[CANCER RESEARCH 40, 298-302, February 1980]0008-5472/80/0040-0000$02.00

Comparative Carcinogenicity in F344 Rats of the Tobacco-specificNitrosamines, N ‘-Nitrosonornicotineand 4-(N-Methyl- N-nitrosamino)-1-(3-pyridyl)-1-butanon&

Stephen S. Hecht,2 Chi-hong B. Chen, Takaaki Ohmori,3 and Dietrich Hoffmann

Division of Environmental Carcinogenesis, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595

ABSTRACT MATERIALS AND METHODS

The tobacco-specific carcinogens, N'-nitrosonornicotine(NNN) and 4-(N-methyl-N-nitrosamino)-1 -(3-pyridyl)-1 -butanone (NNK), were tested for carcinogenicity in F344 rats. Eachnitrosamine in trioctanoin was administered by s.c. injection to12 male and 12 female rats over a period of 20 weeks. Thetotal dose of each nitrosamine was 3.4 mmol. The experimentwas terminated after 12 months. NNK induced nasal cavitytumors in 83% of the males and in 83% of the females, livertumors in 83% of the males and in 100% of the females, andlung tumors in 67% of the males and in 67% of the females.NNN induced nasal cavity tumors in 92% of the males and in75% of the females. Only one liver tumor and no lung tumorswere observed in the NNN-treated rats. These results indicatethat, in the F344 rat, NNK is a more powerful carcinogen thanis NNN.

INTRODUCTION

Cigarette smoking is associated with cancer of the lung,larynx, oral cavity, esophagus, pancreas, and bladder; andtobacco chewing is associated with an increased incidence oforal cavity and esophageal cancer (13, 18, 20). The tobaccospecific nitrosamines, which are derived from the tobaccoalkaloids, are possible causative factors for some of thesecancers. The major tobacco alkaloid is nicotine, which can benitrosated to form NNN,4 NNK, or NNA (Chart 1) (5, 6). NNNand NNK have been identified in mainstream and sidestreamtobacco smoke and in unburned tobacco; their levels arerelatively high (7, 10). Thus, NNN, NNK, and a related tobaccospecific nitrosamine, N'-nitrosoanatabine, are among the mostprevalent of environmental nitrosamines in terms of humanexposure.

NNN induces lung adenomas in mice, esophageal and nasalcavity tumors in rats, and tracheal tumors in hamsters (1, 5, 9,11, 16). In a comparative bioassay in strain A mice, NNKinduced more lung adenomas per mouse than did NNN, whileNNA appeared inactive (5). These results suggested a highercarcinogenicity for NNK than for NNN in other rodent species.In the present study, this expectation has been supported bythe results of a comparative bioassay in F344 rats.

1 This study was supported by USPHS Contract NO1-CP-55666 and Grant

CA-21393 from the Division of Cancer Cause and Prevention, National CancerInstitute. This is Paper 19 in the series, ‘AStudy of Tobacco Carcinogenesis.@

2 Recipient of National Cancer Institute Research Career Development Award

No-5K04-@A0Oi24. To whom requests for reprints should be addressed.:3 visiting scientist from the Department of Pathology, Nara Medical University,

Nara, Japan.4 The abbreviations used are: NNN, N'-nitrosonornicotlne; NNK, 4-(N-methyl

N-nitrosamino)-1-(3-pyridyl)-1 -butanone; NNA, 4-(N-methylM-nitrosamino)-1 -(3-pyridyl)butanal.

Received June 4. 1979: accepted October 26, 1979.

NNN and NNK were synthesized and were greater than 99%pure according to analysis by gas chromatography, high-performance liquid chromatography, and thin-layer chromatography (4, 12). Trioctanoin was obtained from Eastman KodakCompany (Rochester, N. Y.) and was redistilled before use.Six-week-old male and female F344 rats were obtained fromCharles River Breeding Laboratories, North Wilmington, Mass.Rats were housed in solid-bottomed polycarbonate cages withhardwood bedding in groups of 3 and were kept under standardconditions [20 ±2°(S.D.); 50 ±10% relative humidity; 12-hrlight, 1 2-hr-dark cycle]. Animals were given Purina laboratory

chow and tap water ad libitum.The experimental groups were as follows: (a) NNK male; (b)

NNK female; (c) NNN male; (d) NNN female; (e) vehicle controlmale; and ( f) vehicle control female. Each group consisted of12 rats. Each rat received 60 s.c. injections of NNK [1 1.7 mg(0.06 mmol)/0.3 ml trioctanoin], or NNN [10.0 mg (0.06mmol)/0.3 ml trioctanoin], or 0.3 ml trioctanoin. Injections weregiven 3 times weekly for 20 weeks beginning at the age of 7weeks. The total dose of NNK was 702 mg (3.4 mmol), and thetotal dose of NNN was 600 mg (3.4 mmol).

Animals were weighed weekly and moribund animals weresacrificed. The experiment was terminated after 12 months.Gross lesions and representative samples of all major organswere fixed in 10% buffered formalin and were processed formicroscopic examination.

RESULTS

The 50% lethal dose of NNN given s.c. was previously shownto be greater than 1000 mg/kg body weight (11). The dosesof NNN and NNK were equimolar and were chosen for comparison to a previous experiment in which NNN was given inthe drinking water (11). Weight curves indicated that NNK wasslightly more toxic than was NNN. Table 1 summarizes mortalityduring the course of the experiment. Twelve months aftertreatment began, 60% of the animals in the NNK and NNNgroups were dead, and the experiment was terminated.

Both NNK and NNN caused a high incidence of tumors ofthe nasal cavity. Malignant tumors of the nasal cavity wereobserved in 50% of the NNK-treated males, in 33% of theNNK-treated females, in 83% of the NNN-treated males, and in58% of the NNN-treated females. All of these tumors werelocated at the posterior (caudal) portion of the cavity or in theethmoid bone, and some tumors invaded the brain (NNK male,4 of 6; NNK female, I of 4; NNN male, 6 of 10; NNN female, 1of 4). The results are summarized in Table 2.

Histologically, 24 of 27 malignant nasal cavity tumors were

298 CANCERRESEARCHVOL. 40

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Mortalityfollowingtreamentwith NNN orNNKGroupSexTotal

dosea(mmol)No.

ofsurvivorsMo.

5Mo. 6Mo. 7Mo. 8 Mo. 9Mo. 10Mo. 11Mo.12NNKM3.41211ii11

10975NNKF3.4121212121111106NNNM3.412121212111065NNNF3.4121212119853ControlM01212121212ii11iiControlF012121212

12121212

GroupSexNo.of an

ImalsBenignonly8MallgnantbNeoplasticnodule onlyHepatocarcinomaHemangiosarcomaAdenomaonlyCarcinomaNNK

NNKNNNNNNM

FMF12

I 212124

6126C

4g

1O@7k2

o1o5d

111)0o4e

4e.s

0o8

0o5f

00

oControlM120000000ControlF1

20000000

Comparative Carcinogenicity of NNN and NNK

nitrosamines NNN, NNK, and NNA derived “0N'NiOChart I . Structures of the tobacco-specific

from nicotine. In NNN, the a positions are 2'(adjacent to the pyrldlne ring) and 5'.

N CH3

CH3

NNA

NNK

Table I

+CH2O

a Each rat received 60 s.c. injections over a 20-week period of NNK or of NNN in trioctanoin or of

trioctanoin alone.

Table 2Numbers of F344 rats with different types of tumors after exposure to NNN, NNK, or vehicle control

Nasal cavity Liver Lung

:Papillomaoradenomatouspolyporvillouspolyp.Olfactory neuroblastoma or rhabdomyosarcoma.

C One animal also had a benign tumor. Malignant tumors included 5 olfactory neuroblastomas and 1 rhabdomyo

sar@oma(embryonal type).Two animals also had neoplastic nodules and another 2 animals had hemangiosarcoma.

e Includes rats with hepatocellular carcinoma.f Two animals also had adenomas; 4 animals had adenocarcinomas; and 1 animal had an adenosquamous cell

carcinoma.@ Four animals had olfactory neuroblastomas.@ Three rats also had hemangiosarcoma and 1 rat had an anaplastic sarcoma.

I One animal also had a neoplastic nodule.

j Three animals also had benign tumors. Malignant tumors included 9 olfactory neuroblastomas and 1 rhabdomyosarcoma (well-differentiated type).

k Four animals also had benign tumors. Malignant tumors included 6 olfactory neuroblastomas and 1 rhabdomyosarcoma (well-differentiated type).

olfactory neuroblastomas which frequently showed pseudorosettes,truerosettes,and/orpalisadingaroundfinebloodvessels. In 11 of them, the presence of neurofibrils was demonstrated by Bodian's silver impregnation method (see Figs. 1to 8). The remaining 3 cases were rhabdomyosarcomas of thenasal mucosa; 2 were well differentiated and one was of theembryonal type. The benign tumors of the nasal cavity, apparently of multicentric origin, arose from the respiratory epithehumwhich covers the anterior and median regions of the cavity.

Histologically, these tumors were papillomas with or withoutkeratinization and polyps of villous or adenomatous types.Squamous metaplasia was also frequently found in almost allanimals.

Malignant liver tumors developed in 58% of the NNK-treatedmales and in 100% of the females, and malignant lung tumorsoccurred in 42% of the NNK-treated males. Malignant tumorsof the liver and lung were not observed in the animals treatedwith NNN.

FEBRUARY1980 299

NNN

NO

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S. S. Hecht et a!.

should be a major goal of further research on smoking andhealth.

ACKNOWLEDGMENTS

We gratefully acknowledge the expert contributions of Dr. A. Rivenson, Dr. G.Williams, and Dr. M. Kuschner.

We thank Mary Jacobs of the Research Animal Facility and Anne Marrone ofthe Division of Experimental Pathology for their excellent technical assistance.

REFERENCES

1. Boyland, E., Roe, F. J. C., and Gorrod, J. W. Induction of pulmonary tumorsin mice by nitrosonornicotine, a possible constituent of tobacco smoke.Nature (Lond.), 202: 1126, 1964.

2. Chen, C. B., Hecht, S. S., and Hoffmann, D. Metabolic a-hydroxylation ofthe tobacco-specific carcinogen, N'-nitrosonornicotine. Cancer Res., 38:3639-3645, 1978.

3. Dawe, C. J. Neoplasms induced by polyoma virus in the upper respiratorytracts of mice. In: C. S. Muir and K. Shanmagaratnam(eds.), Cancer of theNasopharynx, pp. 179—196. Copenhagen: Ejnar Munksgaard, 1967.

4. Hecht, S. S., Chen, C. B., Dong, M., Omaf, R. M., Hoffmann, D., and Tso,T. C. Studies on non-volatile nitrosamines in tobacco. Beitr. Tabakforsch.,9: 1—6,1977.

5. Hecht, S. S., Chen, C. B., Ornaf, R. M., Hirota, N., Hoffmann, D., and Tso,T. C. Tobacco specific nitrosamines: formation by nitrosation of nicotineduring curing of tobacco and carcinogenicity in strain A mice. J. NatI. CancerInst., 60: 819—824,1978.

6. Hecht, S. S., Chen, C. B., Ornaf, R. M., Jacobs, E., Adams, J. D., andHoffmann, D. Reaction of nicotine and sodium nitrite; formation of nitrosamines and fragmentation of the pyrrolidine ring. J. Org. Chem., 43: 72—76,1978.

7. Hecht, S. S., Ornaf, R. M., and Hoffmann, D. N'-Nitrosonornicotlne intobacco; analy3is of possible contributing factors and biologic implications.J. NatI. Cancer Inst., 54: 1237—1244,1975.

8. Herrold, K. M. Induction of olfactory neuroepithellal tumors in Syrian goldenhamsters by diethylnitrosamine. Cancer (Phila.), 17: 116—121, 1964.

9. Hilfrich, J., Hecht, S. S., and Hoffmann, D. Effects of N'-nitrosonornicotlneand N'-nitrosoanabasine in Syrian golden hamsters. Cancer Left., 2: 169—176, 1977.

10. Hoffmann, D., Adams, J. D., Brunnemann, K. D., and Hecht, S. S. Assessment of tobacco-specific N-nitrosamines in tobacco products. Cancer Res.,39:2505—2509,1979.

11. Hoffmann, D., Raineri, R., Hecht, S. S., Maronpot, A., and Wynder, E. LEffects of N'-nitrosonornlcotine and N'-nitrosoanabasine in rats. J. NatI.Cancer Inst., 55: 977—981, 1975.

12. Hu, M. W., Bondinell, W. E., and Hoffmann, D. Synthesis of carbon-i 4labelled myosmine, nornicotine, and N'-nitrosonornicotine. J. LabelledCompd., 10: 79—88,1974.

13. Jussawalla, D. J., and Deshpande,V. A. Evaluation of cancer risk in tobaccochewers and smokers: an epidemiological assessment. Cancer (Phila.), 28:244—252,1971.

14. Laskin, S., Kuschner, M., Drew, R. T., Cappiello, v. P., and Nelson, N.Tumors of the respiratory tract induced by inhalation ofbis(chloromethyl)ether. Arch. Environ. Health, 23: 135—136, 1971.

15. Pour, P., Althoff, J., and Nagel, 0. Induction of epithelial neoplasms by localapplication of N-nitrosobis(2-hydroxypropyl)amine and N-nltrosobis(2-acetoxypropylamine). Cancer Left., 3: 109—113, 1977.

16. Singer, G. M., and Taylor, H. W. Carcinogenicity of N'-nitrosonornicotine inSprague-Dawley rats. J. Natl. Cancer Inst., 57: 1275—1276, 1976.

17. Tucker, J. M. Carcinogenic action of quinoxaline-1 4-dioxide in rats. J. NatI.Cancer Inst., 55: 137—145,1975.

18. United States Department of Health, Education, and Welfare, Smoking andHealth, a report of the Surgeon General, 284-i 09/661 9. Washington, D.C.:United States Government Printing Office, 1979.

19. Williams, G. M., and Laspia, M. F. The detection of various nitrosamines inthe hepatocyte primary culture/DNA repair test. Cancer Left., 6: 199—206,1979.

20. Wynder, E. L., and Stellman, S. D. Comparative epidemiology of tobaccorelated cancers. Cancer Res., 37: 4608—4622,1977.5 C. B. Chen, R. Young, and S. S. Hecht, manuscript in preparation.

300 CANCERRESEARCHVOL. 40

DISCUSSION

While the percentage of tumor-bearing animals and the survival times were comparable for the rats treated with NNN orNNK, the NNK-treated rats had tumors in the liver and lung aswell as in the nasal cavity; 92% of the NNK-treated animalshad tumors at more than one site. In contrast, a liver tumor wasfound in only one animal treated with NNN. These resultsindicate that, in the F344 rat, NNK is a more powerful carcinogen than NNN. Dose-response studies are needed to establishthese differences quantitatively.

The hepatocarcinogenicity of NNK, but not NNN, is of considerable interest in light of the fact that both are activated toDNA-damaging metabolites by hepatocytes in the hepatocyteprimary culture-DNA repair test (19). Metabolic studies on NNNhave indicated that a-hydroxylation (2'-hydroxylation and 5'-hydroxylation), which leads to 4-oxobutylcarbonium ions, islikely to be a major activation process (2). Parallel studies onNNK have shown that a-hydroxylation at the N-methyl groupgives the same oxobutylcarbonium ion that is formed by 2'-hydroxylation of NNN. However, a-hydroxylation of NNK at theN-methylene group results most likely in the release of amethylating species.5 It is possible that the methylating activityof NNK may be at least partially responsible for the observedliver and lung tumors.

The results obtained with NNN in this study contrast to aprevious experiment in which the same dose of NNN was givento male F344 rats in the drinking water and 12 of the 20 treatedrats developed esophageal tumors; in that experiment, nasalcavity tumors were found in only 3 animals (11). These resultsindicate that NNN may act locally in the esophagus since, whenNNN was administered by s.c. injection as in the present study,esophageal tumors were not observed. Further studies are inprogress to explore the possibility that NNN could inducetumors in the oral cavity, as has been observed for N-nitrosobis(2-hydroxypropyl)amine (15).

The occurrence of esthesioneuroblastomas in 50% of thetreated animals is an important finding of the present study.Esthesioneuroblastomas have been produced previously inlaboratory animals with a variety of agents (3, 8, 14, 17). Dueto its rarity and somewhat confusing morphology, the esthesioneuroblastoma is often overlooked and is often diagnosed asâ€ẫ€˜anaplastic carcinoma' ‘ or â€ẫ€˜adenocarcinoma. , , A careful

study, however, shows the olfactoneurogenic origin of thetumor.

While the role of NNN and NNK in the tobacco-relatedcancers observed in humans is not known, these nitrosaminesshould be considered as potential causative agents. The dailyexposure of a heavy smoker (2 packs of a typical Americannonfilter cigarette) to NNN and NNK can be estimated to be 10and 4 ;.@g,respectively (10). The reduction of such exposures

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@ , @— -@.Fig.1. Olfactoryneuroblastomaof theepitheliumof the ethmoturbinatesin theposteriorregionof thenasalcavity.Noteseptumcoveredwithnormalolfactory

mucosa. H & E, x 80.Fig. 2. HIgher magnification of the tumor in Fig. 1 showing spindle-shaped tumor cells palisading around fine blood vessels and transition from the normal

olfactoryepithellum.H&E,x 160.Fig. 3. Neuroflbril extending from tumor cell (inset) and argyrophil granules in the cytoplasm of some cells in the same tumor. Bodian, X 1280.Fig.4. InvasionIntocranialvaultbythetumorshownin Fig.1. Braintissuein upperleft is in contactwithtumorin whichsomecellsformpseudorosettes.H&E,

x 400.

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Fig. 5. Olfactory neuroblastoma with large cells forming pseudorosettes and true rosettes. H & E, x 400.Fig. 6. Another section of the tumor in Fig. 5. True rosettes with large central tubular structure. H & E, x 400.Fig. 7. Olfactory neuroblastoma. Note incomplete true rosette formation and perlvascular palisading of poorly differentiated tumor cells. H S E, x 160.Fig. 8. Higher magnification of the tumor in Fig. 7. Note olfactory cilia-like structures on the cells of an incomplete true rosette. H & E, x 500.

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1980;40:298-302. Cancer Res Stephen S. Hecht, Chi-hong B. Chen, Takaaki Ohmori, et al.

-nitrosamino)-1-(3-pyridyl)-1-butanoneN-Methyl-N-Nitrosonornicotine and 4-(′NTobacco-specific Nitrosamines,

Comparative Carcinogenicity in F344 Rats of the

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