tumor growth. Also called
antioncogene.
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Chapter 13
Verification of SM Exposure in
Biological Samples
Dirk Steinritz and Horst Thiermann
Contents
13.1 Introduction .................................................................................................................. 350
13.2 Toxicokinetic................................................................................................................ 350
13.3 Detection of Intact SM and SM Biotransformation Products ...................................... 351
13.4 Detection of SM-Protein Adducts ................................................................................ 352
13.5 Detection of SM-DNA Adducts ................................................................................... 353
13.6 Summary and Outlook ................................................................................................. 354
Glossary................................................................................................................................... 354
References ............................................................................................................................... 355
Abstract Sulfur mustard (SM) is a potent vesicant chemical warfare agent. Use of
such agents is considered as crossing a red line. Exposure to SM via inhalational,
cutaneous and ocular route can result in a systemic uptake causing the formation of
specific biomarkers that can be of use for verification. Comprehensive methods for a
free of doubt verification in biological samples do exist that detect either remaining pure
SM in the circulation and tissues, or rely on biomarkers resulting from SM hydrolysis,
SM biotransformation products, SM protein adduct or SM DNA adduct formation.
This chapter provides an overview about existing biomarkers that indicate a SM
exposure and analytical methods for their detection with special focus on the respective
toxicokinetics. Intact SM in urine or blood can be analyzed by GC- or LC-MS methods
in a short time frame after exposure. Specific -lyase metabolites and non-specific TDG
have also been successfully determined by GC- or LC-MS methods. Several specific
protein adducts with SM do occur, including albumin and hemoglobin, and are fre-
quently used for verification purposes. Finally, SM-DNA adducts can be visualized
with immunohistochemical methods or with evidentiary LC-MS based methods.
Keywords Sulfur Mustard Verification Mass spectrometry Biotransformation

products Protein adducts DNA adducts
D. Steinritz H. Thiermann (*)

Bundeswehr Institute of Pharmacology and Toxicology,
Neuherbergstrae 11, Munich 80937, Germany
e-mail: DirkSteinritz@bundeswehr.org; HorstThiermann@bundeswehr.org

350 D. Steinritz and H. Thiermann
13.1 Introduction
Sulfur mustard is a potent vesicant chemical warfare agent. Exposure via inhalation,
cutaneous and ocular route results in typical clinical symptoms (Kehe et al. 2009a, c).
Systemic uptake is commonly observed and is primarily dependent on the exposure
dose, but is independent of the exposure route. Already vapor exposure of the skin is
sufficient to result in significant systemic uptake and the possibility of a reliable veri-
fication (Steinritz et al. 2015).
The use of chemical warfare agents is considered as crossing a red line. Thus,
evidentiary, free of doubt analytical methods for verification are necessary. With
regard to human exposures simply identifying the agent in environmental samples
is insufficient as individual exposures cannot be circumstantiated. Therefore, robust
analytical methods for the investigation of biological samples are required.
Different methods exist that detect either remaining pure SM in the circulation
and tissues, or rely on biomarkers resulting from SM hydrolysis, SM biotransforma-
tion products, SM protein adduct or SM-DNA adduct formation. If biomarkers are
to be used for forensic purposes, they need to meet a number of requirements with
regard to lifetime, chemical stability and specificity (John et al. 2009). Thus a
detailed knowledge is necessary to choose the most applicable biomarker and the
correspondent analytical method with regard to sample type, and sampling time.
In addition to the verification of SM or SM metabolites in biological samples, a
plethora of GC or LC-Ms based analytical methods for the determination of SM in
almost all environmental matrices (air, water and soil) do exist.
13.2 Toxicokinetic
Pure SM not having contact to aqueous environments is considered as stable for

days to weeks. However, in aqueous surroundings the highly reactive compound
reveals a comparably short half-life of 48 min at 37 C in vitro (Vycudilik 1987;
Bartlett and Swain 1949). However, in vivo it was shown that distribution of the
lipophilic SM into adipose tissue after systemic uptake can result in a depot of SM
thus decreasing the hydrolysis rate (Drasch et al. 1987). Intact SM was detected in
rat plasma up to 8 h after exposure (Maisonneuve et al. 1992, 1993). Nevertheless,
a reliable analysis of pure SM is considered to be limited to a short time frame
within the first hours after exposure thus sampling has to be realized as soon as pos-
sible. After sampling SM hydrolysis can be decreased by adding sodium chloride in
excess to the sample and immediately storage at 20 C or below.
The hydrolysis of SM is complex. After intraperitoneal or cutaneous administra-
tion of SM to rats ten different biotransformation products have been described in
the urine (Black et al. 1992a, b) which could also be found in human samples after
SM exposures (Black and Read 1995). Analytical methods have been developed for
four out of this ten biotransformation products. However, only the -lyase
biotransformation products can be used for a verification of SM exposure as other
13 Verification of SM Exposure in Biological Samples 351
metabolites revealed little, but distinct endogenous levels in non-exposed individu-

als (Black and Read 1988). A recent report demonstrated that specific-lyase bio-
transformation products in plasma were found eightfold greater than lower limit of
quantification on day 6 after an accidental exposure (Xu et al. 2014). Nevertheless,
in vivo studies using rats and rabbits point to an extraction of more than 99 % of the
total amount of -lyase products in the first week after exposure (Lin et al. 2014a).
A plethora of reports are available that have investigated the abundance of
SM-protein adducts in vitro (Smith et al. 2008; Noort et al. 2002, 2008) and also
in vivo (Benschop et al. 1997; Xu et al. 2014). However, although most reports have
investigated the biological fate of these SM-protein adducts over time, only a few
reports actually have investigated the biological fate of these SM-protein adducts
from initial peak concentrations until return to zero-levels. A reliable identification
of SM-proteins adducts (N-alkylated valine originated from the N-terminal valine
of hemoglobin) was shown for up to 6 weeks post exposure (Noort et al. 1999).
Recent reports reported a reliable verification of SM exposure by determination of
the albumin adduct even 90 days post exposure (Xu et al. 2014).
Formation of SM-DNA adducts has been intensively investigated. It was shown
in vitro that alkylation of guanine bases at N7 is the most frequent adduct resulting
in the formation of N7-[2-[2-hydroxyethyl)thio[ethyl]guanine (N7-HETEG), fol-
lowed by the inter- and intrastrand diadduct between two guanine bases resulting in
bis[2-(guanine-7-yl)ethyl]sulfide (Bis-G) and N3-[2-[2-hydroxyethyl)thio]ethyl]
adenine (N3-HETEA) was shown to be another major adduct (Fidder et al. 1994).
The frequency of these major adducts may vary to some extend but in general the
N7-guanine monoadduct represents about 60 % of all observed adducts, followed
by N3-adenine monoadduct with about 15 % and the formation of N7-guanine
diadduct is observed in 10 % (Fidder et al. 1994; Ludlum et al. 1986). During DNA
repair SM-DNA adducts are cut out of affected DNA strands, are released into the
circulation and then cleared from the body by renal filtration. N7-HETEG was also
found to the most abundant SM-DNA adduct in vivo (Xu et al. 2014). A detection
of free alkylated nucleoside bases in urine was possible at least to day 14 after
exposure. This is supported by in vivo experiments using mice with topical SM
exposure that revealed a time frame of 21 day for detection of N7-HETEG (Batal
et al. 2013). HETE adducts cleaved from precipitated plasma proteins were detected
at least up to day 40 in an accidental human exposure to SM (Smith et al. 2008).
13.3 Detection of Intact SM and SM Biotransformation

Products
The high abundant availability of urine in combination with a non-invasive sam-

pling and the clean matrix makes urine ideal for analyzing intact SM and its bio-
transformation products. Intact SM in urine or blood can be analyzed using GC-MS
techniques (Vycudilik 1985, 1987; Koller and Szinicz 2009). Sodium chloride
should be added to the samples to decrease SM hydrolysis (John et al. 2009). Later
on, two-dimensional GC-MS/ESI in SIM mode allowed detection of 10 pg SM per

mL blood or per gram tissue (Oostdijk et al. 2007). LC-MS based methods were
also successfully applied to detect SM in blood (Dangi et al. 1994). In general,
reports on the successful analysis of non-hydrolyzed SM in biological samples are
rare most probably due to the limited stability of the highly reactive SM in biologi-
cal matrices. In the majority of all reports, techniques addressing SM biotransfor-
mation products were used.
SM biotransformation products are eliminated via renal filtration. Thus, a detec-
tion of these compounds in urine can easily be achieved. The main SM hydrolysis
product thiodiglycol (TDG) is a polar compound that has to be derivatized into more
suitable compounds for GC-MS analysis (Koller and Szinicz 2009; Jakubowski
et al. 1990). In addition, total TDG can be assessed by acidic or enzymatic hydroly-
sis from glucuronidated TDG. Moreover, TDG bound to blood proteins can be mea-
sured after release by alkaline hydrolysis (Capacio et al. 2004, 2008). The sulphur
atom in SM biotransformation products is oxidized to sulphoxise or sulphone
(Black and Noort 2007). TDGO is best analyzed after reduction to TDG using
GC-MS. However, TDG was found at low levels in unexposed humans (Black and
Noort 2007). Therefore, measurement of TDG only is inappropriate for verification.
Conjugation of SM with glutathion and further transformation results in the forma-
tion of 1,1-Sulfonyl-bis[2-S-(N-acetylcysteinyl)ethane]. Metabolic biotransforma-
tion through -lyase leads to the formation of specific -lyase biotransformation
products (MSMTSE, SBMSE, SBMTE and SBETE) that were found unequivocal
biomarkers of exposure to sulfur mustard (Noort et al. 2002; Koller and Szinicz
2009). GC-MS techniques are suitable to detect -lyase products. As for TDGO, an
initial reduction is necessary. In vivo studies revealed that detection of -lyase prod-
ucts was possible up to 14 days after exposure (Lin et al. 2014b). A simultaneous
detection of seven relevant biomarkers (SMO, TDG, TDGO, SBMTE, MSMTESE,
SBMSE, SBESE and SBSNAE) using UPLC-MS/MS was described (Li et al.
2013). Using that approach verification of a s.c. injection of 3.3 mg SM per kg in
rats was possible. A successful analysis of -lyase products in urine of SM exposed
human individuals using either GC-MS/MS or in urine, blood and blister fluid using
LC-MS/MS was reported (Barr et al. 2008; Xu et al. 2014).
13.4 Detection of SM-Protein Adducts
Methods for detection of SM adducts with cysteine, valine, histidine, aspartate/glu-

tamic acid residues have been reported and have been successfully applied to verify
a SM exposure (Andacht et al. 2014; Black and Noort 2007; Noort et al. 1996, 2002,
2008; John et al. 2009; Fidder et al. 1996a; Steinritz et al. 2015).
Regarding human serum albumin alkylation of the only cysteine 34 residue by
SM has been reported (Andacht et al. 2014; Noort et al. 1999, 2004, 2008). Digestion
of the alkylated albumin by pronase results in the liberation of an alkylated tripeptide
(Cys-Pro-Phe) that can be analyzed by LC-MS/MS methods (Noort et al. 1999,
2004). Recent reports revealed that the current available pronase produced the dipep-
tide (Cys-Pro) instead of the expected tripeptide (Gandor et al. 2015). A LC-ESI MS/
MS method was established for the simultaneous detection of CP dipeptide, CPF
tripeptide and also QCPF tetrapeptide (Gandor et al. 2015). SM albumin adducts
were successfully monitored in four individuals after accidental SM exposure up to
90 days after exposure (Xu et al. 2014).
Sensitive methods are available to detect SM adducts with the N-terminal valine
of hemoglobin. Using a modified Edman degradation the alkylated valine is released.
After further derivatization a detection using GC-MS methods is possible (Fidder
et al. 1996a; Noort et al. 2008). This approach was successfully applied to verify
SM exposure in five human casualties (Black et al. 1997).
SM adducts with proteins cytoskeleton including keratins and actin have been
described and were analyzed by MALDI-TOF technology (Noort et al. 2000; Mol
et al. 2008). However, analysis of these parameters for routine verification purposes
is not established.
13.5 Detection of SM-DNA Adducts
Alkylation of DNA bases after SM exposure does occur frequently resulting in the forma-
tion of four SM-DNA adducts. Alkylation of guanine bases at N7 (N7-[2-[2-hydroxyethyl)
thio[ethyl]guanine (N7-HETEG) is the most frequent adduct. Inter- and intrastrand diad-
ducts between two guanine bases resulting in bis[2-(guanine-7-yl)ethyl]sulfide (Bis-G)
are next frequent. N3-[2-[2-hydroxyethyl)thio]ethyl]adenine (N3-HETEA) was shown
to be another major adduct. A less abundant but biological highly relevant adduct is the
alkylation product of guanine at the O6-residue resulting in the formation of O6-[2-[(2-
hydroxyethyl)thio]ethyl]guanine (O6-HETEG). This adduct was found to account only
for some 0.1 % of all adducts but apparently this lesion is lacking repair via DNA alkyl-
transferases and thus has to be considered as mutagenic (Ludlum et al. 1986). The most
prevalent adduct N7-HETEG can be detected and quantified using LC-MS techniques
(Fidder et al. 1994, 1996b). Comprehensive LC-MS based methods that allow a simulta-
neous quantification of all adducts in urine or blood are reported (Yue et al. 2014; Xu
et al. 2014; Zhang et al. 2014). Alkylated bases were positively detected from day 3 up to
day 32 after exposure, with a maximum peak on days 47 in a human exposure scenario
(Xu et al. 2014).
In addition to analytical techniques that can verify a SM exposure free of doubt,
some indicative methods based on antibody detection of SM-DNA adducts have
been developed (van der Schans et al. 2004; Kehe et al. 2009b, 2013; Noort et al.
2002). In short, DNA is isolated from exposed tissue using standard DNA extraction
kits. The extracted DNA is denaturated, applied to a nitrocellulose membrane and
stained with an antibody directed against the SM-DNA monoadduct 7-HETEG. A
handheld device to identify pure SM was also developed. Synthetic guanine con-
taining oligo-nucleotides were applied to a sample pad. Contact with pure SM
results in the formation of SM-DNA adducts. A lateral flow assay is started.
Positive test and control lines indicate the presence of SM (Kehe et al. 2009b).
Immunohistochemical detection of SM-DNA adducts was successfully developed
(Noort et al. 2002).
13.6 Summary and Outlook
The verification of SM in biological samples is of utmost importance as the evi-

dentiary use of chemical weapons is considered as crossing a red line. Time-
dependent, intact SM, SM hydrolysis products or SM protein and DNA adducts
can be analyzed with chances of successful verification. A plethora of GC- and
LC-MS based methods are available that allow the detection of biomarkers in
vivo. These techniques can also be used for the detection of SM and its hydroly-
sis products in environmental samples. Additional, antibody based assays are
available that can indicate an exposure to SM. Future developments, especially
in the field of analytical methods, aim to the simultaneous detection of almost all
relevant biomarkers. In addition, the objective are to lower the detection limit
and to establish techniques that allow a verification out of tissues other than
blood.
Glossary
-lyase metabolites Specific SM-biotransformation products that can be detected

in urine after sulfur mustard exposure
Biological samples (Non-environmental) samples collected from organisms such
as blood, urine and other tissues
Biomarker Specific analyte that is measured to prove an exposure
Biomedical verification Use of analytical methods (typically mass spectrometry
based methods) for the assessment of biomarkers in order to prove the exposure
towards SM
Biotransformation products Enzymatical, chemical modification of compounds
by an organism
DNA-adducts (N7-HETEG, Bis-G, N3-HETEA, O6-HETEG) Sulfur mustard
induced covalent modification of DNA-bases. N7-HETEG is the alkylation of
guanine at the N7 position, Bis-G represents the a DNA crosslink between two
guanines at the N7 positions, N3-HETEA is the alkylation of adenine at the N3
position and O6-HETEG represents alkylation of guanine at O6 residues
GC-MS Gas chromatographymass spectrometry uses a capillary column for
sample separation and mass spectrometry for analysis
Half-life Time after that the concentration of a compound will drop to 50 % of the
initial concentration
Hydrolysis Cleavage of chemical bounds in aquoues surroundings
Immunohistochemistry Visualization of antigens in tissue by the use of antibodies

LC-MS Liquid chromatographymass spectrometry is an analytical technique
that combines liquid chromatography for separation and mass spectrometry for
analysis
MALDI-TOF Matrix-assisted laser desorption/ionization (MALDI) and time-of-
flight mass spectrometer can be used for the rapid identification of proteins and
their modifications
Sulfur mustard Vesicant chemical warfare agent, first used in World War I.
Sulfur mustard adduct formation Covalent modification of biological targets
by sulfur mustard.
TDG Thiodiglycol is a hydrolysis product of sulfur mustard. In contrast to -lyase
metabolites TDG is also present in the urine of unexposed persons.
Toxicokinetic Fate of a compound after entering the organism (e.g. distribution,
hydrolysis, metabolism, excretion)
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tion of four sulfur mustard-DNA adducts in rabbit urine after dermal exposure by isotope-
dilution liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol
Biomed Life Sci 961:2935
Chapter 14
Occupational and Environmental Mustard
Exposure, Prevention and Chemical Weapons
Convention
Slavica Vucinic, Branka Djurovic, and Biljana Antonijevic
Contents
14.1 Introduction .................................................................................................................. 360
14.2 Occupational Exposure ................................................................................................ 361
14.2.1 Accidental Occupational Exposure ................................................................ 361
14.2.2 Chronic Occupational Exposure..................................................................... 361
14.2.3 Usual Place and Scenario of Exposure ........................................................... 361
14.3 Clinical Effects............................................................................................................. 362
14.3.1 Acute Effects .................................................................................................. 364
14.3.2 Chronic/Delayed/Late Effects ........................................................................ 366
14.3.3 Diagnosis ........................................................................................................ 367
14.3.4 Medical Management and Therapy ................................................................ 368
14.3.5 First Aid on Site ............................................................................................. 368
14.3.6 Hospital Level ................................................................................................ 368
14.3.7 Prognosis and Assessment of Working Ability .............................................. 370
14.3.8 Preventive Measures and Protection............................................................... 370
14.4 Environmental Exposure .............................................................................................. 371
14.4.1 Fate and Behavior in the Environment ........................................................... 372
14.5 Ecotoxicology .............................................................................................................. 374
14.6 The Brief History of the Use of Mustard Compounds and the
Chemical Weapons Convention ................................................................................... 376
14.6.1 The Brief History of Mustard Compounds..................................................... 379
Glossary .................................................................................................................................. 382
References ............................................................................................................................... 384
S. Vucinic (*)
National Poison Control Centre, Military Medical Academy/Medical Faculty,
University of Defense, Crnotravska 17, Belgrade, Serbia
e-mail: zarkovuc@eunet.rs
B. Djurovic
Institute of Occupational Medicine, Military Medical Academy,
Medical Faculty/University of Defense, Crnotravska 17, Belgrade, Serbia
e-mail: djurovic.branka@gmail.com
B. Antonijevic
Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia
e-mail: abiljana@pharmacy.bg.ac.rs

360 S. Vucinic et al.
Abstract Literature on occupational sulfur mustard (SM) exposure is limited,

contrary to extensive data on acute effects of this vesicant as a chemical weapon.
Workers can be occupationally exposed accidentally and/or chronically. Accidental
occupational SM exposure is always short-term exposure to higher concentrations,
depending also on the duration of exposure. Chronic occupational exposure is usu-
ally related to low-level concentration exposure due to leakage or protocol break-
age. It develops as intoxication with mild symptoms that may not manifest
immediately, and also can produce potential health consequences that become evi-
dent months or years after the exposure, as late or delayed effects or just a reduction
in working ability. Environmental releases might occur near the places where SM
is produced and stored, but also due to the disposal of this chemical weapon, by
dumping them into the sea. Fate and behavior of this blistering agent in the environ-
ment is strongly related to its concentration. SM is lipophilic, negligible soluble in
water and relatively stable in the environment. After years of efforts and peace
negotiations, on April 20, 1997, Chemical Weapons Convention entered into force,
and the OPCW began its work providing its implementation, and conditions for a
world safe from chemical weapons.
Keywords Vesicants Sulfur mustard Occupational exposure Environmental

exposure Chemical weapons convention
14.1 Introduction
Before and during the World War I (WWI), over 3000 chemical substances were
investigated as potential chemical weapons in several countries. The best chemists,
three of them future Nobel Laureates in chemistry, found the solution and huge quan-
tities of vesicants were produced on both sides of the front line. One of them, sulfur
mustard (SM), was the most used chemical weapon ever and caused the greatest
number of casualties in the battlefields. On the contrary, the other vesicants, lewisite
and phosgene oxime, even produced in enormous quantities, have never been used.
From the first days of research activities and production, especially after the
WWI, faced with consequences of chemical weapons, community made many
efforts to stop or limit their production and use. Still, they were continuously
produced and were stocked in many countries. Even produced and stocked, they
were not used in WW II, but were used in at least ten other attacks all over the world
(Marrs et al. 1996; Smart 1996; Dacre and Goldman 1996).
In order to prevent further use of chemical weapon like SM, the Chemical
Weapons Convention was adopted in 1997 (OPCW 2014).
Nowadays, stocked vesicants are not completely destroyed and are still real
threat and risk for occupationally exposed personnel, environment and in the case of
accident or malevolent act, for the whole population, as well (WHO 1970; ATSDR
2003; WHO 2004; Szinicz 2005; Saladi et al. 2006).
14 Occupational and Environmental Mustard Exposure 361
14.2 Occupational Exposure
Literature on occupational SM exposure is limited, contrary to data on acute effects

of SM as a chemical weapon. Workers can be occupationally exposed accidentally
and/or chronically.
14.2.1 Accidental Occupational Exposure
Accidental occupational exposure is always short-term exposure to higher concen-

trations and develops as some of clinical forms of acute intoxication depending on
duration of exposure and concentration of SM. Even if it is single or just a repeated
exposure, late effects are possible. Accidental occupational exposure is rare, and it
is usually caused by human factor (mistake or breakage of procedure) or some tech-
nical problem (Henemyre-Harris et al. 2008).
14.2.2 Chronic Occupational Exposure
Chronic occupational SM poisoning is usually related to low-level concentration

exposure due to leakage or protocol breakage. It develops as intoxication with mild
symptoms that may not manifest immediately, and also can produce potential
health consequences that become evident months or years after the exposure, as
late or delayed effects or just a reduction in working ability (Committee on
Toxicology 2005).
14.2.3 Usual Place and Scenario of Exposure
Most of the workers were/are exposed:

During production of vesicants in factories,
In research laboratories, where workers do not follow precautions and safety
measures to prevent exposure;
In stocking facilities, because the storage of chemical warfare agents (CWA)
requires many precautions and safety measures, due to toxicity of vesicants and
their extremely corrosive properties. Therefore, security, safety and monitoring
of these sites are extremely important (Jeffery et al. 2008). One of the occupa-
tional accidents happened in the US CW storage facility where three workers
were collecting samples for routine monitoring of the storage site. They were
protected, but still the same evening they had early symptoms such as itching and
burning. On the second day all of them noticed blisters of the abdominal region
and immediately received the treatment. The chemical agent was SM (Ruhl et al.
1994; Davis and Aspera 2001).
During transportation of a CW stockpile due to unplanned or accidental situa-
tions. Such accident with most casualties happened in Italian harbor Bari in
1943, when German air forces sank 16 US ships stocked with 100 t of mustard
bombs. It caused injuries in 617 US soldiers and an unknown number of Italian
civilian casualties (Dacre and Goldman 1996).
During or after use of vesicants for variety of reasons. During the WWI lack of
proper delivery systems in the battlefield or formulation of the agent, caused
contamination of the allies troops. Several cases were described when accidents
happened on previously contaminated soil or when fisherman caught lumps of
mustard gas dumped in the sea during the war (ATSDR 2003; Jeffery et al. 2008).
Contrary to these cases military personnel can be exposed to low doses of CWA
after its use. Personnel can be exposed when entering the contaminated zone for
different reasons, such as decontamination, destruction of abandoned ammuni-
tion, rescue operation, etc.
During medical procedures and treatment of casualties, including activities on
site to final hospital treatment. This is extremely important for medical personnel
as well as other rescue teams. Training, education, plan and procedures and pro-
tective devices are only a part of measures needed for prevention and protection
(Weibrecht et al. 2012).
During destruction and chemical demilitarization of ammunition containing ves-
icants, because these activities are complicated, multistep processes requiring
high technology and protection of employees. None of processes are specified by
the Convention, but have to provide public and environmental safety, which
makes them expensive and time consuming. A worker was contaminated despite
all the precautions, while he was handling damaged ammunition. Even though he
has noticed brown, oily, smelly liquid leaking from ammunition, he had not
stopped the procedure to protect himself. Just a few hours later he developed skin
lesions on his hands, elbows and left foot. All clinical and laboratory tests for
sulfur mustard confirmed its presence. He developed severe, painful skin injuries
treated for 2 weeks at the department for intensive burn care and following 2
months of treatment till recovery. Beside skin injuries he developed post-traumatic
stress disorder (PTSD), needed psychological support and had to change job
(Manley 2000; Newmark et al. 2007).
14.3 Clinical Effects
Clinical effects of vesicants in occupational exposure depend on properties of vesi-

cants, duration and type of exposure, environmental conditions in the workplace
(temperature, humidity), type of work, extent of protection, and the susceptibility of
the exposed person (Hurst et al. 2008).
Characteristics of vesicants were already discussed, but only some of them that
significantly influence health effects of occupational exposure will be shortly
pointed out.
Mustard gas is actually a liquid chemical at ordinary environmental tempera-
tures. It is colorless, oily, liquid. Usually it contains 70 % SM and the rest of the
30 % are sulfur impurities. At ordinary temperatures it is significantly volatile. It
means that, besides liquid it is always mustard vapor in the air and therefore, haz-
ards of human contact with a working environment are from droplets of liquid and
from mustard vapors (Watson and Griffin 1992).
Mustard gas vapor is 5.5 times heavier than air and may accumulate at ground level
for a long period of time. It is very persistent in the environment because it is slightly
soluble in water. Persistence depends on environmental conditions, especially tem-
perature and humidity. For example, it persists 1248 h at 10 C with rain and a moder-
ate wind, 27 days at 15 C with sun and a light breeze, and 28 weeks at 10 C with
sun, no wind, and a snow cover (CRDEC 1990). The vapor can also be carried long
distances by wind. Its persistence in the soil could be even much longer up to decades.
It is soluble in organic solvents. It means that, working environment with hot, humid
atmosphere are more hazardous, as well as places where significant quantities of vesi-
cant were split even decades ago. These working environments could be hazardous
even for the workers with personal protective equipment. It was described in the case
of soldiers who were severely injured while they were digging ground in areas where
the mustard gas was split more than decade ago (Smart 1996; Marrs et al. 1996).
Sometimes multiple chemical weapons might be simultaneously deployed. In
case of vesicants, SM could be mixed with lewisite. Lewisite (Agent L) as an arseni-
cal vesicant, is ten times more volatile and can be easier to spread over great dis-
tances. It is also liquid at room temperature, slightly soluble in water and because of
that of intermediate persistency in soils. It is classified as a rather stable when stored
free of water contamination (Watson and Griffin 1992). Because of their simultane-
ous effects, the health risk is much higher and therefore it is reasonable to plan all
the precaution measures in accordance to the most potent chemical (Committee on
Toxicology 2005).
Workers are exposed to a blistering agent only when they come into contact with
them at the workplace. Vesicants can be released into the occupational environment
as a liquid or vapor. The most likely routes of exposure are by inhalation or through
the skin and mucous membranes.
Any job associated with physical activity will result in higher systemic exposure
because of the higher respiration, as well as job associated with stress because of
increased adrenergic stimulation. For the same reasons, increased exposure could
also be a consequence of harsh environmental conditions. Physical activity or hot
environment is connected with sweating and the moist skin is more sensitive and
more permeable for vesicants which is why significantly lower mustard concentra-
tions are able to produce adverse skin and systemic effects (Hurst et al. 2008).
Systemic exposure depends on protective equipment, especially personal protec-
tive equipment (PPE). Even designed to protect, it can cause the opposite effect, due
to higher respiration, significant heat stress and decreased working ability (Maibach
et al. 1984).
The risk to human health is mostly a function of the concentration and the expo-
sure duration.
SM can be inhaled or absorbed through dermal or mucosal surfaces easily due
to its lipophylic properties. Depending on the environmental temperature, almost
20 % penetrates the skin in 10 min. Most of it affects epidermal, then dermal tissue
(Renshaw 1946; Hurst et al. 2008), and a small amount (less than 10 %) enters the
blood. From the blood, it is distributed in target organs brain, kidney, liver,
spleen and lungs in different quantities. Major degradation product of SM is thio-
diglycol, which is detected and measurable in urine of exposed persons (Jakubowski
et al. 2000).
Toxic effects of SM are consequences of its chemical reactivity. It is an alkylat-
ing agent that promptly reacts with components of DNA, RNA, and proteins.
Because of similar reactivity to ionizing radiation it is usually called radiomimetic
agent (Watson and Griffin 1992). As a radiation, SM induces cell damage, that
involves DNA damage and cross-link formation. It also disturbs DNA synthesis and
repair, and therefore induces point mutations, chromosomal and chromatid aberra-
tion (Bignold 2006; Jowsey et al. 2009). Further, it induces oxidative damage and
decrease in glutathione activity, allowing damages of enzymes and cell membrane.
All these influences are harmful for mitotic cells, especially quickly dividing cells
of exposed tissues, such as epidermal basal layer cells, epithelial cells of the respira-
tory tract, white cell precursors in bone marrow, etc. In sufficient concentrations SM
can be toxic for all cells (Vogt et al. 1984).
14.3.1 Acute Effects
Acute effects of occupational exposure are similar to other accidental situations

depending on a dose.
If a worker is unprotected and exposed to SM vapor, adverse effects will develop
on the skin and all moist mucous membranes conjunctiva of the eye, airway mucus
from nose to lower airways and upper gastrointestinal tract. The most sensitive
organ is unprotected eye and therefore it is most often injured (86 %). The first
symptoms of intoxication are from eye irritation. These effects could be noticed
after exposure of just one hour to a vapor concentration of 0.5 mg/m3, which is
hardly perceptible by odor and at the same time insufficient to induce the damage of
the airways or the skin (Dahl et al. 1985). A few hours after initial irritation, painful
sensation with tearing, blepharospasm and photophobia, edema of the conjunctiva
can develop. SM penetrates the corneal surface in just a 10 min and can cause necro-
sis of deeper tissues, resulting in recurrent corneal ulceration and rarely even blind-
ness. Recovery is slow and starts after a few weeks.
Changes of other mucosal surfaces start with a short delay after irritation of the
eye mucosa with secretion of nasal mucosa, burning throat and dry persistent cough,
loss of voice, nausea and vomiting. Symptoms may persist for 1 or more years
(Ellenhorn et al. 1997). After higher doses laryngitis, tracheitis and/or bronchitis
could be seen. Effects of SM in the respiratory tract, from the upper nasal mucosa
to the lower terminal bronchioles are dose dependent (Hefazi et al. 2005; Balali-
Mood and Hefazi 2006). With simultaneously developed immunosupression, sec-
ondary infection such as bronchopneumonia is a common consequence. Recovery
follows the recovery of eye injuries.
Skin injuries are local at the contact surface and develop from 23 h to 24 h after
exposure depending on dose (Watson and Griffin 1992; Smith 2002). Since vesi-
cants penetrate the clothing it could be the source of secondary contamination of the
skin, especially if it is moist (Hurst et al. 2008).
Human skin is injured after doses higher than those needed to produce eye
injury. There is no difference in sensitivity related to race or sex, but there are up
to 100-fold individual variations. The most significant source of variation in sus-
ceptibility of skin is individual body region variation. It is related to body site and
skin thickness, so the most sensitive regions are the groin and scrotal region, neck,
scalp, armpit, and area behind the knee. The first reaction is skin erythema,
because of the influence on the superficial blood vessel. Erythema is always fol-
lowed by inflammation and edema. During first 24 h after exposure, skin rashes,
and after that, during the second day, blisters occur. Blisters are almost painless,
and after 56 days the whole area around blisters become painful. Minimal dose
for development of skin erythema is 50 mg/m3 except groin and scrotal region that
are ten times more sensitive and significantly lower minimal doses are needed
(Watson and Griffin 1992). Consequently, injuries of the skin of scrotal region are
the most important in personnel wearing protective clothing. Heavy scars of scro-
tal region are the most important because of their disabling properties. The heal-
ing process is very slow, lasting over 46 weeks. Affected areas, as after radiation
injury, could stay painful, become hypersensitive to most agents (chemical, ther-
mal, mechanical) and undergo hyper or hypo pigmentation. These regions
usually can be heavily scared, too. With simultaneously developed immunosu-
pression, secondary infection is possible (Heinen et al. 1945; Smith 2002; Pita
and Vidal-Asensi 2010).
If SM is mixed with Lewisite, there are some differences in health effects.
Lewisite is more toxic due to high skin absorption and systemic effects, that can be
lethal and cause immediate death, called Lewisite shock. Lethal outcome is also
possible after a week period due to liver dysfunction. Skin injuries are developing
faster, erythema and pain are present from the very beginning and larger blisters are
developing in 23 h (Smith and Dunn 1991; Watson and Griffin 1992). Contrary to
SM, lewisite can penetrate through subcutaneous tissue to muscles and induce
necrosis (IOM 1993).
Systemic effects are described in soldiers exposed in a battlefield as psychologi-
cal changes (depression, followed by anorexia and lethargy, excitation of the ner-
vous system) with convulsions, cardiovascular changes with atrioventricular block
and cardiac arrhythmias, bone marrow depression, etc. They are not recorded in
occupational exposure (Smith et al. 2008).
14.3.2 Chronic/Delayed/Late Effects
It was noticed that the acute SM exposure of soldiers in the battlefield is correlated
with some late effects. Most of affected organs were already injured: skin, eyes,
respiratory system. Therefore, occupational exposure and health status of worker
engaged in production of SM in almost all countries that produced them, were fol-
lowed-up for a long period. Still, many late effects were known after World War II
from studies of ammunitions workers (Pechura and Rall 1993).
The human data considering occupational exposure to low-dose of vesicants are
insufficient for reliable interpretation, and maybe the best description is that all of
these might occur, but there is no data that all have actually occurred (Smith et al.
2008). Chronic occupational exposure to low doses of SM could result in chronic
diseases. With exposure to lower doses, changes are discrete, they appear later, and
have a good prognosis, but with the increase of doses or duration of occupational
exposure, risk and severity of late effects also increase.
Late effects in occupationally exposed personnel can develop after chronic low-
dose exposure or /and after additional accidental single/repeated acute doses. They
include malignant and non-malignant diseases of most vulnerable and most sensi-
tive organs already described in section of acute accidental exposure.
If they occur after single or a repeated acute exposures they are following the devel-
opment of previous injuries. Late respiratory effects include chronic non-malignant
respiratory diseases (asthma, chronic bronchitis, emphysema, chronic obstructive pul-
monary disease, chronic laryngitis) due to previous injuries of epithelial cells
(Papirmeister et al. 1991). Besides that, workers who experienced acute mustard expo-
sure can develop hypersensitivity to smoke, dust, and fumes. These changes positively
correlate with age, smoking, and previous cardiopulmonary disorders. Hypersensitivity
and chronic pulmonary diseases concomitantly reduce working ability.
Most of the studies showed positive correlation between occupational exposure
and respiratory cancers in both types of occupational exposure, acute-accidental
and chronic. First of all, significant increase in cancer rate was confirmed in
Germany (11 vs. 5 expected) (Weiss and Weiss 1975), Japan (33 vs. 0.9 expected)
(Yamada et al. 1953, 1957; Yamada 1963; Wada et al. 1968), and the United
Kingdom (60 % higher risk) (Manning et al. 1981). USA data collected in experi-
ments with volunteers showed no evidence for increased cancer rate even when the
doses were high (Bullman and Kang 2000).
Most of the studies showed the evidence of a dose response relationship
between exposure to SM gas and respiratory cancer, as well as between the duration
of exposure and onset and risk of respiratory cancer (Nishimoto et al. 1983, 1988;
Yamakido et al. 1996).
British study conducted on workers exposed to low doses of mustard gas and
additionally to several accidental releases of mustard gas, showed increased rates
not only for pulmonary cancers, but cancers of the larynx, pharynx, oral mucosa,
esophagus and stomach. Positive correlation with duration of exposure is found
only for lung and pharynx cancers (Easton et al. 1988).
If chronic eye diseases are developed after single or a repeated acute exposures,
they are following the development of previous injuries of the eye and may present
as chronic keratitis and repeated corneal ulceration even decades after injury. Years
after acute exposure, on the basis of previous skin injuries, chronic skin ulceration
and altered pigmentation (hyper- and hypopigmentation) can develop. Wound
repair of these injuries is not regular, and they often heal with severe residual scar
formation. Skin cancer can develop on the scars. As a consequence of scar forma-
tion of the scrotum and penis sexual dysfunction may occur (Ellenhorn et al. 1997).
Workers exposed to low-doses without accidental exposure, can experience
chronic irritation of airway mucosal surfaces with secretion of nasal mucosa,
burning throat and dry, persistent cough, chronic bronchitis and later hypersen-
sitivity to smoke, dust, and fumes (Hurst et al. 2008; Smith et al. 2008). The
signs of chronic eye irritation, with inflammation of conjunctiva, redness and
tearing are also seen (Grant and Schuman 1993), Workers usually complain of
pain, redness, itching followed by desquamation. Even minimal doses can
induce hyper or hypo pigmentation. Except itching, these complications usu-
ally decrease over time. Some effects on the skin, such as eczema, telangiecta-
sia, urticaria, vitiligo, psoriasis, lupus erythematosus are rare and they are not
consequences of direct skin exposure, but immunological disorders induced by
chronic mustard exposure. Skin cancer can develop after chronic exposure too,
but on the contrary to acute exposure, it can occur on any exposed site (IOM
1993; Smith et al. 2008).
Variety of acute neurological and psychiatric symptoms noticed in soldiers
exposed to mustard in a battlefield during WWI, were not noticed in occupationally
exposed workers, so it was concluded that these symptoms are not induced only by
mustard itself. Some authors described anxiety and PTSD in exposed workers
(Smith et al. 2008).
Systemic effects after exposure to low-doses are not expected, but some of them,
such as bone marrow depression, are described.
Experimental data showed that mustard is genotoxic and poses reproductive tox-
icity and teratogenic effect. After exposure of soldiers to acute mustard doses, as
well as occupationally exposed persons, it was not reliably confirmed due to insuf-
ficiency of data (Ellenhorn et al. 1997).
14.3.3 Diagnosis
Diagnosis and differential diagnosis of mustard casualties in the workplace should

not be difficult because toxic agent is known. The symptoms, their onset and sever-
ity, the simultaneous occurrence in the workers from the same working environ-
ment, could point out to the release of significant quantities of CWA. Results of
clinical trials and laboratory tests are useful, but no routine laboratory test for
mustard exposure exists. Some experimental studies demonstrated that analysis of
metabolites, such as thiodiglycol in urine and DNA and protein adducts in blood
(DNA adduct, N7-(2-hydroxyethylthioethyl) -2-deoxyguanosine, as well as

adducts to albumin and hemoglobin) indicate exposure to mustard gas (Jakubowski
et al. 2000).
14.3.4 Medical Management and Therapy
Medical management of workers with acute exposure requires all the procedures
necessary to accidental exposure situations. Before any contact with contaminated
patients, medical staff and members of the rescue teams must protect themselves.
Therefore, they must put on appropriate PPE.
14.3.5 First Aid on Site
The main goal of the first aid is to reduce exposure as soon as possible, because the
cell damage begins within 12 min and quick decontamination will prevent or mini-
mize the injury (Papirmeister et al. 1991).
Before the arrival of the rescue team every worker can help himself a lot with a
few simple things. First of all, leave the contaminated area as soon as possible. If it
is outdoor, the person should remove from the site and get higher position to avoid
vesicant blisters collected in the lower levels. Then, person should remove wet
clothing (if it is liquid blister) and wash the face and exposed (or total) body with a
lot of water, especially the eyes for 510 min and then put sunglasses (if available)
to protect the eyes of the light. This is the first decontamination, and at the same
time, the only highly efficient and helpful for the patient (Willems 1989).
Rescue teams can enter the contaminated area only equipped with PPE including
self-contained breathing apparatus. They have to support vital functions and move
patient to uncontaminated area, undress the patient and perform decontamination. If
it is necessary patient should be transported to hospital.
14.3.6 Hospital Level
Besides themselves, medical personnel have to protect hospital of contamination

and forestall the entry of contaminated patients in the hospital. Therefore, they have
to perform decontamination in decontamination unit, or if it is missing, outdoors,
but not in a emergency department. This second-late decontamination, will not
decontaminate patient, because the mustard is already fixed to the skin cells and
damage is already induced, but will prevent mustard from spreading to medical
personnel and from possible contamination of the hospital (Renshaw 1946).
In a case of emergency with numerous casualties triage should be done. It is

recommended to divide patients in three groups (Willems 1989):
Patients with mild injuries without complications, good prognosis with symp-
tomatic therapy who do not need hospitalization;
Patients with moderate injuries (moderate severity or extent) which require
hospital treatment because they could complicate and became
life-threatening;
Patients with severe life-threatening injuries.
There is no specific therapy for mustard exposure. It is based on long-lasting
supportive and symptomatic medical care (Papirmeister et al. 1991). Further on
patients should be treated according to medical priorities. Patients of highest prior-
ity are with life-threatening injuries. Vital functions must be stabilized according to
medical doctrine. Major therapeutic goals are to treat symptoms, prevent infection,
and aid recovery (Willems 1989).
Dermal injury treatment requires treatment of blisters as burns, including cover-
ing, antibiotic therapy, pain relief therapy, application of corticosteroids and antihis-
taminic, as well as surgical treatment if it is needed. There is no consensus on
treatment of the blisters, especially should they be unroofed or not. Current thera-
peutic protocol includes: (a) application of silver sulfadiazine topical antibacterial
agent, (b) opening and debridment of bullae larger than 2 cm, while smaller than
those should be left intact, (c) collection and forensic analysis of blister fluid, blood
and urine samples, (d) in case of infection, application of 0.20.3 % Chloramine T
solution for disinfection, and targeted antibiotics or antimicotics, (e) wet to dry
dressings should be avoided, (f) in case of hypervolemia, diuretics are needed, (e)
early use of topical steroids, and high calorie and protein diet along with analgesics
and antihistamines (Hall et al. 1999).
Eye treatment requires irrigation for 15 min in all cases except after liquid
mustard contamination, when it is not required and even may increase the severity
of the injury. In any case, ophthalmic treatment is recommended (Marrs et al.
1996).
Specific antidote for lewisite or mustard-lewisite mixture exposure, in the case of
shock or severe pulmonary injuries, is dimercaprol (BAL British Anti Lewisite).
This is kind of chelating therapy for intramuscular use. It should be used only by
experienced and trained personnel, because of its numerous adverse effects. BAL
should not be given if the patient has renal dysfunction, receives iron supplement
therapy or has an allergy history (Hall et al. 1999).
Newer therapeutic agents, recommended for its favorable pharmacokinetic/phar-
macodynamic properties include:
DMSA meso-2,3-dimercaptosuccinic acid HO2CCH(SH)CH(SH)CO2H
DMPS 2,3-dimercapto-1-propanesulfonic acid HSCH2CH(SH)CH2SO3H
DMPS sodium salt HSCH2CH(SH)CH2SO3Na (UnithiolTM)
DMPA the phthalamidic acid o-(HO2C)C6H4CONHCH2CH(SH)CH2SH
14.3.7 Prognosis and Assessment of Working Ability
The assessment of working ability is individual, depending on severity of injuries.

After acute mustard exposure due to injuries, severe infections of wounds and slow
healing process, injured workers are not able to work till recovery, for at least sev-
eral weeks. In case of severe late effects, such as respiratory tract hypersensitivity,
immunosupression or heavily disabling scars, the injured workers should be
removed permanently from specific working environment.
14.3.8 Preventive Measures and Protection
Preventive measures and protection of personnel occupationally exposed to vesi-

cants are complex and specific. Therefore, they require a multidisciplinary approach.
They should include:
Legislations: International community made many efforts to stop proliferation of
chemical warfare. Every nation created its own legislation in accordance with
international fame. Even legislation is specific for every country; they have the
same goal to provide conditions for the maximal level of safety and security.
Technical measures are extremely important. Lack of adequate technical measures
was the reason of occupational exposure of thousands of workers in the first
decades of the twentieth century. They are very expensive but necessary in every
phase of technological process, from production, transportation, storage till use
and demilitarization. Nowadays, lack of adequate technical measures is one of
the biggest problems in the process of demilitarization and presents the highest
occupational and environmental risk of vesicants.
Permanent monitoring of environmental conditions on the site is unavoidable.
Industrial hygiene with specific, frequent and complete measurements of risk
factors is a precondition of prevention. Measurements always must include all
the parameters that can influence health risk. In the case of vesicants, besides the
chemical monitoring, measurements of temperature, humidity, aerosol, noise,
ventilation should be frequent, too. Every change of any of mentioned parameter
that could change, environmental condition should be noticed, registered and
examined.
Medical preventive measures include three different activities: health surveillance
of exposed personnel before deployment in a such risky working environment
called preplacement examination, periodic examinations for already engaged
personnel and medical treatment in a case of emergency situation. Preplacement
examination is an examination before entrance into the working environment
with potential health risk. Occupational medicine physicians should determine
are there any contraindication for work on such a place, meaning, are there any
health condition that could be worsened in the working environment or can limit
the ability to work. In order to assess working ability, occupational medicine
physician has to know risks from the workplace from its description or reports
from industrial hygiene. If such health conditions are found, the patient should be
advised to reconsider that position. Preplacement examination must include a
general physical examination with laboratory tests and specific examination of
target organs respiratory tract, nervous system, skin, and eyes. Additionally, in
this case, the patient should fit to wear personal protective devices and be active
and psychologically stable to resist the psychological pressure due to which these
functions must be examined, too. At the end, if the health condition of a patient
complies with job requirements, he can work. Further on, every change in health
status should be noticed on periodic examinations (Henemyre-Harris et al. 2008).
Unprotected healthcare workers are a special group of occupationally exposed
workers. They can be injured by secondary exposure when they treat contami-
nated patients. There are many disagreements about the risk and protection
needed for the healthcare workers. Exposure of health care workers depends on
numerous factors, such as number of patients, type and quantity of contaminant,
etc. It is very important are they first responders or they receive previously treated
patients (decontaminated and undressed). PPD should be provided in accordance
with the activity (OSHA 2005). Hospitals should provide that just educated and
trained personnel are engaged in the treatment of this kind of patients, to provide
PPE, technical support, etc. (Macintyre et al. 2000; MMWR 2001; Hick et al.
2003; Georgopoulos et al. 2004).
Personal protective equipment (PPE) in an agent vapor atmosphere will be, in the
first place, SCBA masks with full-face protection and chemical protective
clothes. Due to high volatility and penetration ability of vesicants PPE should be
of the highest quality. Workers should be trained and educated to use PPE in a
proper way (Smith 2002).
Protocols and procedures in a workplace, for regular as well as emergency situa-
tions are crucial.
Education and trainings for regular and emergency situation should be mandatory
for all workers. They all should be familiar with risks, first signs of dangers or
first symptoms of intoxication, first aid on site, protocols, PPE, etc.
14.4 Environmental Exposure
Vesicants or blistering agents are a group of chemicals known due to its historical
use as chemical warfare agents for military and terrorist purposes. Vesicant or blis-
ter agents include the following compounds: sulfur mustards (undistilled sulfur
mustard (H), sulfur mustard (HD), and an HD/agent T mixture (HT)); nitrogen mus-
tards [ethylbis(2-chloroethyl)amine (HN 1), methylbis(2-chloroethyl)amine (HN2),
tris(2-chloroethyl)amine (HN3)], and the organic arsenical lewisite.
For the first time, SM was used as a chemical warfare agent in Europe (Ypres,
Belgium) during World War I (WWI) in 1917 (ATSDR 2003; Sidell et al. 1997).
After WWI, it was also used by Italian troops in Ethiopia (19351936), by Egyptian
forces in Yemen (19631967), by the Iraqi regime in Iraqi-Iran war and against
Kurdish in the 1980s (Balali-Mood and Hefazi 2005; Razavi et al. 2012).
SM is found in ocean waters at several sites around the world. In 1943 sulfur
mustard was accidentally released in the harbor of Bary, Italy after the sinking of the
American freighter, S.S. John Harvey that carried large amounts of sulfur mustard.
Disposal of SM at different water sites became standard practice worldwide, includ-
ing the coastal waters around Japan, USA and the Baltic Sea (ATSDR 2003). After
World War II (WWII) (19451948) approximately 11,000 t of highly toxic chemi-
cal agents, were dumped in the Bornholm Basin, east of the island of Bornholm in
the southern Baltic Sea (Sanderson et al. 2010). In a period of 19521965 additional
200300 t of chemical munitions was dumped by East German Authorities. In this
water basin, the main chemical agents dumped were blistering agents and organo-
arsenic compounds (diphenylarsinchlorid) Clark I and adamsite. The total amount
of war material (artillery shells and aircraft bombs) identified in the Bornholm
Basin consists of over 560,000 objects, of which quantity of mustard gas solely has
been estimated at 7027 t (Sanderson et al. 2010). Heavily contaminated water area
in the Bornholm Basin caused accidental exposure of Scandinavian fishermen who
developed characteristic skin reactions, typical for blister agents.
Other environmental releases might occur near the places where vesicants were
produced for military applications, stored in military depots and storage facilities.
For example, although United States has not produced SM since 1968, some data in
the USA have shown that almost 1,500 leaking munitions were identified in the
stockpile since 1982, some of which were leaking sulfur mustard (ATSDR 2003).
Vesicants have been released to the atmosphere after the use as a military weapon,
but their presence in the air is highly temperature related. SM is an oily liquid and
is generally regarded as a persistent chemical agent because of its low volatility.
In cooler weather there is a little vapor; however, mustards evaporation increases as
the temperature increases. Apart from the military use air contamination may poten-
tially occur at stockpile sites and also due to destruction by incineration. However,
mustard vapor has a density 5.4 -fold greater than that of air, causing precipitation
on the ground. From the ground, it slowly evaporates and can be detected in the
layer closer to the ground, but generally its concentration in the air is negligible.
14.4.1 Fate and Behavior in the Environment
Fate and behavior of blister agents in the environment are strongly related to their
physico-chemical properties (Table 14.1). All these agents are lipophilic, negligible
soluble in water and relatively stable in the environment.
Soil Low solubility in water along with low volatility leads to the environmental
persistence of the compounds in the soil. Blister agents can be present from 1 to 2
days on the soil surface under average weather conditions, and from several months
to years under cold conditions (Munro et al. 1999).
SM has been known to persist for weeks to decades in military testing areas and
land dumps where large quantities have been deposited underground. When in a gas
phase, they can be conveyed long distances by air streams, which is strongly
Table 14.1 Identity and physico-chemical properties of mustard compounds

H/HD HT HN1 HN2 HN3
Chemical C6H8Cl2S C6H8Cl2S C6H13Cl2N C5H11Cl2N C6H12Cl3N
formula C8H16Cl2OS2
CAS no. 505-60-2 63918-89-8 538-07-8 51-75-2 555-77-1
Mol. weight 159.08 ND 170.08 156.07 205.54
Physical state Oily liquid Oily liquid Liquid Liquid Liquid
Color Clear/pale Amber/dark Colorless/pale Colorless/ Colorless/pale
yellow brown yellow pale yellow yellow
Density (g/mL) 1.27 1.27 (20 C) 1.09 (25 C) 1.12 1.24
(20 C)
Melting point 1314 C 1 C 34 C 60 C 3.7 C
Boiling point 215 > 228 C Decomposes 75 C 230235 C
217 C
Vapor pressure 0.11 0.10 0.25 0.427 0.011
(mmHg)
Water solubility 0.92 Practically 12 Sparingly 0.16
(g/L) insoluble soluble
Henrys Law 2.1 105 ND ND 8.5 108 3 107
constant
(H, atm m3/
mol)
log Kow 1.37 ND ND 0.9 ND
Log Koc 2.12 ND ND 1.86 2.83
From Munro et al. (1999) and Sidell et al. (1997)
Abbreviations: H undistilled sulfur mustard, HD sulfur mustard, HT HD/agent T mixture, HN1
nitrogen mustards [ethylbis(2-chloroethyl)amine, HN2 methylbis(2-chloroethyl)amine, HN3
(2-chloroethyl)amine
dependent on meteorologic conditions, such as temperature and wind. For example,

SM will evaporate 23 times faster at 20 C than at 5 C (ATSDR 2003). At 25 C,
SM deposited on a surface soil will evaporate within 3050 h (Munro et al. 1999).
With an increase of vapor pressure, volatility of vesicants increases from SM
(0.11 mmHg) to lewisite (0.58 mmHg). Evaporation can be influenced by other fac-
tors such as moisture content, pH, porosity of the surface, and physical constituents
of the soils. Generally, agents with low solubility in water and rapid hydrolysis when
dissolved, are not transported through soil into groundwater (Munro et al. 1999).
Biotic degradation pathway has been identified as relevant for the agents environ-
mental neutralisation or even formation of toxic metabolites, in case of microbial
dehydrohalogenation of lewsite. It was shown that two strains of the bacterial spe-
cies Pseudomonas pickettii and Alcaligenes xylosoxidans use hydrolysis product of
mustard thiodiglycol as a source of carbon for growth (Yang et al. 1992).
Water In water, theoretically, vesicants may undergo chemical transformation,
evaporate from the surface to air, or remain unchanged. At low temperatures and
with minimal turbulences, vesicants will be present in the water for a long time
(Sanderson et al. 2010), particularly in case of SM that freezes at 14 C. Hydrolytic
degradation of vesicants may be slow because of their limited solubility. Under

laboratory conditions, half life of SM at 25 C in distilled water has been reported
to be 48 min. Hydrolysis of HN3 is slower than that of the SM, but the hydrolysis
reactions of HN1 and are probably more rapid. Calculated a hydrolysis half life of
HN2 is about 11 h at 25 C (Munro et al. 1999). HN3 is considered environmentally
persistent, whereas HN1 and HN2 are considered moderately persistent. Hydrolysis
of lewisite is rapid and results in the formation of the hydrosoluble and nonvolatile
2-chlorovinyl arsonous acid.
In seawaters, rate of hydrolysis is slower than in fresh water because high chlo-
rine levels in the water inhibit hydrolitic degradation. The rate of hydrolysis is lim-
ited not only by the slow rate of the solution, but also with intermediate hydrolysis
products. Hydrolysis of SM include complex chemical reactions which all end to
formation of thiodiglycol and hydrochloric acid. Thiodiglycol can be further oxi-
dized to corresponding sulfoxide and sulfone. Additionally, 1,4 oxathiane and
1,4 dithiane are common degradation products of SM that persist in the environ-
ment. 1,4 Oxathiane is formed by dehydrohalogenation of partially hydrolyzed
mustard, whereas 1,4 dithiane is a thermal degradation product of mustard formed
by dechlorination.
The major fate process of the three nitrogen mustards in water or soil is also
hydrolysis, especially under alkaline conditions. The mechanism of hydrolysis is
similar for all three nitrogen mustards, with liberation of chloride and formation of
a cyclic intermediate and several different products reviewed by Munro et al. (1999).
Air When in the atmosphere, it is not expected that photodegradation represents an
important fate process of vesicants. On the other side, reaction with photochemically
produced hydroxyl radicals, or reaction with nitrate radicals are important for the
estimation of the corresponding half times. Based on reaction with hydroxyl radi-
cals (5 105 hydroxyl radicals/m3), it was calculated that sulfur mustard atmo-
spheric half life is about 2.1 days (Meylan and Howard 1993). Nitrogen mustards
may also react with photochemically produced hydroxyl radicals, estimated half
life of HN3 was 5 h (Munro et al. 1999).
14.5 Ecotoxicology
Mammalian acute and prolonged toxicity of vesicants has been studied extensively
providing a number of data on mechanisms of toxicity and toxicological end points
(Watson and Griffin 1992; Ghabili et al. 2010; Razavi et al. 2012; Graham and
Schoneboom 2013). Vesicants, but also their degradation products, are extremely
toxic for terrestrial mammals. Reviewing the toxicological end points of vesicants
Munro et al. (1999) collected the data on median lethal doses (concentrations,
carcinogenicity, genotoxicity, reproductive, systemic and other relevant effects of
mustards and lewisite derivatives. HN1 and SM as typical alkylating agents as well
as their degradation products have been shown to be mutagenic in a wide variety
of species (Fox and Scott 1980). International Agency for Research on Cancer
(IARC) has classified sulfur mustard as carcinogenic to humans (Group 1) based
on sufficient evidence in humans (ATSDR 2003; Wulf et al. 1985).
The toxic military material, often dumped in sea waters worldwide, represents a
serious potential threat to the marine environment. Vesicants are toxic to all aquatic
species, however, their toxic effect is limited by their low water solubility. Toxicity
of degradation products is generally lower than the toxicity of parent compounds.
Estimated lethal concentration of SM in fish amounted in the range of 2550 g/L,
whereas after chronic (30 day) exposure of bluegill sunfish (Lepomis macrochi-
rus), red-eared sunfish (Lepomis microlophus) and black bullheads (Ameiurus
melas) toxicity threshold was assessed at 2 mg/L (Munro et al. 1999). Although dif-
ficult to make direct comparisons of test results, it seems that nitrogen mustards were
less toxic than sulfur mustard for aquatic organisms. Chronic toxicity threshold val-
ues of nitrogen mustards with black bullheads were at least four times higher (HN1
25 mg/L, HN2 10 mg/L, HN3 8 mg/L) than the value of sulfur mustard obtained
for the same exposure duration (30 days). Acute toxicity tests of HN2 performed for
invertebrata Ceriodaphnia dubia and Daphnia magna after 48 h exposure resulted in
LC50 of 1.12 and 2.52 mg/L, respectively, and LC50 of 98.86 mg/L for the fish species
Pimephelas promelas obtained after 96 h exposure (Lan et al. 2005). Based on these
data it can be concluded that HN2 is toxic for invertebrata and harmful for fish spe-
cies. Chronic toxicity tests related to survival and reproduction effect of HN2 showed
the dissimilar susceptibility of the species with the no observed effect concentrations
(NOECs) of 0.0039 and 2.5 mg/L for Ceriodaphnia dubia and Pimephelas prome-
las, respectively. The clear difference in toxicity between species is attributed to the
ability of more complex organisms, such as fish, to detoxify HN2.
In 30 day tests, the thresholds for lethality of lewisite for two aquatic organisms
were 0.2 mg/L (black bullheads) and 0.5 mg/L (bluegill sunfish), indicating much
higher toxicity in relation to mustard agents (Munro et al. 1999).
In 2005, within the EU the Sixth Framework Programme project (FP6), a project
Modeling of Ecological Risks Related to Sea-dumped Chemical Weapons (MERCW)
was launched to evaluate overall chemical war agents (CWA) risks in the Baltic Sea and
also to identify uncertainties and future needs. Data on ecotoxicological risk have been
expressed in toxic units (TU), which represent the ratio between the exposure concen-
tration and fish no observed effect concentration (NOEC). Total calculated TU for all
nine identified CWAs was 0.62, whereas TU of SM alone was 0.083 (Sanderson et al.
2010), indicating no risk for the model applied in the study. There are no data proving
the potential of vesicants to bioconcentrate or biomagnify, due to generally low Kow
values (<2) and probably due to their high in vivo reactivity. For example, results of
MERCW project have shown that models developed to describe CWAs biomagnifica-
tion potential in the Baltic commercial fish, including cod (Gadus morhua), herring
(Clupea harengus), and sprat (Sprattus sprattus) revealed no such potential of SM.
According to review article of Munro et al. (1999), the nitrogen mustards were
less toxic than SM to phytoplankton and higher aquatic plants. At 5 mg/L, Lewisite
inhibited the growth of the phytoplankton, and the water milfoil and water crowfoot
died; at 50 mg/L, all plants died.
The analysis of the microbial community can be used as an indicator of CWA pres-
ence. Some species of microbes are tolerant to hydrolytic products (primarily thiodi-
glycol) and use these as their sole source of carbon and energy (Medvedeva et al.
2009). But for some other microbiota SM and its hydrolysis products have a broad
spectrum of toxic effects reducing thus significantly the near bottom water hetero-
trophic microorganism community and species diversity (Sanderson et al. 2010).
Soil contamination with chemicals can exert toxic effects directly to soil organ-
isms, or indirectly, by altering specific interactions and by disrupting the soil food
chain. Vesicants may be present in soil for a long time and thus induce the reduction
in microbial activity as a consequence of its high toxicity to soil microorganisms.
Results of microcosm assay have shown that the EC50 value of sulfur mustard for
total numbers of microarthropods was 65 and 130 mg/kg for total numbers of nema-
todes (Kuperman et al. 2007). The results also suggested that soil constituents,
including soil organic matter, can affect the partitioning of sulfur mustard from
solid to aqueous phase in soil and thus modify the bioavailability of this chemical to
a specific group of the soil invertebrate community.
14.6 The Brief History of the Use of Mustard Compounds

and the Chemical Weapons Convention
Throughout history, the toxic properties of certain substances have been applied in
armed conflicts. Along with the scientific and industrial progress of a society, the
weapons evolved from poisonous darts to fumigants, or rudimentary Chinese chem-
ical grenades to chemical artillery at the beginning of the industrial revolution.
Although the use of these toxic substances was sometimes advantageous on the
battlefield, the horror and agony of the afflicted echoed in the form of unequivocal
public examining and disapproval for many years to come, far outweighing the
prospect of using them to temporarily gain the upper hand in war (Mayor 2003).
World War I and the German lethal chlorine gas attack against the Allied Forces
at Ypres (April 22, 1915) marked the turning point in the history of chemical war-
fare. The surprise use of 160 tons of chlorine gas, spread over the French trenches,
killing more than 1000 French and Algerian soldiers, and wounding about 4000
more. Whils and effective way of bypassing the trench gunfights and weakening the
enemy defenses, WWI led to over 100,000 deaths and a million injuries. (Fitzegerald
2008). Whilst an effective way of bypassing the trench gunfights and weakening the
enemy defenses, it led to over 100,000 deaths and a million injuries (Fig. 14.1).
However, this did not stop the major military powers from producing and stock-
piling large quantities of chemical arms (200,000 metric tons) after the war, and
they had started to become important parts of many armies arsenals. Most of them
were chlorine, phosgene, diphosgene, chloropicrine, hydrogen cyanide, and vesi-
cants the first generation of chemical weapons (CW) as well as tear gases and
irritant incapacitants. Over time, mustard, lewisite, as well as their various mixtures,
Fig. 14.1 British soldiers

blinded by mustard gas,
1918. www.opcw.org/
index.php
were introduced (propylene mustard, O-mustard, sesquimustard, nitrogen

mustard).
After years of effort and peace negotiations, the greatest Allied Forces managed
to get the international support to ban the use of CW. The protocol for the prohibi-
tion of the use in war of asphyxiating, poisonous or other gases, and of bacteriologi-
cal methods of warfare, commonly known as the Geneva Protocol, was signed by a
number of countries in 1925 and put in motion in 1928. One of the biggest limita-
tions of the protocol was that the countries that had signed it needed only to refrain
from using CW first, but not as a counterattack in case they were attacked by one.
Additionally, it did not restrict the possession and stockpiling of chemical arms,
predict the possibility of sanctions or include a verification mechanism (Hurst et al.
1997; Kenyon 2000; Pitschmann 2014).
The pre-World War II period was marked by the development of new, highly
toxic organophosphorus agents in Leverkusen by IG Farben. These compounds rep-
resent the second generation of CW. They were used in the conflict between Japan
and China in the 1930s, but not during the rest of the WWII (Salem et al. 2008).
Following a period of decreased interest in chemical warfare due to the develop-
ment of nuclear arms, the Geneva Protocol reemerged and was the basis for further
resolutions after the use of tear gases and herbicides in the Vietnam War
(19611973).
The 18th National Committee on Disarmament marked the resumption of the

peace negotiations on the chemical arms ban. During the meeting it was agreed that
the chemical and biological warfare should be regarded independently. At that time,
there was a great divide in the world powers of the West, the countries that were neu-
tral, and the Eastern Bloc, which presented a great hurdle to reaching an agreement.
Besides the stockpiles of the USSR and the USA, there were other states as well that
were interested in acquiring capacities for CW, including South Africa, some of the
Middle East, South Asia, China and the Koreas. Although this was followed by
increased preparedness for chemical defense at the state level, civil defense was not
in the focus of their interest, as at that time, CW were counted as primarily battlefield
weapons. However, as many times before in the history, practice has confuted it.
Bilateral meetings between the USA and the USSR, followed by individual
efforts of some countries in negotiations on chemical disarmament, although with-
out significant progress, at least provided important documents, including the draft
version of the Convention. In the late 1980s, warming of the relations between the
great world powers made it possible to see a global recession from CW as a reality.
The use of CW, nerve agents and mustard, in the Iran-Iraq War from 1983 to 1988,
also changed the climate in favor of the initiative to ban CW. The UN Secretary-
General in March 1984 confirmed in his Report the use of CW, and 1 month later a
working paper (CD/500) with the draft CWC was introduced by the USA. Renewed
diplomatic process between the Soviet Union and the USA, in the period 1986 to
1991, facilitated the exchange of the detailed information on stockpiles of CW, but
also provided a base for the formalized obligation to cease the production of binary
CW and controlled destruction of stockpiles.
Confronted with the danger of chemical warfare during the Persian Gulf War,
albeit CW were not actually used by Iraq, additionally stimulated negotiators at the
Conference on Disarmament to reach the consent on disarmament in September
1992. The draft treaty text of the Convention was submitted to the United Nations
and adopted as its Resolution 47/93 (Kenyon 2000).
In 1993, the Convention on the Prohibition of the Development, Production,
Stockpiling and Use of Chemical Weapons and their Destruction (CWC) was pre-
sented and after a 3 day meeting in Paris, signed by 130 states. The seat of the
Organisation for the Prohibition of the Chemical Weapons (OPCW), as it was
agreed, would be at the Hague, where the Preparatory Commission would meet and
prepare the 1st Session of the Conference of the State Parties (Zanders et al. 1997).
On April 20, 1997, CWC entered into force, and the OPCW began its work pro-
viding implementation of the Convention. Up to now, 190 countries have signed the
Convention. Israel and Myanmar have not ratified it, and Angola, Egypt, South
Sudan and the Korean Peoples Democratic Republic have not yet signed (OPCW
2014). From about 71,000 t of declared chemical toxic substances and precursors,
more than 72 % had been destroyed by the end of 2012, but the destruction of CW
is still not finished, as well as the destruction of old CW. However, one cannot
assume that the CW era is history, especially with the increasing potential of their
further development using new scientific and technological methods that overlap
Table 14.2 The history of chemical disarmament

Year Document Development
1675 The Strasbourg The first international agreement limiting the use of
Agreement chemical weapons (poison bullets).
1874 The Brussels The Brussels Convention prohibited the use of poison
Convention on the or poisoned weapons, and the use of arms, projectiles
Law and Customs of or material to cause unnecessary suffering.
War
1899/1907 Hague Peace Bans the use of poisoned weapons, asphyxiating or
Conferences deleterious gases.
1925 Geneva Protocol Ban on CW use; but no prohibition on development,
interpreted as no first use 132 parties by 2000.
1972 Biological and Toxin Comprehensive BW prohibition 143 parties, 17
Weapons Convention signatories by 2000; but no verification mechanism;
commitment to negotiate on CW.
1993 Chemical Weapons Signing of CWC in Paris; Comprehensive bans on
Convention development, production, stockpiling and use of CW,
with destruction timelines; Establishment of
Preparatory Commission for OPCW.
1997 OPCW, The Hague The CWC enters into force for 87 States Parties; The
OPCW begins its work in The Hague; inspections
began in June 1997.
2007 Tenth Anniversary of 182 Member States. 25,000 metric tons of CW (35 %
the OPCW of the declared stockpiles worldwide) have been
certified by the OPCW as destroyed. 3,000 inspections
have been carried out by OPCW inspection teams
(more than 1,100 military and industrial sites in 80
countries).
2013 190 Member States
Modified from the Basic Fact on Chemical Disarmament (OPCW)
the field of chemistry and biology, or the continuous research of psychoactive sub-
stances. The OPCW is following the scientific achievements, trying to keep abreast
of materials and methods that could have implications on CWC (Table 14.2).
14.6.1 The Brief History of Mustard Compounds
14.6.1.1 Sulfur Mustard
From the military point of view, mustard presents the highest risk, but other vesi-
cant agents also remain the CW of concern, such as Lewisite and phosgene oxime.
The history of mustard begins with the synthesis of SM in 1822 by Despretz. Riche
in 1984, repeated the chemical process and obtained the same substance. A few
years later, Guthrie was the first to report the physico-chemical properties and
clinical effects of SM. Other chemists, such as Niemann in 1960, and Meyer in
1986, were engaged in SM synthesis, with the latter being more successful in
obtaining higher quality mustard (Balali-Mood and Hefazi 2005; Shakarian et al.
2010; Razavi et al. 2012).
The term mustard is derived from its smell (mustard) or color (yellow to dark
brown), while abbreviations are made from terminology used by Allies (Hun Stoffe-
German Stuff, abbreviated HS; later known as H). It contains 2030 % sulfur as
impurity, but in pure form or distilled, it is known as HD. Lommel and Steinkopf,
were German chemists engaged in developing the method for large-scale produc-
tion of mustard in WWI, so the German military used the acronym LOST. The name
yperite is related to its first use on July 1917 near Ypres, Belgium.
Mustard caused more than 20,000 casualties in this attack and about 80 % out of
1,3 million of all chemical casualties in WWI, proving its title King of Battle
Gases. In the post WWI period, the use of mustard (confirmed or alleged) was
reported in 1935, when Italy probably used it against Abyssinia; in the period 1937
to 1944, Japan allegedly used it against Chines, and Egypt against Jemen from
1963 to 1967.
Although CW was not used during World War II, an incident in Bari, where US
ship John Harvey carrying mustard was bombed, resulted in 617 US casualties (83
fatal) and also numerous Italian civilian casualties as a result of toxic gas exposure
(Sidell et al. 1997).
From 1983 to 1988, in the Iran-Iraq war when Iraqi regime used CW, approxi-
mately 5001000 Iranian soldiers were casualties of mustard, along with 45,000
individuals being injured or with chronic medical problems. A number of patients
were treated in hospitals in Ghent, Belgium and other cities of western Europe
(Khateri et al. 2003; Roushan, et al. 2008; Rowell et al. 2009). On March 16, 1988,
Iraqi army used nerve agents, possibly tabun, sarin and VX, as well as mustard gas
against civilians in Halabja, killing 5000 people (Razavi et al. 2012). Further 7000
people were injured or had chronic sequels (Fig. 14.2).
In the aftermath of 1991 Gulf War, in order to eliminate the capacity of Iraq for
the production of CW, the UN Special Commission (UNSCOM) was established.
Over 690 tonnes of vesicants and nerve agents, mainly mustard gas and sarin were
located and destroyed, along with more than 3000 tonnes of chemicals determined
as precursors (Kenyon 2000).
Fig. 14.2 Halabja on March 17, 1988 http://www.kdp.se/old/chemical.html

In past decades, several incidents were recorded in the North Sea, and elsewhere
due to the dumped munition after WWI and WWII. From 1985 till 1995, 350 inci-
dents of CW being found in fishing nets, were reported by Dutch fishermen. In 2002
there were ten situations near Bornholm Island, where people found munitions.
Japan reported more than 830 cases, not only the discoveries of CW, but also 10
deaths, and 400 injuries as a consequence of exposure (Walker 2010). Major mili-
tary powers, such as US, USSR, UK and Japan disposed of CW agents by dumping
them in the seas. US Department of Defense reported that only the U.S military
dumped CW in waters in various locations in the world (North Atlantic, Baltic,
Mediterranean, and Pacific Ocean) over 70 times in the period from 1918 to 1970.
Fortunately, most of the dumping operation were carefully recorded. Russia reported
that at least 160,000 tonnes of CW may be dumped in Russian seas which represents
a threat to environment, safety and public health. (Ong et al. 2009).
14.6.1.2 Nitrogen Mustard
Nitrogen mustard, synthesized in 1930, had similar properties to sulfur mustard.

However, it was not suitable for use as CW. The military production of HN2 started
in Germany in 1941 and USA in 1943. Due to high efficacy, Mustargen (mechlor-
ethamine) (HN2) was for years used as chemotherapeutic for certain types of cancer,
until it was replaced by other, safer agents. Compounds abbreviated HN1, HN2,
HN3, caused severe systemic effects and have never been used in the battlefield.
Mustards are still real threat and risk for occupationally exposed personnel, envi-
ronment and in the case of accident and terrorist act for the general population as
well. Occupational exposure to vesicants can be acute, when high concentrations
are involved, repeated or chronic, that is usually related to low-level concentration
exposure due to leakage or protocol breakage. Workers are expected to strictly com-
ply with hazard-specific safety procedures during work. However, further improve-
ment in protection and preventive measures are needed.
There are still significant uncertainties regarding ecotoxicological risks of the
vesicants, generally due to the lack of precisely estimated environmental concentra-
tions and relevant toxicity data. There is a need to achieve better knowledge about the
fate, behavior and transport properties of vesicants in the environment, establish envi-
ronmental ecotoxicological indicators specific for vesicants that would lead to the
standardized protocols in case of environmental and occupational exposure. Based
on general humanistic principles, CWC prohibits all chemical weapons, and their
proliferation. Although the destruction of most stockpiles is coming to an end, CWC
should remain open to the future in the light of huge potential for new technologies
combining chemistry and biology to be used for the development of new toxic agents
from uncontrolled chemicals.
Glossary
Acute toxicity Describes the adverse effects of a substance that result either from
a single exposure or from multiple exposures in a short space of time (usually
less than 24 h). Adverse ecological effects Refers to any harmful effects on the
environment
Aphonia The inability to produce voice. It is more severe than dysphonia
Bronchospasm Sudden constriction of the muscles in the walls of the bronchi
Chronic bronchitis A chronic inflammatory condition in the lungs that causes
the respiratory passages to be swollen and irritated
CWA:chemical warfare agents A chemical substance whose toxic properties are
used to kill, injure or incapacitate human beings
CWC: Chemical Weapons Convention An international treaty which prohibits
the development, production, stockpiling, transfer and use of chemical weapons
and imposes their destruction
CX:Phosgene oxime A manufactured chemical warfare agent, also called urti-
cant or nettle agent
Dyspnea Shortness of breath or breathlessness is the feeling or feelings associ-
ated with impaired breathing
Ecological risk assessment The process that evaluates the nature and likelihood
Ecotoxicology Study of the effects of toxic chemicals on biological organisms,
especially at the population, community, ecosystem level.
Exposure assessment The determination of the extent of human exposure.
effect.
First Responder Personnel who have responsibility to initially respond to emer-
gencies (firefighters, HAZMAT team members, ambulance attendants)
HN-1, HN-2, HN-3: Nitrogen mustard A class of organic compounds similar to
mustard gas in their molecular structure, important for the treatment of cancer
IARC International Agency for Research on Cancer
Inhalation The flow of air into an organism. In humans, it is the movement of air
from the external environment, through the airways, and into the alveoli.
Inspiratory Relating to the act of breathing in
L: Lewisite A blister agent
Lipophilic The ability of a chemical compound to dissolve in fats, oils, lipids, and
non-polar solvents
Median effective concentration (EC50) A concentration that is statistically
estimated to cause a specified effect in 50 % of a group of test organisms under
specified experimental conditions.
Median lethal concentration (LC50) A concentration that is statistically esti-
mated to be lethal to 50 % of a group of test organisms under specified experi-
mental conditions.
Median lethal dose (LD50)\ A dose that is statistically estimated to be lethal to

50 % of a group of test organisms under specified experimental conditions.
Monitoring of environmental conditions Systematic, complete control and
measurements of risk factors in the environment
Mutagenic Capable of inducing mutation or increasing its rate
No Observed Effect Concentration (NOEC) The concentration of chemical
at which there were no statistically or biologically significant increases in fre-
quency or severity of adverse effects seen between the exposed population and
its appropriate control. Effects may be produced at this dose, but they are not
considered to be adverse.
Occupational exposure Exposure to harmful agent in a workplace.
Octanol-Water Partition Coefficient (Kow) The equilibrium ratio of the con-
centrations of a chemical in n-octanol and water, in dilute solution.
Adverse ecological effects: any adverse effect to the environment Of adverse
ecological effects from exposure to one or more stressors.
Periodic examination Is annual examination of worker exposed to some harmful
agent in a workplace.
Personal Protective Equipment (PPE) Protective suits, gloves, foot covering,
respiratory protection, hoods, safety glasses, goggles, and face shields.
Preplacement examination Is an examination before entrance in the working
environment with potential health risk.
Prolonged toxicity Describes the adverse effects of a substance that result either
from continuous or repeated exposure.
Risk The probability that an adverse effect will occur under a particular condition
of exposure.
Risk assessment A scientifically based process of evaluating the toxic properties
of a chemical and conditions of human exposure to it in order to ascertain the
likelihood that exposed people will be adversely affected and to characterize the
nature of these effects.
Self-contained Breathing Apparatus (SCBA) A respirator that provides fresh air
to the facepiece from a compressed air tank (usually worn on the workers back).
SM:Sulfur mustard A class of related cytotoxic and vesicant chemical warfare
Teratogenic A drug or other substance capable of interfering with the develop-
ment of a fetus, causing birth defects
Threshold Dose or exposure concentration of an agent below that a stated effect
is not observed or expected to occur.
Toxic unit (TU) It represents the ratio between the exposure concentration and
fish no observed effect concentration (NOEC).
Toxicity Inherent property of an agent to cause an adverse biological effect.
Toxicological end point Effect observed in a toxicity study.
Tracheobronchitis A condition involving inflammation of the windpipe orbronchi
Triage The process of screening and classifying sick, wounded, or injured per-
sons to determine priority needs in order to ensure the efficient use of medical
personnel, equipment, and hospitals.
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Chapter 15
Summary and Conclusion
Mahdi Balali-Mood and Mohammad Abdollahi
Contents
15.1 Introduction .................................................................................................................. 390
15.2 The Summary of the Book ........................................................................................... 392
15.2.1 Chemistry of MC Including SM and NM....................................................... 392
15.2.2 History of Use and Epidemiology of Mustard Compounds ........................... 392
15.2.3 Basic and Clinical Toxicology of Mustard Compounds ................................. 393
15.2.4 Clinical Pharmacology and Toxicology of MC .............................................. 394
15.2.5 Delayed Complications and Long-Term
Effects of SM Poisonings Experience of Iran-Iraq War .............................. 394
15.2.6 Upper Respiratory Complications of SM Poisoning ...................................... 395
15.2.7 Lower Airways Complications of SM Exposure ............................................ 395
15.2.8 Dermatologic Aspects of SM Exposure ......................................................... 396
15.2.9 Ocular Injury by Mustard Gas; Early and Late Complications ...................... 396
15.2.10 Immunological and Hematological Complications of
SM Poisoning ................................................................................................. 397
15.2.11 Psychiatric Complications of SM Poisoning .................................................. 397
15.2.12 Genotoxicity, Teratogenicity and Mutagenicity of SM Poisoning ................. 398
15.2.13 Verification of SM Exposure in Biological Samples ...................................... 398
15.2.14 Occupational and Environmental Mustard Exposure,
Prevention and CWC ...................................................................................... 399
15.3 Conclusion and Expert Opinion ................................................................................... 399

e-mail: BalaliMoodM@mums.ac.ir
M. Abdollahi, PharmD, PhD
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical
Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran
Toxicology and Poisoning Research Centre, Tehran University of Medical Sciences,
Tehran, Iran
Endocrinology & Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
e-mail: Mohammad@TUMS.ac.Ir

M. Balali-Mood, M. Abdollahi (eds.), Basic and Clinical Toxicology of Mustard
Compounds, DOI 10.1007/978-3-319-23874-6_15
390 M. Balali-Mood and M. Abdollahi
Abstract Mustard compounds (MC) were initially synthesized in the nineteenth

century and then considered to be used as the chemical warfare agents (CWA). Both
nitrogen (NM) and sulfur mustard (SM) on exposure induce skin blister and thus
called vesicant or blistering agents. But NM has never been used as a CWA; it is
used as an anticancer medicine. Acute toxic effects of SM appear after variable
periods of latency (minutes to weeks) depending on the dose, mode of exposure,
environmental temperature, and the individual. Delayed toxic effects of SM in the
survivors of the WW1 and the Iranian and Kurdish veterans have been evident along
with social problems for both the patients and the governments of the involved coun-
tries. The main objective of the book was to provide a scientific information and
practical guide on MC for the scientists, health professionals, and regulatory bodies
who are involved in teaching, research, medical care of the patients, and policy/regu-
latory making. The International organizations such as the OPCW, UN, WHO, Red
Cross as well as the national authorities of chemical weapon conventions and mili-
tary toxicologists shall also benefit from this book. The most advantage of this book
is that almost all chapters have been written by experts in the field. Meanwhile,
delayed complications and long term effects of SM poisonings is an issue that has
been very well explained according to target organs and tissue injuries in this book.
Keywords Nitrogen compounds sulfur mustard nitrogen mustard chemical

warfare agents poisoning intoxication toxicity
15.1 Introduction
Mustard compounds (MC) were initially synthesized in the nineteenth century and
then considered to be used as the chemical warfare agents (CWA). Both nitrogen
(NM) and sulfur mustard (SM) on exposure induce skin blister and thus called vesi-
cant or blistering agents. SM (but not NM) was used during the world war one
(WW1) and then by the Iraqi army against the Iranian troops and even civilians on
a large scale. NM is used as an anticancer medicine.
SM is an alkylating agent that initially reacts with ocular, respiratory, and cutane-
ous tissues, resulting in acute poisoning and even death. Acute toxic effects of SM
appear after variable periods of latency (minutes to weeks) depending on the dose,
mode of exposure, environmental temperature, and the individual. Delayed toxic
effects of SM in the survivors of the WW1 and the Iranian and Kurdish veterans
have been evident along with social problems for both the patients and the govern-
ments of the involved countries.
The main objective of the book was to provide a scientific information and practi-
cal guide on MC for the scientists, health professionals, and regulatory bodies who
are involved in teaching, research, medical care of the patients, and policy/regulatory
making. The International organizations such as the OPCW, UN, WHO, and Red
Cross as well as the national authorities of chemical weapon conventions and military
15 Summary and Conclusion 391
toxicologists shall also benefit from this book. The most advantage of this book is that
almost all chapters have been written by very expert scientists and clinicians who
have had a real experience of management of patients intoxicated with SM. Meanwhile,
delayed complications and long term effects of SM poisonings is an issue that has
been very well explained according to organ and tissue targets in this book.
Furthermore, the most important features of the book are as follows:
1. Provides basic information on MC for regulatory authorities in different depart-
ments of works, environment, industries, military, and health.
2. A practical guide for the occupational, environmental, toxicological, military,
medical, and health workers on safe use of MC and appropriate treatment of
patients who receive NM and for possible SM exposure.
3. A reference book for the clinical toxicologists, military and emergency physi-
cians who are involved in teaching and research on MC and for all medical and
health professions who are responsible in prevention, diagnosis and treatment of
MC poisonings.
4. Provides updated information on different aspects of MC including basic/clini-
cal pharmacology and toxicology, and clinical management of SM or NM
exposure.
Lack of scientific knowledge on MC in medicine and toxicology have made a lot
of confusion among some health professionals and scientists specially on differen-
tiation between NM and SM. There are problems with NM administration and its
toxic effects in patients who receive this chemical as an antineoplastic agent that
must be clarified. Also, malpractice on the clinical management of patients who
exposed to SM during the chemical war or in occupational settings are important
matters that must be clarified to scientists and health professional. Following the use
of chemical warfare agents (CWA) by the Iraqi army against the Iranian and Kurdish
people during the Iraq-Iran war and the recent claims for the use of CWA in Syria,
many toxicologists and health professionals are now more interested to learn more
about different aspects of the CWA including MC.
In the present book, all basic and clinical aspects from history, chemistry, synthe-
sis, and toxicity of MC to target organs/tissues, biomarkers, epidemiology, occupa-
tional and environmental exposure, risk assessment, clinical management and
guidance on prevention and control have been covered. Therefore, this book is a
practical guide for different occupational, environmental, toxicological, medical
and health professionals and also for the authorities who are involved in different
aspects of mustard compounds. It could be also used as a reference book for MC by
the postgraduate students and researchers in the universities.
The following items have been critically reviewed in this book in separate
chapters:
1. Chemistry
2. History of use and epidemiology
3. Basic pharmacology and toxicology
4. Clinical pharmacology and toxicology
5. Delayed complications and long term effects of sulfur mustard poisonings

Experience of Iraq-Iran war
6. Upper respiratory complications of sulfur mustard poisoning
7. Lower respiratory complications of sulfur mustard poisoning
8. Dermatological complications of sulfur mustard poisoning
9. Ophthalmological complications of sulfur mustard poisoning
10. Immuno-hematological complications of sulfur mustard poisoning
11. Psychiatric complications of sulfur mustard poisoning.
12. Genotoxicity, teratogenicity and mutagenicity of sulfur mustard poisoning.
13. Diagnosis, verification and medical detection of an exposure to sulfur
mustard.
14. Occupational and environmental mustard exposure, prevention and chemical
weapon conventions.
15.2 The Summary of the Book
15.2.1 Chemistry of MC Including SM and NM (Chap. 1)
All NM and SM compounds were well described from chemistry to their applica-
tions. HN-1, HN-2, HN-3 are the most important forms of NM. The HN-2 is chlor-
methine (mechlorethamine) that has been used for treatment of different cancers
such as Hodgkin's disease. SM has the chemical name bis(2-chloroethyl) sulfide
and the IUPAC name 1-chloro-2-(2-chloroethylsulfanyl) ethane. It is also known as
mustard, mustard gas, HD or Yperite. SM was first synthesized from the reaction of
ethylene and sulfur dichloride (Levinstein process) through an electrophilic addi-
tion mechanism. Later, it was prepared by the reaction of thiodiglycol with phos-
phorus trichloride (Meyer reaction) in a substitution reaction. Finally, reaction of
concentrated hydrochloric acid (HCl) and thiodiglycol resulted in the production of
SM. Pure mustard is a viscous, colorless and odorless liquid which evaporates
slowly in the atmosphere. NM and SM slightly differ in properties, but have the
same mechanism of action that described in the book. At chemistry point of view,
decontamination of SM can be achieved via hydrolysis in the presence of aqueous
solutions of sodium hypochlorite and or chloramine-T; in which HD decomposes
into thiodiglycol non-poisonous product.
15.2.2 History of Use and Epidemiology of Mustard

Compounds (Chap. 2)
The term chemical warfare agents (CWAs) refer to any chemical that might have
toxic effects on plants, animals and humans. SM is synthesized by the Belgian
chemist Cesar Mansute Despretz in 1822 for the first time. Victor Meyer, a
Germania chemist, in 1886, completely described the chemical structure of SM. In

the World War I, German army used SM for the first time against British soldiers in
a field near Ypres, Belgium. NM was initially synthesized as a CWA, but has never
been used as a chemical weapon. Different analogues of NM were made during the
early 20th century and some of them have been prescribed as chemotherapeutic
medications. Spain was the first government that used SM against the Rif rebellion
civilian in 19211926. Mussolini also ordered the Italian army to use SM against
unprotected Ethiopian forces and civilian population in 19351936. Through 1963
1967, the Egyptian air force used CWAs and SM in Yaman. Although a large amount
of SM was made during the World War II, fortunately it was not used during that
war. Iraqi army used SM and other CWAs against Iranian forces and Iranian and
Kurdish civilian in 19831988. The result of repeated Iraq's chemical attacks during
the 8 years of war was above 100 thousand casualties, of which almost 5000 were
died. It was estimated that more than half of the chemical casualties were due to SM
poisoning, but 32,000 of them have medical records and around 30,000 of them are
now suffering from the delayed toxic effects of SM. The most tragic use of SM was
the chemical bombardment of the city of Sardasht (a city in the northwestern border
of Iran with Iraq) in spring of 1987 and Halabja (a Kurdish town in Iraq) massacre
in 1988. Recently, it was noted that sarin was used in the Syrian conflict in summer
2012. However, as of 23 June, 2014, all of the Syria's CWAs have been rendered
permanent and removed from Syria.
15.2.3 Basic and Clinical Toxicology of Mustard Compounds

(Chap. 3)
SM is well absorbed through inhalation, dermal, and ocular contacts and tend to
distribute mostly to the lungs, liver, and kidneys. DNA and protein adducts are the
main metabolites of SM which are mainly excreted in the urine along with unchanged
compounds. Since NM has never been used as a chemical warfare, their kinetic
information is mostly related to those which have been used as chemotherapeutic
agents. Upon absorption through intravenous or oral administration, NM is rapidly
converted to the reactive metabolites and distributed so that the highest concentra-
tion can be found in the bone marrows. Mono-alkylation of guanine at N7 and then
N3 makes the main DNA adducts of NM. In an aqueous environment, MC convert
to very active electrophilic metabolites which can attack nucleophilic groups in the
structure of cellular macromolecules. DNA alkylation is known as the main mecha-
nism by which MC exert their both toxic and therapeutic effects. They can also
alkylate other nucleophils, most notably thiol groups in the structure of proteins,
leading to excessive production of reactive oxygen species in the cell. Following the
disruption of such functional macromolecules, a series of maladaptive responses are
activated, including excessive production of reactive oxygen species and inflamma-
tory cytokines, metabolic imbalance in energy production, elevated release of
calcium into the cytosol from intracellular and extracellular sources, and conse-
quently the expression of enzymes involved in necrotic or apoptotic cell death
pathways.
15.2.4 Clinical Pharmacology and Toxicology of MC (Chap. 4)
MC, especially SM as a CWA can cause acute and chronic toxicities, particularly
acute toxic effects and complications in the eyes, lungs, kidneys, skin and other vital
organs in human. Acute, chronic and delayed toxic effects of SM have been exten-
sively studied, particularly on Iranian veterans. Despite the large number of studies,
the mechanistic pathways of SM intoxication in cellular level, as well as clinical
pharmacology and toxicology of MCs are not well understood. In this chapter, toxic
effects of SM on different organs, particularly the skin, lungs, eyes and kidneys have
been comprehensively reviewed. In addition, long-term hematological complica-
tion, neurological impairment, and other delayed immunotoxicity have been dis-
cussed. SM-induced pulmonary toxicity, ocular irritation, and skin disease are the
most common complications of SM poisoning.
Several therapeutic strategies have been proposed so far to treat these complica-
tions, but no specific antidote has been introduced for these health problems.
Conventional medical treatments with antioxidants e.g. N-acetylcysteine and
sodium thiosulfate and some anti-inflammatory drugs such as corticosteroids have
been used, but their effects are not satisfactory. Some new therapeutic strategies
such as antimicrobial peptides, gene and stem cell therapy, and herbal medicines
have been proposed for the treatment of SM complications.
15.2.5 Delayed Complications and Long-Term Effects of SM

Poisonings Experience of Iran-Iraq War (Chap. 5)
Acute and long-term incapacitating properties of SM, in combination with the lack
of an antidote, significant environmental persistence, and relative ease of manufac-
turing, still kept it a potential agent for both military and terrorist use. Even three
decades after SM exposure during the war, around 40,000 Iranian veterans have
complained of delayed effects of SM poisoning. As in the SM veterans of the WWI,
some of the delayed toxic effects in Iranian patients persist for their entire life.
Iranian veterans are still suffering from delayed complications of SM exposure
in different body organs of which the lungs, eyes and skin are the three major
involved organs in delayed SM effects. Respiratory complications are the greatest
cause of long-term disability among Iranian veterans, which exacerbate over time.
Furthermore, neuropsychiatric, reproductive, urogenital, immuno-hematological
and cardiac complications as well as cellular damage and carcinogenicity alongside
with many other delayed complications have been studied and reported among
Iranian SM veterans. Thus, veterans need lifelong medical and nursing care. As
there is no specific treatment for delayed toxic effects of SM, the main therapeutic
approach is symptomatic and supportive therapy. Financial, social, and cultural sup-
port as well as reassurance and supportive love care of the veterans in the family and
society are also important beside medical therapy.
Delayed complications of SM exposure can still be observed in several thou-
sands of Iranian victims of the Iran-Iraq war. Delayed complications of SM have
been reported in several organs, however, the most common delayed complications
have been observed in the respiratory tracts of Iranian chemical veterans. Also, the
skin lesions as well as the eye disorders have been observed in most of Iranian
exposed veterans in the delayed phase of intoxication.
15.2.6 Upper Respiratory Complications of SM Poisoning

(Chap. 6)
SM can be absorbed from skin, eye, mouth and respiratory and the gastrointestinal
systems. Among these organs, respiratory tract and the skin are the main susceptible
organs for SM intoxication and injury. Upper and lower respiratory tract may be
affected by SM, however the acute and chronic effects of SM in upper respiratory
tract has been less studied and most of studies have focused on lung injuries induced
by SM.
The main clinical symptoms of SM in upper respiratory tract in acute phase are
irritative and inflammatory symptoms while in the chronic phase most symptoms
are due to functional damages and cancers. The important parts affected by SM in
the late phase in upper respiratory tract are the larynx and vocal cords as well as the
trachea and bronchioles. DNA alkylation and damage is the most important molecu-
lar mechanism of these effects. Yet there is a gap in knowledge regarding the differ-
ent aspects of early and late effects of SM in upper respiratory tract.
15.2.7 Lower Airways Complications of SM

Exposure (Chap. 7)
The pulmonary effects of SM exposure are the most morbid and fatal complications
in the exposed cases. In acute phase after SM exposure, all initial symptoms are
irritating effects leading to airways and alveolar injuries and cell necrosis. Thus,
treatment should be supportive and sometimes critical care therapy may be manda-
tory. In the chronic phase, productive cough, thick sputum and dyspnea are more
common symptoms. Chronic pulmonary sequels are common in the exposed
patients, but the mechanism of these complications was not clearly described.
However, obstructive and restrictive lung diseases together with recurrent infections
lead to chronic bronchitis, asthma like syndrome, bronchiectasis and finally chronic
obstructive lung disease. Although corticosteroids extensively prescribed for treat-
ment in chronic phase, its efficacy is not curative and its complications are serious.
Imbalance in apoptosis and repair seems to justify these symptoms. Therefore,
treatments include antioxidant and mucolytic (N-acetyl cysteine), bronchodilators
(salbutamol and salmetrol plus fluticasone inhalers), and macrolide antibiotics
(clarithromycin). Lung carcinogenicity of a single exposure to SM was not
confirmed.
15.2.8 Dermatologic Aspects of SM Exposure (Chap. 8)
The skin is one of the important affected target organs by SM as a chemical weapon,
besides the eyes and lungs. Skin exposure with SM results in the onset of a multiple
series of events including a full set of dermal responses for normal wound healing
and their mutual influence on each other, eventually leading to skin toxicity. In this
process, various mediators that have a regulating role in inflammation, apoptosis,
immune responses and some signaling pathways are involved. Despite the large
number of biochemical pathways that have been identified regarding SM injury, the
main difficulty in finding the best effective preventive or countermeasure is to find
which one of these pathways has higher pharmaceutical significance in this respect.
Indeed, among the mentioned therapeutic modalities, only anti-inflammatory
drugs appear to have moderate effectiveness in SM injury treatment. Currently, the
most effective way to enhance the overall efficacy in the management of patients
exposed to SM is combination therapy in order to influence different stages of SM
injury.
Also, in case of the possibility of using some topical preparations such as anti-
oxidants, protease inhibitors or scavengers, the possible problems related to drug
penetration in the skin should be taken into account.
15.2.9 Ocular Injury by Mustard Gas; Early and Late

Complications (Chap. 9)
SM rapidly reacts with ocular tissues. The eyes are the most sensitive organs to SM
exposure. In the acute phase, it results in conjunctivitis, blepharospasm, corneal
swelling and edema with severe pain and. Gradual spontaneous recovery usually
occurs, the pain reduces within a few days, regeneration of the corneal epithelium
appearing within 57 days. In late stages, severe dry eye, limbal stem cell defi-
ciency, corneal vascularization and corneal ulcers and probably retinochoroidal
changes may complicate the course of the disease several years after exposure.
Copious irrigation, topical steroids, artificial tears, cycloplegics and corneal protec-
tion are the mainstay of treatment in the acute phase. Late complications occur in a
small percent of those initially severely intoxicated. In the chronic phase, current
approaches are chiefly conservative and symptomatic by nature: wearing contact
basis, tarsorrhaphy and blepharorrhapy. Inflammation can be partially reduced
using topical as well as systemic steroids. More drastic action, i.e. corneal trans-
plantation, is needed in cases of scar formation, abnormal deposition, vasculariza-
tion loss of eye sight and severe stromal thinning that eventually leading to loss of
global integrity.
15.2.10 Immunological and Hematological Complications

of SM Poisoning (Chap. 10)
In addition to the skin, lungs, eyes, nervous system and gastrointestinal manifesta-
tions, SM induces hematological and immunological complications. Evidence from
experimental studies confirms that SM induces adverse effects on immune system
functions. Animal models are precious in the investigation of the physiological and
molecular mechanisms involved in SM hematological and immunological effects.
Clinical and immunological investigations of human cases have provided the most
direct evidence of the immunologic effects of MC. Clinical studies as a whole sup-
port a close synchrony between animal and clinical observations regarding the
immunosuppressive properties of SM. The immunological complications of the SM
exposed patients reflect the dysfunctions in the immune systems, both at cellular
and molecular levels. Such disruptions would, in turn, result in an increased risk of
infection and possible development of certain cancers. As the toxic effects of SM
are progressive and the clinical outcome of veterans can worsen over time, develop-
ing additional therapeutic strategies to increase the immunity of the patients are
required.
15.2.11 Psychiatric Complications of SM Poisoning (Chap. 11)
SM effects on the central nervous system and its neuro-psychiatric complications

have been proven difficult to deal with. The common neuro-psychiatric complica-
tions attributed to SM exposure include headache, fatigue, peripheral polyneuropa-
thy, impaired memory and concentration, anxiety and depression. The peripheral
polyneuropathy is usually the sensory type based on the electrophysiological inves-
tigation, including nerve conduction velocity measurement. Post-traumatic stress
disorder (PTSD) is one of the most common psychiatric disorders due to combat
experience, especially CWA including SM. Other psychiatric complications com-

monly observed in the victims of SM exposure are sleep disorders and sexual dys-
functions. Apart from loss of libido and penis dysfunction on erection, cases of
oligospermia and azospermia have been observed that are mostly due to SM expo-
sure and the PTSD following the exposure. The last but not the least are the changes
in quality of life of these patients due to chronic medical and psychological compli-
cations of SM exposure.
15.2.12 Genotoxicity, Teratogenicity and Mutagenicity

of SM Poisoning (Chap. 12)
Different in vitro and in vivo studies showed DNA damage and mutations follow-
ing SM exposure. These findings along with the other reported delayed complica-
tions as cancer following SM toxicity, suggest instability in the genetic system.
DNA is the main target for SM toxicity and DNA cross links and adducts consti-
tute 15 % and 85 % of DNA damages, respectively. Several studies have docu-
mented the mutagenic effects of SM in mammalian cells, in vivo and in vitro test
systems. Comet assay, western blotting, chromosomal aberration, immunohisto-
chemistry, quantitative PCR and Ames test were found to be the main experimen-
tal tools in the investigation of SM genotoxicity. Measurement of DNA damage
and proteins involved in DNA damage and repair signaling, evaluation of markers
of oxidative stress and chromosomal aberration are among the most important
tests that confirmed SM genotoxicity. Future studies, including evaluations of
enzymes involved in DNA methylation and quantitative miRNA expression pat-
tern may facilitate better understanding of the regulators of protein-coding gene
expression and epigenetic mechanisms in SM toxicity. New advancement might
help for a novel therapeutic agent for SM toxicity. Few studies are available regard-
ing the reproductive effects of SM in animals and humans and the results are
controversial.
15.2.13 Verification of SM Exposure in Biological Samples

(Chap. 13)
SM is a potent vesicant chemical warfare agent and use of such agents is considered
as crossing a red line. Exposure to SM via inhalation, cutaneous and ocular route
can result in a systemic uptake causing the formation of specific biomarkers that can
be of use for verification. Comprehensive methods for a free of doubt verification in
biological samples do exist that detect either remaining pure SM in the circulation
and tissues, or rely on biomarkers resulting from SM hydrolysis, SM biotransforma-
tion products, SM protein adduct or SM DNA adduct formation.
Intact SM in urine or blood can be analyzed by GC- or LC-MS methods in a

short time frame after exposure. Specific -lyase metabolites and non-specific TDG
have also been successfully determined by GC- or LC-MS methods. Several spe-
cific protein adducts with SM do occur, including albumin and hemoglobin that are
frequently used for verification purposes. SM-DNA adducts can be also visualized
with immunohistochemical methods or with evidentiary LC-MS based methods.
15.2.14 Occupational and Environmental Mustard Exposure,

Prevention and CWC (Chap. 14)
Literature on occupational SM exposure is limited, contrary to extensive data on

acute effects of this vesicant as a chemical weapon. Workers can be occupationally
exposed by an accident or intoxicated chronically at low does daily exposure during
their works. Accidental occupational SM exposure is always short-term exposure to
higher concentrations, depending also on the duration of exposure. Chronic occupa-
tional exposure is usually related to low-level concentration exposure due to leakage
or protocol breakage. It develops as intoxication with mild symptoms that may not
manifest immediately, and also can produce potential health consequences that
become evident months or years after the exposure, as late or delayed effects or just
a reduction in working ability. Environmental releases might occur near the places
where SM is produced and stored, but also due to the disposal of this chemical
weapon, by dumping them into the sea. Fate and behavior of this blistering agent in
the environment is strongly related to its concentration. SM is lipophilic, negligible
soluble in water and relatively stable in the environment. After years of efforts and
peace negotiations, on April 20, 1997, Chemical Weapons Convention entered into
force, and the OPCW (http://www.opcw.org/) began its work providing its imple-
mentation, and conditions for a safe world from chemical weapons.
15.3 Conclusion and Expert Opinion
The potential of SM toxicity in both acute and chronic is very clear. SM can be
dangerous to human depending on dose, duration, and the route of exposure. The
skin, eyes, and the respiratory tracts are the main targets of SM. Regarding the
potential of SM to interact with human genetic elements; it is not surprising to intro-
duce the cancer as one of the delayed consequences in the exposed patients and
more frequently in occupational chronic exposures. Effects of SM on the reproduc-
tive system, birth defects, and misdevelopment of children are issues that have
rarely been reported in SM victims, although were shown in some animal studies.
The potent stability of MC in the environment if released in any amount is the
main concern that leads scientists to think about large scale decontamination
methods. If MC is released into a large area from any sources such as an industrial
plant, from a container, or a bottle, it enters the environment. This release does not
necessarily always lead to human exposure, but the pollution of the soil, water sup-
ply, or its vapor matter. SM disappears from the outward soil easily by evaporation,
particularly hot environments or hydrolyzes in humid condition, but if SM stored in
the underground, it may be detected after several decades. SM in exposure to water
or in an extra wet environment reacts slowly leading to half mustard and finally to
thiodiglycole, which much less toxic.
Human might be exposed by breathing, eating, or drinking the substance, or by
skin and or eye contacts. The degree of toxicity depends on the dose, the duration,
and the route of exposure.
NM as a pharmaceutical compound is categorized as a chemotherapeutic medi-
cine. There are few reports on the possible long-term risk of occupational exposure
to such medicines that may induce carcinogenic, mutagenic, and teratogenic effects
or spontaneous abortions and increasing the risk of congenital malformations and
skin disturbances. There are specific guidelines that pharmaceutical companies
must adhere to prevent from any possible occupational exposure of the workers or
pollution of the environment. There are very specific rules for the waste disposal of
these materials that are implied by the pharmaceutical companies.
At the moment, the long term delayed toxic effects of SM are a serious medical
concern that cannot be ignored. Treatment of delayed SM toxicity is different from
the acute one and thus medical health professional and the related personnel should
be familiar with the SM toxicity, signs and symptoms, and management of both
acute and delayed toxic effects. Training of the medical staff at the world level to
make them prepared how to manage the acute and chronic effects of possible expo-
sure to SM is now a mandate that can be followed by the world responsible organi-
zations such as the OPCW. The destruction of already-made and stocked CWA is
one of the duties of the OPCW that is being followed up seriously. Currently, most
of the sulfur mustard at army bases is being destroyed by burning or neutralization.
The process of destruction has its own concerns and needs specially trained experts
and needs special care to protect the environment from any probable adverse effects
to the biological life. The hope and wish of the authors are to see all countries
adhere to regulatory rules and prohibit making or stocking of the CWA and try to
destroy any trace of already-prepared ones. The editors of this book who have been
involved in the OPCW scientific advisory board trust that a world of free from CWA
will bring peace to all human beings and other creatures.
Index
A C
Air trapping, 110 Calmodulin 1 (CAM1), 218
AMPs. See Antimicrobial peptides (AMPs) Carcinogenicity, 85, 323324
Anemia, 116 Cartas de un soldado, 35
Antimicrobial peptides (AMPs), 89 Cell cycle arrest, 59
Antitumor drug, 74 Cell death suppressing, 8990
Anxiety Chemical warfare agents (CWAs)
definition, 303 blistering agents or vesicants, 3031
and depression chemical compounds, 36
traumatic and stressful accidents, EGYPT-Yaman War (1963-1967), 36
302303 gas, liquid/solid forms, 30
and war, 303305 highly toxic agents, 3
Apolipoprotein A1 (APOA1), 178 mustard exposure, prevention, 399
Apoptosis, 59 Rif War in Morocco (1921-1926), 32, 35
Aziridinium ion, 21 Syrian Arab Republic, 3940
World War One (WWI), 30
Chemical weapons convention
B chemical disarmament, history, 379
BAL fluid analysis, 110 chemical warfare danger, 378
Bendamustine, 72, 7475 Geneva Protocol, 377378
Biotransformation products, 351352 German lethal chlorine gas attack, 376
Bronchiolitis obliterans (BO) hazard-specific safety procedures, 381
apoptosis, 178180 NM, 381
asthma and COPD, 195 pre-World War II period, 377
bronchiectasis, 196 SM gas, 379381
cardiopulmonary involvement, 197 18th National Committee on Disarmament,
chronic inflammation, 176177 378
clinical and pathological findings, 180182 verification mechanism, 377
histopathological changes, 110 World War I, 376
host response, 110 Cherry angioma, 113
lung cancer, 196197 Chest radiography, 109
oxidative stress, 178 Chlorambucil, 7374
proteolysis, 177 Chromosomal aberration
pulmonary fibrosis, 196 genotoxicity, 327328
Bronchoalveolar lavage (BAL), 176 in HPRT, 321

M. Balali-Mood, M. Abdollahi (eds.), Basic and Clinical Toxicology of Mustard
Compounds, DOI 10.1007/978-3-319-23874-6
402 Index
Chronic intoxication, 8082 Electromyography (EMG), 115

Corneal transplantation techniques, 122 Emphysema, 185186
Coronary artery disease (CAD), 84 Environmental exposure
Corticosteroids, 88 air, 374
Curcuma longa, 90 soil, 372373
Cyclophosphamide, 73 water, 374
Cytoxan, 67 Erythema, 221, 222
Eschar, 221
D
Delayed toxicity G
angiographic changes, 84 Gamma-glutamyl-transpeptidase (GGT), 187
CAD, 84 Geneva Protocol, 36
carcinogenicity, 85 Genotoxicants, 319
dermal complications, 8283 Genotoxicity
eyes effects, 83 acute and chronic injuries, 320
genotoxicity, 84 chemical injuries, 320321
reproductive system, 84 2-chloro-ethylethylsulfide (CEES), 322
respiratory effects, 83 DNA alkylation, 322
Depression DNA and genetic materials, 319
and anxiety DNA repair enzymes, 322
traumatic and stressful accidents, in vitro and in vivo tests, 319320
301302 in vitro studies in prokaryotic organisms,
war, 303305 321, 329333
CNS in-vivo studies in Drosophila, 321,
chronic effects, SM exposure, 293 334337
fatigue and lethargy, 294 laboratory tests, application
headaches, 293 chromosomal aberration, 327328
impaired memory and concentration, DNA damage, 324326
294295 DNA repair signalling, 326
neuropathy, 293294 oxidative stress measurement, 326327
neurotoxic complications, 292 metabolites and electrophiles, 319
seizure, 295 Nitrogen mustard, 322
tremor, 295 Genotoxins, 319
Vertigo, 295 Global Severity Index (GSI), 115
definition, 300301 Glutathione, 58
mood/affective disorders, 301
psychotherapy, 307
Dermal injuries, 76. See also Skin H
Desmosome, 227 Headache
Discoid lupus erythematosus, 112 depression, CNS, 293
Distilled mustard, 50 Health-Related Quality Of Life (HRQOL),
DNA adducts with SM, 353354 309
DNA damage Hematocrit (Hct), 116
Comet assay, 325 Hematological complication
DNA mono adducts/cross links, 324325 in animal sudies, 276277
measurement, 324325 biological effects, 275276
protein evaluation, 325326 in human studies
bone marrow cells, 278
hemolytic anemia, 279
E patients_T reticulocytes, 278279
Ecotoxicology, 374376 mechanisms of action, 276
Efferocytosis, 179 Hemidesmosomes, 217, 227
EGYPT-Yaman War (1963-1967), 36 Hemoptysis, 187
Index 403
Herbal medicine, 90 J
Hexamethylenetetramine (HMT), 162 Japan-China War (1939-19450), 37
High Resolution Computed Tomography
(HRCT)
BO, 181, 182 K
delayed destructive pulmonary sequelae, Keratinocyte suspension, 89
109 Keratolimbal allograft (KLAL), 122
diagnostic approaches, upper respiratory "King of the Battle Gases," 3, 51
tract, 155156
emphysema, 185, 186
lung, 188190 L
Hypercapnia, 109 Laryngitis, 146
Hypoxemia, 109 Laryngoscopy, 156
Late asthmatic responses (LAR), 201
Levinstein process, 11, 65
I Living-related conjunctival-limbal allograft
Air trapping, 183 (lrCLAL), 122
Ifosfamide, 74 L-phenylalanine. See Melphalan
Immuno-hematological complications, Lung
116117 acute phase
Immunological complication symptoms, 183184
in animal sudies, 276277 treatments, 197198
biological effects, 275276 air trapping, 183
in human studies BO (see (Bronchiolitis obliterans
delayed outcomes, 282286 (BO)))
short-term outcomes, 280282 chronic coughing, 184185
mechanisms of action, 276 chronic phase
physiology of, 274275 bronchodilators, 200
Interferon gamma-1b (INFI3-1b), 200201 corticosteroids, 199
International Agency for Research on Cancer GERD, 201202
(IARC), 8 interferon gamma-1b, 200201
Intoxication Macrolids, 199
accidental occupational exposure, 361 morphine, 200
chronic occupational SM poisoning, 361 NAC, 199200
mechanistic pathways, 394 protease inhibitors, 200
Iodine/povidone, 88 surfactant therapy, 201
Iran-Iraq war (1981-1988) symptoms, 184
delayed complications of SM exposure emphysema, 185186
body organs, 106108 lung parenchyma, 186187
dermal complications (see (Skin)) pulmonary toxicity, 173175
immuno-hematological complications, radiologic data
116117 chest X-ray, 188
neuropsychiatric complications, 113, lung HRCT, 188190
115116 respiratory ducts, 182183
ophthalmologic complications (see respiratory function
(Ocular delayed complications)) polysomnography, 191
respiratory complications (see pulmonary function tests, 191194
(Respiratory delayed complications,
Iranian veterans))
urogenital and reproductive, 117 M
ventricular diastolic abnormalities, 117 Mass spectrometry (MS), 352
reminiscence of, 105106 Mechlorethamine (HN2), 10, 51, 72, 73
use and epidemiology, 35, 3739 Mechlorethamine (mustargen), 42
Italian-Ethiopian War (1935-1936), 3536 Melphalan, 74
404 Index
Mustard compounds (MC) pharmaco- and toxicokinetics

chemistry, 392 absorption, 55
clinical toxicology, 393394 distribution, 55
CWA, 390 elimination, 56
epidemiology, 392393 metabolism, 5556
medicine and toxicology, 391 physical properties, 8, 17, 18
molecular molecular, 5153 reduce tumor growth in mice, 10
nitrogen (see (Nitrogen mustard (NM))) spiromustine, 51
nomenclature, 5153 structures of, 8
pharmacology, 394 synthesis of, 1315
physico-chemical properties, 51, 54 therapeutic uses of
structure, chemical, 5153 bendamustine, 72, 7475
sulfur (see (Sulfur mustard (SM))) chlorambucil, 7374
Mustard gas cyclophosphamide, 73
ambient temperature, 50 HN2, 72, 73
colorless and an oily liquid, 50 ifosfamide, 74
Mustard gas keratopathy (MGK), 77 melphalan, 74
Mustard Lung (ML), 175, 179 Prochlorperazine, 72
Mustargen. See Mechlorethamine (HN2) uramustine, 74
Mutagenicity warfare agents, 51
DNA alkylation, 328 Non-steroidal anti-inflammatory drugs
lung cancer biopsies, 322323 (NSAIDs), 86
point mutations, 322
in repair enzymes, mutations, 322
O
Occupational exposure
N accidental, 361
N-acetyl-cysteine (NAC), 199200, 236 causes, 361362
Nerve Conduction Velocity (NCV), 115 chronic, 361
Neuropathy, depression, 293294 vesicants, clinical effects (see (Vesicants))
Neuropsychiatric complications, 113, 115116 Ocular delayed complications
Niacinamide, 237 intoxication, 112
Nicotinamide adenine dinucleotide (NAD), 71 management of, 122
Nitrogen compounds. See Nitrogen mustard opacification, 111112
(NM) SM exposure, 111
Nitrogen mustard (NM) symptoms, 111
antimitotic and anticancer medication, Ocular injury by mustard gas
66, 137 anatomy
antineoplastic drugs, 8 choroid (choroidea/choroid coat), 257
apoptosis, 59 conjunctiva, 255256
aziridinium ion, 21 cornea, 256
cancer treatment, 3, 10 crystalline lens, 256257
chemistry, 392 eyelid, 255
chemotherapeutic medicine, 400 optic nerve, 258
chemotherapy, 7, 10, 4142 retina, 257258
colorless to pale yellow, 7 sclera, 257
combats, 7 anterior segment
DNA damage, 5658 acute phase, 260262
harmful chemical materials, 65 chronic phase, 263266
HN-1, HN-2 and HN-3, 7 mild eyelid erythema and conjunctival
Iso- Pr-N(EtCl)2, 7 congestion, 261
mechanism of action and symptoms, 8 chronic smoldering inflammation, 270
oily liquids, 7 immediate damage, 269270
Index 405
posterior segment, 266267 symptom, 80

tissue toxic effect treatment, 87, 88
cell DNA alkylation, 258 upper respiratory tract (see (Upper
epithelial migration and endothelial respiratory tract))
redistribution, 259 Poly (ADP-ribose) polymerases (PARPs),
human mononuclear leukocytes, 260 215216
oxidative stress, 259 Positive Symptom Distress Index (PSDI), 115
SM-DNA adducts, 259 Post-traumatic stress disorder (PTSD)
treatment auditory pseudo-hallucinations, 296
acute phase, 267268 depersonalization, 296
chronic phase, 268269 derealization, 296
Ocular surface. See Ocular injury by mustard emotional and behavioral symptoms, 297
gas pharmacotherapy, 298299
Organization for Prohibition of Chemical psychological reactions, 298
Weapons (OPCW), 64 psychosocial interventions, 299
risk factors, 296
Survivor guilt, 297
P toxic exposure, 297
Papillary dermis, 219, 228 Presbyopia, 111
Penetrating keratoplasty (PK), 122 Prochlorperazine, 72
Poisoning, SM. See also SM poisoning Prostaglandin E (PGE), 76
acute effects in eyes, 80, 81 Protein adducts with SM, 352353
AMPs, 89 Pruritus, 90
cell death suppressing, 8990 Psychological complications, SM
dermatology CNS and peripheral complications of
symptoms, 79 patients, 292295
treatment of, 87, 88 depression and anxiety, 300307
epidermal wound healing, 89 HRQOL, 309310
epigenetic and gene therapy, 89 PTSD, 296299
gastrointestinal, 7980 QOL, 309310
herbal medicine, 90 sexual dysfunction, 307
Iranian veterans, delayed complications sleep disorders, 307309
body organs, 106108 PTSD. See Post-traumatic stress disorder
dermal complications (see (Skin)) (PTSD)
immuno-hematological complications, Pulmonary fibrosis (PF), 176
116117 Pulmonary function tests (PFT), 109, 191194
neuropsychiatric complications, 113,
115116
ophthalmologic complications (see R
(Ocular delayed complications)) Reactive skin decontamination lotion (RSDL),
respiratory complications (see 23, 230
(Respiratory delayed complications, Respiratory delayed complications, Iranian
Iranian veterans)) veterans
urogenital and reproductive, 117 air trapping, 110
ventricular diastolic abnormalitie, 117 BAL fluid analysis, 110
medications, 87, 88 bronchiectasis, 109110
non-occupational exposure, 138 bronchodilators, 119120
occupational exposure, 138 cause of, 107
primary and secondary prevention, 86 chest radiography, 109
psychiatric complications (see corticosteroids, 120
(Psychological complications, SM)) curcuminoids, 121
recombinant protein technology, 89 cutaneous and ocular injuries, 107
respiratory system gamma interferon, 120121
406 Index
Respiratory delayed complications, Iranian acute skin lesions, 231, 232

veterans (Cont.) anti-inflammatory drugs, 238
internal factors, 121 antioxidants and scavengers, 234236
macrolides, 120 calmodulin antagonist, 237
mucolytic agents, 119 chronic skin complications, 232235
mutagenic alkylating agent, 110 conventional therapy, 230
nonsteroidal anti-inflammatory agents, 120 decontamination, 229230
physical therapies, 119 deleterious effects reduction, 238239
pulmonary artery hypertension, 121 PARP inhibitors, 237
spirometry, 109 prophylaxis, 229
symptoms, 107, 109 protease inhibitors, 236237
Respiratory ducts, 182183 Sleep disorders
Rif War in Morocco (1921_"1926), 32, 35 interrupted sleep/early morning awakening,
Roxithromycin, 88 307
sleep-wake disturbance, 308309
SM-DNA adducts, 353354
S SM poisoning
Sardasht, 39 acute and long-term incapacitating
Serum cytokines profiles, 284285 properties, 394395
Single-cell microgel electrophoresis technique, acute toxic effects, 390
118 in biological samples, 398399
Skin carcinogenicity, 323324
acute phase, 220223 chemistry, 392
carcinogenesis, 226227 cholinergic syndromes, 292
delayed complications, war veterans, dermatologic aspects, 396
223226 environmental mustard exposure, 399
diseases, 65 genetic toxicology (see (Genotoxicity))
histopathology, 227229 genotoxicity, 398
inflammation 7-(2-hydroxy-ethylthioethyl) guanine, 328
epithelial damage/protease activation, immunological and hematological
220 complications, 397
inflammatory mediators, 218219 lower airways complications, 395396
signal transduction pathway, 219 mutagenic effects (see (Mutagenicity))
Iranian veterans mutagenicity, 398
blisters, 112 neurotoxic complications, CWA, 292
histopathological examinatio, 113 occupational chronic exposures, 399
hyper and hypo-pigmentaions, 113, 114 ocular injury by mustard gas, 396397
lipophilic substances, 112 prevention and CWC, 399
management of, 122123 psychiatric complications, 397398
symptoms, 112 spermatogenesis reduction, 340
lipophilic substances, 214 teratogenicity, 324, 398
occupational exposure, 226 upper respiratory complications, 395
penetration of, 214 SM protein adducts, 352353
SM induced cytotoxicity Sparse delayed neurological complication, 115
calcium homeostasis, 217218 Spirometry, 109, 191194
DNA damage, 215, 216 Spiromustine, 51
glutathion/lipid peroxidation reaction, "Sprhbchse 37," 40
217 Stem cell technology, 89
human keratinocytes, 216217 Stratum corneum, 76
nitric oxide signaling, 218 Sulfanilamide disaster, 7
oxidative stress, 218 Sulfur, 7
PARPs, 215216 Sulfur mustard (SM)
vesicant injury management acute and chronic health impairments, 3
Index 407
acute toxic effects hematology (see (Hematological

eyes, 77 complication))
hematological effects, 78 histidine, 2021
immunotoxicity, 78 immunology (see (Immunological
nervous system injury, 77 complication))
oral and gastrointestinal (GI) tract, 77 inflammation, 5859
renal dysfunctio, 7778 intermolecular cyclization of, 19, 20
respiratory system, 7677 Iran-Iraq war (see (Iran-Iraq war
skin, 7576 (1981-1988)))
alkylating site of DNA, 20 Italian-Ethiopian War (1935-1936), 3536
analytical methods, 18 Japan-China War (1939-19450), 37
antidotes, 2223 lethal dose 50 % (LD50), 104105
battlefields, 51 lung (see (Lung))
bombs, 40 mechanism of action, 104
calcium homeostasis, 59 metabolism, 104
carcinogenic, 3 military purposes, 50
chemical agent, 46, 8 mode of cytotoxic action of, 19
chemical formula, 65, 136 molecular weight, 65
chemical properties, 17, 18, 103, pharmaco- and toxicokinetics
137, 138 absorption, 51, 54
chemical structure, 103 distribution, 54
chemical weapon, 137 elimination, 55
chronic toxicity, 8082 metabolism, 5455
clinical pharmacology physical properties, 1516, 137
Ca ion calmodulin signaling pathway, psoriasis, 65
7172 Rif War in Morocco (1921-1926), 32, 35
dermal exposure, 6768 skin (see (Skin))
DNA alkylation, 7071 spectroscopic properties, 16, 17
eye contact, 67 synthesis of
hydrolysis of, 69 bis(2-chloroethyl) polysulfides, 1213
inflammatory factors, 70 chlorine powder/bleach powder, 11
inhalation, 6667 crude compound purification, 12
intravenous injection of [14C], 69 ethylene and sulfur dichloride, 1011
metabolic processes, 68 history, 31, 32
NAD+ and ATP depletion, 71 olefinic bond, 11
oral ingestion, 67 peroxides, 12
oxidative stress, 72 photochemical preparation, 12
oxidising agent, 69 thiodiglycol with phosphorus
radioisotope labelling of, 68 trichloride, 11
compounds in, 8, 9 Syrian Arab Republic, 3940
conflicts, 40, 41 therapeutic uses, 72
creation and usage, 102103 thiol depletion, 58
cytotoxic and powerful vesicant World War I, 3
characteristics, 3 World War II (1939-1945), 36
decontamination of, 2122 World War One (WWI), 3235
delayed toxicity (see (Delayed toxicity))
development events, 31, 32
disposal factory, 40 T
distilled mustard, 50 Telomere, 84
DNA damage, 5658 Teratogenicity, 324, 328, 338339
effective battle gas, 8 Toxicity, SM, 390
EGYPT-Yaman War (1963-1967), 36 Trans-epidermal water loss (TEWL), 224
H agent, 65 Trichlormethine or trimustine, 51
408 Index
U SM-DNA adducts, 353354

Upper respiratory tract SM protein adducts, 352353
acute complications, 143145, 150 GC- and LC-MS based methods, 354
aerosol/vapor, 142 intact SM and biotransformation products,
anatomy of, 139 351352
in animal models toxicokinetic, 350351
acute effects, 158159 Vesicants
chronic effects, 159160 characteristics, 363
bidirectional effects, 142 chemical weapons, 363
cancer, 150152 in occupational exposure
chronic complications acute effects, 364365
airway narrowing, 146 chronic/delayed/late effects, 366367
chronic bronchiti, 147 diagnosis and differential diagnosis,
delayed toxic effects, 147 367368
demographic and clinical feature of, eye treatment, 369
147149 first aid on site, 368
Iraq-Iran conflict, 146 hospital level, 368369
laryngeal effects, 147 medical management and therapy, 368
laryngitis, 146 preventive measures and protection,
symptoms, 147 370371
epithelial cells of, 142 working ability, prognosis and
HRCT, 155156 assessment, 370
larynx, 141 risk to human health, 364
molecular mechanisms, 152155 stocked, 360
nasal mucosa, 142 Video assisted trachoscopy (VAT), 182
nasal sinuses, 140
SM-induced damages
acute phase management, 160161 W
antidotal therapy, 162 World War II (1939_"1945), 36
chronic phase management, 161 World War One (WWI), 3235
organ specific treatments, 162
SM induced injuries
HRCT, 155156 X
laryngoscopy, 156 Xerosis, 224, 234
speech evaluatio, 157158
stroboscopy, 157
video laryngoscopy, 157 Y
Uramustine, 74 Yellow rain, 37
Yperite. See Sulfur mustard (SM)
V
Verification, SM
detection

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Mahdi Balali-Mood

Mohammad Abdollahi Editors

Basic and Clinical

ISBN 978-3-319-23873-9 ISBN 978-3-319-23874-6 (eBook)

Springer Cham Heidelberg New York Dordrecht London

Printed on acid-free paper

Springer International Publishing AG Switzerland is part of Springer Science+Business Media (www.

Mahdi Balali-Mood, MD, PhD

Mohammad Abdollahi, Pharm D, PhD

Mohammad Abdollahi (MA) acquired a PharmD in 1988 from the University of

1 Chemistry of Mustard Compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . 1

11 Psychiatric Complications of Sulfur Mustard (SM) Poisoning . . . . 291

Mohammad Abdollahi, PharmD, PhD Department of Toxicology and

Leila Etemad Pharmaceutical Research Center, Mashhad University of Medical

Dirk Steinritz Bundeswehr Institute of Pharmacology and Toxicology, Munich,

Springer International Publishing Switzerland 2015 1

Keywords Sulfur mustard Nitrogen mustards HN-1 HN-2 HN-3

1.1.1 Commonly Used Chemical Warfare Agents

Toxic chemical compounds in munitions/devices causing death or harm to human

Table 1.1 The most common chemical warfare agents

Ethyl N,N-dimethylphosphoroamidocya Tabun Nerve agent C5H11N2O2P CH3

1.2.1 Nitrogen Mustard

Fig. 1.1 Nitrogen mustards

1.2.2 Sulfur Mustards

1.3 Applications ofMustard Compounds

1.3.1 Medicinal Uses

1.4.1 Synthesis ofSulfur Mustard

SCl2 + CH 2 = CH 2 ClCH 2CH 2SCl

3S ( CH 2CH 2OH )2 + 2PCl3 3S ( CH 2CH 2Cl )2 + 2H 3PO3

S ( CH 2CH 2OH )2 + 2HCl S ( CH 2CH 2Cl )2 + 2H 2O

The treatment of bis(2-hydroxyethyl) sulfide with hydrogen chloride (above) was

2CH 2 = CHCl + H 2S S ( CH 2CH 2Cl )2

1.4.1.1 Bis(2-Chloroethyl) Polysulfides

Levinstein mustard gas composes a considerable amount of bis(2-chloroethyl) poly-

Fig. 1.2 Structures of bis(2-chloroethyl) tri and penta sulfides

1.4.2 Synthesis ofNitrogen Mustard Compounds

N ( CH 2 CH 2 OH )3 + 3SOCl2 N ( CH 2 CH 2 Cl )3 + 3SO 2 + 3HCl

Another synthetic route is to prepare alkanolamines without employing ethylene

1. HCN + HCHO CH 2 OHCN

H3C CH3 H3C CH3 H3C CH3 H3C CH3

Fig. 1.3 Examples of nitrogen mustard analogs

Fig. 1.4 Preparations of analogs of nitrogen mustard

Containing a mechlorethamine group and a benzimidazole heterocyclic ring with

1.5 Physical Properties

1.5.1 Spectroscopic andPhysical Properties ofSulfur Mustard

water is in marked contrast to organic solvents. Around 20C, 0.06% of mustard

Table 1.3 Physical properties of sulfur mustard

1.6 Chemical Properties

Table 1.5 Physical properties of nitrogen mustards

covalently bond to nucleophilic groups such as amine, carboxyl, sulfhydryl and

1.7 Analysis andDetection ofSulfur Mustard

1.8 Interactions withBiological Molecules

1.8.1 Interactions withDNA andMode ofCytotoxicity

an intermolecular cyclization of SM which leads to formation of an intermediate ion

1.8.2 Interaction withImidazole

Sulfur mustards reaction with histidine; an imidazole-based amino acid demon-

Fig. 1.7 Cross-linking of guanine by sulfur mustard

1.9 Decontamination ofSulfur Mustard Compounds

The best way to detoxify sulfur mustard is to destroy it irreversibly. Incineration is

Fig. 1.8 Formation of aziridinium ion and bonding to guanine

ClCH 2 CH 2SCH 2 CH 2 Cl hydrolysis

1.10 Antidotes forSulfur Mustard

P-chloroperbenzoic acid and disinfectant chloramine-T detoxify sulfur mustard

1.1.1 Commonly Used Chemical Warfare Agents

1.2.1 Nitrogen Mustard

1.2.2 Sulfur Mustards

1.3 Applications ofMustard Compounds

1.3.1 Medicinal Uses

1.4.1 Synthesis ofSulfur Mustard

1.4.1.1 Bis(2-Chloroethyl) Polysulfides

1.4.2 Synthesis ofNitrogen Mustard Compounds

1.5 Physical Properties

1.5.1 Spectroscopic andPhysical Properties ofSulfur Mustard

1.6 Chemical Properties

1.7 Analysis andDetection ofSulfur Mustard

1.8 Interactions withBiological Molecules

1.8.1 Interactions withDNA andMode ofCytotoxicity

1.8.2 Interaction withImidazole

1.9 Decontamination ofSulfur Mustard Compounds

1.10 Antidotes forSulfur Mustard