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122! D. Hoffmannl C.B. Chen, and,S:S. Heoht the methylaarboniumi ion may be responsible for the occurrence of liver, tumors. Currently; we are studying this concept with, ["4C]NNQC Iabeled, at the methyl group or at the carbonyl gnoup.. In addition to «-hydroxylation, we detected other metabolites of NNN that are )3-hydroxylated. Pyridine-N-oxides were also identified as metabolites of both NNN' and NNK. At present, we are detenmining, the biological signifi- cance of these N-oxides in bioassays. Furthermore, we are exploring methods of chemoprevention by inhibiting, metabolic activation of tobacco carcinogens and'by increasing the :detoxificatiom TOBACCO-SPECIFI6!N-MITROSA'MINES IN THE SAL'IVA OF'TO'BACCIO CHEWERS The study of the metabolic activation of the tobacco~specific carcinogens is na merely of acadenua interest to us. It gives us the opportunity to examine fluids of smokers and chewers for metabolites of NNN and NNK and, thus, too evaluate whether the metabolism of these carcinogens in man is similar to that in theexperi'mentale animal. ft is also important to, explore why the degree ofmetabol'ic activation of these tobacco carcinogens differs widely in individual chewers and smokers. In vitro experiments have shown tha N-nitrosamines can be formed from the : tobacco alkaloids by incubation of snuff wit'h~ saliva (Hecht et al'. 19°75). We therefore investigated'tihis phenomenon in tobacco chewers and& smokers. In a preliminary study, we were also able to show that concentrations of nonvolatile N-nitrosamines in the saliiva of individual tobacco chewers vary signifiaantNy. This supports the concept that in vivo nitrosation of the tobacco: alkaloids is involved in the formation of these nitrosamines; We have initiated a large-scale controtledl study on the formation ofINNN, NNK, and NAtB in the oral cavityofl chewers and smokers, including analysis of saliva for metabolites of NNN and NNK., SUMMARY Chemical-analyt'ical studies have shown that tobacco smoke contains small amounts of volatile N-nitrosamines. In the mainstream smoke of' ai cigarette, theseearcinogenscan be reduced significantly by utilization of tobacco lonvin nitrate content and especially by filtration through cellulose acetate filter tipsthat~ selectively , retain volatile: N-nitrosamines. Cured and fermented tobacco and tobacebsmoke contain three majQr nonvolatile N-nitrosamines. These:are formed from nicotine, nornicotiine, and anatabine. In mice, rats, and Syrian golden hamsters, NNN' and NNIfK are proven carcinogens. They are organ-specific carcinogens, burtmay also be contact carcinogens. NIAtB', the third identified totiacconitrosamine, is cuo- rentlybe:ing bi~~oassaye~d, for its carcinogenic activity. :<x>SSss ~ is.{it~ti9a~"'

Nitrosamines in Tobacco Products /123 ~•;~t!$?dtSt#~3y3Y?iif f~3'r?t h'~ii' ~3?i NNN and NNK are metabolically activated by a-hydroxylation. The a-hydroxynitrosamines are unstable and decompose with formation of car- bonium ions, the probable ultimate carcinogenic form of these tiobacco,speci'fic compounds. N-nitrosation of the alkaloids occurs during the processing of tobacco and ditring, smoking, but may also take place in vivo. This has been demonstrated by evidence for the formation of NNN, NNK and NAtB' during tobacco chewing,. Detailed studies on the in, vivo formation of these tobacco carcinogens in chewers and smokers has begun. These studies will be folltnwed by the development of methods that inhibit the formation of these compounds. ACKNOWLEDGMENTS Our studies are supported, in part, by Public Health Service contract NO 1-CP' 55666 and by grant CA-21393 from the Divisioni of Cancer Cause and Prevention of the National Cancer Institute andl by American! Cancer Society research grant BC-56. S. S. H. is a recipient of National Cancer Institute research career development awardl 5K04CA00124. C. B. C. is recipient of National Institute of Environmental Health Sciences development award no: ESO12236. REFERENCES Boyland, E., F.J.C. Roe, and J.W. Gortod. 1964. [nductioni of pulmonary tumors in mice by nitrosonornicotine, a possible constituent! of tobacco smoke. Nature 2a2:111z6_ Brunnemann, K.D. and D. Hoffmann. 197$' Analysis of volatile nitrosamines in tobacco smoke and polluted'indoor environments. /nt: Agency Sci. Publ. 9:3431 Brunnemann„ K.D., L. Yu, and D. Hoffmann. 11977„ Assessment of carcinogenic volatdlo N=nitrosamines in tobacco and in mainstreamiand sidestream smoke from cigarettes. CancerRes., 37:3218. Chen, B.C., SS. Hecht, and D! Hoffmann. 1978. Metabolic a-hydroxylatiorr of the tobacco-specific N-nitrosonornicotine. Cancer Res. 36:3639. Chen„B.C., S.S. Hecht, R. Young, T. Ohmori, and D: Hoffmann 1979. Comparative carcinogenicity and metabolism of the tobacco-specific caricinogens, NNN and NNK. Pkoc. Am. Assoc. Cancer Res. 20:81. Dtuckrey, H. and R. Preussmann. 1'962. Zur Entstehung karzinogener N8trosamine am Beispielldes Tabakrauches. Naturwissenschaften 49:498. Enzell, C.R., I. Wahlberg, and AS. Aasen. 1977. lsoprenoids and alkaloids of tobacco. Fortschr. Chem, Org. Naturst: 34:1. Fine, D.H., F. Rufeh, D. Lieb, and D.P. Rbunbehlcr. 1975. Description of the thermad energy analyzer (TEA)fartrace determination of volatile and narnvolatileN-nitroso compounds. AhaP. Chem. 47c l L88. Hecht, S.S., R.M. Ornaf, and D. Hoflfmann. 1975. N-nitrosonornicotine in tobacco; analysis of possible contributing factors andlbioloQic implications.1. Natl. Cancer Liant. 54i 12-37: Hechti, S.S., 1. Schmeltz, andl D; Hoffmann. 1977. Ni'trogenouscompounds in cigarette smoke and their precursors. Recent Adt'. Tobacco Sd,. 3:59! vrr ~ fa:t.i~ii?! "i i~,i::}t;•

1'24l D. Hoffmann, C.B. Chen, and S.S. Hecht li.'tisfirt~t?~slt3}i;?i~ s~n, rY;~ Hecht, S.S., C:B. Chen, and D! Hoffmann. 1979. Tobacco-specific nitrosamines: Occurrence, formation, carcinogenicity and metabolism. Acc. Chem, Res. 12:92'. Hecht, S.S., C.B: Chen,, T. Ohmori, andl D. Hoffmann. 1!980. Comparative carcino- genicity in F~34k1 rats of the tobacco-specific nittnsamines N'-nitrosonomicotine and 4-(iN-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res, (in press): Hecht, S,S:,, C.B:. Chen, R.M. Ornaf, E. Jacobs, J.D. Adams, and' D: Hbffmanm 1978a. Reaction of nicotine and sodium initrite: Formation,of' nitrosamines and fragmentation of the pyrrplidine ring. J. Org. Chem. 43:72. Hecht, S'.S., C.B. Chen, N. Hirota, R.M. Onnaf; T.C. Tso, and!D, Hoffmann. 1978b Tobacoo>specific nitrosamines: Formation from nicotine in vitro and during to- bacco curing and1 carcinogenicity in1 strain A mice. J. Natl: Cancer Inst. 60:819. Hilfiich„ J., S.S, Hecht,, and D. Hbffimann. 1977. Effects of N'-nittosonornicotine and N'-nitrosoanabasine in Syrian golden hamsters. Cancer Lett,. 2:1169. Hoffrnann, D., 0. Dong, andl S:S'. Hecht. 1977. Origin in tobacco smoke of N'- nitrosonornicotine„a tobacco-specific carcinogen. J. Nat1: Cancer Ivrsr. 58i l'84L. Hoffmann, D., S.S. Hecht, R.M. Ornaf, and E:L. Wynderi. 1'976; N'-nitrosonor- nicotine in tobacco: Formation and careinogenicity. Intern. Agency Sci: Publ.. 14:307. Hoffmann, D:,, 1 Schmeltz, S.S~: Hecht, and E.L. Wynder. 1978. Tobacco car- cinogenesis. In Polycyclic hydrocarbons and cancer (ed. H. V. Gelboin and P. O. P. Ts'o),, vol. 1, p:85 L Academic Pness, New York. Hoffmann, D., R. Raineri, S.SI Hecht, R. Maronpot, and E.L. Wynder: 1975. Effects of N'-nitrosonomicotine and N'-nitrosoanabasine in rats. J. Natl. Cancer Inst: 55:977: Hoffmann, D., J.C. Adams, K.D. Brunnemann, and S.S. Hecht. 1979. Assessment of tobacco-specific N-nirtrosamines in tobacco products. Cancer Res. 39:2505. Jasko, W.J. 1'979! Influence of' cigarette smoking, on drug metabolism in man. Drug Metab. Rev: 9:22. Magee, P.N., R'. Mbntesano, and R. Preussmann. 1'976: N-Nitroso compounds and related carcinogens. Avn: Chem. Soc. Monogr. 173:491. Royal College of: Physicians. 1977. Smoking and h'ealth. Pitman Medical Publishing Co., Ltd., London. Public Health Service 1979. , SSmoking and, health: A report to the Surgeon GeneraL DHEW publication number 79-50066. Government Printing Office; Washington, ID.C: Schmeltz, I: and D. Hoffmann. 1977. Nitrogen-containing compounds in,tobacco and tobacco smoke. Chem: Rev. 71:295. Singer, G,.[vt. and H.W. Taylor. 1976. Carcinogenicity of N'-nitrosonomicotine in Snrasue-Dawley rats: J. NatL Cancer Inst. 57:1275. Wynder; E. L. and L.J: Bross. 19611. A study of etiological factors in cancer of the esophagus. Cancer 14:385: Wynder, E:,L. and R. Goldsmith. 1977. The epidemiology of bladder cancer. A second'' look. Cancer 40:1246. Wynderi, E.L., I.J. Bross, and K.ML Feldman., 1957. A: study, of etiological factors in cancer ofl the mouth. Cancer 10:13I00, Wynderi, E. L.:, K. Mabuchi, N. MaruQhi, and J. G. Fortner. 19731 Epidemiology of cancer of the pancreas: J: Nat. Canc•er Inst. 50:50.

Nitrosamines in Tobacco Productsl125 CO'MMEN1rS' GORi: Have you foundi any compounds that may be inhibitory of the car- cinogenic activity of nitrosamines? HOFFMANN: We have no data on the in vivo inhibition of the formation of tobacco-specific N-nitrosamines. We have found the opposite. This aspect was already suggested by Boyland et al. (19Cn4) many years ago. Thiocyan+ ate catalytically increases,tihe in vivo nitrosamine formationiduring chewing andl smoking. We found the thiocyanate concentration in the saliva, to be correlated witbi the q}iantity of tobacco smoked. This was expected, since thiocyanate is the major detoxification product of' HCN, a major smoke constituentL As discussed before, N-nitrosamines are procarcinogens and, thus, they have to be activatedl metabolically. Thus far„ our bioassay data are not completed, however, in vitro studies by Chen and Hiecht'. (1978) from our group have shown t'hatone can inhibit the metabolic activation of N-nitrosonornicotine. The best approach towards reduction of the nitrosamines in smoke wouldd obviously be to use low-nitrate tobacco. However, when, we reduce the nitrate content of tobacco, we have increased formation of carcinogenic polycyclic hydrocarbons. At this moment we regard selective filtration as the best approach towards reducing volatile and tobacco-specific N- nitrosarnines in the smoke. WYNDER 11 wonder whether you could comment on those experiments which again support our interplay between epidemiological observation and ex- perimental stWdies. Hurnandatalhave shown that high alcohol consumption increases the risk of cancer of the esophagus, and the experimental work done at our institute by McCoy et al. (1i979) showed that the metabolicc activation of nit'rosamines via a-hydroxylation is increasedL HOFFMANN: Yes, one of our studies has shown that Syrian golden hamsters maintainedion anialcoholic diet had increased a-hydroxylatiionin the liver and cheek pouch of N-nirtrosonornicotine, compared to the a-hydroxylation occurring,inthe same organs of hamsters oni a standard diet. Human studies, especially in France and the U.S., have documented that drinking, in additioni to smoking, increases the risk for cancer of the esophagus andloral cavity over that for nondrinking,smokers (Tuyns 1970; Kissin et al, 1973; Wynder et all 1977). Therefore, model studies with the Syrianigolden hamster were: initiated limourinstitute. As mentfioned before, Dr. McCoyhasshown~that whenihamsters, wereonanalcoholicdiet,,themetabolic activation of the carcinogenic nitrosamines was significantly increased in both the liver and the hamster pouch. We have an ongoing experiment in which: weapplytobacco-s~pecificn'itrosamines by swab totheoratcavity;of hamsters. Onegpoup of hamsters i,

1261 @. FHoffmann, C.B. Chen, and S:S, Mecht 1 i~ll~t~4iitn~s?fti?!?£ it~'r=S 3'~?1i1 ° > ~~.~;' is on ainormal dietand the second group on an alcoholic diet. The technique of painting the hamster pouch is relatively simple and by simple manipuba- tionione can readily observe the treated tissue. There is no questioni that! alcohol andl nitrosamine together induce more tumors ini the hamster pouch than the nitrosamine in conjunction with the standkrd alcohol-free diet. At present, however, these results arebasedlon macroscopic observations only. Upon histopathological confirmationof the macroscopic observation, we expect this study to fully support the epidemiological data, namely that smoking andldrinking together represent a high risk for cancer of'the oral cavity and esophagus. BATTISTA: Over the years we have been talking about nitrates; an increase in, nitrates results in the decrease of the carcinogenicity of tobacco smoke (when applied to the skin): You were finding that increasing the level of nitrate increases the nitrosamine levels, which one wouldI expect, to give a higher incidence ofl cancer. Now, in none of the data presented here have we looked directly at effects of nitrosamines in smoke on the lung. We apparently have two diametrically opposite effects to nitrate. Would you like to clarify this point?' Hio,FFMAIwN: Nios we: have done it. I'have not ment'ionedI it because of our time limits today. We have painted NNN together withitobacco tar on the skin of mice. This resulted in an increase of lung adenomas„but not of skin tumors,, again suggesting that N-nitrosamines are organ-specific carcinogens. Obvi'- ously, one goal in tobaccocarcinogenesis is to develop low-tar, low niicotine cigarettes. Tobacco which is high in nittatehas increased combustibility andl the nitrogen oxides generated from, the nitrate dL+ring smoking serve as scavengers of C,H-radicals and„ thus, inhibit the pyrosynthesis of the carcinogenic pollycyclic hydrocarbons. This is, of course, desirable since reductionof polycyclic aromatic hydrocarbons in the smoke leads to reduced tumorigenic activity as measured on mouseskim Inhalation experiments with hamsters by Dontenwill et al. (1973) have shown; thatsrnoke fromnitrate-rich cigarette tobaccos is also less, tumorigeniic i~nthe larynx. Thus, we believe that the use :of nitrate-rich tobacco has been and is a step forward inithe directioniof the less harmful cigarette. What we have to try now is to reduce the nitrosamines in the smoke of these cigarettes. In the case of the volatile nitrosamines, this has already been accomplished in the smoke of U.S. cigarettes. Ninety percent of all U.S. cigarettes have celQuloseaaetate filter tips. These are capable ofseiecti'velyrernovingt'~he volatile nitrosamines in the smoke up to: 85%. In that respect there wouldl be two ways to reducetobaeco-specific carcinoge~ns~ First! is selective fiiltratiion, and preliminary data in this area appear quite promising. A second approach would be to keep:the alkaloids low in the tobacco, because without the alkaloids, these nitrosamines cannot be formedl. That is why we are very much opposed to hssk`s:

Mitrosamines in Tobacco Products / 127 the introduction of low-tar„high-niicotine cigarettes. One thing we are sure of is when you have a high alkaloid content in tobacco, youi have : a high yield ofl tobacco-specific carcinogens in the tobacco and in the smoke. Thus, the suggcstion;of low-tar, high-nicotine cigarettes as discussed inithe United Kingdom,, is counterproductive because the higher levels of nicotine in the smoke will continue to support habituation of smokers and will also result in higher nitrosamine content ofthesrnoke, unless methods for their selective filtration are found. References Bioyland'4 E., F.J.C. Roe, and J.W. Gorrod. 1'964. Inductionof pulmonary tumors in mice by nitrosonornicotine, a possible constituent of tobacco smoke. Nature 202:1126. Chen, B.C:, S.S. Hecht, and D. Hoflfrttann. 1978. Metabolic a-hydroxylationi of the tobacco-specific N'-nitrosonomicotine. Cancer Res. 36:3639: Dontenwill, W., H. Chevalier, H. Harke, U. Lafrenz, G'. Reckzeh, and B. Schreider. 1973. Investigatinnion the effects of chronic cigarette-smoke inhalation in Syrian golden hamsters. J. Natl: Cancer Inst. 51i:17$'1. Kissins B., M.M. Kaley, W.H. Su, and R. Lermer: 1!973. Head and neck cancer ini alcoholics. The relationshipof drinking, smoking and dietary patterns. J. Am. Med. Assoc. 224:1174, McCoy, G.D., C-h. B. Chens S:S: Hecht, and E.C. McCoy. 1979. Enhancement of metabolismiand mutagenesis of nitrosopyrrolidine in liver fractions isolated from chronic ethanol-consuming hamsters. Cancer Res. 39:793. Tuyns, A.J. 1'970. Cancer of the esophagus. Further evidence of the relation of drinking habits inFrance. Intl. J. Cancer 5: 1I52'. Wynder, E. L. ,, IVI_ Mushinski, and J,C. Spivak. 1977. Tobacco and alcohol consumption in relationito the development of multiple primary cancers. Cancer 40:1872. ki<.uxN>oooSka ~~;p~,ysyM.+