Int. J. Exp. Path.

(I99I), 72, I-7 ADONIS 0959967391000015

ik I-

Increased production of oxygen free radicals in

tcienc.s
Library
cigarette smokers
J. Kalra, A.K. Chaudhary and K. Prasad
Departments of Pathology and Physiology, College of Medicine, University of Saskatchewan and
University Hospital, Saskatoon, Saskatchewan, Canada
Received for publication 25 April I990
Accepted for publication 7 August I990

Summary. Oxygen free radicals are known to produce damage in many biological tissues.
Cigarette smoking is a major risk factor for various diseases. It is possible that oxygen free
radical producing activity of polymorphonuclear (PMN) leucocytes is increased by cigarette
smoking. We studied the oxygen free radical producing (luminol-dependent chemilumines-
cent) activity of PMN leucocytes in blood and the malondialdehyde (lipid peroxidation product)
content of blood and serum in nonsmokers and smokers. The zymosan-induced chemilumines-
cent activity was measured on a LKB I251 luminometer. The malondialdehyde (MDA) was
measured as thiobarbituric acid (TBA) reactive substances. The chemiluminescent activity due
to oxygen-derived free radicals (superoxide anion, hydrogen peroxide, hydroxyl radical) and
superoxide dismutase (SOD)-inhibitable (superoxide anion) in nonsmokers were
1215.I±9i.i and 849.3±72.3 mV minm/il PMN leucocytes respectively. There was a
significant increase in the oxygen-derived free radicals and SOD-inhibitable chemilumines-
cence in smokers. The values of blood and serum MDA were I7I.7±6.i and 222.2±5.6
nmoles/l respectively in nonsmokers. There was an increase in both blood and serum MDA in
smokers. These results suggest that tjhe increased generation of oxygen free radicals by PMN
leucocytes might be responsible for an enhanced risk of various diseases related to cigarette
smoking.
Keywords: oxygen free radicals, PMN leucocytes, cigarette smoke, chemiluminescence,
malondialdehyde

Cigarette smoking has been implicated in the
pathogenesis of ischaemic heart disease
(USDHEW 1975; Kennel I98I; Timmis
I985), emphysema and obstructive lung
disease (Anderson & Ferris I962; Hoidal &
Niewoehner I983), and neoplastic disorders
(USPHS 197I; Doll & Peto I978). The
evidence incriminating cigarette smoking in
coronary artery disease and occlusive per-
Correspondence: DrJ. Kalra, Department of Pathology, University Hospital, Saskatoon, Saskatchewan,
Canada S7N OXO.
ipheral arterial disease is now substantial
(USDHEW 19 75). Any one of the major risk
factors-cigarette smoking, hypercholester-
olaemia and hypertension-approximately
doubles the risk of coronary heart disease
(Kennel I98 i). Coronary artery athero-
sclerosis has been related to smoking (Auer-
bach et al. I971, 1976, 1977). The ingre-
dients thought to be associated with the
I
2 J. Kalra et al.
development of atherosclerosis in cigarette
smoke are carbon monoxide (Webster et a].
1970) and nicotine (Auerbach et al. I97I)
but the pathophysiologic mechanisms are
unclear. Recently Prasad and Kalra (I989)
have reported the involvement of oxygen free
radicals in experimental atherosclerosis.
Cigarette smoke is known to contain reactive
peroxy radicals (Church & Pryor I985;
Pryor et al. I983). Polymorphonuclear
(PMN) leucocytes are another source of
oxygen free radicals (Fantone & Ward
I982). Cigarette smoke can activate PMN
leucocytes to produce oxygen free radicals
through activated complement C5 (C5a).
Activated complement C5 induces leucocyte
chemotaxis, autoaggregation, increased
adherence, and oxygen free radicals genera-
tion (Webster et al. I980; Craddock et al.
I977). Cigarette smoke can directly activate
the alternate pathway of complement in vitro
(Kew et al. I985; Firpo I985). It has been
reported that hydrogen peroxide can gener-
ate chemotactic activity from C5 (Shingu &
Nobunga I984) and that leucocytes when
activated by smoking release hydrogen per-
oxide in relatively large amounts (Hoidal et
al. I98I). Totti et al. (I984) have shown that
physiological concentrations of nicotine
enhance PMN leucocyte responsiveness to
C^a. Oxygen free radicals exert their cytotoxic
effect by causing peroxidation of membrane
phospholipids which can result in alterations
in membrane fluidity, increasing permeabi-
lity, and loss of membrane integrity (Free-
man & Crapo I982; Meerson et al. I982).
If oxygen free radicals play a role in the
enhanced risk of various pathogenic pro-
cesses including ischaemic heart and occlu-
sive peripheral arterial disease in cigarette
smokers, then there may be an increase in
oxygen free radical producing activity of
PMN leucocytes and the content of blood
malondialdehyde (MDA), a lipid peroxida-
tion product in such individuals. We there-
fore investigated the oxygen free radical
producing activity of PMN leucocytes and
the blood MDA in smokers and nonsmokers.
Materials and methods
Study subjects
This study included a total of 71 individuals.
The subjects were divided into two groups.
Group I comprised 48 healthy nonsmokers
who never smoked cigarettes and were not
exposed to any passive smoking in their
environment. This group consisted of I9
males and 29 females with an average age of
30.8 ± I.2 years (range 19-5 6 years). Group
II included 23 smokers (six males and 17
females) with an average age of 36.2±2.0
years (range 21-56 years). Entrance criteria
in this group included subjects who smoked
at least Io cigarettes per day for 8-I 5 years.
All the subjects in this study were healthy
volunteers recruited from hospital employees
or their friends and acquaintances. Informed
consent was obtained from all subjects. They
were not suffering from any disease and were
not on any medications including oral con-
traceptives. Venous blood was collected into
tubes containing ethylene diaminetetraace-
tic acid (EDTA) for determination of total
white blood cells (WBC) and PMN leucocyte
counts, blood MDA and oxygen free radical
producing activity of PMN leucocytes. Blood
was also collected for the determination of
serum MDA.
WBC Counts
Total WBC and PMN leucocyte counts were
made using a Technicon H6ooo System.
Preparation of opsonized zymosan
Opsonized zymosan was prepared by the
method described in the manual of Wallace
(I985) for chemiluminescence and by Pra-
sad et a). (I989). In short, zymosan A (Sigma
Chemical Company) was opsonized by addi-
tion of i ml of zymosan suspension (50 mg/
ml) in Hank's balanced salt solution (HBSS)
to 3 ml of serum. The mixture was incubated
for 40 min at 3 70C in a shaker bath and then
centrifuged at 3000 r.p.m. for io min at
ambient temperature (I8-20°C). The super-
 Cigarette smoke and oxygen free radicals 3
natant was removed and the pellet was (CL) for each sample was determined (a)
suspended in 4 ml ofHBSS and centrifuged at without activation by zymosan (resting), (b)
3000 r.p.m. for I0 min at ambient tempera- with activation by zymosan, and (c) with
ture. The pellet was suspended in 5 ml of activation by zymosan in the presence of
HBSS to make the final concentration of superoxide dismutase (SOD) as shown in Fig.
zymosan io mg/ml. i. The area under each curve was integrated
to give the total CL response during the
Chemiluminescence studies period of monitoring. The difference in areas
under zymosan-activated and resting curves
Luminol-dependent chemiluminescence pro- is designated as oxygen-derived free radicals
vides a sensitive indicator of production of CL, while that under zymosan-activated in
oxygen free radicals by resting and zymosan- the absence and presence of SOD is desig-
stimulated PMN leucocytes. The method of nated as SOD-inhibitable oxygen free radi-
chemiluminescence measurement was cals CL. The integrated area under the curve
essentially similar to that of Tono-Oko et al. is in mV x min (mV min). The absolute
(I983) and as described by Prasad et al. values are expressed as mV min per i16 PMN
(i989). The chemiluminescence was moni- leucocytes.
tored for 6o min using a LKB I25I lumin-
ometer.
Luminol-dependent chemiluminescence Malondialdehyde (MDA) assay
The MDA level in the blood and serum was
32 - measured as thiobarbituric acid (TBA) reac-
tive substances. The assay method for the
measurement of MDA was essentially the
same as Yagi (I976) and as previously
described by Prasad and Kalra (I989).
24j
E
0) Statistical analysis
0

-Ii
c
0. The results are expressed as mean ± s.e.m.
0
16 The data were analysed by unpaired Stu-
C._
Ea) dent's t-test using BMDP software. Probabi-
0
lity (P) values < 0.05 were considered statis-
0
i tically significant.
8
Results
.,1
I --
'
Ii Total WBC and PMN leucocyte counts
n L-
-

16
0 32
Time (min)
Fig. i. Typical tracing of zymosan-incluced chemi-
luminescence of PMN leucocytes of bilood from an
individual in the presence and absenc eofsuperox-
ide dismutase (SOD). Resting ch
cence (without-zymosan activatio
,
48 60 Total WBC counts in nonsmokers and
48 60
smokers were 6.65±0.22 and 6.42±0.51
giga-cells/l respectively. Mean polymorpho-
nuclear (PMN) leucocyte counts in blood of
nonsmokers and smokers were found to be
3.95±0.20 and 3.76±0.i8 giga-cells/l

eemilumines- respectively. There were no significant differ-

zymosan-induced chemiluminescerne in the ences in the total WBC and PMN leucocyte
absence of SOD; -, zymosan-indu ced chemilu-
- -
counts in the blood of smokers and non-
minescence in the presence of SOD. smokers.
4 J. Kaira et al.
0)
0, _ cytes in nonsmokers and smokers are
3000 r- summarized in Fig. 3. The SOD-inhibitable
Co
0 chemiluminescent activity of PMN leuco-
cytes in nonsmokers was 849.3 ± 72.3 mV
0 2000 r min/io6 PMN leucocytes. There was a
m -r-
marked increase in SOD-inhibitable chemilu-
a)
CD0
10001
minescent activity of PMN leucocytes of
LL _ smokers as compared to that of nonsmokers.
ow
. 0
These results suggest that PMN leucocytes of
o smokers have increased capacity to produce
oxygen free radicals.
Fig. 2. Polymorphonuclear leucocyte chemilumi-
nescence due to oxygen-derived free radicals Malondialdehyde (MDA) levels
(OFR) in 0, nonsmokers and smokers. The
U,

results are expressed as mean±s.e.m. * P<o.o5.
Chemiluminescence studies
Chemiluminescent activity of PMN leuco-
cytes in blood was measured in 48 healthy
nonsmokers and 23 smokers. Total oxygen-
derived free radical chemiluminescent acti-
vity of PMN leucocytes in nonsmokers and
smokers are summarized in Fig. 2. The total
oxygen-derived free radical chemilumines-
cence in healthy nonsmokers was found to
be 1215.I±9I.I mV min/i06 PMN leuco-
cytes. The values for chemiluminescent acti-
vity of PMN leucocyte in smokers were
significantly higher than those in non-
smokers. Superoxide dismutase (SOD)-inhi-
bitable chemiluminescence of PMN leuco-
0)
C
Blood and serum MDA content were mea-
sured in 48 healthy nonsmokers and I2
smokers. The MDA was measured in only I 2
smokers because initially we were mainly
interested in PMN chemiluminescence and
not in MDA in smokers. The values for blood
and serum MDA of both the groups are
summarized in Fig. 4. Blood and serum MDA
in nonsmokers were 17I.7±6.i and
222.2 ± 5.6 nmoles/l respectively. The levels
of blood and serum MDA were significantly
(P < o.o0 5) greater in smokers as compared to
nonsmokers. These results suggest that lipid
peroxidation is increased in smokers, indicat-
ing cellular damage.
Discussion
The results of the present study showed that
there was approximately a twofold increase
in the chemiluminescent activity of zymo-

0c.
0

2000 400 *
-0Z
300-
0)
1000
200
200
C>

E 0 0
0

Fig. 3. Superoxide dismutase (SOD)-inhibitable
PMN leucocyte chemiluminescence in 0, non-
smokers and smokers. The results are
U,
expressed as mean±s.e.m. * P<o.o5.
Blood Serum
Fig. 4. Blood and serum malondialdehyde (MDA)
in 0, nonsmokers and U, smokers. The results are
expressed as mean ± s.e.m. * P < o.o 5.
 Cigarette smoke and oxygen free radicals
san-stimulated PMN leucocytes in smokers smokers. The mechanism for this increase in
as compared to that in nonsmokers. Also the PMN chemiluminescence might be an inher-
blood and serum MDA content were higher ent change in the property of the PMN
in the smokers than in nonsmokers, indicat- leucocytes in smokers. The possible mecha-
ing an increase in lipid peroxidation in
smokers.
In this study opsonized zymosan was used
to stimulate PMN leucocytes as has been
used by other investigators (Prasad et al.
I 989; Hastings et al. I 982; Holt et al. I 984;
Nelson et al. 1 977). Whole blood was used to
investigate the PMN leucocyte chemilumi-
nescence in this study. No attempt was made
to isolate PMN leucocytes. However, the
chemiluminescence of blood was expressed
in terms of the PMN leucocyte count of the
blood. Whole blood has been used by us and
various investigators in the past for chemilu-
minescent studies (Tono-Oko et al. i983;
Prasad et al. i989; Dechatelet & Shirley
I 98 I; Selvaraj et al. I 982). Polymorphonuc-
lear leucocyte stimulation is accompanied by
increased oxygen consumption (Dewald et al.
1979) leading to the production of superox-
ide anion (O°) and hydrogen peroxide
(H202) which may react to form hydroxyl
radical (-OH) and singlet oxygen ('02) (Del
Maestro et al. I980). Subsequent involve-
ment of leucocyte-associated myeloperoxi-
dase generates hypochlorous acid (HOCI)
from H202 and Cl- (Fantone & Ward I982).
No significant differences in the total WBC
and PMN leucocyte counts were observed in
nonsmokers and smokers in this study. How-
ever, other investigators (Abboud et al.
I986; Bridges et al. I985) have reported an
increase in total WBC and PMN leucocyte
count in smokers. These differences in the
findings might be due to consumption of a
large number of cigarettes for prolonged
periods in their studies.
In the present study oxygen free radical
producing activity of PMN leucocytes was
found to be increased in smokers. There is no
information available regarding the PMN
leucocyte chemiluminescent activity in
smokers. The results suggest that somehow
or other there is an increase in the chemilu-
minescent activity of PMN leucocytes in
5
nism for increased activation of PMN leuco-
cytes and hence increased production of
oxygen free radicals in smokers might be due
to the following reasons: (a) cigarette smoke
can activate an alternate pathway of comple-
ment formation (Kew et al. I985; Firpo et al.
I985) which would induce leucocyte
chemotaxis, autoaggregation, increased
adherence and oxygen free radicals genera-
tion (Webster et al. I980; Craddock et al.
I977); (b) smoking is known to release
hydrogen peroxide from leucocytes (Hoidal
et al. I 98 I) which can generate chemotactic
activity from complement C5 (Shingu &
Nobunga I984); (c) nicotine in cigarette
smoke is known to enhance PMN leucocyte
responsiveness to C5a (Totti et al. I984).
Oxygen free radicals have been reported to
produce tissue and microvascular injury
(Mullane et al. I987; Hernandez et al. I987;
Lucchesi & Mullane I986). Oxygen free
radicals have been suggested to exert their
cytotoxic effect by causing peroxidation of
membrane phospholipids, which can result
in alterations in membrane fluidity, increas-
ing permeability and loss of membrane integ-
rity (Freeman & Crapo I982; Meerson et al.
I982). The increase in lipid peroxidation
product (MDA) in smokers in this study then
may be due to an increased production of
oxygen free radicals by PMN leucocytes. The
possible endothelial cell injury would lead to
a host of changes in the endothelial cell
lining which could lead to a critical sequence
of cellular interactions, culminating in for-
mation of lesions of atherosclerosis (Ross
I986). As such, oxygen free radicals have
been implicated in the genesis and mainte-
nance of atherosclerosis (Prasad & Kalra
I989). Atherosclerosis would lead to coron-
ary artery and other occlusive peripheral
arterial diseases.
In conclusion, there was an increase in the
oxygen free radical producing activity of
PMN leucocytes associated with an increase
6 1. Kaira et al.
in the lipid peroxidation product (MDA) in leukostasis and leukopenia. J. Clin. Invest. 6o,
smokers. Polymorphonuclear leucocyte- 260-264.
derived oxygen free radicals may damage the DECHATELET C.R. & SHIRLEY P.S. (I98I) Evalu-
endothelial cells, which would lead to coron- ation of chronic granulomatous diseases by a
ary artery and occlusive peripheral artery chemiluminescent assay of microlitre quanti-
ties of whole blood. Clin. Chem. 27,
disease. These results suggest that the risk of I739-I 74'.
cigarette smoking in heart disease might be DEL MAESTRO R.F., THAW H.H., BJORK J., PLANKER
related to an increased production of oxygen M. & ARFORS K.E. (I980) Free radicals as
free radicals by PMN leucocytes. mediators of tissue injury. Acta Physiol. Scand.
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Acknowledgements DEWALD B., BAGGIOLINI M., CURNUTTE J.T. &
BABIOR B.M. (I979) Subcellular localization of
This work was supported in part by grants the superoxide forming system in human neu-
from Saskatchewan Health Research Board trophils. J. Clin. Invest. 63, 2I-29.
and Saskatchewan Heart and Stroke Foun- DOLL R. & PETO R. (I978) Cigarette smoking and
dation. bronchial carcinoma: dose and time rela-
tionship among regular smokers and lifelong
The authors thank D. Duncan and P.K. non-smokers. J. Epidemiol. Commun. Hlth 32,
Chattopadhyay for their technical assistance 303-3 I 3.
and D. Rennie for assistance in preparation of FANTONE J.C. & WARD P.A. (I982) Role of
the manuscript. oxygen-derived free radicals and metabolites in
leukocyte dependent inflammatory reactions.
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