Carrinogenesis vol.13 no. 11 pp.2041-2045, 1992
Cigarette smoking related poly cyclic aromatic hydrocarbon—DNA adducts in peripheral mononuclear cells
Regjna M.Santella, Ricardo A.Grinberg-Funes1, Tie Lan Young, Christopher Dickey, Vishwa Nath Singh1, Lian Wen Wang and Frederica P.Perera Cancer Center/Division of Environmental Science, School of Publk Health, Columbia University, New York, NY 10032 and 'Hoffmann-La Roche, Nutley, NJ 07110, USA
Material and methods Study subjects A total of 63 male smokeTs employed at Hoffmann-La Roche, Nuttey, NJ, volunteered for the study. Twenty seven non-smoking controls were age- and sex-matched volunteers recruited at Hoffmann-La Roche and Columbia University. At the time of fasting blood collection a questionnaire was administered eliciting information on current active and passive smoking, occupational, residential, familial cancer incidence history as well as dietary intake of charcoal-broiled foods over the past year. The standardized self-administered diet assessment
Table I. Comparison of assay results and questionnaire data in smokers and non-smokers
Introduction A number of studies have investigated cigarette smoking related DNA adducts in peripheral blood samples. These studies have utilized either total white blood cells or fractionated subpopulations of specific cell types. Two studies investigating total white blood cell polycyclic aromatic hydrocarbon (PAH*)-DNA adducts by F.I .ISA were unable to detect a significant increase in smokers compared to non-smokers (1,2) while two other studies found an increase (3,4). In the study of van Schooten et al. (4) mean adducts in smokers (3.3/108) were 1.8-fold higher than in non-smokers (1.9/108). While means were not reported in the study by Liou et al. (3), smoking was positively associated with PAH—DNA antigenicity (OR = 2.44, 95% CI = 0.96-6.26, P = 0.09). By the postlabeling assay no effect of smoking was observed in three studies (5-7), including two in which occupational exposure resulted in elevated adducts (6,7) and one in which increased adducts were detectable in bronchial tissues (8).
Number
Smokers
Non-smokers
63
27
47.6 ± 8.0 27-74
42.2 ± 9.6 30-62
38.1 ± 16.6 15.0-100
-
26.4 ± 16.0 10-50
-
286 ± 90 130-500
4.4 ± 3.4b 3.0-17.5
70% 4.38 ± 4.29 1.0-24.1
22% 1.35 ± 0.78* 1.0-4.06
Age Mean Range Pack years Mean Range Cigarettes/day Mean Range Cotinine Mean* Range* PAH-DNA % positive Mean ± SO* Range*1 •«/!•
•Abbreviations: PAH, polycyclic aromatic hydrocarbon; BPDE-L benzo(a]pvrene diolepoxide.
© Oxford University Press
b P < 0.001. 'Adducts/lO8 nucleotides.
2041
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
Studies on cigarette smoking related polycyclk aromatic hydrocarbon—DNA adducts in blood have produced conflicting results. To determine whether a subset of specific white blood cells is a useful marker for monitoring exposure to cigarette smoke, blood was obtained from 63 heavy smokers and 27 non-smokers. Adduct levels were determined by competitive enzyme-linked immunosorbent assay with a polyclonal antiserum recognizing benzo[a]pyrene and structurally related diolepoxide-DNA adducts. Analysis of the lymphocyte plus monocyte fraction from smokers indicated 70% had detectable adducts with a mean of 4.38 ± 4.29 adducts/108 nuckotides, while in non-smokers the corresponding values were 22% and 1.35 ± 0.78/108 (P < 0.001). Plasma cotinine levels differed significantly in smokers (286 ± 90 /xg/1) compared to non-smokers (4.4 ± 3.3 /ig/I) (P < 0.001). However, cotinine was not correlated with self-reported smoking history in these heavy smokers. Nor were DNA adducts in smokers correlated with cigarettes per day, pack-years and plasma cotinine, indicating large interindividual variation in DNA adduct formation. These data demonstrate lymphocytes plus monocytes from smokers have elevated levels of polycyclic aromatic hydrocarbon diolepoxide-DNA adduct levels compared to non-smokers.
Studies in specific white blood cell populations have also produced conflicting results. One study of a cell population reportedly containing >90% lymphocytes by 32P-postlabeling with the nuclease PI procedure demonstrated no difference widi smoking exposure (9). Two other studies isolated total mononuclear cells by Ficoll (—15% monocytes and - 8 5 % lymphocytes) and found adducts in smokers 1.4- to 2.4-fold higher than in non-smokers (10,11). Another isolated monocytes from active and passive smokers and demonstrated smokingspecific adducts which disappeared after 40 h of non-smoking (12). There have been no prior studies of adduct detection in specific white blood cells by ELJSA. Therefore, we investigated whether cigarette smoking related DNA adducts could be detected in lymphocytes plus monocytes by competitive ELISA utilizing a polyclonal antiserum that recognizes benzo[a]pyrene diolepoxide I (BPDE-I)-DNA adducts (13). This antiserum has been shown to crossreact with structurally related PAH diolepoxide-DNA adducts and thus provides a relative measure of this class of adducts.
R.M^antella et al.
Table n . Correlation of PAH—DNA adducts in smokers with indices of smoking exposure
Cig./day Pack-years Cod nine Daily tar Lifetime tar Daily nicotine Lifetime nicotine
PAH-DNA
Cig./day
0.0569 P = 0.658 -0.0926 P = 0.470 -0.0691 P = 0.594 -0.0532 P = 0.679 -0.101 p = 0.428 -0.0348 p = 0.786 -0.107 p = 0.404
l
Cotininc
Lifetime tar
Daily tar
Daily nicotine
1
0.0357 P = 0.778 0.541 p < 0.001 0.706 p < 0.001 0.489 p < 0.001 0.753 p < 0.001
1
0.0614 p = 0.622 0.0573 p = 0.643 0.0269 p = 0.829 0.0392 p = 0.757
1
0.860 p < 0.001 0.982 p < 0.001 0.882 p < 0.001
1
0.828 p < 0.001 0.971 p < 0.001
1
0.865 p < 0.001
Ann/
•
NONSMOKERS
El SMOKERS
Mil N.D.
2-4
5-8
9-12
VTA 13-24
ADDUCTS/108 NUCLEOTIDES Fig. 1. Histogram of PAH-DNA adducts in lymphocytes. Samples having < 15% inhibition were considered non-detectable and assigned a value of 1 adduct/10* nucleotides. Harvard/Willett questionnaire (14,15) was also administered, to be reported separately. Table I gives information on age distribution and cigarette smoking status in smokers and non-smokers. Daily and lifetime tar and nicotine exposure was calculated from the 1991 Federal Trade Commission report on tar, nicotine and carbon monoxide in cigarettes. Environmental tobacco smoke exposure was calculated based on questionnaire data. Blood sampling and analysis Blood (40 ml) was collected into heparinized tubes and separated within 4 h of sampling. Mononuclear cells were separated by Ficoll (Sigma Chemical Co., St Louis, MO) and washed twice with PBS. Pelleted cells were frozen at -70°C until DNA isolation by standard phenol and chlorofomi/isoamyl alcohol extractions and RNase treatment. DNA yields were >200 jig. PAH diolepoxide-DNA adducts were analyzed by competitive ELJSA essentially as described previously (16). Briefly, % microwell black plates (MicroFLUOR ' B \ Dynatech Laboratories, Alexandria, VA) were coated with 0.2 ng BPDE-I-DNA (5 adducts/103 nucleotides). A previously characterized rabbit antiserum (17) was used at 1:1 600 000 dilution. A standard curve was constructed by mixing 50 id diluted antiserum with BPDE-I-DNA (1.5 adduct/105 nucleotides) in carrier non-modified calf thymus DNA such that 50 /d contained 5-150 fmol BPDEI-deoxyguanosine adduct in 50 jig DNA. Test samples were assayed at 50 /ig/well after sonication and denaturatkn by boiling for 3 min and cooling on ice. Goat anti-rabbit IgG-alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN)
2042
was used at 1:400 dilution and the substrate was 4-methylumbeUiferyl phosphate (100 fil, 50 jig/ml 0.1 M diethanolamine, pH 9.6). Fluorescence was read on a Microfluor reader (Dynatech Laboratories). Samples were run in triplicate and median values used for determination of percentage inhibition. For analytical purposes, those samples with < 15% inhibition were considered non-detectable and assigned a value of 1/108, an amount midway between the lowest positive value and zero. Plasma cotinine was analyzed by HPLC as reported (18). Statistical analysis The initial statistical analysis was performed using die Wilcoxon rank-sum nonparametric test. Adduct data were then log transformed to normalize the distribution. Pearson's method was used to analyze correlations between logtransformed adduct levels and various exogenous and host variables including active and passive smoking (cigarettes/day, pack-years, daily and cumulative lifetime tar and average total hours exposed to passive smoke/day), age and dietary exposure to charcoal-broiled foods.
Results The mean age of smokers was 47.6 ± 8.0 and of non-smokers 42.2 ± 9.6 (Table I). About 90% of the smokers were currently consuming at least 20 cigarettes/day (mean 26.4 ± 16.0) and
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
•1
0.638 P < 0.001 0.0838 P = 0.500 0.510 p < 0.001 0.419 p < 0.001 0.667 p < 0.001 0.471 p < 0.001
Pack-years
Cigarette smoking related PAH-DNA adducts
25
CO
20
•o
o 0
o 15 3 C
o
3 •o
20 cigarettes/day (23,24). About 90% of the smokers in our study reported smoking 20 or more cigarettes/day. At the time of blood collection subjects reported that 5 min—8 h had elapsed since they smoked their last cigarette. While there was no correlation between plasma cotinine and time since last cigarette in these subjects, the reported half-life of cotinine in 2043
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
10
R.M-Santella et al.
Acknowledgements The assistance of J.N.Burling, MD, Director of Hoffmann-LaRoche Employee Health and his staff is greatly appreciated. Cotinine assays were performed by Dr E.Norkas and M.Agrawal, Our Lady of Mercy Medical Center, Bronx, NY. The secretarial assistance of Admas Aberra is gratefully acknowledged. This work was supported by NIH grants ESO5249, CA21111, American Cancer Society Sig 13 and an award from Hoffmann-La Roche.
References l.Perera.F.P., Santella.R.M., Brenner.D., Poirier.M.C, Munshi.A.A., Fischman.H.K. and VanRyzin J . (1987) DNA adducts, protein adducts and SCE in cigarette smokers and nonsmokers. J. Natl CancerInst., 79,449-456. 2.Perera,F.P., Mayer J., Jaretzki.A., Hearne.S., Brenner.D., Young.T.L., Fischman.H.K., Grimes.M., Grantham.S., Tang.M.X., Tsai,W.-Y. and Santella.R.M. (1989) Comparison of DNA adducts and sister chromatid exchange in hing canceT cases and controls. Cancer Res., 49, 4446-4451. 3.Liou,S.H., Jacobson-Kram.D., Poirier.M.C, Nguyen.D., Strickland,P.T. and Tockman.M.S. (1989) Biological monitoring of fire fighters: sister chromatid exchange and polycyclic aromatic hydrocarbon-DNA adducts in peripheral blood cells. Cancer Res., 49, 4929-4935. 4.vanSchooten,F., vanLeeuwen.F.E., Hillebrand.M.J.X., deRijke.M.E., Hart.A.A.M., vanVeen.H.G., Oosterink.S. and Kriek.E. (1990) Determination of benzo(a)pyrene dkrf epoxide—DNA adducts in white blood cell DNA from coke-oven workers: the impact of smoking. / . NatL Cancer Inst., 82, 927-933. 5.Phillips,D.H., Hewer^A. and Grover.P.L. (1986) Aromatic DNA adducts in human bone marrow and peripheral Wood leukocytes. Carcinogenesis, 7, 2071-2075. 6.Savela,K., Hemminki,K., Hewer.A., Phillips.D.H., Putman.K.L. and Randeralh.K. (1989) Interlaboratory comparison of the ^P-postlabeling assay for aromatic DNA adducts in white blood cells of iron foundry workers. Mutat. Res., 224, 485-492. 7.Reddy,M.V., Hemminki.K. and Randcrath.K. (1991) Postlabeling analysis of polycyclic aromatic hydrocarbon - DNA adducts in white blood cells of foundry workers. J. ToxicoL Envir. Health, 34, 177-185. 8. Phillips.D.H., Schoket,B., Hewer.A., Bailey,E., Kosuc.S. and Vincze.I. (1990) Influence of cigarette smoking on the levels of DNA adducts in human
2044
bronchial epithelium and white blood cells. Int. J. Cancer, 46, 569-575. 9. Jahnke.G.D., Thompson.C.L., Walker.M.P., GaUagherJ.E., Lucier.G.W. and DiAugustine.R.P. (1990) Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis. Carcinogenesis, 11, 205-211. lO.Savela.K. and Hemminki.K. (1991) DNA adducts in lymphocytes and granuloctyes of smokers and nonsmokers detected by the ^P-postlabehng assay. Carcinogenesis, 12, 503-508. ll.Schoket.B., Phillips.D.H., Hewer.A. and Vincze.I. (1991) "P-Postlabeling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminum production plant workers. Mutat. Res., 260, 89—98. 12. Holz,O., Krause.T., Scherer.G., Schmktt-Preub.U. and Rudiger.H.W. (1990) ^P-Postlabeling analysis of DNA adducts in monocytes of smokers and passive smokers. Int. Arch. Occup. Environ. Health, 62, 299—303. 13.Santella,R.M., Gasparro.F.P. and Hsieh.L.L. (1987) Quantitation of carcinogen-DNA adducts with monoclonal antibodies. Prog. Exp. Tumor Res., 31, 6 3 - 7 5 . 14.Willet,W.C, Polk.B.F., Underwood.B.A., Stampfer.M.J., Pressel.S., Rosner.B., TaylorJ.O., Schruder.K. and Hames,C.G. (1984) Relation of serum vitamins A and E and carotenioids to the risk of cancer. New Engl. J. Med., 310, 430-434. 15. WiUet.W.C, Stampfer.MJ., Underwood.B.A., Spdzer.F.E., Rosner.B., and Hennekcns.C.H. (1983) Validation of a dietary questionnaire with plasma carotenoid and alpha-tocopherol levels. Am. J. Clin. Nutr., 38, 631-639. 16. Perera.F.P., Hemminki.K., Young.T.L., Santella.R.M., Brenner.D. and Kelly ,G. (1988) Detection of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers. Cancer Res., 48, 2288-2291. 17.Poirier.M.C., Santella.R., Weinstein.I.B., Grunberger.D. and Yuspa.S.H. (1980) Quantitation of benzo[a]pyrene-deoxyguanosine adducts by radioimmunoassay. Cancer Res., 40, 412-416. 18.Machacek,D.A. and Jiang,N.S. (1986) Quantitation of cotinine in plasma by liquid chromatography. Clin. Chem., 32, 979-982. 19. Hemminki.K., Grzybowska.E., Chorazy.M., Twardowska-Saucha.K., SroczynskiJ.W., Putnam,K.L., Randerath.K., Phillips.D.H., Hewer.A., Santella.R.M., Young.T.L. and Perera.F.P. (1990) DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis, 11, 1229-1231. 20. Buckton.K.E., Brown.W.M.C. and Smith.P.G. (1967) Lymphocyte survival in men treated with x-rays for ankytosing spoodylitis. Nature, 214, 470-473. 21.Meuret,G. (1974) Human monocytopoiesis. Exp. Hematol., 2, 238-249. 22.Holz,O., Krause.T. and Rudiger.H.W. (1991) Differences in DNA adduct formation between monocytes and lymphocytes after in vivo incubation with benzo{a]pyrene. Carcinogenesis, 12, 2181-2183. 23.Hill,P., Haley,N.J. and Wynder.E.L. (1983) Cigarette smoking: carboxyhemoglobin, plasma nicotine, cotinine and thiocyanate vs self-reported smoking data and cardiovascular disease. J. Chron. Dis., 36, 439-449. 24.Woodward,M., Tunstall-Pedoe.H., Smith.W.C. and Tavendale.R. (1991) Smoking characteristics and inhalation biochemistry in the Scottish population. J. Clin. Epidemiol., 44, 1411-1414. 25. Haley ,N.J., Sepkovic.D.W. and Hoffmann.D. (1989) Elimination of cotinine from body fluids: disposition in smokers and nonsmokers. Am. J. Public Health, 79, 1046-1048. 26.Perera.F.P., MayerJ., Santella.R.M., Brenner.D., Jeffrey,A., Latriano.L., Smith.S., Warburton.D., Young.T.L., Tsai.W.Y., Hemminki.K. and BrandtRauf.P. (1991) Biologic markers in risk assessment for environmental carcinogens. Environ. Health Perspea., 90, 247-254. 27.Nebert,D.W. (1991) Role of genetics and drug metabolism in human cancer risk. Mutat. Res., 247, 267-281. 28.Kawajiri,K. and Fujii-Kuriyama.Y. (1991) P450 and human cancer. Jpn. J. Cancer Res., 82, 1325-1335. 29.Geneste,O., Camus,A.-M., Castegnaro.M., Petruzzelli.S., Macchiarine.P., Angeleti.C.A., Giuntini.C. and Bartscri,H. (1991) Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers. Carcinogenesis, 12, 1301-1305. 30. Manchester,D.K., Bowman.E.D., Parker.N.B., Caporaso,N.E. and Weston.A. (1992) Determinants of polycyclic aromatic hydrocarbon-DNA adducts in human placenta. Cancer Res., 52, 1499-1503. 31.Ayesh,R., IdleJ.R., Rhchie^.C, Crothers.M.J. and Hetzel.M.R. (1984) Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature, 312, 169-170. 32.Caporaso,N., Hayes.R.B., Dosemeci.M., Hoover.R., Ayesh.R., Hetzel.M. and Idle^l. (1989) Lung cancer risk, occupational exposure, and the debrisoquine metabolic phenotype. Cancer Res., 49, 3675—3679. 33.Kellermann,G., Shaw.C.R. and Luyten-KeUermann.M. (1973) Aryl
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
heavy smokers (16.5 ± 1.2 h) may have influenced plasma cotinine levels (25). When data from smokers and non-smokers were combined there were correlations between PAH — DNA adducts and plasma cotinine and other indices of smoking. However, there was no correlation between PAH-DNA adducts and cigarettes/day, pack-years and plasma cotinine in smokers alone. These results were not unexpected given the large interindividual variation in adduct levels which have been measured in workers with similar exposures (reviewed in 26). Genetic differences in metabolic capacity for activation and inactivation of carcinogens are important factors controlling interindividual variation in adduct formation (reviewed in 27,28). For example, a correlation has been seen between in vivo DNA adducts measured by n P postlabeling in lung tissue (29) or synchronous fluorescence spectroscopy in placenta (30) and aryl hydrocarbon hydroxylase activity in the same tissues measured in vitro. Epidemiologic studies have implicated genetic differences in cytochrome P450 2D6 (debrisoquin) and 1A1 as risk factors in lung cancer (31,34). Susceptibility also increased in smokers lacking expression of glutathione S-transferase /x, an enzyme that inactivates carcinogen metabolites (34). In parallel studies, we are investigating the role of these factors as well as genetic differences in DNA repair capacity in determining steady state adduct levels. Understanding the relationship of genotype to adduct formation will improve risk estimation at the individual level. But this work also indicates the importance of analyzing biomarkers in specific fractions of white blood cells.
Cigarette smoking related PAH-DNA adducts hydrocarbon hydroxylase indudbility and bronchogenic carcinoma. New EngL J. Med., 289, 934-937. 34.Kouri,R.E., McKinney.C.E., Slomiany.D.J., Snodgrass.D.R., Wray.N.P. and McLcmore.T.L. (1982) Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes. Cancer Res., 42, 5030-5037. 35-SeideganU., Pero.R.W., Markowitz.M.M., Roush.G., Miller.D.G. and Beatne.EJ. (1990) Isoeozyme(s) of glutathkne transferase (class Mu) as a matter for the susceptibility to lung cancer a follow up study. Cardnogenesis, 11, 33-36. Received on April 17, 1992; revised on June 15, 1992; accepted on July 10, 1992.
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
2045
Cigarette smoking related poly cyclic aromatic hydrocarbon—DNA adducts in peripheral mononuclear cells
Regjna M.Santella, Ricardo A.Grinberg-Funes1, Tie Lan Young, Christopher Dickey, Vishwa Nath Singh1, Lian Wen Wang and Frederica P.Perera Cancer Center/Division of Environmental Science, School of Publk Health, Columbia University, New York, NY 10032 and 'Hoffmann-La Roche, Nutley, NJ 07110, USA
Material and methods Study subjects A total of 63 male smokeTs employed at Hoffmann-La Roche, Nuttey, NJ, volunteered for the study. Twenty seven non-smoking controls were age- and sex-matched volunteers recruited at Hoffmann-La Roche and Columbia University. At the time of fasting blood collection a questionnaire was administered eliciting information on current active and passive smoking, occupational, residential, familial cancer incidence history as well as dietary intake of charcoal-broiled foods over the past year. The standardized self-administered diet assessment
Table I. Comparison of assay results and questionnaire data in smokers and non-smokers
Introduction A number of studies have investigated cigarette smoking related DNA adducts in peripheral blood samples. These studies have utilized either total white blood cells or fractionated subpopulations of specific cell types. Two studies investigating total white blood cell polycyclic aromatic hydrocarbon (PAH*)-DNA adducts by F.I .ISA were unable to detect a significant increase in smokers compared to non-smokers (1,2) while two other studies found an increase (3,4). In the study of van Schooten et al. (4) mean adducts in smokers (3.3/108) were 1.8-fold higher than in non-smokers (1.9/108). While means were not reported in the study by Liou et al. (3), smoking was positively associated with PAH—DNA antigenicity (OR = 2.44, 95% CI = 0.96-6.26, P = 0.09). By the postlabeling assay no effect of smoking was observed in three studies (5-7), including two in which occupational exposure resulted in elevated adducts (6,7) and one in which increased adducts were detectable in bronchial tissues (8).
Number
Smokers
Non-smokers
63
27
47.6 ± 8.0 27-74
42.2 ± 9.6 30-62
38.1 ± 16.6 15.0-100
-
26.4 ± 16.0 10-50
-
286 ± 90 130-500
4.4 ± 3.4b 3.0-17.5
70% 4.38 ± 4.29 1.0-24.1
22% 1.35 ± 0.78* 1.0-4.06
Age Mean Range Pack years Mean Range Cigarettes/day Mean Range Cotinine Mean* Range* PAH-DNA % positive Mean ± SO* Range*1 •«/!•
•Abbreviations: PAH, polycyclic aromatic hydrocarbon; BPDE-L benzo(a]pvrene diolepoxide.
© Oxford University Press
b P < 0.001. 'Adducts/lO8 nucleotides.
2041
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
Studies on cigarette smoking related polycyclk aromatic hydrocarbon—DNA adducts in blood have produced conflicting results. To determine whether a subset of specific white blood cells is a useful marker for monitoring exposure to cigarette smoke, blood was obtained from 63 heavy smokers and 27 non-smokers. Adduct levels were determined by competitive enzyme-linked immunosorbent assay with a polyclonal antiserum recognizing benzo[a]pyrene and structurally related diolepoxide-DNA adducts. Analysis of the lymphocyte plus monocyte fraction from smokers indicated 70% had detectable adducts with a mean of 4.38 ± 4.29 adducts/108 nuckotides, while in non-smokers the corresponding values were 22% and 1.35 ± 0.78/108 (P < 0.001). Plasma cotinine levels differed significantly in smokers (286 ± 90 /xg/1) compared to non-smokers (4.4 ± 3.3 /ig/I) (P < 0.001). However, cotinine was not correlated with self-reported smoking history in these heavy smokers. Nor were DNA adducts in smokers correlated with cigarettes per day, pack-years and plasma cotinine, indicating large interindividual variation in DNA adduct formation. These data demonstrate lymphocytes plus monocytes from smokers have elevated levels of polycyclic aromatic hydrocarbon diolepoxide-DNA adduct levels compared to non-smokers.
Studies in specific white blood cell populations have also produced conflicting results. One study of a cell population reportedly containing >90% lymphocytes by 32P-postlabeling with the nuclease PI procedure demonstrated no difference widi smoking exposure (9). Two other studies isolated total mononuclear cells by Ficoll (—15% monocytes and - 8 5 % lymphocytes) and found adducts in smokers 1.4- to 2.4-fold higher than in non-smokers (10,11). Another isolated monocytes from active and passive smokers and demonstrated smokingspecific adducts which disappeared after 40 h of non-smoking (12). There have been no prior studies of adduct detection in specific white blood cells by ELJSA. Therefore, we investigated whether cigarette smoking related DNA adducts could be detected in lymphocytes plus monocytes by competitive ELISA utilizing a polyclonal antiserum that recognizes benzo[a]pyrene diolepoxide I (BPDE-I)-DNA adducts (13). This antiserum has been shown to crossreact with structurally related PAH diolepoxide-DNA adducts and thus provides a relative measure of this class of adducts.
R.M^antella et al.
Table n . Correlation of PAH—DNA adducts in smokers with indices of smoking exposure
Cig./day Pack-years Cod nine Daily tar Lifetime tar Daily nicotine Lifetime nicotine
PAH-DNA
Cig./day
0.0569 P = 0.658 -0.0926 P = 0.470 -0.0691 P = 0.594 -0.0532 P = 0.679 -0.101 p = 0.428 -0.0348 p = 0.786 -0.107 p = 0.404
l
Cotininc
Lifetime tar
Daily tar
Daily nicotine
1
0.0357 P = 0.778 0.541 p < 0.001 0.706 p < 0.001 0.489 p < 0.001 0.753 p < 0.001
1
0.0614 p = 0.622 0.0573 p = 0.643 0.0269 p = 0.829 0.0392 p = 0.757
1
0.860 p < 0.001 0.982 p < 0.001 0.882 p < 0.001
1
0.828 p < 0.001 0.971 p < 0.001
1
0.865 p < 0.001
Ann/
•
NONSMOKERS
El SMOKERS
Mil N.D.
2-4
5-8
9-12
VTA 13-24
ADDUCTS/108 NUCLEOTIDES Fig. 1. Histogram of PAH-DNA adducts in lymphocytes. Samples having < 15% inhibition were considered non-detectable and assigned a value of 1 adduct/10* nucleotides. Harvard/Willett questionnaire (14,15) was also administered, to be reported separately. Table I gives information on age distribution and cigarette smoking status in smokers and non-smokers. Daily and lifetime tar and nicotine exposure was calculated from the 1991 Federal Trade Commission report on tar, nicotine and carbon monoxide in cigarettes. Environmental tobacco smoke exposure was calculated based on questionnaire data. Blood sampling and analysis Blood (40 ml) was collected into heparinized tubes and separated within 4 h of sampling. Mononuclear cells were separated by Ficoll (Sigma Chemical Co., St Louis, MO) and washed twice with PBS. Pelleted cells were frozen at -70°C until DNA isolation by standard phenol and chlorofomi/isoamyl alcohol extractions and RNase treatment. DNA yields were >200 jig. PAH diolepoxide-DNA adducts were analyzed by competitive ELJSA essentially as described previously (16). Briefly, % microwell black plates (MicroFLUOR ' B \ Dynatech Laboratories, Alexandria, VA) were coated with 0.2 ng BPDE-I-DNA (5 adducts/103 nucleotides). A previously characterized rabbit antiserum (17) was used at 1:1 600 000 dilution. A standard curve was constructed by mixing 50 id diluted antiserum with BPDE-I-DNA (1.5 adduct/105 nucleotides) in carrier non-modified calf thymus DNA such that 50 /d contained 5-150 fmol BPDEI-deoxyguanosine adduct in 50 jig DNA. Test samples were assayed at 50 /ig/well after sonication and denaturatkn by boiling for 3 min and cooling on ice. Goat anti-rabbit IgG-alkaline phosphatase (Boehringer Mannheim, Indianapolis, IN)
2042
was used at 1:400 dilution and the substrate was 4-methylumbeUiferyl phosphate (100 fil, 50 jig/ml 0.1 M diethanolamine, pH 9.6). Fluorescence was read on a Microfluor reader (Dynatech Laboratories). Samples were run in triplicate and median values used for determination of percentage inhibition. For analytical purposes, those samples with < 15% inhibition were considered non-detectable and assigned a value of 1/108, an amount midway between the lowest positive value and zero. Plasma cotinine was analyzed by HPLC as reported (18). Statistical analysis The initial statistical analysis was performed using die Wilcoxon rank-sum nonparametric test. Adduct data were then log transformed to normalize the distribution. Pearson's method was used to analyze correlations between logtransformed adduct levels and various exogenous and host variables including active and passive smoking (cigarettes/day, pack-years, daily and cumulative lifetime tar and average total hours exposed to passive smoke/day), age and dietary exposure to charcoal-broiled foods.
Results The mean age of smokers was 47.6 ± 8.0 and of non-smokers 42.2 ± 9.6 (Table I). About 90% of the smokers were currently consuming at least 20 cigarettes/day (mean 26.4 ± 16.0) and
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
•1
0.638 P < 0.001 0.0838 P = 0.500 0.510 p < 0.001 0.419 p < 0.001 0.667 p < 0.001 0.471 p < 0.001
Pack-years
Cigarette smoking related PAH-DNA adducts
25
CO
20
•o
o 0
o 15 3 C
o
3 •o
20 cigarettes/day (23,24). About 90% of the smokers in our study reported smoking 20 or more cigarettes/day. At the time of blood collection subjects reported that 5 min—8 h had elapsed since they smoked their last cigarette. While there was no correlation between plasma cotinine and time since last cigarette in these subjects, the reported half-life of cotinine in 2043
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
10
R.M-Santella et al.
Acknowledgements The assistance of J.N.Burling, MD, Director of Hoffmann-LaRoche Employee Health and his staff is greatly appreciated. Cotinine assays were performed by Dr E.Norkas and M.Agrawal, Our Lady of Mercy Medical Center, Bronx, NY. The secretarial assistance of Admas Aberra is gratefully acknowledged. This work was supported by NIH grants ESO5249, CA21111, American Cancer Society Sig 13 and an award from Hoffmann-La Roche.
References l.Perera.F.P., Santella.R.M., Brenner.D., Poirier.M.C, Munshi.A.A., Fischman.H.K. and VanRyzin J . (1987) DNA adducts, protein adducts and SCE in cigarette smokers and nonsmokers. J. Natl CancerInst., 79,449-456. 2.Perera,F.P., Mayer J., Jaretzki.A., Hearne.S., Brenner.D., Young.T.L., Fischman.H.K., Grimes.M., Grantham.S., Tang.M.X., Tsai,W.-Y. and Santella.R.M. (1989) Comparison of DNA adducts and sister chromatid exchange in hing canceT cases and controls. Cancer Res., 49, 4446-4451. 3.Liou,S.H., Jacobson-Kram.D., Poirier.M.C, Nguyen.D., Strickland,P.T. and Tockman.M.S. (1989) Biological monitoring of fire fighters: sister chromatid exchange and polycyclic aromatic hydrocarbon-DNA adducts in peripheral blood cells. Cancer Res., 49, 4929-4935. 4.vanSchooten,F., vanLeeuwen.F.E., Hillebrand.M.J.X., deRijke.M.E., Hart.A.A.M., vanVeen.H.G., Oosterink.S. and Kriek.E. (1990) Determination of benzo(a)pyrene dkrf epoxide—DNA adducts in white blood cell DNA from coke-oven workers: the impact of smoking. / . NatL Cancer Inst., 82, 927-933. 5.Phillips,D.H., Hewer^A. and Grover.P.L. (1986) Aromatic DNA adducts in human bone marrow and peripheral Wood leukocytes. Carcinogenesis, 7, 2071-2075. 6.Savela,K., Hemminki,K., Hewer.A., Phillips.D.H., Putman.K.L. and Randeralh.K. (1989) Interlaboratory comparison of the ^P-postlabeling assay for aromatic DNA adducts in white blood cells of iron foundry workers. Mutat. Res., 224, 485-492. 7.Reddy,M.V., Hemminki.K. and Randcrath.K. (1991) Postlabeling analysis of polycyclic aromatic hydrocarbon - DNA adducts in white blood cells of foundry workers. J. ToxicoL Envir. Health, 34, 177-185. 8. Phillips.D.H., Schoket,B., Hewer.A., Bailey,E., Kosuc.S. and Vincze.I. (1990) Influence of cigarette smoking on the levels of DNA adducts in human
2044
bronchial epithelium and white blood cells. Int. J. Cancer, 46, 569-575. 9. Jahnke.G.D., Thompson.C.L., Walker.M.P., GaUagherJ.E., Lucier.G.W. and DiAugustine.R.P. (1990) Multiple DNA adducts in lymphocytes of smokers and nonsmokers determined by 32P-postlabeling analysis. Carcinogenesis, 11, 205-211. lO.Savela.K. and Hemminki.K. (1991) DNA adducts in lymphocytes and granuloctyes of smokers and nonsmokers detected by the ^P-postlabehng assay. Carcinogenesis, 12, 503-508. ll.Schoket.B., Phillips.D.H., Hewer.A. and Vincze.I. (1991) "P-Postlabeling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminum production plant workers. Mutat. Res., 260, 89—98. 12. Holz,O., Krause.T., Scherer.G., Schmktt-Preub.U. and Rudiger.H.W. (1990) ^P-Postlabeling analysis of DNA adducts in monocytes of smokers and passive smokers. Int. Arch. Occup. Environ. Health, 62, 299—303. 13.Santella,R.M., Gasparro.F.P. and Hsieh.L.L. (1987) Quantitation of carcinogen-DNA adducts with monoclonal antibodies. Prog. Exp. Tumor Res., 31, 6 3 - 7 5 . 14.Willet,W.C, Polk.B.F., Underwood.B.A., Stampfer.M.J., Pressel.S., Rosner.B., TaylorJ.O., Schruder.K. and Hames,C.G. (1984) Relation of serum vitamins A and E and carotenioids to the risk of cancer. New Engl. J. Med., 310, 430-434. 15. WiUet.W.C, Stampfer.MJ., Underwood.B.A., Spdzer.F.E., Rosner.B., and Hennekcns.C.H. (1983) Validation of a dietary questionnaire with plasma carotenoid and alpha-tocopherol levels. Am. J. Clin. Nutr., 38, 631-639. 16. Perera.F.P., Hemminki.K., Young.T.L., Santella.R.M., Brenner.D. and Kelly ,G. (1988) Detection of polycyclic aromatic hydrocarbon-DNA adducts in white blood cells of foundry workers. Cancer Res., 48, 2288-2291. 17.Poirier.M.C., Santella.R., Weinstein.I.B., Grunberger.D. and Yuspa.S.H. (1980) Quantitation of benzo[a]pyrene-deoxyguanosine adducts by radioimmunoassay. Cancer Res., 40, 412-416. 18.Machacek,D.A. and Jiang,N.S. (1986) Quantitation of cotinine in plasma by liquid chromatography. Clin. Chem., 32, 979-982. 19. Hemminki.K., Grzybowska.E., Chorazy.M., Twardowska-Saucha.K., SroczynskiJ.W., Putnam,K.L., Randerath.K., Phillips.D.H., Hewer.A., Santella.R.M., Young.T.L. and Perera.F.P. (1990) DNA adducts in humans environmentally exposed to aromatic compounds in an industrial area of Poland. Carcinogenesis, 11, 1229-1231. 20. Buckton.K.E., Brown.W.M.C. and Smith.P.G. (1967) Lymphocyte survival in men treated with x-rays for ankytosing spoodylitis. Nature, 214, 470-473. 21.Meuret,G. (1974) Human monocytopoiesis. Exp. Hematol., 2, 238-249. 22.Holz,O., Krause.T. and Rudiger.H.W. (1991) Differences in DNA adduct formation between monocytes and lymphocytes after in vivo incubation with benzo{a]pyrene. Carcinogenesis, 12, 2181-2183. 23.Hill,P., Haley,N.J. and Wynder.E.L. (1983) Cigarette smoking: carboxyhemoglobin, plasma nicotine, cotinine and thiocyanate vs self-reported smoking data and cardiovascular disease. J. Chron. Dis., 36, 439-449. 24.Woodward,M., Tunstall-Pedoe.H., Smith.W.C. and Tavendale.R. (1991) Smoking characteristics and inhalation biochemistry in the Scottish population. J. Clin. Epidemiol., 44, 1411-1414. 25. Haley ,N.J., Sepkovic.D.W. and Hoffmann.D. (1989) Elimination of cotinine from body fluids: disposition in smokers and nonsmokers. Am. J. Public Health, 79, 1046-1048. 26.Perera.F.P., MayerJ., Santella.R.M., Brenner.D., Jeffrey,A., Latriano.L., Smith.S., Warburton.D., Young.T.L., Tsai.W.Y., Hemminki.K. and BrandtRauf.P. (1991) Biologic markers in risk assessment for environmental carcinogens. Environ. Health Perspea., 90, 247-254. 27.Nebert,D.W. (1991) Role of genetics and drug metabolism in human cancer risk. Mutat. Res., 247, 267-281. 28.Kawajiri,K. and Fujii-Kuriyama.Y. (1991) P450 and human cancer. Jpn. J. Cancer Res., 82, 1325-1335. 29.Geneste,O., Camus,A.-M., Castegnaro.M., Petruzzelli.S., Macchiarine.P., Angeleti.C.A., Giuntini.C. and Bartscri,H. (1991) Comparison of pulmonary DNA adduct levels, measured by 32P-postlabelling and aryl hydrocarbon hydroxylase activity in lung parenchyma of smokers and ex-smokers. Carcinogenesis, 12, 1301-1305. 30. Manchester,D.K., Bowman.E.D., Parker.N.B., Caporaso,N.E. and Weston.A. (1992) Determinants of polycyclic aromatic hydrocarbon-DNA adducts in human placenta. Cancer Res., 52, 1499-1503. 31.Ayesh,R., IdleJ.R., Rhchie^.C, Crothers.M.J. and Hetzel.M.R. (1984) Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature, 312, 169-170. 32.Caporaso,N., Hayes.R.B., Dosemeci.M., Hoover.R., Ayesh.R., Hetzel.M. and Idle^l. (1989) Lung cancer risk, occupational exposure, and the debrisoquine metabolic phenotype. Cancer Res., 49, 3675—3679. 33.Kellermann,G., Shaw.C.R. and Luyten-KeUermann.M. (1973) Aryl
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
heavy smokers (16.5 ± 1.2 h) may have influenced plasma cotinine levels (25). When data from smokers and non-smokers were combined there were correlations between PAH — DNA adducts and plasma cotinine and other indices of smoking. However, there was no correlation between PAH-DNA adducts and cigarettes/day, pack-years and plasma cotinine in smokers alone. These results were not unexpected given the large interindividual variation in adduct levels which have been measured in workers with similar exposures (reviewed in 26). Genetic differences in metabolic capacity for activation and inactivation of carcinogens are important factors controlling interindividual variation in adduct formation (reviewed in 27,28). For example, a correlation has been seen between in vivo DNA adducts measured by n P postlabeling in lung tissue (29) or synchronous fluorescence spectroscopy in placenta (30) and aryl hydrocarbon hydroxylase activity in the same tissues measured in vitro. Epidemiologic studies have implicated genetic differences in cytochrome P450 2D6 (debrisoquin) and 1A1 as risk factors in lung cancer (31,34). Susceptibility also increased in smokers lacking expression of glutathione S-transferase /x, an enzyme that inactivates carcinogen metabolites (34). In parallel studies, we are investigating the role of these factors as well as genetic differences in DNA repair capacity in determining steady state adduct levels. Understanding the relationship of genotype to adduct formation will improve risk estimation at the individual level. But this work also indicates the importance of analyzing biomarkers in specific fractions of white blood cells.
Cigarette smoking related PAH-DNA adducts hydrocarbon hydroxylase indudbility and bronchogenic carcinoma. New EngL J. Med., 289, 934-937. 34.Kouri,R.E., McKinney.C.E., Slomiany.D.J., Snodgrass.D.R., Wray.N.P. and McLcmore.T.L. (1982) Positive correlation between high aryl hydrocarbon hydroxylase activity and primary lung cancer as analyzed in cryopreserved lymphocytes. Cancer Res., 42, 5030-5037. 35-SeideganU., Pero.R.W., Markowitz.M.M., Roush.G., Miller.D.G. and Beatne.EJ. (1990) Isoeozyme(s) of glutathkne transferase (class Mu) as a matter for the susceptibility to lung cancer a follow up study. Cardnogenesis, 11, 33-36. Received on April 17, 1992; revised on June 15, 1992; accepted on July 10, 1992.
Downloaded from http://carcin.oxfordjournals.org/ at New York University on June 2, 2015
2045
