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I An interlaboratory comparison of data from 11 laboratories in 6 countries has demonstrated that GC and RIA techniques can reliably quantitate nicotine and cotinine in urine and plasma samples. An excellent correlation of laboratory methods was observed in plasma samples and in urine samples to which cotinine had been added as a tracer. However, in urine samples without tracer, the RIA values for cotinine were found to be slightly higher than those observed by GC. This could be due to a cross reaction of the antibody with another compound present in urine, or the discrepancy could arise from a loss of urinary cotinine during GC extraction. The former explanation is more likely to apply here. All methods have led to perfect distinction between nonsmokers and active smokers (26). Table 2 presents data from model studies on the uptake of ETS by nonsmokers under acute exposure conditions (27-30). The main purpose of these assays was to develop the methodology for field studies and to compare the uptake of nicotine from environments with various degrees of pollution and different types of pollutants under controlled conditions. It has been shown that the equilibrium of nicotine between vapor phase and particulate phase of ETS depends greatly on the concentration and pH of the emitted smokestream (31) and, thus, influences the uptake of nicotine by inhalation. After repeated exposure to ETS under controlled conditions, such as twice daily 80-minute exposure on 3 consecutive days to the diluted pollutants of 4 concurrently smoked cigarettes (32), the measurements in 4 nonsmokers have shown that except for nicotine in the saliva, the physiological fluids do not reflect maximal concentrations of nicotine and cotinine until at least 24 hours later. This observation has led to comparisons of the elimination of cotinine in smokers and nonsmokers exposed to ETS (33). In the first study, the half-life (t1/2) of cotinine elimination from plasma of smokers was 18.5 hours; in the case of passive smokers, it was 49.7 hours. The corresponding disappearance (tl/z)of cotinine from the urine took 21.9 hours and 32.7 hours, respectively. In a second assay, five cigarette smokers were asked to abstain from tobacco use for 5 days and were then given nicotine gum for three days. After another 8 days of abstinence from nicotine, the volunteers were exposed to sidestream smoke. At this point, the cotinine elimination (tj/Z) from urine (ng/ml) by smokers took 15.4 hours, by nicotine gum users 18.2 hours, by 8-day exsmokers 27.5 hours, and by the never-smokers 25.6 hours (33). These findings suggest that the residence times of nicotine, cotinine and other tobacco alkaloids, are likely related to the route of nicotine uptake as well as to possible differences in metabolism between smokers and nonsmokers. The longer elimination time for cotinine in nonsmokers has been confirmed by other study groups (35-37), however, the observation has also been challenged (38,39). A longer residence time of nicotine metabolites in nonsmokers could conceivably increase the possibility of endogenous formation of carcinogenic N-nitrosamines (40). 98

REFERENCES 1. Pillsbury, H.C., Bright, C.C., O'Connor, K.J., and Irish, F.W. Tar and nicotine in cigarette smoke. J. Assoc. Offic. Anal. Chem. 52:458-462, 1969. 2. Dube, M.F. and Green, C.R. Methods of collection of smoke for analytical purposes. Recent Advan. Tobacco Sci. Jt: 42- 102, 1982. 3. Herning, R.I., Jones, R.T., Bachman, J., and Mines, A.H. Puff volume increases when low-nicotine cigarettes are smoked. Brit. Med. J. 283: 187-189, 1981. 4. Haley, N.J., Sepkovic, D.W., Hoffmann, D., and Wynder, E.L. Cigarette smoking as Risk for Cardiovascular Disease. Part VI. Compensation with nicotine availability as a single variable. Clin. Pharmacol. Ther. 38: 164-170, 1985. 5. Chamberlain, A.T. and Higgenbottam, T.W. Nicotine and cigarette smoking: An alternative hypothesis. Med. Hypotheses 17: 285-297, 1985. 6. Kozlowski, L.T., Frecker, R.C., Khouro, P., and Pope,M.A. The misuse of "less hazardous" cigarettes and its detection: Hole-blocking of ventilated filters. Am. J. Publ. Health 70: 1202-1203, 1980. 7. Federal Trade Commission. Report of tar and nicotine con- tent of the smoke of 208 varieties of domestic cigarettes, 1954-1983. U.S. Govt. Printing Office, Washington, DC, 1983. 8. Great Britain Laboratory of the Government Chemist. Report of the Government Chemist, 1981. Her Majesty's Stationery Office, London, p. 109, 1982. 9. Toxic and carcinogenic agents in undiluted mainstrea$ smoke and sidestream smoke of different types of cigarettes. Carcinogenesis 8: 729-731, 1987. 10. National Research Council. "Environmental Tobacco Smoke. Measuring Exposures and Assessing Health Effects." National Academy Press, Washington, DC, 1986. 337 pp. 11. U.S. Surgeon General. "The Health Consequences of Involuntary Smoking." U.S. Dept. Health and Human services. DHHS (CDC) 87-8398, 1987, 359 pp. 107

others (65) and also by a controlled chamber assay (61). One study in which significant elevations of COHb were found used controlled exposure to tobacco smoke at a level of 25 ppm CO for 8 hours. This intense exposure resulted in an average increase of COHb levels by 2.5% (85). However, such results are not applicable to free-living situations in field studies (67). 3. Thiocyanate. Smoke is detoxified in the liver to thiocyanate (SCN-). Measurement of SCN- has been used to differentiate smokers from nonsmokers or, as mentioned earlier, in combination with nicotine-cotinine assays to distinguish smokers from chewers of tobacco. Thiocyanate can also be derived from the diet, cruciferous vegetables being an excellent source (68). The specificity of SCN as a marker of tobacco smoke inhalation is poor and it is generally difficult to distinouish light smokers from nonsmokers. This lack of specificity makes SCN- unsuitable for the evaluation of ETS uptake by nonsmoking subjects. 4. Hydroxyproline. Japanese investigators have studied the excretion of hydroxyproline in persons exposed to ETS as well as in active smokers and in persons exposed to high levels of air pollutants (69). The rationale for these studies is that the inhalation of nitrogen dioxide causes degradation of lung collagen and elastin which results in urinary excretion of hydroxyproline. The investigations of the Japanese group suggested an elevated excretion of hydroxyproline by children of smoking parents as well as by wives of smoking husbands, active smokers, and individuals exposed to vehicle emissions. Since NOx levels in ETS are relatively low by comparison to mainstream smoke or vehicle emissions (56,70,71), such increased elimination of hydroxyproline in passively exposed persons seemed surprising. In fact, another group of investigators has been unable to confirm this finding (72). Additional investigations, under controlled exposure conditions and field studies are needed before this compound can be properly evaluated as a marker for ETS uptake. 5. N-Nitroso-Amino Acids. The occurrence of endogenous nitrosation reactions in cigarette smokers has been demonstrated in several studies. This phenomenon entails the risk of endogenous formation of carcinogenic N-nitrosamines. Endogenous formation of N-nitrosamines has been proven by urinary excretion of the noncarcinogenic N-nitrosoproline (NPRO), N- nitrosothioproline (NTPRO), and N-nitrosomethylthioproline (NNTPRO). Whereas the average excretion of NPRO in nonsmokers amounted to 2.0±1.5 ug/24 hrs, cigarette smokers excreted an average of 7.0±4.0 ug/24 hrs (73-77). In the case of NTPRO, the average urinary excretion by nonsmokers (ug/24 hrs) was 5.9, that by cigarette smokers 8.7 and that of NMTPRO was 5.6 and 8.5, respectively (75). Only two studies have explored the possibility that endogenous formation of N-nitrosamino acids may 102

B. Genotoxicity of Physiological Fluids Several studies have explored the possibility that physiological fluids of cigarette smokers contain significantly higher amounts of genotoxic agents than those of nonsmokers (81). The most extensive data base in this field has shown significantly higher mutaqenicity in the Salmonella thvohimurium assay of urine of cigarette smokers compared to those of nonsmokers. Since the original study by Yamasaki and Ames in 1977 (83) at least 20 investigations have shown that the urine of cigarette smokers is significantly more mutagenic than the urine of nonsmokers w#o are not ex#osed to genotoxic agents in occupational environments. But it has also been shown that the mutagenicity of the urine of smokers can be effected by diet (84). It has further been surmized that exposure of nonsmokers to ETS may lead to increased urinary excretion of mutagens. Of the 6 published studies in which the urine of passive smokers was tested for mutagenicity with the Ames test, 3 showed increased activity and 3 showed no increase or, at the most an insignificant increase in mutagenic activity (81,85-87). It appears likely that the presently widely used methodology by Yamasaki and Ames (83) can be significantly refined (86,88). This may then enable investigators to assay the urine of involuntary smokers for their exposure to genotoxic agents or their precursors due to exposure to ETS. C. Adduct Formation of Carcinogens in Passive Smokers. Measurements in physiological fluids of nicotine and its major metabolite, cotinine, have been shown to be objective indicators of the uptake of ETS. It appears also that, upon refinement of the methodology, the assay for mutagenicity of the urine will reflect the uptake of genotoxic ETS constituents by nonsmokers. However, these assays will not reflect an individual's response to specific ETS carcinogens. That information is best obtained by assessing levels of macromolecular adducts with carcinogens or their metabolites. Development of such assays is based an examining the mechanisms of metabolic activation and detoxification of tobacco smoke carcinogens. 1. Benzo(a)pyrene. In the case of active smokers, adducts of at least 4 types of tobacco carcinogens or procarcinogens have been studied. These adducts are formed by reaction of specific metabolites of tobacco smoke constituents with DNA and/or hemoglobin. Benzo(a)pyrene (BaP), a carcinogenic representative of the polynuclear aromatic hydrocarbons in tobacco smoke is known to be metabolized to bay region diol epoxides (e.g. 7,8- dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBaP). Such diol epoxides can bind to DNA in human tissues and lymphocytes. Antibodies developed against the major BPDE-DNA adduct have been used to 104

methylbutyramide. Methods Enzymol. 84: 628-640, 1982. 25. Gritz, E.R., Baer-Weiss, V., Benowitz, N.L., Van Vunakis, H., and Jarvik, M.E. Plasma nicotine and cotinine concentrations in habitual smokeless tobacco users. Clin. Pharmacol. Ther. 30: 201- 205, 1981. 26. Biber, A., Scherer, G., Hoepfner, I., Adlkofer, F., Heller, W.-D., Haddow, J.E., and Knight, G.J. Determination of nicotine and cotinine in human serum and urine: an interlaboratory study. Toxicol. Lett. 35: 45-52, 1987. 27. Harke, H.P. Zum Problem des Passiv-Rauchens. Muench. Med. Wochenschr. 112: 2328-2334, 1970. 28. Cano, J.P., Catalin, J., Badre, R., Duma, C., Viala, A., and Guillerme, R. Determination de la nicotine par chromatographie en phase gazeuse. II. Appl. Ann. Pharm. France 28: 633-640, 1970. 29. Russell, M.A.H. and Feyerabend, C. Blood and urinary nicotine in nonsmokers. Lancet 1: 179-181, 1975. 30. Hoffmann, D., Haley, N.J., Adams, J.D., and Brunnemann, K.D. Tobacco sidestream smoke. Uptake by nonsmokers. Prev. Med. 13: 608-617,1984. 31. Eudy, L.W., Thome, F.A., Heavner, D.L., Green, C.R., and Ingebrethsen, B.J. Studies on the vapor-particulate phase distribution of environmental nicotine by selective trapping and detection methods. Proc. 79th Ann. Mtg. Air Pollution Control Association, Minneapolis, June 22-27, 14 p., 1986. 32. Hoffmann, Brunnemann, K.D., Haley, N.J., Sepkovic, D.W., and Adams, J.D. Nicotine uptake by nonsmokers exposed to passive smoking under controlled conditions and the elimination of cotinine. Proc. 4th International Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87^, Volume 2: 13- 17,1987. 33. Greenberg, R.A., Haley, N.J., Etzel, R.A, and Loda, F.A. Measuring the exposure of infants to tobacco smoke. New Engl. J. Med. 310: 1075-1078, 1984. 34. Haley, N.J., Sepkovic, D.W., Louis, E.T., and Hoffmann, D. Absorption and elimination of nicotine by smokers, nonsmokers and chewers of nicotine gum. In: The Pharmacology of Nicotine, Rand, M.J. and Thurau, K., eds., IRL Press, Washington, DC, 1988, pp. 20- 21. 35. Goldstein, G.M., Collier, A., Etzel, R., Lewtas, J., and Haley, N.J. Elimination of urinary cotinine in children exposed to known levels of sidestream cigarette smoke. Proc. 4th 109

48. Jarvis, M.J., Russell, M.A.H., Feyerabend, Eiser, J.R., Morgan, P., Gammage, P., and Gray, E.M. Passive exposure to tobacco smoke: saliva cotinine concentrations in a representative population sample of nonsmoking school children. Brit. Med. J. 291: 927-929, 1985. 49. Luck, W. and Nau, H. Nicotine and cotinine concentrations in serum and urine of infants exposed via passive smoking or milk from smoking mothers. J. Pedriatr. 107: 816-820, 1985. 50. Pattishall, E.N., Strope, G.L., Etzel, R.A., Helms, R.W., Haley, N.J., and Denny, F.W. Serum cotinine as a measure of tobacco smoke exposure in children. Am. J. Dis. Children 139:1101- 1104, 1985. 51. Schwartz-Bickenbach, Schulte-Hobein, Abt, Plum, C., and Nau, H. Smoking and passive smoking during pregnancy and early infancy: effects on birth weight, lactation period, and cotinine concentrations in mother's milk and infant's urine. Toxicol. Lett. 35: 73-81, 1987. 52. Sepkovic, D.W., Axelrad, C.M., Colosimo, S.G., and Haley, N.J. Measuring tobacco smoke exposure: clinical applications and passive smoking. Presented at the Both Ann. Mtq. Air Pollution Control Association 1987, New York, NY, Abstr. 87-80-2, 1987. 53. Jarvis, M.J., McNeill, A.D., Russell, M.A.H., W4est, R.J., Bryant, A. and Feyerabend, C. Passive smoking in adolescents: One year stability of exposure in the home. Lancet 1: 1324-1325, 1987. 54. Coultas, D.B., Howard, C.A., Peake, G.T. Salivary cotinine levels and involuntary tobacco smoke exposure in children and adults in New Mexico. Am. Rev. Resp. Dis. 136: 305-309, 1987. 55. Muranka, H., Higashi, E., Itani, S., and Shimiza, Y. Evaluation of nicotine, cotinine, thiocyanate, carboxyhemoglobin, and expired carbon monoxide as biochemical tobacco smoke uptake parameters. Int. Arch. Occup. Environ. Health 60: 37-41, 1988. 56. U.S. Department of Health and Human Services. "The Health Consequences of Involuntary Smoking". A report of the Surgeon General. DHHS (CDC) 87-8398, 1986, 359 p. 57. Palladino, G., Adams, J.D., Brunnemann, K.D., Haley, N.J., Hoffmann, D. Snuff-dipping in college students: a clinical profile. Milit. Med. 151: 342-346, 1986. 58. Haley, N.J. and Hoffmann, D. Analysis of nicotine and cotinine in hair to determine cigarette smoker status. Clin. Chem. 31: 1598-1600, 1985. 59. Sepkovic, D.W. and Haley, N.J. Biomedical applications of ill

95. Bryant, M.S., Skipper, P.L., Tannenbaum, S.R., and Maclure, M. Hemoglobin adducts of 4-aminobiphenyl in smokers and nonsmokers. Cancer Res. 47: 602-608, 1987. 96. Wynder, E.L. and Hoffmann, D. "Tobacco and Tobacco Smoke. Studies in Experimental Tobacco Carcinogenesis." Academic Press, New York, NY, 1967, 730 p. 97. Binder, H. and Lindner, W. Bestimmung von Aethylenoxyd im Rauch garantiert unbegaster Zigaretten. Fachliche Mitt. Oesterr. Tabakregie 13: 215-220, 1972. 98. International Agency for Research on Cancer. "Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs, Volume 1-42." IARC Monogr. Suppl. 7: 1987, 440 p. 99. Tornqvist, M., osterman-Golkars, S., Kautiainen, A., Jensen, S., Farmer, P.B., and Ehrenberg, L. Tissue doses of ethylene oxide in cigarette smokers determined from adduct levels in hemoglobin. Carcinogenesis 7: 1519-1521, 1986. 100. Hecht, S.S., Carmella, S.G., Trushin, N., Spratt, T.E., Foiles, P.G., and Hoffmann, D. Approaches to the development of assays for interaction of tobacco-specific nitrosamines with hemoglobin and DNA. IARC Sci. Publ. 89: 121- 128, 1988. 101. Benner, C.L., Bayona, J.M., Caka, F.M., Tang, H., Lewis, L., Crawford, J., Lamb, J.D., Lee, M.L., Lewis, E.A., Hansen, L.D., and Eatouqh, D.J. Chemical Composition of Tobacco Smoke. 2. Particulate Phase Compounds. Environ. Sci. Technol. 23: 688-699, 1989. 115

12. Saxena, K. and Scheman, A. A suicide plan by nicotine poisoning: A review of nicotine toxicity. Vet. Hum. Toxicol. 27: 495-497, 1985. 13. Gehlbach, S.H., Williams, W.A., Perry, L.D., Freeman, J.H., Langone, J.J., Peta, L.V., and Van Vunakis, H. Nicotine absorption by workers harvesting green tobacco. Lancet 1: 478- 480, 1975. 14. Pomerleau, O.F. and Pomerleau, C.S. "Nicotine Replacement - A Critical Evaluation". Pro#r. Clin. Biol. Res. 261: 1-317, 1988. 15. Fayerabend, C. Determination of nicotine in physiological fluids by gas chromatography. IARC Sci. Publ. 81: 299307, 1987. 16. Feyerabend, C. and Bryant, A.E. Determination in physiological fluids by gas chromatography. IARC Sci. Pubi. 81: 309-316, 1987. 17. Van Vunakis, H., Gjika, H.B., and Langone, J.J. Radioimmunoassay for nicotine and cotinine. IARC Sci. Publ. 81: 317-330, 1987. 18. Machacek, D.A. and Jiang, N. Quantification of cotinine in plasma and saliva by liquid chromatography. Clin. Chem. 32: 979- 982, 1986. 19. Chien, C-Y., Diana, J.N., and Crooks, P.A. Determination of nicotine in plasma by high performance liquid chromatography with electrochemical detection. LC-GC ¢: 53-55, 1988. 20. Bjercke, R.J., Cook, G., Rychlik, N., Gjika, H.B., Van Vunakis, H., and Langone, J.J. Stereospecific monoclonal antibodies'to nicotine and cotinine and their use in enzyme- linked immunosorbent assays. J. Immunol. Methods 90: 202-213, 1986. 21. Neurath, G.B., Duenger, M., Orth, D., and Pein, F.G. trans-3'-hydroxycotinine as a main metabolite in urine of smokers. Internatl. Arch. Occup. Environ. Health 59:199-201, 1987. 22. Neurath, G.B., Pein, F.G. Gas chromatographic determination of trans-3'-hydroxycotinine, a major metabolite of nicotine in smokers. J. Chromatog. Biomed. Appl. 415: 400-406, 1987. 23. Adlkofer, F., Scherer, G., Jarczyk, L., Heller, W.D., and Neurath, G.B. Pharmacokinetics of 3-hydroxycotinine. In: The Pharmacology of Nicotine. M.J. Rand and K. Thurau, eds. IRL Press, Washington, DC 1988, pp. 25-28. 24. Langone, J.J. and Van Vunakis, H. Radioimmunoassay of nicotine, cotinine, and gamma-(3-pyridyl)-gamma-oxo-N- 108

cotinine quantitation in smoking related research. Am. J. Public Health 75: 663-664, 1985. 60. U.S. Department of Health and Human Services. "The Health Consequences of Smoking - Nicotine Addiction". A report of the Surgeon General, DHHS (CDC) 88-8406, 1988, 618 p. 61. Mumford, J.L., Forehand, L., Burton, R., Lewtas, J., Hammond, S.K., and Haley, N.J. Serum and urine cotinine as quantitative measures of passive tobacco smoke exposure in young children. Proc. 4th Internatinal Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87", Volume 2: 18-21, 1987. 62. Hill, P., Haley, N.J., and Wynder, E.L. Cigarette smoking: carboxyhemoglobin, plasma nicotine, cotinine and thiocyanate vs. self-reported smoking data and cardiovascular disease. J. Chron. Dis. 36: 439-449, 1983. 63. Wald, N., Idle, M., Smith, P.G., and Bailey, A. Carboxyhemoglobin levels in smokers of filtered and plain cigarettes. Lancet 1: 110-112, 1977. 64. Jarvis, M.J. and Russell, M.A.H. Measurement and estimation of smoke dosage to nonsmokers from environmental tobacco smoke. Eur. J. Respirat. Dis. (Suppl) 133: 68-75, 1984. 65. Jarvis, M.J. Uptake of environmental tobacco smoke. IARC Sci. Publ. 81: 43-58, 1987. 66. Hoffmann, D., Brunnemann, K.D., Adams, J.D., and Haley, N.J. Indoor air pollution by tobacco smoke: model studies on the uptake by nonsmokers. Proc. 3rd International Conference on Indoor Air Quality and Climate, Stockholm, "Indoor Air", Volume 2: 313-338, 1984. 67. Scherer, G., Westphal, K., Hoepfner, I., Adlkofer, F., and Sorsa, M. Biomonitoring of exposure to potentially mutagenic substances from environmental tobacco smoke. Proc. of the 4th International Conference on Indoor Air Quality and Climate, Berlin, "Indoor Air '87", Volume 2: 109-114, 1987. 68. Haley, N.J. Axelrad, C.M., and Tilton, K.A. Validation of self-reported smoking behavior: biochemical analysis of cotinine and thiocyanate. Am. J. Publ. Health 73: 1204-1207, 1983. 69. Kasuga, H., Matsuki, H., Osaka, F., and Inoue, M. The study on the relationship between urinary hydroxyproline and creatinine ratio from the viewpoint of public health. Tokai J. Exp. Clin. Med. 4: 343-351, 1979. 70. Guerin, M.R. Formation and physico-chemical nature of sidestream smoke. IARC: Sci. Publ. 81: 11-24, 1987. 112

assess its presence in surgical specimens of lung tissue, in human placenta, and in peripheral blood lymphocytes (89-91). Evidence for the presence of such adducts in samples from smokers has been ascertained but significant differences between smokers and nonsmokers have not been observed. 2. Aromatic Amines. 4-Aminobiphenyl and 2-naphthylamine are the known tobacco smoke constituents which are most likely to contribute to the increased risk of bladder cancer of cigarette smokers. The mechanisms by which these compounds are metabolically activated and produce DNA adducts in the bladder epithelium have been extensively studied (92). These studies have shown that the corresponding hydroxylamines are key intermediates in DNA and protei-n modification. The hydroxylamines also react with hemoglobin, in the case of 4- aminobiphenyl, a sulfinic acid amide of the beta-cysteine (93- 95). This adduct.readily releases 4-aminobiphenyl upon treatment with dilute acid. A method was developed to analyze the released 4-aminobiphenyl by gas chromatography with detection by negative ion chemical ionization mass spectrometry (95). Application of this method to smokers showed that adduct levels were higher than in nonsmokers, and decreased upon smoking cessation. The method may be further refined for assessing the uptake of carcinogenic aromatic amines from ETS by nonsmokers. 3. Ethylene. This volatile unsaturated hydrocarbon is present in both mainstream smoke (200-400 ug/cigarette) and sidestream smoke of cigarettes (96). Cigarette smoke contains also traces of the carcinogenic ethylene oxide (7.0 ug/cigarette; 97,98). Upon absorption, ethylene is metabolized to the reactive ethylene oxide. The latter binds to cellular macromolecules and to hemoglobin. The alkylated valine is cleaved off of the isolated hemoglobin and the derivatized hydroxyethylvaline is analyzed by GC-MS. Cigarette smokers showed significantly higher hydroxyethylvaline levels (389±138 pg/g hemoglobin) than nonsmokers (58±25 pg/g; 99). So far the method has not been applied to estimates of exposure of involuntary smokers to the procarcinogen ethylene. 4. Tobacco-Specific N-Nitrosamines. During tobacco processing and during smoking tobacco alkaloids give rise to tobacco-specific N-nitrosamines (TSNA). The nicotine-derived N- nitrosamines N'-nitrosonornicotine (NNN) and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are powerful carcinogans. They occur in relatively high concentrations in cigarette mainstream smoke (NNN, 0.12-3.7 ug/cigarette; NNK, 0.08-0.77 ug/cigarette) and sidestream smoke (NNN, 0.15-1.7 ug/cigarette; NNK, 0.2-1.4 ug/cigarette; 40). These agents are metabolically activated by aipha-hydroxylation, leading to a highly reactive intermediate which forms DNA adducts and protein adducts (Fig. I). Metabolic activation of NNN and NNK also leads to the formation of hemoglobin adducts. Acid or base hydrolysis 105