Cancer:Letters, 2 (1977).169--176 © Elsevier/North-HollandScieafifiePublishing Ltd.

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NITRosoNoRNICOTINE :ANDN.!:NITROSOANABAS~NE INSYRIAN GOLDEN' HAMSTERS JORN H!LFRICH, STEPHENS. HECHTand DIETRICH HOFFMANN* Naylor Dana Institute fOr Disease P~euention, American Health Foundation, Valhalla, New York 10595 (U.S.A.)

(Received 24 August 1976) SUMMARY N'-Nitrosonomicotine (NNN) and N'-nitrosoanabasine (NAB) were synthesized •and injected subcutaneously three time~l weekly in male and female ' Syrian golden hamsters for 25 weeks (total dose 375 rag). N-Nitr0sopiperidine (I~[PD) served as positive control (total dose 150 mg)i Wifl~in 83 weeks 12 out of 19 hamsters ~ven NNN had developed t~mcheal tumors, 1 had.a Carcinoma o~!the nasalcavity. During the same t2me n o n e o f the.hamsters;given NAB. : developed tumors, whereas all 20 hamsters ~,4ven.NPD developedtracheal. tumors and about 50% had additional neoplasms in: the nasal Cavity: under: the described condi%i0ns and in comparison to NPD; NNN appears t o ' b e a. moderately active tumorigenic agent in~he.'upper.respiratory tract of syrian. h~wasters, whe~:eas NAB is inactive. INTRODUCTION Tobacco chewers and snuff users faceau increased risk for developing c ~ c~r of the lip~ oral cavity and esophagus, and possibly',eancer of~he nasopharynx [21,23--26]. Cigarette, :cigar and pipe ~mok~r~ also have an increased risk for cancer o f the upper digestive tract and cigarette smokers~ in particular, have a higher risk for developing cancer of the lung [23]. In experimental animals, tobacco extract and smoke have been showll: to be tumorigenic [4,7,27]'. Thus, the characterization and reduction o£ the ca~.~cinogenicagents in tobacco and tobacco smoke are important. N~,Nitrosonornico~ine (NNN; Fig.t) in processed tobacco is thus far the most prevalent, carcinogenic nitrosaminefound in animal and plant matel~als destined f0r human consumption [22]~ Its c:,~ncentrations range from 0.3--90 ppm m smoking tobaccos, chewing tobaccc~i andsnuff [3,10,!3,17]. in tobac*Addra~s correspondence to: JSrn J{ilfrich at the al:ove address.

170

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Fig.1. Structures of the cyclic N-N]trosamines NNN, NAB and NPD. co smoke NNN ranges from 140--250 ng per cigarette; about half of this amount is directly transferred from the tobacco and the rest is pyrosynthesized durir, g smoking [11,14]. Although N'-nitrosoanabasine (NAB; Fig.l) was expected to be present in tobacco products, especially in anabasine-rich tobacco varieties, it has thus far neither been d e t e c t e d i n processed tobacco nor in tobacco smoke [10]. NNN and NAB induce tumors of the esophagus, pharyn.~: and the nasal cavity in rats [6,15] and NNN also induces lung adenomas in mice [5,12]. NAB app~axs to be less carcinogenic in the rat than NNN [ I 5 ] . Although the Syrian. go~derL hamster has been widely used for tobacco smoke inhalation studies [2,7,18], the carcinogenic activity of NNN and NAB in the Syrian hamster has thus far n o t been reported. MATERIALS AND METHODS NNN was synthesized according to the method of ]:-Iuet al. [16]. NAB was prepared by nitrosation of anabasine [ 10]. The purit T and chemical integrity of the nitrosamines were assured by thin-layer chromatography, gas-liquid chromatography (GLC), high pressure liquid chromatography, ultraviolet, nuclear magnetic resonance, infrared, and mass spectrometry [10,1t,16]. F o r use in the positive control group, nitrosopiperidine (NPD; Fig.l) was obtained from Eastman Organic Chemicals, Rochester, N.Y. and was found to be of high purity by GLC analysis. The saline solutions of the nitrosamines were prepared weekly and were stored in the dark between injections, Forty male and 40 female Syrian golden hamsters {random bred, 8--10 weeks old) obtained from Lakeview Hamster Colony, Newfield, New Jersey were divided into 4 groups of 10 females and 10 males each. Each animal wa~ given a subcutaneous injection three times weekly for 25 weeks of 0.5 ml o f a saline solution containing the ni~osamine to be assayed. The LDs~ for NNN and NAB, which was higher than 1000 mg/kg body weight in rats injected subcutaneou~fly, was not tested; the dose of NNN and

171 NAB was based on the availability of the substance. The four groups were treated as follows: Group I. 1.0% of NNN, single dose 5 mg, ~otal dose 375 mg; Greup II. 1.0% of NAB, single dose 5 rag, total dose 375 rag; Group III. 0.4% of NPD, single dose 2 rag, total dose 150 mg (positive control group), according to Dontenwill and Mohr [S]; Group IV. Saline solution alone (negative control group). Moribund animals were killed during the experimental period. Eighty-three weeks after the first injection the experiment was terminated and the remaining animals were killed. All animals were necropsied except those lost through cannibalism. Gross lesions and representative samples of all mz'or organs were fizzed in 10% formalin and processed by standard procedures. ~.ach trachea and[ esophagus was opened lon~tudinally and prepared f,>r histological examination. Cross sections of the nasal cavity were also examined histologically. RESULTS AND DISCUSSION The weight curves for the males and females of each group were similar; therefore, the curves for both sexes are plotted together in Fig.2. A considerable weigh~ loss was observed after treatment with NPD. Weight curves for NNN and NAB treated animals were similar to the control group.

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173 of the trachea, of which only one~.(NPD grnup.male, 35 weeks) showed infiltrat• ing growth. T h e firSt.tracheal tumors were found in. the NPD grovp 20 weeks a f t e r treatment was initiated and in the NNN group, after 38 Weeks..Furthermore; all NPD-treated animals showed nmitipie tumors.in the trachea, while in the NNN-treated animalsonlysingleneoplasms, or in a few cases, up r~ three tumors, •were observed, tn addition, neoplasms of t h e nasal c a v i ~ were detected primarily in the NPD-treated hamsters, •first appearing at 27. weeks after treatment was begun. Histologically, these tumors were papillomas (2 females) or adenocarcinomas of varied differentiation (2 females, 5 males). In the NNN group, only one male hamster showed an adenocarcinoma of the nasal cavity which• appeared after 45 weeks. The relatively strong carcinogenic effect of NPD on the respiratory system of Syrian golden hamsters has been reported [1,8,9] and was confirmed by our results. In this experiment, NNN, in c o m p a r i s o n t o NPD, appeared to be a moderately active tumorigenic agent.in the upper respiratory tract of Syrian hamsters, whereas NAB appeared to b~ inactive, Thus, NNN was me,re carcinogenic than NAB in both the rat [15] a n d ~he Syrian golden hamster. This observation is of interest in view of the minor structural differences between these two compounds (Fig.l). In addition, substitution o f the a-position .~f NPD to give N A B resulted in loss of carcinogenic activity in the Syrian hamster. A si'mflax effect was found when methylated nitrosopiperidines were tested in rats; 2-methyl-NPD was somewhat less active than NPD and 2,6-diraethyl NPD was inactive [19]. Our observations on the carcinogenieity of NNN in the respiratory tract of the Syrian golden hamster s u p p o r t the concept•that this tobacco-specific nitrosamine may be related to the increased incidence of cancer in .tobacco smokers and.chewers. NNN occurs, at high levels in both tobacco and tobacco smoke and is formed during tobacco chewing. This compound (as Well as NAB) might also be formed in rive since the corresponding amines ace easily nit~osated [20].

ACKNOWLEDGEMENTS The authors thank Mr Chang Choi for his excellent assistance. This study was supported by Core Cancer Center Program CA-I7613 and by National Cancer Institute ConJxact N01-CP-55666. REFERENCES I Althoff, J., Wilson, R., Cardesa, A. and Pour, P. (1974) Comparative studies of ncopl~stie response to a single dose of nitroso compounds. Z. I~rebsforsch.,81,251--259. 2 Bernfeld, P., Homburger, F. and Rusfield, A.B ~1974) Strain differences in the response of inbred Syrian hamsters to cigarette.';moke L..b,~latlon.J. Nail. Cancer Inst.~53,

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175 25 Wynder , E.L. and Bro~, I J, (1961) A s¢,udy of etiologic 'factors in cancer of the esophagus. Cancer, 14, 389-~413. 26 WYnder E.L, Bro~v, LJ. and Feldman, R.M. (1957) A study of the etiological factom in carmer of the mouth. Cancer, 10 , 13-0-1323. 27 Wynder, E:L, and Hoffmann, D. (1957) Tobacco and Tobacco Smoke. Studies in Experimental Carcinogenesis, Academic Press, New York.