ORIGINAL ARTICLE
Cardiotoxic (Arrhythmogenic) Effects of
1,1-Difluoroethane Due to Electrolyte Imbalance
and Cardiomyocyte Damage
Kaushal Joshi, MS, Michael Barletta, MS, PhD, and John Wurpel, MS, PhD
Abstract: Inhalant abuse is the intentional inhalation of chemical vapors
to attain euphoric effects. Many common household products are abused
by inhalation and one is 1,1-difluoroethane (DFE), which is a halogenated
hydrocarbon used in refrigeration, dust-off spray, and airbrush painting.
Although many human DFE abuse cases have been studied, the etiology
and mechanism of sudden death is still unknown. In this study, an animal
model was used to simulate the human conditions of DFE inhalation abuse
that results in sudden death.
Current research targets mechanistic studies involving electrolyte
changes and cardiomyocyte damage after DFE administration in vivo. To in-
vestigate these changes, Sprague Dawley rats (N = 6) were exposed to
30 seconds of 20 L/min of DFE in multiple doses. Isoflurane acted as a
control. Two additional groups, epinephrine and epinephrine + DFE,
were included to simulate the clinical condition of DFE abuse. Plasma
sodium, potassium, calcium, and magnesium levels were measured,
followed by lactate dehydrogenase, creatine kinase, and cardiac troponin I
levels. In addition, oxidative stress markers were also evaluated in all animal
groups. Electrolyte levels showed a significant rise in plasma potassium
and magnesium levels for the treated groups. In addition, lactate dehydro-
genase, creatine kinase, and cardiac troponin I levels in DFE and epineph-
rine + DFE administered rats were significantly elevated as compared
with control. Some oxidative stress makers were also elevated signifi-
cantly in treatment groups. Furthermore, histopathological analysis
showed hyperemia/congestion in treated rats.
These results support cardiotoxic effects indicating that DFE results
in fatal arrhythmias, and the study can be important during clinical cases
involving inhalant abuse.
Key Words: inhalant abuse, 1,1-difluroethane, arrhythmia, toxicity,
myocardial damage
(Am J Forensic Med Pathol 2017;38: 115125)
I nhalant abuse is the intentional inhalation of chemical vapors
by sniffing, snorting, bagging, or huffing the substance to attain
a euphoric effect. Studies have shown that inhalant abuse causes
rapid intoxication, which can be short-lived, but continuous self-
administration of these inhalants can maintain the preferred level
of intoxication.1,2 Spray paints, shoe polish, dust-off spray, glue,
and lighter fluids are some products commonly abused by peo-
ple.3 One of the important factors that attract people to inhalant
abuse is the fact that these products are easily accessible at home,
school, and workplace, and they are legal for all age groups. Thus,
it is important to identify such hazardous chemicals and analyze
them to know the mechanism and to prevent or cure any clinical
conditions that might occur.
Manuscript received March 10, 2016; accepted June 23, 2016.
From the St. John's University, Queens, NY.
The authors report no conflict of interest.
Reprints: John Wurpel, MS, PhD, Pharmaceutical Sciences, St. John's
University, 8000 Utopia Pkwy, Queens, NY 11439.
E-mail: wurpelj@stjohns.edu.
Copyright 2017 Wolters Kluwer Health, Inc. All rights reserved.
ISSN: 0195-7910/17/38020115
DOI: 10.1097/PAF.0000000000000262
Am J Forensic Med Pathol Volume 38, Number 2, June 2017
Copyright 2017 Wolters Kluwer Health, Inc. All rights reserved.
Many common household products are abused by inhalation;
these have chemicals such as trichloroethylene, 1,1,1-trichloroethane,
methylene chloride, toluene, and 1,1-difluoroethane (DFE).2 Even
though it is difficult to classify these inhalants, they can be broadly
categorized in 4 main groups. The National Institute on Drug
Abuse classified these inhalants under aerosols, volatile solvents,
nitrates, and gases. 1,1-Difluoroethane falls under 2 groups, aero-
sols and gases.3 In 2004, the SAMSHA survey revealed that
19 million of US residents aged 12 years or older were illicit drug
users.4 In 2006, the number was around 20.4 million,5 and current
data (2013) suggest that 24.6 million US residents aged 12 years
or older were illicit drug users.6 Many young people inhale vapors
from different sources in search of quick intoxication without
being aware that using inhalants, even once, can have serious
health consequences.3
1,1-Difluoroethane, also known as Freon 152A, is a haloge-
nated hydrocarbon used as a propellant in dust-off spray. 1,1-
Difluoroethane is colorless gas, which is liquefied under pressure.
It has a melting point of 117C and boiling point of 25C.
Apart from aerosol propellants, other potential uses of DFE in-
clude refrigeration blends and catalyst regeneration. In recent
years, inhalant abuse is rising because there has been an increase
in drug abuse from readily available sources such as lighter fluid,
spray paints, cleaning fluids, and so on.7 1,1-Difluoroethane is
readily available because it is a key ingredient in dust-off sprays,
and thus, there are many cases where DFE has been abused either
through sniffing fumes directly from the container or by using
plastic bags. In addition, DFE has gained notoriety because of
its ability to rapidly induce intoxication after inhalation.8
In a recent inhalant abuse case, a man in his 30s was found
dead with a can of air duster beside him.7 It was confirmed that
the man had abused an inhalant for euphoric effect, and later, it
was determined that he had inhaled DFE through a can by wearing
a gas mask connected to a plastic bag. An empty DFE canister was
found in one of the plastic bags, and toxicological analysis by gas
chromatography proved the presence of DFE. Another case in-
volving a vehicle accident due to DFE abuse has been reported.9
In this case, a 24-year-old female driver was found dead after
a car accident. Investigation showed that the female had inhaled
dust-off spray just before and while operating the vehicle.
Blood and tissue samples showed varied amount of DFE pres-
ent throughout the body, which was related to the cause of
death. Similarly, there are several other cases related to DFE
abuse and related to halogenated hydrocarbons, which can be
life-threatening and dangerous.
Arrhythmias are irregularities in the electrical system of the
heart, which produce abnormal heart rhythms. Arrhythmias can
cause the heart to pump less effectively; hence, the heart rate
can be too fast or too slow.10,11 Arrhythmias such as skipped beat,
premature ventricular contractions (PVCs), ventricular tachy-
cardia, and ventricular fibrillation can be observed. If a point is
attained where ventricular tachycardia or ventricular fibrillation
occurs, then restoring a normal rhythm becomes very difficult
resulting in death.
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Joshi et al
Different mechanisms have been proposed with respect to
the occurrence of arrhythmias. One of the important theories
related to arrhythmias suggests that the number of impulse-
generating foci or a single rapid firing focus gives rise to fibril-
lation.12 This theory is known as ectopic foci theory, where
there are abnormal pacemaker sites within the heart apart from
sino-atrial node that display automaticity.13 Thus, ectopic foci can
be a region other than sinus node where impulse formation can
lead to cardiac arrhythmias.14 Abnormal focal activity or ectopic
foci can arise from afterdepolarization. Afterdepolarization refers
to increase in intracellular calcium concentration and decrease in
potassium and magnesium concentrations.15 Apart from electro-
lyte changes, cardiomyocyte damage can be other reason that
can affect changes in normal rhythm.14
Another important mechanism for arrhythmias is the re-entry
theory. Re-entry can take place within a small local region in the
heart.13 Re-entry arrhythmias can be caused by electrophysiolog-
ical abnormalities, but arrhythmias resulting from this mechanism
do not need to depend on it.15 Many antiarrhythmic drugs alter the
effective refractory period or conduction velocity and thus reverse
the effects of re-entry mechanisms.
Electrolyte imbalance plays an important role in the genesis
of arrhythmias. Electrolyte imbalance can alter cardiac ionic cur-
rents, which can increase arrhythmogenic effects.16 Experiments
have shown that combination of increased potassium levels in se-
rum and decreased calcium levels have cumulative effects on car-
diac conduction and may result in ventricular tachycardia and
ventricular fibrillation.17,18 Magnesium is the second most abun-
dant intracellular ion found in cardiomyocytes after potassium.16
Magnesium has opposing effects to calcium because it has been
shown that magnesium blocks the calcium ion channel. Thus, an
increase in serum magnesium levels is associated with decreased
calcium levels.
Cardiac biomarkers are biological parameters that can be
measured and quantified as an indicator of changes occurring
in the heart. These substances are first released into blood when
there is cardiac damage or stress. The markers can be indicators
of myocardial infarction, acute coronary syndrome, cardiac ische-
mia, and necrosis.19 Critical markers of early tissue necrosis or
myocardial ischemia are cardiac troponin I (cTnI), creatine ki-
nase (CK), and lactate dehydrogenase (LDH). Troponin is con-
sidered to be the standard for diagnosis of any acute injury with
additional information from patient history and electrocardiogram
(EKG) recordings.20
Cardiac arrhythmias can cause sudden cardiac death, and
some mechanistic studies and pathological processes link the
cause to oxidative stress caused by reactive oxygen species (ROSs).
In excitable cardiomyocytes, ROSs regulate both cellular metabo-
lism and ion homeostasis.21 Reactive oxygen species such as sin-
glet oxygen, superoxide radical, and hydroxyl radical are highly
unstable and highly reactive. A study on isolated heart showed that
exposure to oxygen radicals for brief time intervals caused a de-
crease in high-energy phosphates, loss of contractile function of
heart, and some structural abnormalities.22 To counter the effects
of free radicals, enzymatic and nonenzymatic antioxidants are
usually present within the cell. Three important cellular enzymes
are superoxide dismutase (SOD), glutathione peroxidase (Gpx),
and catalase (CAT). Some ex vivo studies showed that there was
impaired energy production, depressed contractile function, and
increased lipid peroxidation when isolated heart was exposed to
free radicals.23
Enzyme leakage (LDH, CK, and troponin) and oxidative
stress markers can be due to cardiomyocyte damage or histopath-
ological changes in the heart. Apoptosis, necrosis, disorientation
of cells, uneven pigmentation of cells, appearance of granules,
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017
and hyperemia/congestion are some of the histopathological
changes observed in the heart.24,25 During acute cardiac complica-
tions or sudden death scenarios, tissue does not undergo necrosis
or apoptosis because it usually takes time to reach that point of tis-
sue damage. In these cases, preliminary changes such as hyper-
emia or congestion can occur. Both hyperemia and congestion
are the result of excessive blood in a tissue. Hyperemia, if ob-
served under microscope, gives bright red and swollen appear-
ance. Tissue damage may not be evident, but capillaries are
engorged with blood. Physiologically, hyperemia/congestion can
occur, because there is increase in demand of nutrients because
of cardiac work or stimulus associated with irritation of tissue.
Later, tissue may develop edema, thrombus, weakened ventricular
muscle, and necrosis.
Based on recent increase in DFE abuse and related complica-
tions to it, we established a rat model to simulate the clinical con-
ditions of inhalation abuse that resulted in sudden death. In this
study, the effects of multiple doses of DFE on the cardiovascular
system were monitored.
1,1-Difluoroethane is abused by direct inhalation. Thus, we
mimicked the same route of administration in rats and evaluated
the ability of DFE to elicit arrhythmias. Furthermore, to elucidate
mechanisms behind these arrhythmias, electrolyte levels, biomarker
levels, oxidative stress markers, and pathological changes were
evaluated. It was hypothesized that these arrhythmogenic effects
of 1,1-difluoroethane were due to electrolyte changes, enzyme
leakage, stress parameters, and pathological changes in vivo.
MATERIALS AND METHODS
Chemicals and Instrumentation
1,1-Difluoroethane of 98% (600 g) was obtained from Sigma-
Aldrich chemical (St. Louis, Mo). Propranolol, epinephrine, lido-
caine, amiodarone, verapamil, and metoprolol were purchased
from VWR chemicals. Isoflurane 5 % acted as control for all the
experiments. Grass polygraph, electrocardiogram (ECG) pream-
plifier, and subdermal needle electrodes were obtained from Grass
Technology (West Warwick, RI). Microtome for pathology exper-
iments was obtained from Reichert-Jung (Depew, NY).
Experimental Animals
Male Sprague Dawley rats (275300 g) were purchased from
Taconic farms, Germantown, New York. The animals were main-
tained at the St. John's University Animal Facility. All procedures
mentioned were approved by the Institutional Animal Care and
Use Committee of St. John's University. The research was con-
ducted in compliance with the Animal Welfare Act and other
federal statutes.
Plasma Sample and Heart Collection
Blood samples from rats were collected by cardiac puncture
either under isoflurane anesthesia for control group or DFE for
treatment group and were immediately stored in lithium heparin
tubes, followed by centrifugation at 1500g and 4C for 10 minutes
to separate plasma fraction from blood. Plasma was transferred to a
capped polypropylene 2-mL tubes and stored at 80C until needed.
1,1-Difluoroethane Exposure
Rats were exposed to a fixed amount of DFE using a flow
meter designed to deliver gas to a plastic chamber with an area
of 2300 cm3.2 The chamber had an inlet through which DFE
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017
was administered and an outlet connected to charcoal canister
for safety.
The DFE flow was regulated by a flow meter, which was set
at a rate of 20 L/min, and this flow rate was kept constant for all
the experiments. All the animals were given a constant multiple
DFE doses for 30 seconds at 20-L/min flow rate with 1-minute
break between each dose.2 After multiple exposures, the animals
were immediately removed and blood was collected by cardiac
puncture. For pathology experiments, the hearts were placed in
10% formalin buffer and fixed using paraffin after 3 to 4 days.
Isoflurane acted as control for all the experiments, because we
wanted to use a halogenated compound, which has no cardiac ef-
fects and can also be used as anesthesia.
Electrocardiogram Monitoring and Onset of Death
In an EKG test, the electrical impulses made while the heart
is beating are recorded and usually shown on a piece of paper
known as electrocardiograph. Rats were divided into 7 groups.
The first group was treated with multiple DFE doses as discussed
earlier. The remaining groups were treated with propranolol + DFE,
verapamil + DFE, amiodarone + DFE, lidocaine + DFE, and met-
oprolol + DFE, the last group was given a dose of isoflurane,
which acted as control for the experiment.
For DFE administration, 1 rat was placed at a time in the
chamber and was given a dose of 20-L/min DFE for 30 seconds.
Immediately, pin electrodes were inserted subcutaneously in 4
limbs of the rat, and ECG machine was started, which was
followed by ECG recording for 30 seconds. After 30-second re-
cording, the rats were given a second dose of DFE. Similarly, this
procedure was repeated 4 to 5 times or until death occurred. Thus,
30-second EKG recording was obtained after every DFE. Thus,
every animal had a total of 2- to 3-minute overall recording on
the electrocardiograph. In addition, time of death for all the animal
groups was recorded.
For the remaining groups, the same procedure for DFE ad-
ministration was followed except that antiarrhythmic agents were
administered via intraperitoneal route before DFE administration.
Antiarrhythmic agents such as propranolol, metoprolol, verapamil,
lidocaine, and amiodarone were administered at 5-, 25-, 10-, 1-, and
50-mg/kg doses, respectively. Last group acted as a control, and
isoflurane 5 % was used for all the animals.
Electrolyte Panel
Sodium, calcium, potassium, and magnesium levels were mea-
sured using kits purchased from Stanbio Laboratory (Boerne, Tex).
Assay for Sodium
For measuring the sodium levels, 0.5-mL plasma or standard
was added to test tube and mixed with 0.5-mL precipitating re-
agent (aqueous trichloraacetic acid). The mixture is centrifuged
at high speed for 5 to 10 minutes. A total of 0.5 mL of supernatant
is then added to 2.5-mL coloring reagent (uranyl acetate). The
mixture is incubated at room temperature for 10 minutes and cen-
trifuged at high speed for 5 minutes. Supernatant is transferred to
cuvette and read at 420 nm against blank.
Assay for Calcium
For calcium levels, 0.5 mL of specimen is added to coloring
reagent containing orthocresolphthalein complexone. The mixture
is incubated at room temperature for 5 minutes. Absorbance is
read at 550 nm.
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Cardiotoxic (Arrhythmogenic) Effects of DFE
Assay for Potassium
A total of 0.05 mL of specimen is added to trichloroacetic
acid solution, followed by centrifugation at high speed for 5 to
10 minutes. The supernatant added to mixture of potassium boron
and sodium hydroxide. Sample is transferred to cuvette and incu-
bated at room temperature for 5 minutes. The cuvette is read
at 580 nm.
Assay for Magnesium
A total of 0.01 mL of sample or standard is mixed with 1-mL
Xylidyl Blue-1, and the mixture is incubated at room temperature
of 10 minutes and read at 520 nm.
Cardiac Biomarkers
Cardiac biomarkers, LDH and CK, were measured using
Stanbio assay kits, whereas troponin I was measured by perform-
ing enzyme-linked immunosorbent assay (ELISA), using Life
Diagnostic ELISA kit.
Assay for LDH
Specimens mixed with working reagent prepared from LDH
buffer and nicotinamide adenine dinucleotide (NAD). The cuvette
is incubated at 37C for a minute and absorbance is recorded at
1 minute. Furthermore, continuing the incubation absorbance is
again recorded at 2- and 3-minute mark. Average absorbance is
determined per minute (A/min) and multiplied by the factor
3376 to get amount of LDH in unit per liter.
Assay for CK
Specimens mixed with working reagent are prepared from
imidazole buffer and CK enzyme reagent containing nicotinamide
adenine dinucleotide phosphate (NADP) and adenosine diphos-
phate. The cuvette is incubated at 37C for a minute, and absor-
bance is recorded at 1 minute. Furthermore, continuing the
incubation absorbance is again recorded at 2- and 3-minute mark.
Average absorbance is determined per minute (A/min) and mul-
tiplied by the factor 3376 to get amount of LDH in unit per liter.
Assay for cTnI
The ELISA uses 2 different affinity purified antibodies. First
is used for immobilization on well plate, and the second is conju-
gated to horseradish peroxidase (HRP). Plasma sample is diluted
with plasma diluent and allowed to react simultaneously with the
2 antibodies, resulting in cTnI being sandwiched between solid
phase and HRP-conjugated antibodies. After 1 hour of incubation
on the shaker, the plates are washed to remove any unbound HRP-
conjugated antibodies. A solution of HRP substrate, tetramethyl-
benzidine, is added, and plates are incubated for 20 minutes
resulting in development of blue color. The color development is
stopped by addition of 1 N HCl, and the concentration on cTnI
is proportional to the absorbance at 450 nm. The amount of cTnI
is calculated by using a standard curve and graph.
Oxidative Stress Assay
Assay for CAT
The CAT activity was measured according to a well-known
method, which is based on the rate of disappearance of exogenous
H2O2 added to the sample.26
One-milliliter sample was mixed with 1 mL of 30 mM H2O2
and 1 mL of 0.01 M PBS pH 7.4, in a spectrophotometric quartz
cuvette. The changes in absorbance of reaction mixture was
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Joshi et al
measured at 240 nm twice, once immediately after mixing and
again 1 minute later.
The enzyme activity was calculated as nM of H2O2 reduced/
mg protein/min.
Assay of GPx
Glutathione peroxidase catalyzes the conversion of glutathi-
one to glutathione disulfide. In the presence of an excess of
-NADPH and glutathione reductase, glutathione disulfide is re-
duced to GSH and NADPH is reoxidized to NADP+. The rate of
decrease in absorbance at 340 nm as a result of the conversion
of NADPH to NADP+ reflects the activity of GPx. The GPx
activity was measured according to the method of Paglia and Val-
entine27 as modified by Greenwald.28 To a 1-mL spectrophoto-
metric quartz cuvette, 500 L of 0.01 M PBS pH 7.4, 100 L of
10 U/mL glutathione reductase solution, and 100 L of 10 mM
GSH were added in succession. After mixing and standing at
room temperature for 10 minutes, the reaction mixture was mixed
with 100 L of 15 mM -NADPH and incubated at 37C for
3 minutes. After the addition of 100 L of 5 M H2O2, the decrease
in absorbance of the reaction mixture was monitored at 340 nm
for 1 minute. The enzyme activity was calculated as nM product
oxidized/mg of protein/min.
Assay of SOD
The activity SOD was measured by the spectrophotometric
method.28 This method measures the ability of SOD to prevent
the autoxidation of epinephrine by O2 to a pink adrenochrome
showing a strong absorbance at 480 nm. Hence, the extent of
inhibition of autoxidation, as reflected by the dismutation of
O2 by SOD, is taken as measure of catalytic activity. A 100-L al-
iquot of sample was diluted with 1.8 mL of NaHCO3 Na2CO3
buffer pH 10.2, mixed with 100 L of 0.01 M ()-epinephrine,
and allowed to stand at room temperature for 1 minute. The
change in absorbance of the reaction mixture was read at 480 nm
for 1 minute. The enzyme activity was calculated as nM product
oxidized/mg of protein/min.
FIGURE 1. Effect of DFE on EKG monitoring. A, Regular sinus rhythm after isoflurane treatment. B, Premature ventricular contractions
after DFE treatment. Ventricular tachycardia (C) and ventricular fibrillation (D) after DFE treatment.
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017
Adenosine Triphosphate Measurement
Adenosine triphosphate (ATP) is primary energy currency of
the living system. Virtually, all energy-dependent processes use
the chemical energy stored in phosphate bond of ATP. Principle
in ATP ELISA involves a simple method, which uses the phos-
phorylation of glycerol to product that is easily quantified by
colorimetric method.
The plasma samples are first deproteinized. Ten microliter of
deproteinized sample is added to 96 well plate, and volume is ad-
justed to 50 L by ATP assay buffer. A reaction mixture consisting
of ATP probe, ATP converter, and developer is made, and 50 L of
the reaction mixture is added to all the plates. Simultaneously,
ATP standard curve is prepared by following the same procedure.
Samples and standards are incubated for 30 minutes and protected
from light. Absorbance is measured at 570 nm in a micro-plate
reader. Amount in ATP was calculated by the following equation:
ATP concentration (C) = B/V D (nmol/L), where B is ATP
amount from standard curve, V is sample volume added into sam-
ple wells, and D is dilution factor.
Epinephrine Measurement
Epinephrine was assayed using competitive ELISA kit by
Abnova. Upon acylation and extraction as per the assay protocol,
the samples and standards were added to 25-L enzyme solution
in 96 well plate. Furthermore, 50-L adrenaline antiserum was
added, and the samples and standards were incubated on a shaker
at room temperature for 2 hours. The contents of the well were
discarded, and plate was washed thrice using wash solution. One
hundred microliter of enzyme conjugate was added, and plate
was incubated for 30 minutes on a shaker at room temperature.
After another wash, 100 L of substrate was added to all the
wells and incubated for 30 minutes at room temperature. Finally,
100-L stop solution was added and absorbance was read be-
tween 620 and 650 nm within 10 minutes. Values for unknown
samples were obtained after using nonlinear regression curve
fitting for standards.
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017 Cardiotoxic (Arrhythmogenic) Effects of DFE

Cardiac Tissue Histopathology

To check morphology and pathophysiology, the heart was
surgically removed and fixed in 10% formalin buffer, which was
around 5 to 10 of tissue volume. The fixed tissue was cut into
appropriate portions and placed in embedding cassette. Further-
more, the tissue was dehydrated with 2 changes of 70%, 95%,
100% ethanol and xylene for an hour. Lastly, the cassettes with
tissue in it were placed in paraffin at 56C to 58C for 2 hours.
Tissue embedding was done into paraffin blocks. The paraf-
fin blocks were used to take 4- to 5-m sagittal sections using mi-
crotome and blade. The paraffin-embedded tissue sections were
subjected to hematoxylin and eosin staining and observed under
light microscope at 40 for any morphological changes.

FIGURE 2. Effect of DFE on epinephrine release. Plasma epinephrine Statistical Analysis

levels for treatment and control group. Data are expressed as
mean SEM, N = 6 rats per group, P 0.05 was considered
statistically significant and was denoted as * for EPI + isoflurane
and + for isoflurane group.
Results for the experiments are expressed as mean SEM for
6 and were analyzed by 1-way analysis of variance, followed by
Newman Keuls post hoc test. Differences from the groups were
considered to be significant when P value is 0.05 or less. The
Mann-Whitney U test was used for histopathology experiments,
and the grading for groups was considered to be significant when
P value is 0.05 or less.

FIGURE 3. Effect of DFE on electrolyte levels plasma sodium (A), plasma calcium (B), plasma potassium (C), and plasma magnesium (D)
for treatment and control groups. Data are expressed as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant
and was denoted as * for EPI + isoflurane and + for isoflurane group.

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Joshi et al Am J Forensic Med Pathol Volume 38, Number 2, June 2017

RESULTS Effect of DFE on Electrolyte Levels

Effect of DFE on EKG
Effect of multiple DFE doses on rat EKG was observed.
Figure 1 shows comparison between EKG after isoflurane and
DFE treatments. Panel A shows regular sinus rhythm after iso-
flurane treatment. 1,1-Difluoroethane treatment showed various
arrhythmias such as PVCs, ventricular tachycardia, and ventricu-
lar fibrillation as shown in panel B, C, and D, respectively. In ad-
dition to these EKG recordings, it was observed that animals
treated with DFE died after 4 to 5 doses of DFE, which is approx-
imately around 6 minutes post-DFE treatment.
Effect of DFE on Epinephrine Release
Endogenous epinephrine levels are important, because the
halogenated hydrocarbons are believed to sensitize the adrenal
gland to release catecholamines. As described in Figure 2, plasma
epinephrine levels showed a rise in epinephrine levels for the
DFE-treated group as compared with control.
Plasma electrolyte levels were evaluated for sodium, potas-
sium, magnesium, and calcium for all the groups. No significant
changes were observed for sodium and calcium levels as shown
in Figure 3, A and B between treatment and control groups,
whereas potassium and magnesium levels were significantly ele-
vated as shown in Figure 3, C and D.
Effects on Cardiac Biological Markers and ATP
Levels After DFE Treatment
As shown in Figure 4, plasma biomarker levels were evalu-
ated for LDH, CK, and troponin I (cTnI). Significant changes
were observed for both the treatment groups for all the biomarkers
as compared with control. In addition, ATP levels were measured
in plasma. ATP levels significantly decreased in DFE- and
EPI + DFEtreated groups as compared with control.
Effect of DFE on Oxidative Stress Markers
Plasma samples were analyzed for enzymatic oxidative
stress markers (Fig. 5). The SOD, CAT, and Gpx were analyzed.
Plasma SOD and Gpx levels were not affected significantly as

FIGURE 4. Effects on cardiac biological markers and ATP levels after DFE treatment LDH (A), CK (B), troponin I (C), and ATP levels (D)
for treatment and control groups. Data are expressed as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant
and was denoted as * for EPI + isoflurane and + for isoflurane group.

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Am J Forensic Med Pathol Volume 38, Number 2, June 2017 Cardiotoxic (Arrhythmogenic) Effects of DFE

FIGURE 5. Effect of DFE on oxidative stress markers SOD (A), CAT (B), and Gpx levels (C) for treatment and control groups. Data are expressed
as mean SEM, N = 6 rats per group, P 0.05 was considered statistically significant and was denoted as * for EPI + isoflurane and + for
isoflurane group.

shown in Figure 5, A and C whereas plasma CAT levels were sig- amiodarone showed that time for onset of death was increased
nificantly elevated in DFE- and EPI + DFEtreated groups as seen as compared with DFE-treated animals.
in Figure 5B.
DISCUSSION
Histopathological Analysis After DFE Treatment
In the last few years, clinical cases and reports related to DFE
The heart sagittal sections for treatment and control groups
abuse have gone up markedly. With its intended use, very little
were evaluated for histopathological changes. All the sections
risk is involved; on the contrary, DFE-containing products have
were analyzed after treating with hematoxylin and eosin stain.
become abuse substrates and have been diverted from their origi-
Changes with respect to congestion or hyperemia were checked
nal intended use. Toxicokinetic studies have been recently per-
for all the sections. Isoflurane and EPI + isoflurane sections did
formed,2 but the mechanism of DFE action still remains unknown.
not see any changes as described in Figure 6, A and C respectively,
The current study determines the arrhythmogenic effects of DFE
whereas DFE and EPI + DFE treatment showed congestion/
and reasons behind it, which might answer the cause of death
hyperemia as shown with black arrows in Figure 6, B and D re-
due to DFE and determine the mechanism of action occurring be-
spectively. Furthermore, the sections were graded on the basis of
cause of DFE intoxication. 1,1-Difluoroethane has also been seen
a scale from 0 to 3, with 0 being least severe damage and 3 being
to cause lethargy in accidents involving its abuse; hence, giving a
highest damage. As shown in Table 1, there was significant difference
clue to its central nervous system effects. In a pharmacokinetic
in the grading for treated and control groups. 1,1-Difluoroethane
study in vivo, tissue levels of DFE were determined at different
and EPI + DFE got grading of 2.8 and 2.7, respectively, whereas
time points after its exposure. The brain and heart showed DFE
control groups got a grading of 0.8.
in tissues at different levels, but DFE level in the heart remained
higher than the brain tissue after approximately 60-second post-
Protection Experiment DFE withdrawal.2 A previous report also had speculated that
To check if antiarrhythmic agents have an effect on onset of DFE elimination is rapid in the brain as compared with the heart.29
death, protection experiment was performed in vivo. As shown in This may further result in abuser inhaling more DFE to sustain eu-
Figure 7, antiarrhythmic agents, propranolol, metoprolol, and phoric effect because central nervous system effects are reduced

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Joshi et al
FIGURE 6. Histopathological analysis after DFE treatment. Histopathology of heart. Isoflurane (A), DFE (B), isoflurane + EPI (C), and
EPI + DFE (D). Black arrows showing hyperemia/congestion.
and thus lead to accumulation in the heart. In another case report, a
24-year-old woman was pronounced dead after a vehicle accident
involving inhalation of DFE.9 Aortic blood showed highest DFE
concentration, which was around 85.6 mg/L. Lower blood levels
were calculated in sites, which were further from the lung and
heart. This demonstrated the difference in absorption of DFE
through the body tissues. As DFE or similar volatile compounds
have low solubility in water, it is highly unlikely to be absorbed
in nasopharyngeal region or trachea. Furthermore, most DFE
absorption occurs through alveoli, where it equilibrates with
the blood of the pulmonary capillaries. Thus, DFE diffuses into
the blood and can be well distributed throughout the different
organs in the body.2
The present study deals with arrhythmogenic effects and the
possible mechanism behind DFE toxicity. Electrocardiographic
monitoring was performed to see changes in cardiac rhythms,
and several arrhythmias were observed ranging from low to high
severity. After multiple DFE doses in rats, arrhythmias such as
skipped beat, PVCs, coupled beats, ventricular tachycardia, and
ventricular fibrillation were observed. This confirms previous
data in which 42-year-old man was found dead after repeated ex-
posure to DFE.29
Studies have shown cardiac arrhythmias to be the most com-
mon cause of deaths due to volatile solvent abuse.30 The evidence
for this has been shown during the early 1900s when some pa-
tients under chloroform anesthesia suddenly die because of car-
diac arrhythmias.31 In that study, the group showed that when
myocardium was exposed to chloroform, it was sensitive to epi-
nephrine. Epinephrine and sympathetic nervous system stimula-
tion later resulted in abnormal cardiac muscle contraction.31 Later,
many such studies were reported with sensitization of myocardium
to epinephrine by volatile solvents during their use in hospitals,
factories, industrial environment, or abuse.30 Hence, it is impor-
tant to know the amount of epinephrine released endogenously
during abuse cases. Thus, as shown in Figure 2, the release of en-
dogenous epinephrine in plasma was checked for isoflurane- and
122 www.amjforensicmedicine.com
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017
DFE-treated animals. 1,1-Difluoroethanetreated animals had sig-
nificantly higher amount of epinephrine release than the control
group, which confirms the process happening in the clinical cases
as shown in some studies. While performing in vivo studies, it has
been observed that insufficient epinephrine is usually released,
and hence, a common procedure is to administer epinephrine ex-
ternally to mimic conditions similar to clinical cases or accidents
during inhalation or volatile substance abuse. Hence, apart from
isoflurane and DFE groups, 2 other experimental groups were
used in our study. Epinephrine + isoflurane and epinephrine + DFE
were 2 additional groups, which relate to clinical conditions dur-
ing such abuse cases.
Two important mechanisms behind arrhythmias are ectopic
foci and re-entry phenomena. As discussed earlier, changes in
electrolyte levels play an important role in the genesis of these ar-
rhythmias. Many studies have shown an increase in extracellular
potassium and a decrease in extracellular calcium (or increase in
intracellular calcium) can cause arrhythmias.17,18
We checked electrolyte levels for sodium, calcium, potas-
sium, and magnesium in rat plasma. Results showed no significant
change in plasma sodium and plasma calcium levels. Plasma
TABLE 1. Histopathology Grading for Treatment and Control
Groups
Treatment Groups Grading
Isoflurane 0.8
DFE 2.8+*
EPI + isoflurane 0.8
EPI + DFE 2.7+*
Data are expressed as mean SEM, N = 6 rats per group analyzed by
Mann Whitney U test, P 0.05 was considered statistically significant
and was denoted as * for EPI + isoflurane and + for isoflurane group.
2017 Wolters Kluwer Health, Inc. All rights reserved.
Am J Forensic Med Pathol Volume 38, Number 2, June 2017
FIGURE 7. Protection experiment after antiarrhythmic treatment.
Data are expressed as mean SEM, P 0.05 was considered
statistically significant and was denoted as * for EPI + isoflurane
and + for isoflurane group.
potassium and magnesium levels were significantly higher in DFE
and EPI + DFE groups. Studies have shown normal serum K+ is
around 4 to 5 mmol/L. With more than 5.5 mmol/L, it is believed
to cause problems in regular cardiac rhythm.17 Normal plasma
magnesium values range from 1.3 to 2.1 mEq/L,16 and levels
higher than that, it may cause arrhythmias. Correlation between
plasma electrolyte levels and EKG recordings has also been made
in some studies.32 It was suggested that an increase in extracellular
potassium levels cause changes in the normal EKG. In addition,
several different clinical cases were discussed with increased po-
tassium and EKG changes, followed by increased chance of myo-
cardial infarction or heart attack.32 In another study, experiments
focused on cause of ventricular fibrillation and simultaneous elec-
trolyte imbalance were performed.18 The study concluded that
ventricular fibrillation and ventricular tachycardia resulted in elec-
trolyte imbalance with potassium levels increasing and calcium
levels decreasing. In addition, it has been shown that abnormal fo-
cal activity or ectopic foci can arise from afterdepolarization,
which is increase in intracellular calcium concentration and de-
crease in potassium and magnesium concentration.15
The LDH, cTnI, CK, and myoglobin are considered to be im-
portant biomarkers for cardiotoxicity, myocardial infarction, and
early detection for cardiac tissue damage or necrosis.19 A recent
case study showed abnormalities in EKG, followed by changes
in troponin and CK levels after accidental ingestion of toluene
and xylene by a 65-year-old person.33 The person was further re-
ported to have conditions, which demonstrated all the criteria for
diagnosis of myocardial infarction. In addition, in vivo experi-
ments on rats have also shown changes in LDH and CK levels.34
Significantly elevated levels of LDH and CK were found in Wistar
rats, when the animals were exposed to carbon tetrachloride. Even
though liver toxicity of carbon tetrachloride is well-known, the
study was focused on cardiotoxic effects of carbon tetrachloride.
After DFE administration, plasma levels for LDH, troponin I,
and CK were checked. All 3 biomarkers were significantly ele-
vated, which may indicate that there was cardiomyocyte damage.
Furthermore, epinephrine pretreated animals also showed elevated
levels of all the 3 biomarkers as compared with epinephrine con-
trol group. In addition to these cardiac biomarkers, ATP levels
during arrhythmias are also important. ATP is required because
there is a high demand of energy for contraction of the heart.35
In addition, ion homeostasis depends on sufficient energy supply
2017 Wolters Kluwer Health, Inc. All rights reserved.
Copyright 2017 Wolters Kluwer Health, Inc. All rights reserved.
Cardiotoxic (Arrhythmogenic) Effects of DFE
and, if not enough, may cause pathophysiological changes as di-
verse as myocardial infarction, ventricular hypertrophy, or ische-
mia. In such complications, there is mismatch in ATP supply
and ATP use, which further affects electric and mechanical stabil-
ity of the heart.35 Furthermore, studies have also shown that con-
centration of ATP may fall by more than 70% in 20 minutes after
onset of ischemia, arrhythmias, or myocardial infarction.36 Thus,
apart from cardiac biomarkers, ATP levels in rats were measured
with or without DFE treatment. Treatment groups showed sig-
nificant decrease in ATP levels as compared with control
groups, which relates to some of the previous studies. Together
with CK, LDH, and cTnI, these results definitely indicate
cardiomyocyte damage.
Oxidative stress markers and antioxidant levels can change
because of volatile solvents or inhalants. Chronic and acute stud-
ies on such inhalants have been done earlier to check the effects on
organ systems and tissues.34,37 To directly check the relation be-
tween inhalant abuse and oxidative stress, a study was conducted
on approximately 50 to 60 adolescents aged between 11 and
19 years who had tendency to abuse inhalants and were admitted
by their parents or relatives. Inhalant abuse by these adolescents
was done by using different products such as glue, thinners, and
some other solvents.37 None of the volunteers were administered
antioxidants before they were admitted. Around 30 were healthy
volunteers, never exposed to inhalants. The study showed that
there was an increase in lipid peroxidation and the antioxidant en-
zymes SOD and GPx were significantly elevated in blood levels.
Similarly, in our study, the animals treated with DFE showed
significant elevation in SOD, CAT, and GPx. In another study,
endogenous protective mechanisms due to carbon tetrachloride
cardiotoxicity were analyzed in vivo.34 In this study, carbon tetra-
chloride induced myocardial endogenous enzymes in rat plasma.
Catalase, SOD, and Gpx levels were significantly elevated in rats
treated with carbon tetrachloride. Similarly, DFE and EPI + DFE
were seen to induce these enzymes endogenously.
Changes in biomarker levels, oxidative stress, and histopath-
ological alterations are linked together when there is tissue or cell
damage.34 A clinical scenario describing solvent abuse case was
reported, whereas biomarker levels, EKG data, and histopathol-
ogy were analyzed.38 This solvent abuse case involved a person
admitted to hospital who later died on the 23rd day. The patient
had a history of abusing shoe cleaning fluid containing a mixture
of trichloroethylene, methylene chloride, and dipropylene glycol.
Electrocardiogram showed changes in normal sinus rhythm, and
blood levels had high CK levels. Furthermore, in addition to
EKG and biomarker levels, histopathological analysis was also
performed. Cardiac sections showed hypertrophied myocardial
fibers, focal thickening, and congestive cardiomyopathy.38 In an-
other study, the effect of carbon tetrachloride on oxidative stress,
cardiac biomarkers, and histopathology studies were performed.34
As discussed earlier, in addition to changes in oxidative stress
parameters and cardiac biomarkers, histopathological studies
also showed significant changes. While control rats showed
normal cardiac architecture, carbon tetrachloride exposed rats
TABLE 2. Grading for Hyperemia/Congestion
Number Grading
0 Baseline vessels (least severe)
1 Single area dilated vessels
2 Patches, scattered foci
3 Most of the slide showed dilated
vessels (most severe)
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Joshi et al
FIGURE 8. Summary: Cardiotoxic (Arrhythmogenic) effects of
DFE in vivo.
showed inflammation, pathological changes, and edema. In the
current study, acute effects of DFE on histopathology of heart
were analyzed. 1,1-Difluoroethane and EPI + DFE groups showed
hyperemia and congestion as compared with control rats. Hyper-
emia or congestion is considered to be preliminary change, which
usually occurs in cardiac tissue before necrosis or apoptosis. The
cardiac sections were graded, and scoring was based on blind
study conducted by certified pathologist. A score of 0 was consid-
ered to be least severe and 3 was considered to be most severe, as
described in Table 2. The grading was done for changes occurring
because of hyperemia or congestion. The treatment groups got
grading more than 2.5 for hyperemia/congestion and overall dis-
orientation of cells, whereas control groups got grading less than
1 on both occasions. These changes suggest that treatment groups
showed severe effects as compared with control. As the animals
die within minutes after the DFE treatment, necrotic or apoptotic
cells were not expected because cardiac tissue is tough and may
need more time for prominent morphological changes. On the
other hand, just some primary changes in cell structure (hyperemia
or congestion) were thought to be possible markers.
Because treated animals showed significant changes in elec-
trolyte levels, biomarker, and histology, the final experiment in
this study involved the use of known antiarrhythmic agents to
check whether these drugs delay arrhythmia occurrence and on-
set of death. Antiarrhythmic agents selected were propranolol,
metoprolol, amiodarone, lidocane, and verapamil. The purpose
of selecting a wide variety of established antiarrhythmic drugs
was to check their effects on different ion channels and how it
would reflect on EKG and onset of death. In addition, DFE had
significantly affected electrolyte levels, and hence, it was impor-
tant to check whether these ion channel blockers decrease the
number and severity of arrhythmias. Lidocaine is a sodium chan-
nel blocker, aniodarone is a potassium channel blocker, verapamil
is a calcium channel blocker, propranolol is a nonselective -blocker,
and metoprolol is a selective 1 blocker. Each antiarrhythmic
agent was administered separately, followed by DFE treatment.
It was observed that propranolol, metoprolol, and amiodarone pre-
treatment significantly delayed onset of death and compared with
DFE-treated animals.
Thus, considering the data for electrolyte changes, biomarker
level elevation, oxidative stress markers, histopathology, and
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Am J Forensic Med Pathol Volume 38, Number 2, June 2017
protection experiments, Figure 8 represents a summary of mecha-
nism that might be occurring because of DFE administration.
As described in the Figure 8, electrolyte changes followed
by decrease in arrhythmias due to action of antiarrhythmic agents
provide primary information about mechanism behind DFE toxic-
ity. Electrolyte changes and action of antiarrhythmic agents can
relate to both ectopic foci theory and re-entry mechanism of ar-
rhythmias. Secondary effects such as changes in cardiac bio-
marker levels (CK, LDH, and cTnI) and oxidative stress markers
(CAT, SOD, and Gpx) can cause damage to cardiomyocytes. Damage
in cardiomyocytes can affect pathophysiology of the heart. Be-
cause time required for death of the animals due to DFE toxicity
is quick, no major changes in histopathology were observed.
Preliminary histopathological changes such as hyperemia/congestion
were observed. Considering all the previous changes, it gives
indication of possible cardiomyocyte damage or myocardial in-
farction. Furthermore, taking into consideration all the ob-
served changes, that is, electrolyte changes, antiarrhythmic
action of known agents, and pathophysiological changes, ec-
topic foci theory seems to be the possible mechanism through
which DFE exerts its effect.
In conclusion, the study was focused on arrhythmogenic ef-
fects of 1,1-difluoroethane, which is present in commonly used
commercial products. With recent increase in drug abuse cases,
it is important to address this issue and determine the cause and
mechanism behind deaths happening due to it. Thus, we tried to
mimic the exact situation that would arise if DFE is abused and
then evaluate the effects in a rat model. Electrocardiogram studies
had shown severe arrhythmias such as ventricular fibrillation and
ventricular tachycardia after DFE treatment, which cause death.
Furthermore, electrolyte imbalance, cardiac biomarkers, oxidative
stress markers, and histopathology were significantly affected. Fi-
nally, the findings suggest that DFE has cardiotoxic effects caus-
ing fatal arrhythmias, and the study can be important during
sudden clinical deaths, forensic testing, and evaluation.
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