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lReprinted from ACCOUNTS OF CHEMICAL RESEARCH, 12,92 (1979).] Copyright © 1979 by the American ChemicaliSucietp and reprinted by permission of the cupyrightowner. obacco-Specific Nitrosamines: "Occurrence, Formation, Uarcinogenicity, and Metabolism ,STEPHEN S. HECHT,* CHI-HONG B. CHEN, and DIETRICH HOFFMANN ;. Division of Ent;ironmental Carcinogenesis, ><'aylor Dano Irzstilutr for Disease Pteuention, American Health Foundation, Valhalla, Neu, York 10595 Receiied April 17, 1978 It is now widely accepted that cigarette smoking is causally associated with lung cancer.t' It is less widel.•v known that smoking is also correlated with an~ increased incidence of cancer of the oral cavity,, esophagus, pancreas, and bladder.'4 Tobacco chewing can also cause oral cavity and esophageal cancer.3~4'" In fact, cancer of the mouth is a major cancer among men in India, where the habit of chewing the betel quid! con* taining tobacco is widespread.8 Cigarette smoke is known to contain tumor initiators, such as the poly- nuclear aromatic hydrocarbons, and tumor promoters and cocarcinogens, such as catechol.9' These agents can explain many of the observed effects of cigarette smokee condensates in experimental animals and most likely are involved in some of the human cancers associated Dietrich hloffmann is a member ot the American Heatah Foundation and'Chief, of its Division of Environmental ICarcinogenesis. He received his B.S. and M.S: degrees from the University of'Kieliand the Ph.D: from the IJlax Ptanck Institute for Biochemistry, Munich;, Germany. He was an Associate Member of the Sloan-Kettering Institute for Cancer Fesearch, Stephen S. Heeht received the Phi.D: degree from Massachusetts Institute - of Technology in 1968. He is an associate member of the American Health Foundation. - Chi-hong B. Chen is a research associate at the Foundation. She earned her Ph D. degree from New York University. - The investigators are Interested in the isolation: identification, and reduction of carcinogens in materialsv,rtlich~.have been associated with human cancer, as welli as in the metabolic activation of polynuclear aromatic hydrocarbons. N-nitrosamirres, ottxr carcinogens, and the reaction of the active species of carcinogens with cellular macromolecules. This paper is no. 17 in,the series 'A Study of Tobacco Carcinopenesis`. with smoking. However, nitrosamines may also be causative factors in the tobacco-related cancers, es- pecially in those organs which are remote from direct contact with tobacco or tobacco smoke. Thus it is known that nitrosamines can cause esophageal, pan- creas, and' bladder cancer in experimental animals as well as affect the lung and oral cavityao-lz Since the first report on the carcinogenicity of di- methylnitrosamine,13 a wide variety of nitrosamines have been tested in various experimental animals.1o,1< (1) U.S. Department of Health, Education and Welfare: U.S: Publ. H1th, Serv. Publ. tioi 1103; 196a; HEW Pub1i No. (HSM) 71-7513, 1971;, DHE1N' Publ. No. (CDC) 76-8704 (1975). (21 1t',vnder, E. L.: Stellman. S. D: Cancer Res. 1977, 37, 4608. (3) Wynder, E. L.: Bross, 1. J.;,Feld'man„R. M. Cancer 1957,,10„1300. (4) \4'vnder, E. L.; Bross. I. J. Cancer 1961. 14, 389. (5) Wynder, E. L.: Mabuchii K.; Maruchii N.: Fortner, J. G. J. Natl, Cancer lnst: 1973. 50, 645. (6) 11'ynder, E. L.: Goldsmith. R. Cancer 1977, -00, 1246. (~~) \Tucve, G. E.: Bissinger, L. L.; Proehl. E. C. J. Am: Geriatr. Soc. 1953, 1; 497. (8) Javant., K.;,Balakri.chnan4 V.; Sanghva,L. D.; Jussaw•a11a;,D. J, Brii. J. Canc-er 1977, 35, 232. (9) 1i'.•nder, E., L.; Hoffmann, D. "Tobacco and Tobacco Smoke'; Academic Press: New York, 1967. (101 Magee, P. N.; Montesano. R; Preussmann„R ACS Monogr. 1976, No. 173, 491. . (11) Pour, P.; Althoff, J.; Krilger, F.; Schraahl. D.; Mohr. U. Cancer Lert. 1975. 1, 3. 4 (1a Pour, P.; Althoff, J.; Nagel, D. Cancer Lett. 1977, 3, 109. (13) Magee, P. N.; Barnes, J. M. Brit. J. Cancer 1956, 10, 11{.

1 1....1t.(.U lILLI Uu(L1JLl, L.. NNN NICOTYRINE ANABASINE ANATABINE BIPYRIDYL Figure 1. Common tobacco alkaloids in tobacco and/or tobacco smoke. . Most nitrosamines with available hydrogens on~ the carbons a to the nitrosamine nitrogen are carcinogenic. Nitrosamines generally affect specific organs in a given experimental animal, and the effect is often inde- pendent of the route of administration. In animals` generally used for bioassay the target organs are fre- quently the esophagus and liver in the rat, the re- spiratory tract in the Syrian golden hamster, and the lungs and liver imthe mouse:10•" However, other organs such as pancreas and bladder are also affected by ni- trosamines with particular structural characteris- tics.1o,12.15 Because of the structural' diversity of nitrosamines which can cause cancer in experimental animals and the potency of many of these agents, their occurrence and! formation in human environments have been studied' intensively.10 Analytical methods are now available for the routine determination of trace amounts of nitros- amines. Important among these is the methodology involving the "thermal energy analyzer" (TEA), a sensitive and specific detector for nitrosamines, which can be coupled to a gas or liquid chromatograph.l6' By use of this technique and conventional analytical methods, nitrosamines have been identified most -commonly in processed meats, cheese, air pollution,, unburned tobacco, mainstream and sidestream tobacco smoke, cosmetics, and industrial cutting fluids.'1`21' Concentrations are often in the parts per billion range, although much higher levels of certain nitrosamines (14) Druckrey, H.; Preussmann, R.; Ivankovic, S.; Schmahl;, D. 2. Krebsforsch. 1967, 69, 103: (15) Druckrey, H.;, Preussmann4 , R.; 1.•ankovic, S:; Schmidt. C. H.; Mennel, H. D.; Stab], K. W. Z. Krebsforsch. 1964, 66, 280. (16) Fine, D.; Rufeh, F.; Lieb, D.;,Rounbehler, D. P.,Anal. Chem. 1975, 47, 1188. (17) Sen, N. P. In "Toxic Constituents in Animal Foudstuffs";,Liener~ H. E., Ed.; Academic Press: New York, 1974,p 131. (18), Fine, D. E; Rounbehler, D. P.; Pellizzari;, E. D.; Bunch, J. E.; Berkley, R W:;,McCrae, J.; Bu~'sey, J. T.; Sawicki, E; Krost,lG; DeMarrai.s, G. Bull. Environ. Contam. Toricol: 1976, 15, 739. '(19) Brunnemann, K. D.; Yu+,L.; Hoffmann, D. Cancer Res. 1977, 37, 32I8. (20)' Fan, T. Y.; Goff, U.; Song, L; Fine, D. H.; Arsenault, G. P.; Biernann; K. Food Cosmet. Toxicol. 1977, 15, 423: (21) Fan, T. Y.; Morrison, J.; Rounbehler, D P.; Ross, R Fine D H.; ;: itfiles, W.; Sen. N: P. Science 1977, 196, 70! NPr NAB 9s Figure 2. Some nitrosamines which can be derived from the tobacco alkaloids. have been found' in cosmetics, cutting fluids, tobacco, and tobacco smoke. Nitrosamines are also formed in vivo by nitrosation of amines. This route of exposure is of particular concern since nitrite and amines are widely distributed.22 Since tobacco and tobacco smoke have specific car- cinogenic effects in man, one can hypothesize that there may be unique carcinogenic agents in tobacco and tobacco smoke. The tobacco-specific nitrosamines are such a group. These nitrosamines are derived from the tobacco alkaloids (see Figure 1). The most prevalent alkaloid' is nicotine, which. occurs in general in con- centrations of 1-2% in commercial tobacco products. Both nicotine and nornicotine could give rise to the prototype of tobacco-specific nitrosamines, N''-nitro- sonornicotine (NNN). Nicotine could also be nitrosated to form 4-(N-methyl-N! nitrosamino)-11-(3-pyridyl)-1- butanone (NNK) or 4-(A'-methyl-N-nitrosamino)-4- (3-pyridyl)butanal (NNA). In addition, N-nitroso- pyrrolidine (NPy), which is formed during smoking, could be d'erived' from nicotine and nornicotine.19 Nitrosation of anabasine would give nitrosoanabasine (NAB). The structures of the nitrosamines whi&will be considered in this Account are shown in Figure 2. Of course, inspection of Figure 1 reveals other inter- esting possibilities for nitrosation of the tobacco al- kaloids; some of these possibilities are the subjects of ongoing studies. Occurrence and Formation of Tobacco-Specific Nitrosamines The prototype of the tobacco-specific nitrosamines, NNN, has been detected in both tobacco smoke and unburned tobacco. Various analytical methods have been used, including gas chromatography (GLC),23-26 combined GLC-mass spectrometry,27 thin-layer chro- matography,28 high-pressure liquid chromatography (LC),'-` an& combined LC-thermal energy analysis.31' (22) Mirvish„S. S. Toxico! Appl. Pharmocol. 1975„31, 325. (23) Hoffmann, Di; Rathkamp, G.; Liu, Y. Y: Int. Ag. Res. Cancer Sti: Pub'1: 1974, No. 9,159. 251 (24) Klus, H.; Kuhn, H. Fachliche Mitt. Oesterr. Tabakregie 1973,14, . (25) Hecht. S. S.; Ornaf„R: M.; Hoffmann, D. J. Notl: Cancer Inst. 1975. 54„1237. Also see note added'in proof. (26) Bharadwaj, V P.; Takayama, S.; Yamada, T.; Tanimura, A. Gann 1976. 66; 585. (2i) Munson, J. 11'.;:Abdine, H. Anal. Lett: 1977, 10, 777. (28) hlus: H.; Kuhn4 H. Fachliche Mitt. 0esterr. Tabakregie 1975, 16, 307. (29) Hecht, S. S.; Ornaf, R. M.; Hoffmann„D. Anal, Chem. 1975, 47, 2046. - (30) Hoffmann, D.; Dong, M.; Hecht, S. S. J. Natl. Cancer Inst. 1977, .58,1841. (31) Brunnemann, K. D.; Adams, J.; Hoffmann D Abstr. Tob. Chem R C 3 on/ ex /at Greensboro NC 1977: ~;~~ ..,,,..,. 0,,,,4 ~ _Apa~

FRESH HARVESTED NICOTINE NORNICOTINE TOBACCO NITRITE CURED TOBACCO NICOTINE NNN NORNICOTINE MAINSTREAM CIGARETTE SMOKE NITROGEN I OXIDES Figure 3. Origins of NNN in tobacco and tobacco smoke (tobacco is known to contain nitrate and nitriteu), NhTN' levels in the smoke of a typical American 85-mm nonfilter cigarette range from 140 to 240 ng/cigarette. Surprisingly high levels of NNNI were found in un- burned tobacco (0.3-9.0 ppm in cigarette tobacco, 3.0-45.3 ppm in cigar tobacco, 3.5-90.6 ppm in chewing tobacco, and 12.1-29.1 ppm in snuff). These levels are among the highest for an environmental nitrosamine in terms of occurrence and, human exposure.32 Thus,, rather detailed studies were carried out to determine the origins of NNN in tobacco and tobacco smoke. To study the formation of NNN in tobacco, plants were analyzed at various stages of growth and curing.33 NNN' was not detected prior to harvest or in freshly harvested Burley tobacco but only during and after air curing (0.5-1.1 ppm), which is the process normally employed before use of Burley tobacco in market -products. Since either nicotine or nornicotine could have been a precursor to NNN in tobacco, tobacco leaves were fed nicotine-2`-"C or nornicotine-2'-14C.34 The yield of NNN from nicotine was 0.009% and from nornicotine, 0.007%. These results showed that both nicotine and~ nornicotine could be precursors to NNN in tobacco. However, the greater abundance of nicotine in tobacco leaf (20-100 times the concentration of nornicotine) favored nicotine as the major precursor of NNN in tobacco. The transfer of NNN from cigarette tobacco to mainstream smoke was also studied.' For this purpose, NNN-2'-"C was added to cigarettes and the smoke was analyzed, The transfer rate was found to be 11.3%. Since, in this experiment, the tobacco column smoked contained 974 ng of NNN', 110 ng was transfered to the mainstream smoke. Analysis of the mainstream smoke revealed 238 ng of NNN; thus the remaining 12S'ng was formed during smoking. Therefore,,about 50% of the NNN in mainstream smoke originated byy transfer from tobacco while the remainder was formed'' during smoking, Either nicotine or nornicotine could be a precursor to NNN formed during smokinr. To examine this question, nicotine or nornicotine was added to cigarettes and the smoke was analyzed for NNN.=1 ' In each case, NNN'' concentration in smoke increased, indicating that (32)'Fine, D. H.14HO Cconference Proceedings 197S:1ARC Sci. PuhL No: 19;, 267, (33)' Hecht, S. S.; Chen. C. B.; Dong. M.; Ornaf, R. M.; Hoffmann. D.: ""Tso, T. C. f3eitr TobokJursch. 1977. 9, 1. • (34)' Hecht, S. S.; Chen, C. B.;,Ornaf; R. M.;,Hirota, N.: Hoffmann. D.; Tso. T. C. J. Notl. Cancer In.ct. 1975, 60, 819. from NNA both alkaloids are precursors to NNN' formed during smoking. However, nicotine is considered the more important precursor due to its higher concentration in tobacco. The results of these studies on the formation of NNN during curing, its transfer to smoke, and its formation during smoking are summarized in Figure 3. In~ tobacco samples examined so far, the levels of NAB are significantly less than those of NNN. In fact, NAB has not yet been detected with certainty in un- burned tobacco.5 These findings are in line with the major role of nicotine rather than nornicotine as a precursor to NNN since kinetic studies showed that nornicotine and anabasine were nitrosated at similar rates.3S The fact that these rates are relatively high suggests that the formation of NNN and NAB could be favored in vivo: When chewing tobacco was incu- bated with human saliva for 3 h at 37 °C and the mixture analyzed for NNN, the concentration,of NNN increased~ by 44% over that in the chewing tobacco,, presumably as a result of further nitrosation.25 Thus, in vivo formation of NNN and NAB could constitute an addit.ionali exposure of smokers or chewers to these tobacco-specific nitrosamines. Since nicotine is the major precursor to NNN in tobacco and tobacco smoke, the reaction of nicotine with sodium nitrite was studied to provide information on formation of other tobacco-specific nitr~osamines, especially. NNK and NNA, which coul6 arise by oxi- dative cleavage of the; 1'-2' bond or 1'-5' bond of nicotine followed b_v nitrosation.3e The reaction was investigated under a variety of conditions. All three nitrosamines were formed when the reaction was done under relatively mild conditions (17 h,, 20 °Cl. The yields (0.1-2.8%) were typicaL of those for formation of nitrosamines from tert.iarvy amines.3' At 90 °C, with a fivefold! excess of nitrite, only NNN and NNK were detected. Under these conditions„both NNK and NNA -gave secondary products. NNK was nitrosated a to the carbonyl to yield 4-(N-rnethyl-N-nitrosamino)-2-oxi- mino-l-(3-pyridyl)-1-butanone (1),while NNA under- went cyclization followed byy oxidat~ion„decarboxylation, and dehydration to give 1-methyl-5-(3-pyridyl)pyrazole (2) as shown in Scheme I. Extensive fragmentation and oxidation of the pyrrolidine ring were also observed under these conditions. The major prodllcts.resulting from fragmentation of the pyrrolidine ring were cis- and' (35) ASirvish, S. S.; Sams, J.; Hecht. S. S. J. Natl. Cancer,lnst. 1977, $9, 121'1. (36) Hecht. S. S.; Chen, C. B.; Ornaf, R. M.; Jacobs, E.; Adams: J. D.;. Hoffmann. D. J. Org. Chem, 1978, 43. 72. (a7 ) Lijinsky; W.; Keefer, L.: Conrad. E:: van de Bnprd, R. J.:1'6d. ( irncer Inst. 197:. ./9, 1239. Scheme I Formation of 1-Methyl•5-(3-pyridyl)pyrazole (2)

. va. ar., i./ I J _,Na a, No NONO 1 uuu(.cu /vbcrUaurjtirbea trarl..s-3-(3-pyridy,l)acrylonitrile and N-methylnicotin- amide. Z-:The formation of NNN, NNK, and NNA from riic- I otine probably involves the intermediacy of cyclic iminium salts, as shown in Scheme 11.38 These salts .'can* undergo hydrolysis to the free amines which are nitrosated, or, at near neutral pH, can~ be directly ni- trosated to give nitrosamines. The formation~ of ni- trosamines trosamines from iminium salts under neutral conditions has been demonstrated in~at least two studies and is of interest because iminium salts are known to be inter- mediates in the mammalian metabolism of nico- tine.36,39~'' The possibility that tobacco bacteria could nitrosate nicotine via this pathway is currently under investigation. The formation of NNK and NNA from nicotine inn these model'studies encouraged us to search for these :. nitrosamines in tobacco and tobacco smoke. In studies . undertaken so far, NNK, but not NNA, has been de- tected. tected. NNK was most readily analyzed by combined ' LC-TEA, although conventional LC methods have also been used.31-" Levels of NNN and NNK in tobacco and mainstream cigarette smoke are summarized in Table I. These data are significant because carcinogenicity studies indicated activity for both NNK and NNN. The analytical studies discussed in this section were all done using NNN-2'-14C as internal standard. Tobacco was extracted with~ aqueous ascorbic acid and smoke was collected in traps containing ascorbic acid, to prevent artefactual formation of nitrosamines. Carcinogenicity of Tobacco-Specific - Nitrosamines • The first studies on the carcinogenicity of NAB and NNN were done by Boyland and' co-workers., who demonstrated that"IvTAB caused esophageal tumors in `(38) Smith, P. A. S.;,Loeppky, R: A1. J. Am. Chem. Soc: 1967.8R ,1147. (89) Keefm L. K.; Roller, P. P. Science 1973, 181, 1245. (40) Nguyen. T. L.; Gruenke„L. D.; Castairnoli, N. J:11led.,Chem: 1976.. 19„ 1168. i& (41) I<furphy, P. J. J. Biol: Chem. 1973, 248, 2796, . Scheme II Formation of Tobacco-Specific Nitrosamines from Nicotine Table I NNN and NNK in Tobacco and Tobacco Smoke 95 mainstream, sidestream, tobacco, ug/cig µg/cig ppm product NNN NNK NNN 'NNK NNN NNK Burley, NF 3.7 0.32 6.1 0.66 7.0 - Bright, NF 0.62 0.42 1.7 0.50 0.22 0.37 commercial, 0.24 .0:111 1.7 0.41 1.7 0.74 NF commercial, F 0.31 0.19 nd° nd 1.4 0.70 . Kentucky„ 0.39 0.16 nd nd .. 0.63 0.13 ` 1R1, NF little cigar, F 5.5 4.2 . 0.88 0.81 45.3 ' 35.4 large cigar, F 3.6 2.4 nd nd 5.0 2.2 Columbia cigar 3.2 1.9 16.6 15.7' , nd nd ° nd = not' determined. rats and that NNN induced lung adenomas in mice.*zt3 In our own~ studies, the carcinogenicity of NNN and NAB was first compared in Fischer rats:'" In the NNN group, 14 of 20 animals developed esophageal tumors after a total dose of 3.6 mmol/rat given in the drinking water. By contrast, NAB at this dose gave only 2 of 20 tumor-bearing animals. NNN also induced'tumors of the olfactory epithelium in Sprague-Dawley rats, as demonstrated by Singer and Taylbr!s The tumorigenic activities of NNN and NAB were then compared in Syrian Golden hamsters.'6 . In this experiment, NNN and NAB were each given by sub- cutaneous injection, the total dose being 2 mmol/' hamster. Within 83' weeks, 12 of 19 hamsters given~ NNN developed tumors of the trachea, which is a typicaI target organ for nitrosamines in this species. No tumors were observed in the animals treate&with NAB. To compare NNN, NNK, and' NNA, bioassays were done in strain A mice, whi& are unusuallv susceptible to lung adenomas.'i The number of these tumors observed in treated vs. control', groups can be used as an~ indicator of carcinogenic activity. Each compound was given i.n a total dose of 0.1 mmol/mouse. As judged by multiplicityy of lung tumors, both NNN and NNK showed significant activity (P < 0:05) compared to controls, and NNTK was significantly more active (P < 0:0b) than NNN. NNA did not show significant tu- morigenic activity. The greater tumorigenicity of NNK and NNN in this strain of mice suggests potentially higher carcinogenicityy in other rodent species; these bioassays are currently in progress. Metabolic Studies on NPy and NNN Nitrosamines, like many other classes of chemical carcinogens, must' undergo metabolic transformation to be converted into reactive electrophilic species which can alkvlate nucleophilic cellular macromolecules. This process of metabolic activation has been studied ex- tensively by the Millers, who were pioneers in devel- oping these concepts. According to their scheme, an inactive procarcinogen is metabolically transformed to a proximate carcinogen and finally to an ultimate carcinogen: the latter is a reactive electrophile su& as (42) &ovland! E.; Rue;,F. J. C.: Gurrod. J. \C.: Mitchley, B. C. V. Brit. J. Ca n c cr 1964. 23. 265. (4fi1 Boyland. E.; Roe. F. Ji C.; Gorrod; J.11'. Nature (London) 1964, 202. 1126. (441 Huffmann, D::Raineri. R.: Hecht. S. S.; Maronpot. R.;1tynder. E. L. J. Narl: Concer Ina. 1975.5.5.977. . - .. (45) Singer. G. M.: Ta>•lur, H.11'. J. 1'atl. Cancer ln,ct. 1976. 37. 1275. (461 Hilfrich. J.; Hecht, S. S.; Hnffmann. D. CanccrLett. 1977.2, 169. I

' a carbonium ion.17 Such a scheme can be applied to ' dialkyl or cyclic nitrosamines in severali ways, and ~'various critical initial steps have been suggested4 in- ' -cludjng o ~hs droxylation, ~3-hydroxylation, and d-oxi- -,,,,dation. Since the intermediates generated ~: metabolically may be unstable, indirect means have ' been used to gain evidence supporting the various pathways. Most studies to date on both cyclic and acyclic nitrosamines support the hypothesis that an initial a-hydroxylation is a critical step in carcinogenesis by nitrosamines. For cyclic nitrosamines„substitution at the a positions often results in decreased carcino- genicity, as demonstrated in studies by Lijinsky, Keefer, and Taylor. For example, 2,5-Me2NPy was significantly less carcinogenic in the rat than an equimolar dose of NPy.SO' Similar results were obtained with nitroso- piperidlne-S1 Substitution of deuterium atoms a to the nitrosamine function of nitrosomorpholine decreases activity. Thus, 3,3,5,5-tetradeuterionitrosomorpholinee was less carcinogenic than nitrosomorpholine.52 This reduction in activity was consistent with the slower rate of C-D bond breaking in a-hydroxylation of the deu- terated compound! Further information on the role of a-hydroxy- nitrosamines as active intermediates in nitrosamine carcinogenesis has been obtained in recent years by the use of a-acetbxynitrosamines as model compounds.53:~t Numerous a-acetoxynitrosamines have been synthes- ized. and most, including 2-(Ac0)NPv„were mutagenic in Solnlonel'la t~yphimurium without enzymatic acti- vation.5:~-'° . In addition, meth.l(acetoxymethti•1)- nitrosamine and 1-acetoxvpropvlpropylnitrosamine both showed primarily local carcinogenic effects in experimental aniirla]s.6"°'-' These results are consistent %~-ith the role of a-h}-droxylation in activation of di. alkylnitrosamines. since in the presence of esterase, the a-acetox~~~ compounds are readily hydrolyzed to the corresponding a=hvdroxvnitrosamines. Despite the apparent importance of a-hydrox~~lation as an activation step for cyclic nitrosamines, limited information was available on,metabolism of these compounds b.• this process.' This was due, in part., 147) Miller. E. C.; Miller. J. A. .4CS Monu;r. 1976, 173: (48) Kruger, F. W: In: "Prcicedings of the Second International Symposium of the Princess Takamat.cu Cancer Research Fund", Nakahara. W., Takayama, S., Sugimura, T.. and Odashima: S.. Eds.; Tokyo: Tokyo Ptess. 1972, p 213. (49) Schoental, R. Brit. J:,Concer 1973. 28, 436. (50) Lijinsky, W.; Taylor, H. W. Cancer Res. 1976, 36. 1988. (51) Lijinsky, W.; Taylor, H. Want. J. Cancer 1975. 16. 318. (52)~Lijinsk.: W.; Ta.•lor. H. 1t'.: Keefer. L. 1:. J. Nor1: Cancer Inrt. 19,6. 57.1311. (53) Rbller„P. P.; Shimp: D. R.: Keefer. L. K. Tctrahcdron Lett: 1975. , 2065. (Fial Wiessler, M. Angeu. Chem. 1974. ?A6. 61-. (55) Tannenbaum. S. R.: Kraft. P.: Baldwin..J.: Branz. S. C'anrcr Lctt: 1977, 2, 305. (56) Baldwin, J. E.; Branz. S. E.: Gomez. R. F.: Jiraft. P.: Sinske.•, A. J.; Tannenhaum„S. R: Trlrchedrun Lctt. 19,6; 333. (i7) Shuoter. K. V.; Wiessler. N1: Chem: Rir,L li7tcrcLr: 1y76. 14 . 1. t58/ Okada. \i:; Suzuki'i E.: Takaku. a.: Atochizuki. AlI Gonn 1975. 66: 457. /591 Camus, A. \3.;1t'iessler. \l.: Mala.•eille. C.; Bartsch. H. 9futation Res. 1978, 39; 187: ,r (60) J1"ard. J. hi.; Rice. Ji W Roller. P. P.: Wenk, M. L. Cancer Res. 1977, 37. 3046. (61) Wiessler, M.;,SchmahJ, D. Z. Krebb[urcch. 1976. S.i. 47. (62) Althuff, J.; Grandjean. C.; Pour. P.;,Godd. B. Z. An•b.,fur.ch. 1977. 5(R, 127. (63) Gtandjetn; C. J. J. \'atl: Cancer Ina. 1976. 57. lNl' e-Hydroxylation of NPy° Intermediates and Products Resulting from Scheme III `M~=OEi0!C \N `0' 5 N 1-N- N"W +;n 8 9 10 ° Reproduced with permission from Cancer Research.•° to the inherentt instability of the a-hydroxynitros- amines. In our studies on the metabolism of cyclic nitrosamines. we have used model compounds to de- terrnine the probable products of metabolic a- hvdrox-Oation and have then searched for these products as metabolites. In this way, metabolic a- hYdroxvlation of NPy and'NNN was demonatrated.66-,E-' ' The approach for NPy is outlined in Scheme III. n-Hvdro.vlatiom of NPy ' would give 2-(HO)NPy (4) which is expected to undergo spontaneous ring opening to 3'-formvl-l-propanediazohvdroxide (6); this inter- mediate would! lose I*1, and hvdroxide to give oxo- carbonjum ion 7. This oxocarbonium ion could react with cellular macromolecules as we10 as be trapped' by water to give -1-hvdtox~.butvrald'ehvde (9), which exists predorninantl\• as the cyclic hemiacetal. 2-h.ld'roxv- tetxahydrofilran (10). To validate this scheme, 2- (Ac0)NP\.'6 (3) and -1-(.N'-carbethoay-j\'-nitros- amino)butanal (5) were s%-nthesized as precursors to the unstable intermediates 4 and' 6. Hydrol~~sis of both 3 and 5 gave '?-h~•drox~~tetrah~•.drofuran (10) as the major product. in support of the intermediacy of 4, 6, and' 7 (64) kruger. F. 1C;: Bertram. B.: Eisenbrand. G. Z. tirebF/ursch'. 1976, 125. (6i1 Rugs. A. E.: Uin•ich. S. S. J. .\'arL Cancer Inst. 1977. 68, 651. (luir Hecht: S. S.: Chen. C. B.; Hoffmann. D. Cancer Res, 191 8, 38.215. . (6'1 Chen. C. B.: Hecht. S. S.: HaffSnnnn_ D" Prur" Am. _a..ar- t'n.- Rc.-: 1978. 1.9; 116. (6Si Chen. C. B.: Hecht. S. S.; Hoffmann. D. Cancer Re•c. 1978.38.3639.

Tobacco Nitrosamines Scheme IV Intermediates and Products Resulting from c-Hydroxylation of NNN° N•0 r \ /NY/ \\.iYO /M N7R0 . ° Reprinted .vith permission from Cancer Research.se as shown in Scheme IIL Both 3 and 5 were mut.agenic in S. typhimurium strains TA 100 and TA 1535without enzymatic activations presumably because of in situ hydrolysis to 4, 6, and 7. Metabolic a-hydroxylation of NPy was then dem- onstrated in vitro by isolation of 4-hydroxybutyr- aldehyde 2,4-dinitrophenylh.•drazone (8) after incu- bation of NPy with liver microsomes. The formation of 10 was also demonstrated in vivo b}• isolation of 8 from the urine of rats injected wit.h, NPy. Metabolic a-hv.droxylittion of the tobacco-specific carcinogen NNN was studied using a similar approach. Despite the apparent similarity of NNN and NPv. some differences were apparent in the model and metabolic studies. Scheme IV summarizes the main features of these eaperiments..Both 2'-(AcO)NNN (11) and 5'- (Ac0)NNN (14) were synthesized' as model precursors to 2'-(HO)NNN (12) and 5'-(HO)NNN ('13). The syntheses were done by procedures reported for 2- (Ac0)NPy.'s Hydrolvsis of the 2'-acetate i l gave, as .:>:; major products, myosmine (50-60%) and the keto -~.alcohol 20 (5-10 70. The 5'-acetate 14 gave predomi- nantlv the lacto122 (60-70%). Formation~of20 and 22 from the 2'-acetate and the 5'-acetate can be ration- alized as shown in Scheme IV. However, the high yield' of myosmine in the solvolysis of 11 and the rapid rate of decomposition of 11 in~ H~0 (half-life at 37 °C = 10 min vs. 180 min for 14) indicated that loss of'OAc and N0+ was a competing pathway to formation of 12. Therefore. nitrosourethane 15 was also prepared' as a model compound for the 2'-h~~~dro.ylation pathway. Hydrolysis of 15 gave predominantly. 20. When tested in S. typhrmurium TA 100, all three model compounds were mutagenic without activation. The ?'-acetate 11 was weakly mutagenic, the 5=acetate 14 was moderately mutagenic, and the nitrosourethane 15 was hinhih~ mutagenic. These differences in muta- aenicity may be partially due to differing rates of hy- drolvsis of"11, 1•3, and 15. When NNN was tested at comparable doses in the presence of hepatic super- natants, no sianificant activity was observed. However, NNN was mutagenic at higher doses. Evidence for metabolic c%-hvdroxvlation of NNN was obtained in vitro or in vivo. When NIkN was incubated with rat liver micrc,somes, t.he formation of 20 and 21 was demonst.rated hti isolatiun.'of the 2.4-dinitro~

~ phenylhydrazones 24 and 25. When rats were treated _11~ with NNN, 73-85% of the dose was excreted in the urine, but 20 and 21 were not detected. However, .products of further oxidation of 20 and 21, the keto acid ~ 23, hydroxy acid 26, and lactone 27 were isolated. The formation of 20 and 21 in vitro is most readily explained by initial 2'-hydroxylation or 5'-hydroxylation of 1jNN, .as indicated in Scheme IV. The metabolites 23, 26, and 27 were formed, at least partially, by metabolic oxi- dation of 20 and 21. Pathways other than an initial :` cr-hydroxylation of NTNN could have been involved in -: the formation of 23, 26, and 27, which are also observed '°~ in the metabolism of nicotine.69 . The results of these in vitro and in vivo experiments demonstrate that both NPy and NNN undergo met- abolic a-hydroxylation in the rat. The mutagenicity data discussed above are consistent with the involve- ment of a-hydroxylation as the critical step in the metabolic activation of NPy and NNN. Further evi- dence on the role of R-hydroxylation in the activation of these compounds is currently being sought. through carcinogenicity studies of a-deuterated NNN deriva- tives and through studies of the binding of NPy and NNN to DNA and RNA of target tissues. Prospects :- ~._; ,: .. : .• °.. . . . The results described in this review indicate that the tobacco-specific, carcinogens, NNN and NNIi, may be causative factors in the various cancers associated with ' tobacco usage. These nitrosamines are derived pre- dominanth' from the major tobacco alkaluid. niccrtine, by nitrosatiun during the curing and smoking of to- bacco. Other tobacco alkaloids maN • also be precursors to carcinogenic nitrosamines. Since NNIN and NNK form during curing, it is feasible to reduce their con- centrations in tobacco by appropriate management of 169) Gorrod. J. %C.: Jemier. P. In: "Essa}•s in Toxicology", Ha.•es. \i'. .1., Ed.; Vol. 6: Academic Press: New York. 1975. p 35. the curing and related processes. Formation during smoking can also be inhibited. The reduction of these and related nitrosamines in tobacco and tobacco smoke js one approach to reduce tobacco-related cancers. " A second approach begins with an understanding of the metabolic activation and detoxification of tobac- co-specific nitrosamines. The enzymes that mediate ' these transformations can be induced or inhibited by environmental modifiers. Such modifiers may increase or decrease the carcinogenic effects of these nitros- amines. NNN and NPy, as well as NNK, all undergo metabolic a-hydroxylation which is a likely activation process. Specific induction of a-hydroxylation could lead to greater carcinogenic activity; the modifier causing this would _act as a cocarcinogen. Similarly, specific inhibition of a-hydroxylation could have a protective effect against carcinogenesis by these ni- trosamines. The identification of these modifiers through metabolic studies and bioassays is important for a more complete characterization of the causative ' factors in tobacco carcinogenesis and for the prevention of tobacco-related cancer. Note Added in Proof. Recently N'-nitrosoan- atabine, another tobacco-specific N-nitrosamine, has been identified in tobacco (0.6-13 ppm), cigarette smoke (0.33-4.6 ug/cig), and cigarette sidestream smoke (0.16-1.5 jug/cig).'° Data on tha carcinogenic activity of this,nitrosamine are not yet. available. :- This uork tra.d supparted b.•National Cancer Institute Grants No. 1-CR-.55666. C.-t-11123i6. and CA-?1393. S.S.H. is recipient of .Vatiunal Cancer Institute Research Career Det•elopntent Au•ard No. 5KlyCAO0121. We are grateful to Dr. Edmond La 1'uie fur mutagenicit.• assal•s and to Dr. G. Darid ;I1cCoy for hi>lpful discussions un the metabolism experiments in vitro. We ;;rc•ath• appreciate the cooperation of Dr. T. C. Tso, USDA. &•Itst•ille. MD. for providing us tr.ith experimental tobacco samples. i . (70) Hoffmann. D.: et al.. submitted for publication.