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Increased frequency of oxidant-mediated DNA strand breaks in mononuclear leucocytes exposed to activated neutrophils from cigarette smokers
Mutation Research, 225 (1989) 95-99
95
Elsevier MTRL 0176
Increased frequency of oxidant-mediated DNA strand breaks in mononuclear leucocytes exposed to activated neutrophils from cigarette smokers Gwen Schwalb and Ronald Anderson Division of Immunology, Departmentof Medical Microbiology, Institutefor Pathology, Universityof Pretoria (Republic of South Africa)
(Accepted26 October 1988)
Keywords: Smoking;Phagocytes;Neutrophils;Oxidants,reactive;DNA damage
Summary Following activation with the synthetic chemotactic tripeptide FMLP, potentiated by cytochalasin B (CB), blood neutrophils from cigarette smokers generated greater amounts of both extracellular and intracellular reactive oxidants than cells from non-smoking control subjects. FMLP/CB-activated neutrophils from cigarette smokers also inflicted increased oxidant-mediated damage to the DNA of cocultured mononuclear leucocytes, which was prevented by the inclusion of superoxide dismutase and catalase individually and in combination. These observations demonstrate that cigarette smoking primes phagocytes to generate increased amounts of potentially carcinogenic reactive oxidants.
Cigarette smoking causes a peripheral leucocytosis (Cone et al., 1971; Helman and Rubinstein, 1975) and the lungs of cigarette smokers are populated by up to 3 times more neutrophils and macrophages than the lungs of non-smokers (Ludwig et al., 1985; Hunninghake and Crystal, 1983). However, cigarette smoking does not only increase the numbers of phagocytes in the circulation and lungs, it also activates these cells to generate increased amounts of reactive oxidants (Hoidal et al., 1981; Hoidal and Niewoehner, 1982; Ludwig and Hoidal, 1982; Anderson et al., 1987). Since phagocyte-derived
reactive oxidants are potential carcinogens and induce chromosomal aberrations in bystander cells (Weitberg et al., 1983; Weitberg, 1987) it is possible that they are involved in the aetiology of bronchial carcinoma in cigarette smokers. In the present study the relative capacities of blood neutrophils from cigarette smokers and nonsmoking control subjects to generate reactive oxidants and to inflict DNA strand breaks on cocultured mononuclear leucocytes have been investigated.
Correspondence: Dr. R. Anderson, Institute for Pathology,
S m o k e r s and non-smoking control subjects. 6
P.O. Box 2034, Pretoria 0001 (SouthAfrica).
Methods
volunteer, asymptomatic cigarette smokers (mean
0165-7992/89/$03.50 © 1989Elsevier SciencePublishers B.V. (BiomedicalDivision)
96 age 33 +_ 4 years; 4 males and 2 females) with an average smoking history of 27 + 8 pack years, and 6 age- (33 +_ 3 years) and sex-matched, nonsmoking subjects were recruited to the study.
Chemicals and reagents. All chemicals were purchased from the Sigma Chemical Co., St. Louis, MO (U.S.A.). Indicator-free Hanks balanced salt solution (HBSS), pH 7.4, was used as the cellsuspending medium. Preparation of neutrophils and mononuclear leucocytes (MNL). These were prepared from heparinised venous blood as described elsewhere (Schwalb et al., 1988).
Reactive oxidant generation by neutrophils. Neutrophils (105) from cigarette smokers and paired, matched, non-smoking controls were preincubated with 0.1 mM luminol (5-amino-2,3-dihydro-l,4-phthalizinedione) for 5 rain at 37°C in 800 /~1 of HBSS. Activation of membraneassociated oxidative metabolism was initiated by the addition of 100 #1 of cytochalasin B (CB, final concentration 1/zg/ml) followed 10 sec later by 100 ttl of the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine(FMLP, final concentration 1 #M). Luminol-enhanced chemiluminescence (LECL) responses of unstimulated and FMLP/CB activated neutrophils were measured at 10-sec intervals for 1 min and thereafter at l-rain intervals using a Lumac g Biocounter (Model 2010; Lumac Systems Inc., Titusville, FL, U.S.A.). The results are expressed as relative light units (r.l.u.). FMLP/CB-activated LECL is a useful method to measure the generation of reactive oxidants by neutrophils since it detects distinct early (within 1 min) and later-occurring (after 5-10 min) oxidative events, which are of extracellular and intracellular origin respectively (Bender and Van Epps, 1983).
Measurement of DNA-strand breaks. These were measured by alkali unwinding and determination of ethidium bromide fluorescence using a Hitachi (Tokyo, Japan) fluorescence spectro-
photometer with excitation at 520 nm and emission at 590 nm according to the method of Birnboim and Jevcak (1981). Under the conditions employed ethidium bromide binds preferentially to doublestranded DNA. Neutrophils (4 × 107) were preincubated at 37°C for 15 min in a volume of 3.4 ml HBSS in 9-mm plastic tissue culture dishes followed by addition of 2 × 10 7 MNL. Neutrophilfree control systems were also included. The neutrophils were activated by the addition of FMLP/CB (1/~M and l ~g/ml final concentrations respectively) and the dishes incubated for 15 min at 37°C, after which the non-adherent MNL were separated from the adherent neutrophils, enumerated and resuspended to 107/ml in 250 mM mesoinositol, 10 mM sodium phosphate and 1 mM MgCl2 (pH 7.2). 200 ~tl of MNL suspension were lysed in alkaline medium containing 9 M urea, l0 mM NaOH, 2.5 mM cyclohexanediaminetetracetate and 0.1070 sodium dodecyl sulphate. Ethidium bromide fluorescence was determined after a 60-min incubation period at 15°C. The results were calculated according to the formula, D (percentage of double-stranded D N A ) = (F-Fmin)/(Fma~Fmin) X 100, where F is the fluorescence of the sample, Fmin the background fluorescence determined in samples that were sonicated at the beginning of the unwinding period in order to induce maximal unwinding, and Fma~ is the fluorescence of samples kept at pH 11.0, which is below the pH level needed to induce unwinding of double-stranded DNA. This system was also used to test the effects of catalase (200 units/ml) and superoxide dismutase (SOD, 100 units/ml) on the frequency of DNA-strand breaks in MNL exposed to FMLP/CB activated neutrophils.
Results
Expression and statistical analyses of results. The results are expressed as the mean values _+ the standard error of the mean (S.E.M.) for each series of experiments and statistical analyses were performed using the paired t-statistic by comparison of the values of each investigation obtained with
97 TABLE 1 M E A S U R E M E N T OF P E A K L E C L RESPONSES A N D T H E F R E Q U E N C Y OF D N A S T R A N D BREAKS IN C O C U L T U R E D M N L D U R I N G F M L P / C B A C T I V A T I O N OF NEUTROPI-IILS F R O M C I G A R E T T E SMOKERS A N D C O N T R O L SUBJECTS Test system
Peak L E C L responses in relative light units Extracellular
lntracellular
928 ± 170
1196 ± 1058
69 ± 2
1757 _+ 295 (/9 < 0.025)
3090 ± 1058 (p < 0.05)
49 ± 2 (p < 0.005)
FMLP/CB-activated neutrophils from non-smokers FMLP/CB-activated neutrophils from cigarette smokers
Percentage of doublestranded D N A remaining in cocultured M N L
Results are expressed as the m e a n values + S E M ' s for neutrophils from 6 cigarette smokers and 6 matched, non-smoking control subjects. The back-ground L E C L values for unstimulated neutrophils from smokers and non-smokers were 111 ± 7 and 110 ± 27 respectively. In the absence of F M L P / C B the percentages of double-stranded D N A remaining in M N L cocultured with smokers' and nonsmokers' neutrophils were 74 _+ 4 and 78 _+ 4 respectively.
TABLE 2 M E A S U R E M E N T OF T H E E F F E C T S OF C A T A L A S E A N D SOD I N D I V I D U A L L Y A N D IN C O M B I N A T I O N ON T H E F R E Q U E N C Y OF D N A S T R A N D BREAKS IN M N L EXPOSED TO F M L P / C B - A C T I V A T E D NEUTROPHILS F R O M C I G A R E T T E SMOKERS Test system
M N L + neutrophils only MNL + FMLP/CB-activated neutrophils MNL + FMLP/CB-activated neutrophils + catalase MNL + FMLP/CB-activated neutrophils + SOD M N L + FMLP/CB-activated neutrophils + catalase + SOD
Percentage of doublestranded D N A remaining in cocultured M N L 72 ± 4 a 51 ± 4 68 _+ 4 63 ± 4 72 _+ 3
a T h e results are expressed as the m e a n value _+ S E M ' s of 3 separate experiments using neutrophils from 3 different smokers.
neutrophils from a smoker and the corresponding matched, non-smoking control subject. FMLP/CB-activated L E C L responses. The peak extracellular and intracellular FMLP/CB-activated LECL responses of FMLP/CB-activated neutro-
phils from cigarette smokers and non-smoking control subjects are shown in Table 1. Neutrophils from smokers generated greater amounts of both extracellular (p<0.025) and intracellular (p<0.05) reactive oxidants than those of non-smokers. DNA-strand breaks. These results are also shown in Table 1. Exposure of MNL to the FMLP/CB-activated neutrophils of smokers was accompanied by a higher frequency of DNA strand breaks (17< 0.005) than MNL exposed to activated neutrophils from non-smokers. Both catalase and SOD individually and in combination prevented the oxidant-mediated strand breaks in MNL cocultured with smokers' FMLP/CB-activated neutrophils (Table 2).
Discussion Cigarette smoking primes circulating neutrophils and pulmonary macrophages to release increased amounts of reactive oxidants on exposure to stimuli of membrane-associated oxidative metabolism (Hoidal et al., 1981; Hoidal and Niewoehner, 1982; Ludwig and Hoidal, 1982; Anderson et al., 1987). The mechanism of this
98 smoking-related priming of oxidant release by phagocytes has not been established, but the importance of acute smoke exposure (i.e. cigarettes smoked per day) has been described (Richards et al., 1988). Priming of superoxide generation by PMA (phorbol myristate acetate)-activated neutrophils f r o m rats occurs within 15 weeks of chronic smoke exposure or 1 h after intraperitoneal injection of nicotine (0.02 and 0.2 m g / k g ) and it has been proposed that this agent (nicotine) is the smoke-derived, priming factor (Gillespie et al., 1987). However, a circulating, priming agent has not been detected in the plasma of cigarette smokers (Ludwig and Hoidal, 1982; Anderson et al., 1987). It is possible that priming of neutrophil membrane-associated oxidative metabolism m a y occur at an earlier stage, during cellular differentiation. Interestingly, T-lymphocyte derived granulocyte-macrophage colony stimulating factor (GM-CSF) primes neutrophils to generate increased amounts of superoxide during activation with F M L P by altering the affinities and increasing the numbers of F M L P receptors (Weisbart et al., 1986). Irrespective of the biochemical mechanisms of the priming process it is clear that cigarette smoking increases the potential hazard of oxygen toxicity to tissue cells by inducing chronic inflammation and release o f reactive oxidants by hyperactive phagocytes. In the present study we have confirmed previous reports that neutrophils f r o m cigarette smokers generate increased levels of reactive oxidants, especially extraceUular oxidants, when activated with stimuli such as F M L P / C B (Ludwig and Hoidal, 1982; Anderson et al., 1987). Oxidants released by activated phagocytes are potential carcinogens and inflict c h r o m o s o m a l damage on bystander cells (Weitberg et al., 1983; Weitberg, 1987). In the present study we have observed a much higher frequency of oxidantmediated D N A strand breaks in M N L exposed to activated neutrophils f r o m cigarette smokers than during coculture o f these cells with neutrophils f r o m non-smokers. The M N L were protected from phagocyte-derived oxidants by catalase and SOD individually and in combination, which indicates
that H202, superoxide and possibly hydroxyl radical p r o m o t e the damage to the D N A of MNL. In conclusion, neutrophils f r o m cigarette smokers release increased amounts of toxic extracellular reactive oxidants which damage the D N A o f bystander cells. Sustained oxidative damage to the D N A of lung tissue ceils, chronically exposed to hyperactive phagocytes, may contribute to the development of bronchial carcinoma in cigarette smokers.
Acknowledgement This investigation was supported by a research grant awarded by the National Cancer Association of South Africa.
References Anderson, R., A.J. Theron and G.J. Ras (1987) Regulation by the anti-oxidants ascorbate, cysteine and dapsone of the increased extracellular and intra-cellular generation of reactive oxidants by activated phagocytes from cigarette smokers, Am. Rev. Resp. Dis., 135, 1027-1032. Bender, J.G., and D.E. Van Epps (1983) Analysis of the bimodal chemiluminescence pattern stimulated in human neutrophils by chemotactic factors, Infect. Immunol., 41, 1062-1070. Birnboim, H.C., and J.J. Jevcak (1981) Fluorimetric method for the rapid detection of DNA strand breaks in human white blood ceils produced by low doses of radiation, Cancer Res., 41, 1889-1892. Cone, F., J. Lellouch and D. Schwartz (1971) Smoking and leukocyte counts: results of an epidemiologicalstudy, Lancet, 2, 632-634. Helman, N., and L.S. Rubinstein (1975) The effects of age, sex and smoking on erythrocytes and leukocytes, Am. J. Clin. Pathol., 63, 35-44. Hoidal, J.R., and D.E. Niewoehner (1982) Lung phagocyte recruitment and metabolic alterations induced by cigarette smoke in humans and hamsters, Am. Rev. Resp. Dis., 126, 548-552. Hoidal, J.R., R.B. Fox, P.A. le Barbre, R. Perri and J.E. Repine (1981) Altered oxidative metabolism responses in vitro of alveolar macrophages from asymptomatic smokers, Am. Rev. Resp. Dis., 123, 85-89. Hunninghake, G.W., and R.G. Crystal (1983) Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers, Am, Rev. Resp. Dis., 128, 833-838.
99 Gillespie, M.N., J.O. Owasoyo, S. Kojima and M. Jay (1987) Enhanced chemotaxis and superoxide anion production by polymorphonuclear leukocytes from nicotine-treated and smoke-exposed rats, Toxicology, 45, 45-52. Ludwig, R.W., and J.R. Hoidal (1982) Alterations in leukocyte oxidative metabolism in cigarette smokers, Am. Rev. Resp. Dis., 126, 977-980. Ludwig, R.W., B.A. Schwartz, J.R. Hoidal and D.E. Niewoehner (1985) Cigarette smoking causes accumulation of polymorphonuclear leukocytes in alveolar septum, Am. Rev. Resp. Dis., 131, 828-830. Richards, G.A., A.J. Theron, t~.A. Van der Merwe and R. Anderson (1988) Spirometric abnormalities in young smokers correlate with increased chemiluminescence responses of activated blood phagocytes, Am. Rev. Resp. Dis., in press. Schwalb, G., A.D. Beyers, R. Anderson and A.E. Nel (1988) Promotion of DNA strand breaks in cocultured mononuclear
leukocytes by protein kinase C-dependent pro-oxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation, Cancer Res., 48, 3094-3099. Weisbart, R.H., D.W. Golde and J.C. Gasson (1986) Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leu-phe receptors, J. Immunol., 137, 3584-3587. Weitberg, A.B. (1987) Chloramine-induced sister chromatid exchanges, Mutation Res., 190, 277-280. Weitberg, A.B., S.A. Weitzman, M. Destrempes, S.A. Latt and T.P. Stossel (1983) Stimulated human phagocytes produce cytogenetic damage in cultured mammalian cells, N. Engl. J. Med. 308, 26-30.
Communicated by D.R. Davies
95
Elsevier MTRL 0176
Increased frequency of oxidant-mediated DNA strand breaks in mononuclear leucocytes exposed to activated neutrophils from cigarette smokers Gwen Schwalb and Ronald Anderson Division of Immunology, Departmentof Medical Microbiology, Institutefor Pathology, Universityof Pretoria (Republic of South Africa)
(Accepted26 October 1988)
Keywords: Smoking;Phagocytes;Neutrophils;Oxidants,reactive;DNA damage
Summary Following activation with the synthetic chemotactic tripeptide FMLP, potentiated by cytochalasin B (CB), blood neutrophils from cigarette smokers generated greater amounts of both extracellular and intracellular reactive oxidants than cells from non-smoking control subjects. FMLP/CB-activated neutrophils from cigarette smokers also inflicted increased oxidant-mediated damage to the DNA of cocultured mononuclear leucocytes, which was prevented by the inclusion of superoxide dismutase and catalase individually and in combination. These observations demonstrate that cigarette smoking primes phagocytes to generate increased amounts of potentially carcinogenic reactive oxidants.
Cigarette smoking causes a peripheral leucocytosis (Cone et al., 1971; Helman and Rubinstein, 1975) and the lungs of cigarette smokers are populated by up to 3 times more neutrophils and macrophages than the lungs of non-smokers (Ludwig et al., 1985; Hunninghake and Crystal, 1983). However, cigarette smoking does not only increase the numbers of phagocytes in the circulation and lungs, it also activates these cells to generate increased amounts of reactive oxidants (Hoidal et al., 1981; Hoidal and Niewoehner, 1982; Ludwig and Hoidal, 1982; Anderson et al., 1987). Since phagocyte-derived
reactive oxidants are potential carcinogens and induce chromosomal aberrations in bystander cells (Weitberg et al., 1983; Weitberg, 1987) it is possible that they are involved in the aetiology of bronchial carcinoma in cigarette smokers. In the present study the relative capacities of blood neutrophils from cigarette smokers and nonsmoking control subjects to generate reactive oxidants and to inflict DNA strand breaks on cocultured mononuclear leucocytes have been investigated.
Correspondence: Dr. R. Anderson, Institute for Pathology,
S m o k e r s and non-smoking control subjects. 6
P.O. Box 2034, Pretoria 0001 (SouthAfrica).
Methods
volunteer, asymptomatic cigarette smokers (mean
0165-7992/89/$03.50 © 1989Elsevier SciencePublishers B.V. (BiomedicalDivision)
96 age 33 +_ 4 years; 4 males and 2 females) with an average smoking history of 27 + 8 pack years, and 6 age- (33 +_ 3 years) and sex-matched, nonsmoking subjects were recruited to the study.
Chemicals and reagents. All chemicals were purchased from the Sigma Chemical Co., St. Louis, MO (U.S.A.). Indicator-free Hanks balanced salt solution (HBSS), pH 7.4, was used as the cellsuspending medium. Preparation of neutrophils and mononuclear leucocytes (MNL). These were prepared from heparinised venous blood as described elsewhere (Schwalb et al., 1988).
Reactive oxidant generation by neutrophils. Neutrophils (105) from cigarette smokers and paired, matched, non-smoking controls were preincubated with 0.1 mM luminol (5-amino-2,3-dihydro-l,4-phthalizinedione) for 5 rain at 37°C in 800 /~1 of HBSS. Activation of membraneassociated oxidative metabolism was initiated by the addition of 100 #1 of cytochalasin B (CB, final concentration 1/zg/ml) followed 10 sec later by 100 ttl of the synthetic chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine(FMLP, final concentration 1 #M). Luminol-enhanced chemiluminescence (LECL) responses of unstimulated and FMLP/CB activated neutrophils were measured at 10-sec intervals for 1 min and thereafter at l-rain intervals using a Lumac g Biocounter (Model 2010; Lumac Systems Inc., Titusville, FL, U.S.A.). The results are expressed as relative light units (r.l.u.). FMLP/CB-activated LECL is a useful method to measure the generation of reactive oxidants by neutrophils since it detects distinct early (within 1 min) and later-occurring (after 5-10 min) oxidative events, which are of extracellular and intracellular origin respectively (Bender and Van Epps, 1983).
Measurement of DNA-strand breaks. These were measured by alkali unwinding and determination of ethidium bromide fluorescence using a Hitachi (Tokyo, Japan) fluorescence spectro-
photometer with excitation at 520 nm and emission at 590 nm according to the method of Birnboim and Jevcak (1981). Under the conditions employed ethidium bromide binds preferentially to doublestranded DNA. Neutrophils (4 × 107) were preincubated at 37°C for 15 min in a volume of 3.4 ml HBSS in 9-mm plastic tissue culture dishes followed by addition of 2 × 10 7 MNL. Neutrophilfree control systems were also included. The neutrophils were activated by the addition of FMLP/CB (1/~M and l ~g/ml final concentrations respectively) and the dishes incubated for 15 min at 37°C, after which the non-adherent MNL were separated from the adherent neutrophils, enumerated and resuspended to 107/ml in 250 mM mesoinositol, 10 mM sodium phosphate and 1 mM MgCl2 (pH 7.2). 200 ~tl of MNL suspension were lysed in alkaline medium containing 9 M urea, l0 mM NaOH, 2.5 mM cyclohexanediaminetetracetate and 0.1070 sodium dodecyl sulphate. Ethidium bromide fluorescence was determined after a 60-min incubation period at 15°C. The results were calculated according to the formula, D (percentage of double-stranded D N A ) = (F-Fmin)/(Fma~Fmin) X 100, where F is the fluorescence of the sample, Fmin the background fluorescence determined in samples that were sonicated at the beginning of the unwinding period in order to induce maximal unwinding, and Fma~ is the fluorescence of samples kept at pH 11.0, which is below the pH level needed to induce unwinding of double-stranded DNA. This system was also used to test the effects of catalase (200 units/ml) and superoxide dismutase (SOD, 100 units/ml) on the frequency of DNA-strand breaks in MNL exposed to FMLP/CB activated neutrophils.
Results
Expression and statistical analyses of results. The results are expressed as the mean values _+ the standard error of the mean (S.E.M.) for each series of experiments and statistical analyses were performed using the paired t-statistic by comparison of the values of each investigation obtained with
97 TABLE 1 M E A S U R E M E N T OF P E A K L E C L RESPONSES A N D T H E F R E Q U E N C Y OF D N A S T R A N D BREAKS IN C O C U L T U R E D M N L D U R I N G F M L P / C B A C T I V A T I O N OF NEUTROPI-IILS F R O M C I G A R E T T E SMOKERS A N D C O N T R O L SUBJECTS Test system
Peak L E C L responses in relative light units Extracellular
lntracellular
928 ± 170
1196 ± 1058
69 ± 2
1757 _+ 295 (/9 < 0.025)
3090 ± 1058 (p < 0.05)
49 ± 2 (p < 0.005)
FMLP/CB-activated neutrophils from non-smokers FMLP/CB-activated neutrophils from cigarette smokers
Percentage of doublestranded D N A remaining in cocultured M N L
Results are expressed as the m e a n values + S E M ' s for neutrophils from 6 cigarette smokers and 6 matched, non-smoking control subjects. The back-ground L E C L values for unstimulated neutrophils from smokers and non-smokers were 111 ± 7 and 110 ± 27 respectively. In the absence of F M L P / C B the percentages of double-stranded D N A remaining in M N L cocultured with smokers' and nonsmokers' neutrophils were 74 _+ 4 and 78 _+ 4 respectively.
TABLE 2 M E A S U R E M E N T OF T H E E F F E C T S OF C A T A L A S E A N D SOD I N D I V I D U A L L Y A N D IN C O M B I N A T I O N ON T H E F R E Q U E N C Y OF D N A S T R A N D BREAKS IN M N L EXPOSED TO F M L P / C B - A C T I V A T E D NEUTROPHILS F R O M C I G A R E T T E SMOKERS Test system
M N L + neutrophils only MNL + FMLP/CB-activated neutrophils MNL + FMLP/CB-activated neutrophils + catalase MNL + FMLP/CB-activated neutrophils + SOD M N L + FMLP/CB-activated neutrophils + catalase + SOD
Percentage of doublestranded D N A remaining in cocultured M N L 72 ± 4 a 51 ± 4 68 _+ 4 63 ± 4 72 _+ 3
a T h e results are expressed as the m e a n value _+ S E M ' s of 3 separate experiments using neutrophils from 3 different smokers.
neutrophils from a smoker and the corresponding matched, non-smoking control subject. FMLP/CB-activated L E C L responses. The peak extracellular and intracellular FMLP/CB-activated LECL responses of FMLP/CB-activated neutro-
phils from cigarette smokers and non-smoking control subjects are shown in Table 1. Neutrophils from smokers generated greater amounts of both extracellular (p<0.025) and intracellular (p<0.05) reactive oxidants than those of non-smokers. DNA-strand breaks. These results are also shown in Table 1. Exposure of MNL to the FMLP/CB-activated neutrophils of smokers was accompanied by a higher frequency of DNA strand breaks (17< 0.005) than MNL exposed to activated neutrophils from non-smokers. Both catalase and SOD individually and in combination prevented the oxidant-mediated strand breaks in MNL cocultured with smokers' FMLP/CB-activated neutrophils (Table 2).
Discussion Cigarette smoking primes circulating neutrophils and pulmonary macrophages to release increased amounts of reactive oxidants on exposure to stimuli of membrane-associated oxidative metabolism (Hoidal et al., 1981; Hoidal and Niewoehner, 1982; Ludwig and Hoidal, 1982; Anderson et al., 1987). The mechanism of this
98 smoking-related priming of oxidant release by phagocytes has not been established, but the importance of acute smoke exposure (i.e. cigarettes smoked per day) has been described (Richards et al., 1988). Priming of superoxide generation by PMA (phorbol myristate acetate)-activated neutrophils f r o m rats occurs within 15 weeks of chronic smoke exposure or 1 h after intraperitoneal injection of nicotine (0.02 and 0.2 m g / k g ) and it has been proposed that this agent (nicotine) is the smoke-derived, priming factor (Gillespie et al., 1987). However, a circulating, priming agent has not been detected in the plasma of cigarette smokers (Ludwig and Hoidal, 1982; Anderson et al., 1987). It is possible that priming of neutrophil membrane-associated oxidative metabolism m a y occur at an earlier stage, during cellular differentiation. Interestingly, T-lymphocyte derived granulocyte-macrophage colony stimulating factor (GM-CSF) primes neutrophils to generate increased amounts of superoxide during activation with F M L P by altering the affinities and increasing the numbers of F M L P receptors (Weisbart et al., 1986). Irrespective of the biochemical mechanisms of the priming process it is clear that cigarette smoking increases the potential hazard of oxygen toxicity to tissue cells by inducing chronic inflammation and release o f reactive oxidants by hyperactive phagocytes. In the present study we have confirmed previous reports that neutrophils f r o m cigarette smokers generate increased levels of reactive oxidants, especially extraceUular oxidants, when activated with stimuli such as F M L P / C B (Ludwig and Hoidal, 1982; Anderson et al., 1987). Oxidants released by activated phagocytes are potential carcinogens and inflict c h r o m o s o m a l damage on bystander cells (Weitberg et al., 1983; Weitberg, 1987). In the present study we have observed a much higher frequency of oxidantmediated D N A strand breaks in M N L exposed to activated neutrophils f r o m cigarette smokers than during coculture o f these cells with neutrophils f r o m non-smokers. The M N L were protected from phagocyte-derived oxidants by catalase and SOD individually and in combination, which indicates
that H202, superoxide and possibly hydroxyl radical p r o m o t e the damage to the D N A of MNL. In conclusion, neutrophils f r o m cigarette smokers release increased amounts of toxic extracellular reactive oxidants which damage the D N A o f bystander cells. Sustained oxidative damage to the D N A of lung tissue ceils, chronically exposed to hyperactive phagocytes, may contribute to the development of bronchial carcinoma in cigarette smokers.
Acknowledgement This investigation was supported by a research grant awarded by the National Cancer Association of South Africa.
References Anderson, R., A.J. Theron and G.J. Ras (1987) Regulation by the anti-oxidants ascorbate, cysteine and dapsone of the increased extracellular and intra-cellular generation of reactive oxidants by activated phagocytes from cigarette smokers, Am. Rev. Resp. Dis., 135, 1027-1032. Bender, J.G., and D.E. Van Epps (1983) Analysis of the bimodal chemiluminescence pattern stimulated in human neutrophils by chemotactic factors, Infect. Immunol., 41, 1062-1070. Birnboim, H.C., and J.J. Jevcak (1981) Fluorimetric method for the rapid detection of DNA strand breaks in human white blood ceils produced by low doses of radiation, Cancer Res., 41, 1889-1892. Cone, F., J. Lellouch and D. Schwartz (1971) Smoking and leukocyte counts: results of an epidemiologicalstudy, Lancet, 2, 632-634. Helman, N., and L.S. Rubinstein (1975) The effects of age, sex and smoking on erythrocytes and leukocytes, Am. J. Clin. Pathol., 63, 35-44. Hoidal, J.R., and D.E. Niewoehner (1982) Lung phagocyte recruitment and metabolic alterations induced by cigarette smoke in humans and hamsters, Am. Rev. Resp. Dis., 126, 548-552. Hoidal, J.R., R.B. Fox, P.A. le Barbre, R. Perri and J.E. Repine (1981) Altered oxidative metabolism responses in vitro of alveolar macrophages from asymptomatic smokers, Am. Rev. Resp. Dis., 123, 85-89. Hunninghake, G.W., and R.G. Crystal (1983) Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers, Am, Rev. Resp. Dis., 128, 833-838.
99 Gillespie, M.N., J.O. Owasoyo, S. Kojima and M. Jay (1987) Enhanced chemotaxis and superoxide anion production by polymorphonuclear leukocytes from nicotine-treated and smoke-exposed rats, Toxicology, 45, 45-52. Ludwig, R.W., and J.R. Hoidal (1982) Alterations in leukocyte oxidative metabolism in cigarette smokers, Am. Rev. Resp. Dis., 126, 977-980. Ludwig, R.W., B.A. Schwartz, J.R. Hoidal and D.E. Niewoehner (1985) Cigarette smoking causes accumulation of polymorphonuclear leukocytes in alveolar septum, Am. Rev. Resp. Dis., 131, 828-830. Richards, G.A., A.J. Theron, t~.A. Van der Merwe and R. Anderson (1988) Spirometric abnormalities in young smokers correlate with increased chemiluminescence responses of activated blood phagocytes, Am. Rev. Resp. Dis., in press. Schwalb, G., A.D. Beyers, R. Anderson and A.E. Nel (1988) Promotion of DNA strand breaks in cocultured mononuclear
leukocytes by protein kinase C-dependent pro-oxidative interactions of benoxaprofen, human polymorphonuclear leukocytes, and ultraviolet radiation, Cancer Res., 48, 3094-3099. Weisbart, R.H., D.W. Golde and J.C. Gasson (1986) Biosynthetic human GM-CSF modulates the number and affinity of neutrophil f-met-leu-phe receptors, J. Immunol., 137, 3584-3587. Weitberg, A.B. (1987) Chloramine-induced sister chromatid exchanges, Mutation Res., 190, 277-280. Weitberg, A.B., S.A. Weitzman, M. Destrempes, S.A. Latt and T.P. Stossel (1983) Stimulated human phagocytes produce cytogenetic damage in cultured mammalian cells, N. Engl. J. Med. 308, 26-30.
Communicated by D.R. Davies