Int. J. Exp. Path. (I99I), 72, I-7
ADONIS
0959967391000015
ik I.
tcienc.s Library
Increased production of oxygen free radicals in 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 chemiluminescent) activity of PMN leucocytes in blood and the malondialdehyde (lipid peroxidation product) content of blood and serum in nonsmokers and smokers. The zymosan-induced chemiluminescent 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 chemiluminescence 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-
ipheral arterial disease is now substantial (USDHEW 19 75). Any one of the major risk factors-cigarette smoking, hypercholesterolaemia and hypertension-approximately doubles the risk of coronary heart disease (Kennel I98 i). Coronary artery atherosclerosis has been related to smoking (Auerbach et al. I971, 1976, 1977). The ingredients thought to be associated with the
Correspondence: DrJ. Kalra, Department of Pathology, University Hospital, Saskatoon, Saskatchewan, Canada S7N OXO. 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 generation (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 generate chemotactic activity from C5 (Shingu & Nobunga I984) and that leucocytes when activated by smoking release hydrogen peroxide 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 permeability, and loss of membrane integrity (Freeman & Crapo I982; Meerson et al. I982). If oxygen free radicals play a role in the enhanced risk of various pathogenic processes including ischaemic heart and occlusive 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 peroxidation product in such individuals. We therefore 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 contraceptives. Venous blood was collected into tubes containing ethylene diaminetetraacetic 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 Prasad et a). (I989). In short, zymosan A (Sigma Chemical Company) was opsonized by addition 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 Luminol-dependent chemiluminescence provides a sensitive indicator of production of oxygen free radicals by resting and zymosanstimulated PMN leucocytes. The method of chemiluminescence measurement was essentially similar to that of Tono-Oko et al. (I983) and as described by Prasad et al. (i989). The chemiluminescence was monitored for 6o min using a LKB I25I luminometer. Luminol-dependent chemiluminescence 32
24j E 0
16
C._
Ea)
leucocytes. Malondialdehyde (MDA) assay
Statistical analysis The results are expressed as mean ± s.e.m. The data were analysed by unpaired Student's t-test using BMDP software. Probability (P) values < 0.05 were considered statistically significant.
-Ii
0.
under zymosan-activated and resting curves is designated as oxygen-derived free radicals CL, while that under zymosan-activated in the absence and presence of SOD is designated as SOD-inhibitable oxygen free radicals CL. The integrated area under the curve is in mV x min (mV min). The absolute values are expressed as mV min per i16 PMN
The MDA level in the blood and serum was measured as thiobarbituric acid (TBA) reactive 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).
-
0) 0 c
period of monitoring. The difference in areas
0
i
0
8 .,1 I
Results --
'
Ii n
L-
16
0
32
48 60
48
60
Time (min)
Fig. i. Typical tracing of zymosan-incluced chemiluminescence of PMN leucocytes of bilood from an individual in the presence and absenc eofsuperoxide dismutase (SOD). Resting ch cence (without-zymosan activatio zymosan-induced chemiluminescerne in the absence of SOD; -, zymosan-indu ced chemiluminescence in the presence of SOD. ,
- -
eemilumines-
Total WBC and PMN leucocyte counts Total WBC counts in nonsmokers and smokers were 6.65±0.22 and 6.42±0.51 giga-cells/l respectively. Mean polymorphonuclear (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 respectively. There were no significant differences in the total WBC and PMN leucocyte counts in the blood of smokers and nonsmokers.
J. Kaira et al.
4 0) 0,
_
cytes in nonsmokers and smokers are summarized in Fig. 3. The SOD-inhibitable chemiluminescent activity of PMN leucocytes in nonsmokers was 849.3 ± 72.3 mV min/io6 PMN leucocytes. There was a marked increase in SOD-inhibitable chemiluminescent activity of PMN leucocytes of smokers as compared to that of nonsmokers. These results suggest that PMN leucocytes of smokers have increased capacity to produce oxygen free radicals.
3000 r-
Co 0
2000 r
0
m
a) CD0 LL
-r-
10001
_
ow
. 0
o
Fig. 2. Polymorphonuclear leucocyte chemiluminescence due to oxygen-derived free radicals smokers. The (OFR) in 0, nonsmokers and results are expressed as mean±s.e.m. * P
E
0
0
0
Fig. 3. Superoxide dismutase (SOD)-inhibitable PMN leucocyte chemiluminescence in 0, nonsmokers. The results are smokers and expressed as mean±s.e.m. * P
ADONIS
0959967391000015
ik I.
tcienc.s Library
Increased production of oxygen free radicals in 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 chemiluminescent) activity of PMN leucocytes in blood and the malondialdehyde (lipid peroxidation product) content of blood and serum in nonsmokers and smokers. The zymosan-induced chemiluminescent 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 chemiluminescence 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-
ipheral arterial disease is now substantial (USDHEW 19 75). Any one of the major risk factors-cigarette smoking, hypercholesterolaemia and hypertension-approximately doubles the risk of coronary heart disease (Kennel I98 i). Coronary artery atherosclerosis has been related to smoking (Auerbach et al. I971, 1976, 1977). The ingredients thought to be associated with the
Correspondence: DrJ. Kalra, Department of Pathology, University Hospital, Saskatoon, Saskatchewan, Canada S7N OXO. 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 generation (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 generate chemotactic activity from C5 (Shingu & Nobunga I984) and that leucocytes when activated by smoking release hydrogen peroxide 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 permeability, and loss of membrane integrity (Freeman & Crapo I982; Meerson et al. I982). If oxygen free radicals play a role in the enhanced risk of various pathogenic processes including ischaemic heart and occlusive 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 peroxidation product in such individuals. We therefore 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 contraceptives. Venous blood was collected into tubes containing ethylene diaminetetraacetic 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 Prasad et a). (I989). In short, zymosan A (Sigma Chemical Company) was opsonized by addition 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 Luminol-dependent chemiluminescence provides a sensitive indicator of production of oxygen free radicals by resting and zymosanstimulated PMN leucocytes. The method of chemiluminescence measurement was essentially similar to that of Tono-Oko et al. (I983) and as described by Prasad et al. (i989). The chemiluminescence was monitored for 6o min using a LKB I25I luminometer. Luminol-dependent chemiluminescence 32
24j E 0
16
C._
Ea)
leucocytes. Malondialdehyde (MDA) assay
Statistical analysis The results are expressed as mean ± s.e.m. The data were analysed by unpaired Student's t-test using BMDP software. Probability (P) values < 0.05 were considered statistically significant.
-Ii
0.
under zymosan-activated and resting curves is designated as oxygen-derived free radicals CL, while that under zymosan-activated in the absence and presence of SOD is designated as SOD-inhibitable oxygen free radicals CL. The integrated area under the curve is in mV x min (mV min). The absolute values are expressed as mV min per i16 PMN
The MDA level in the blood and serum was measured as thiobarbituric acid (TBA) reactive 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).
-
0) 0 c
period of monitoring. The difference in areas
0
i
0
8 .,1 I
Results --
'
Ii n
L-
16
0
32
48 60
48
60
Time (min)
Fig. i. Typical tracing of zymosan-incluced chemiluminescence of PMN leucocytes of bilood from an individual in the presence and absenc eofsuperoxide dismutase (SOD). Resting ch cence (without-zymosan activatio zymosan-induced chemiluminescerne in the absence of SOD; -, zymosan-indu ced chemiluminescence in the presence of SOD. ,
- -
eemilumines-
Total WBC and PMN leucocyte counts Total WBC counts in nonsmokers and smokers were 6.65±0.22 and 6.42±0.51 giga-cells/l respectively. Mean polymorphonuclear (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 respectively. There were no significant differences in the total WBC and PMN leucocyte counts in the blood of smokers and nonsmokers.
J. Kaira et al.
4 0) 0,
_
cytes in nonsmokers and smokers are summarized in Fig. 3. The SOD-inhibitable chemiluminescent activity of PMN leucocytes in nonsmokers was 849.3 ± 72.3 mV min/io6 PMN leucocytes. There was a marked increase in SOD-inhibitable chemiluminescent activity of PMN leucocytes of smokers as compared to that of nonsmokers. These results suggest that PMN leucocytes of smokers have increased capacity to produce oxygen free radicals.
3000 r-
Co 0
2000 r
0
m
a) CD0 LL
-r-
10001
_
ow
. 0
o
Fig. 2. Polymorphonuclear leucocyte chemiluminescence due to oxygen-derived free radicals smokers. The (OFR) in 0, nonsmokers and results are expressed as mean±s.e.m. * P
E
0
0
0
Fig. 3. Superoxide dismutase (SOD)-inhibitable PMN leucocyte chemiluminescence in 0, nonsmokers. The results are smokers and expressed as mean±s.e.m. * P
