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Cancer L&t. 20: 333-340,1983. (31) Hecht, S.S., Chen, C.B., McCoy, G.D., Hoffmann, D., and Domellof, L. Alpha-hydroxylation of N-nitrosopyrrolidine and N'-nitrosonomicotine by human liver microsomes. Cancer L&t. 8: 35-41, 1979. (3`2) Castonguay, A., Rivenson, A., `B-u&in, N., Reinhardt, J., Stathopoulos, S., Weiss, C.J., Reiss, B., and Hecht, S.S. Effects of chronic ethanol consumption on the metabolism and carcinogenicity of N'nitrosonomi- coke in F344 rats. Cancer Res. 44: 2285-2290, 1984. (33) He&t, S.S., and Lin, D. Comparative mutagenicity of 4&rbethoxy- nitrosamino)-4-(3-pyridyl)-butanol and 4-(carbethoxynitrosamino)-l- (3-pyridyl)-l-but, model compounds for alpha-hydroxylation of N'nitrosonornicotine. Carcinogenesis (in press). (34) Hecht, S.S., Castonguay, A., Rivenson, A., Mu, B., and Hoffman, D. Tobacco-specific nitrosamines: Carcinogenicity, metabolism, and possi- ble role in human cancer. J. Environ. Health Sci. 1: l-54, 1983. (35) Brittebo, E.B., and Tjiilve H. Autoradiographic observations in the dis- tribution and metabolism of N-[14C] nitrosonomicotine in mice. J. Cancer Res. Clin. Oncol. 98: 233-242, 1980. 77 (36) Waddell, W.J., and Marlowe, C. Localization of [14C] nitrosonomico tine in tissues of the mouse. Cancer Res. 40: 3518-3520, 1980. (37) Brittebo E.B., and Tjl%lve, H. Formation of tissue-bound N-nitrosonor- nicotine metabolites by target tissues of Sprague-Dawley and Fischer rats. Carcinogenesis 2: 959-963, 1981. (38J McCoy, G.D., Chen. C.B., and Hecht, S.S. Influence of modifiers of MFO activity on the in vitro metabolism of cyclic nitrossmines. In: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. GeIboin, J.R. Gillette, and P.J. O'Brien (eds.). Microsomes, Drug Oxidations, and Chemical Carcinogene sis, Vol. II. New York, Academic Press, 1980, pp. 11841192. (39) McCoy, G.D., Katayama, S., Young, R., Wyatt, M., and Hecht, S.S. In- fluence of chronic ethanol consumption on the metabolism and car- cinogenicity of tebaccerelated nitro samines. In: H. Bartsch, I.K. O'NeilI, M. Castegnaro, M. Okada, and L. Davis &is.). N-Nitroso Compounds: Occurrence and Biological Effects. IARC Sci. PubL 41: 309318,1982. (4OJ Waddell, W.J., and Marlowe, C. Inhibition by alcohols of the car&o genicity of radioactive nitrosonomicotine in sites of tumor formation. Science 221: 51-52, 1983. (41) Chung, F-L., Juchatz, A., Vitarius, J., and Hecht, S.S. Effects of dietary compounds on target tissue alpha-hydroxylation of N-nitrosopyrrohdine and N'-nitrosonornicotine. Cancer Res. 44: 2924-2928,1984. (42) Chung, F-L., Wang, M., and H&t, S.S. Effects of dietary indoles and isothiocyanates on N-nitrosodimethylamine and 4-(methylnitro- saminokld3-pyridyl)-l-butauone alpha-hydroxylation and DNA meth- ylation in rat liver. Carcinogenesis 6: 534543, 1985. (43) Swarm, P.F. Effect of ethanol on nitrosamine metabolism and distribu- tion. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence. IARC Sci. Publ. 5? 501-512, 1984. (44) Manson, D., Cox, P.J., and Jarman, M. Metabolism of N-nitrosomorpho line by the rat in viuo and by rat liver microsomes and its oxidation by the Fenton system. Chem. Biol. Interact. 20: 341-354,1978. (45) Hecht, S.S., and Young, R. Metabolic alpha-hydroxylation of N-nitro somorpholine and 3,3,5,5,-tetradueteroNnitrosomorphoI.ine in the F-344 rat. Cancer Res. 41: 5039-5043, 1981. (46) Hecht, S.S. N-Nitroso2hydroxymorpholine, a mutagenic metabolite of N-nitroscdiethanokunine. Carcinogenesis 5: 1745-1747,1984. (47) Lofberg, B., and Tjgve, H. Tissue specificity of N-nitrosmorpholine metabolism in SpragueDawley rats. Food Chem. Ibxicol. 23 647657, 1985. (48) Linjinsky, W., Taylor, H.W., and Keefer, L.K. Reduction of rat liver car- cinogenicity of 4-nitrosomorphohne by alphadeuterium substitution. J. Natl. Cancer Inst. 57: 1311-1313, 1976. (49) Chung, F-L., Palladino, G., and Hecht, S.S. Reactions of N-nitrosomor- pholine metabohtes with deoxyguanosine and DNA. Proc. Am. Assoc. Cancer Res. 26: 89, 1985. 78 (50) Chung, F-L., and Hecht, S.S. Formation of the cyclic l,NZ-glyoxal- deoxyguanosine adduct upon reaction of N-nitroso-2-hydroxymorphe line with deoxyguanosine. Carcinogenesis 6: 1671-1673,1985. (51) Bartsch, H., Camus, A., and Malaveille, C. Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat liver tissue fractions. Mutat. Res. 37: 149-162, 1976. EXPERIMENTAL STUDIES INVOLVING EXPOSING LABORATORY ANIMALS TO SMOKELESS TOBACCO OR ITS CONSTITUENTS This section reviews bioassays evaluating the carcinogenicity in ani- mals of smokeless tobacco and its constituents, particularly the tobaccospecific nitrosamines (TSNA) described in the section on the chemical constituents of smokeless tobacco. The bioassays involved multiple routes of administration of chewing tobacco, snuff, or extracts of these products and of several TSNA. Studies of chewing tobacco, snuff, and TSNA are summarized in tables 1 to 3 respectively, with comments on the individual investiga- tions provided below. Bioassays With Chewing Tobacco Oral Administration An alcohol extract of Indian chewing tobacco diluted 150 (group 1) or 1:25 (group 2) was gavagefed to male Swiss mice over 15 to 20 months. In another group of mice, a mixture of the tobacco extract with stan- dard laboratory diet was administered over 21 to 25 months (group 3). This treatment produced tumors in 8 of 15 mice at risk in group 1, in- cluding 5 mice with lung tumors and 2 with liver tumors; 4 of 10 mice at risk in group 2 developed lung and liver tumors. The feeding experiment (group 3) resulted in 8 of 10 mice with tumors, specifically 4 with tumors of the lung and 4 with liver tumors. Despite the high toxicity of the tobacco extracts and certain short-comings of the methodology, these assays indicate that the extract of chewing tobacco is carcinogenic in mice (1). Application to the Oral Mucosa and Cheek Pouch Three different extracts of an Indian chewing tobacco were applied daily for up to 18 months to the buccal mucosa of strain A and Swiss mice. No excess of tumors was observed (2). The oral mucosa of a group of weanling Wistar rats was painted twice weekly with a 2-percent alkaloid-free extract of an Indian chewing tobacco. No tumors were observed at the application site even though applications were con- tinued throughout the lifespan of the rats 13). 79 E TABLE l.-Bioassays for Carcinogenic Activity of Chewing `lbbaceo or Chewing `lbbacco Extracts* Duration of Fraction of Animals With `lbmors Route of Species, Exposure Application sex `l&t Material and Dose (Months) EXpOSed Controls Reference Oral- intubation Oral-feeding Skin- topical Oral- swabbing Oral- swabbing Oral-pouch implantation Oral-pouch Oral-pouch swabbing Oral-pouch swabbing Subcutaneous injection mice, M mice, M mice, M+F mice, M+F rats (NS)$ hamsters UW hamsters (NS) hamsters, M hamsters. F mice (NS) extract diluted I:25 4/l/2 diluted 1:50 15-20 0.2% extract in diet 21-25 DMSO extract (dose ?) 21-22 extracts applied daily, dose not given 2% alkaloid-free extract, dose not given- + time 2cm3 plug up to 18 Lifespan up to 30 DMSOextract three times weekly DMSOextract three times weekly, dose not given 2% tobacco extract in water, twice daily application 2% tobacco extract partially 18-24 5 6 IO-23 4110t lung adenocarcinoma 8115f and liver carcinoma 8llOt lung adenocarcinoma 0110 011 no excess tumors compared to controls o/10 0112 o/50 o/20 l/20 0110 0114 o/12 0112 3117 1117 squamouscell carcinoma or completely free of alkaloids, 25 solution once a month (site not specified) o/7 0111 0110 o Abbmviskm: DMSO. dimethyl sulfoxide t Animals at risk. $ INS) = not stated. TABLE 2.iBioassays for Carcinogenic Activity of Snuff or Snuff Extracts* Route of AoDlication Species, Sex `ht Material and Dose Duration of Fraction of Animals With `hmors EXpOEUre R&r- Amdications lMontbsl EXDO!%Xl Controls ence Oral-feedinz Hamsters. M S. 20% of diet Once dailv 24 0/1OOt Oil00 17 Lies-oaintinn Mice. M SE. dose not liven 3 times dailv 2 o/20 0120 18 Oral-swabbing Rats, M SE+H 0.5 ml daily 2 SE (approx. 30%) 0.5 ml daily up to 30 SE (approx. 30%) 0.5 ml daily up to 30 + (NNN+NNK) NNN+NNK 0.5 ml daily up to 30 Lip canal- Rats, F S 200 mg twice daily 9-22 instillation S 200 mg twice daily 18 H 200 mg 18 S+H twice daily 18 Lip canal- Rats. M S 50 mg daily up to 30 instillation s+se ES 50 mg daily 50 mn daily up to 30 UP to 30 0120 o/30 5/30 (3 papilloma in oral cavity. 2 lung adenoma) 13/30 (8 papilloma in oral cavity, 5 lung adenoma) 1142t (oral carcinoma) l/l0 (oral carcinoma) 017 217 (2 oral carcinoma) 0120 18 l/21 (lung adenoma) 20 l/21 (lung adenoma) 20 1121 (lung adenoma) 20 o/20 Oil0 0110 0110 21 22 22 22 3132 (papilloma and 1 Oil0 20 carcinoma in test canal, 1 oral papilloma) 1132 (oral papilloma) Oil0 20 2121 (oral papilloma) Oil0 20 Cheek pouch- Hamsters 10 ml paste once up to 30 0150 0150 instillation lNS)S ? 6 0125 2: H ? 6 0125 27 SfH ? 6 11125 (papilloma and carcinoma 27 of the oral cavity) Subcutaneous Rats, M+F SE 50 mg. 84 weekly 26 0182 0182 28 injection applications Rats (NS) TE 45 mg. 70 weekly 21 + 4 18175 1175 29 aoolications * Abbreviations: ES, extracted snuff; H. infected with herpes simplex virus; NNK. 4.(methylnitrosamino)-I-(-3.pyrldyl).I-butanone: NNN. N'-nitrosonornicotine. S. snuff. SE, snuff extract t NO tumors of the oral cavity, esophagus. nasopharynx and larynx; all other tumors nearly identical to those in control animals. a3 $ INS) = not stated. TABLE 3.-Csrcinogenicity of Tobacco-Specific Nitrosammes* Nitrw Species and samine Strains Route of Principal Application lhrget Organs Doee NNN NNK NAT NAB NNA A/J mouse F344 rat SpragueDawley rat Syrian golden hamster AIJ mouse ip. F344 rat S.C. Syrian golden hamster S.C. F344 rat F344 rat Syrian golden hamster A!J mouse i.p. S.C. p.0. p-0. S.C. S.C. p.0. S.C. i.p. lung nasal cavity esophagus esophagus nasal cavity nasal cavity trachea nasal cavity lung nasal cavity lung, liver trachea, lung, nasal cavity esophagus none none 0.12 mmollmouse 0.2-3.4 mmokat 1.0-3.6 mmokat 8.8 mmoUrat 0.9-2.1 mmol/harnster 0.12 mmol/mouse 0.1-2.8 mrnokat 0.9 mmolhamatm 0.005 mmol/hamst.er 0.2-2.8 mmoUrat 3-12 mmolkat 2 mmol/hamster 0.12 mmollmouse ' Hoffmann and Hecht (11) A group of 12 male Syrian golden hamsters received topical applica- tions on the buccaI mucosa of a dimethyl suboxide (DMSO) extract of an Indian chewing tobacco three times weekly for 21 weeks. None of the treated hamsters developed tumors in the oral mucosa; however, 8 of 12 treated animaIs had 1eukopIakia. These changes were not seen in the oral mucosa of the animals treated with DMSO alone (4). In another bioassay, 12 male hamsters received applications to the cheek pouch of a DMSC extract of Indian chewing tobacco three times weekly over their entire lifespan. Tumors were not observed in the treated group or the control group (5). When 1 mg of a paste made of a chewing tobacco extract was applied topicaky to the mucosa of the cheek pouches twice daily over a 6-month period, and animals were maintained without fur- ther treatment for another 6 months, the incidence of hyperplasia in the buccaI pouches was 17.6 percent, that of dysplasia was 29.4 percent, and that of squamous ceil papilloma or carcinoma was 17.6 percent in 17 hamsters. There were no tumors in the 20 control animaIs (6). Fifty hamsters received implantations of a 2 cm3 plug of chewing tobacco in their cheek pouches. The opening of the cheek pouch was ligated and the animals were observed for 18 months. After 13 months, 21 of 50 animals had survived. No tumors were recorded upon termina- tion of the assays (7). Although the studies cited above had some inherent weaknesses due to short application time or low dose, it appears, nevertheless, that both the oral mucosa of rats and the cheek pouches of Syrian golden 82 hamsters are relatively resistant to the carcinogenic activity of the extracts of chewing tobacco. Subcutaneous Application Seventeen C57 black mice were subcutaneously injected with 1 ml of a 2-percent solution of either partly or completely alkaloid-free extracts of an Indian chewing tobacco once a month for 1 to 24 months. One squamous carcinoma at an unspecified site developed in one mouse receiving the partly alkaloid-free extract (8). Skin Application A large number of studies have been published regarding the tumori- genicity on mouse skin of various extracts of chewing tobacco. Most of these bioassays failed to produce skin tumors. The negative results ap pear to be due primarily to the low dose applied or the short duration of the applications (9,101. The negative results indicate also that the con- centrations of TSNA and PAH in these extracts do not suffice to induce tumors upon topical application (11). However, the application of meth- anol or DMSC extracts of cigarette tobacco induced a low but signifi- cant number of benign tumors in the skin of CAFl and Swiss mice when these extracts were applied three times weekly for up to 24 months to the shaved backs of the mice (12,13). A number of studies have reported tumor-promoting activity of the extracts of chewing tobacco when these were applied to mouse epidermis previously treated with a tumor initiator @&?,1416). The bioassay data with chewing tobacco are sum- marized in table 1. Bioassays With Snuff Oral Administration For 2 years, 50 male BIO 15.16 and 50 male BIO 87.20 hamsters were each maintained on a standard diet containing 20 percent moist, fresh snuff. Controls consisted of 50 male BIO 15.16 hamsters and 50 male BIO 87.20 hamsters on a diet containing 20 percent cellulose (of caloric value similar to the snuff-containing diet). The spectrum of tumors ob served was nearly identical in both groups. Hamsters of both strains gavaged 60 times with 5 mg of the carcinogen 3-methylcholanthrene (MC) had a significantly increased incidence of both benign and malig- nant tumors of the forestomach and large intestine. Hamsters of the BIO 87.20 strain also had an increased incidence of stomach cancers while the BIO 15.16 strain developed tumors of the skin. lo assay the cocarcinogenic activity of snuff, 50 hamsters of each strain received the diet containing 20 percent snuff plus 50 times 0.5 mg of MC. Compared to the control group (diet containing 20 percent cellulose), the tumor yield was not increased in the two experimental groups indicating a lack 83 of carcinogenic activity as well as of cocarcinogenic activity of the snuff in this setting (17). Application to the Lip, Oral Mucosa, or Cheek Pouch The upper lips of 20 male BALB mice were painted 3 times a day for 5 days weekly over a 2-month period with a concentrated water extract of snuff (group 1). In another group of 20 male mice, the upper lips were in- oculated with herpes simplex virus type 1 (HSV-1) and were subse quently painted with a concentrated snuff extract for 2 months (group 2). A control group of 20 male mice received inoculation of the upper lips with HSV-1 and painting with water (group 3). Wo months' exposure to snuff extract (group 1) or HSV-1 inoculation (group 3) alone did not induce dysplasia in the epithelium of the labial mucosa, while HSV-1 in- oculation combined with painting of snuff extract produced epithelial dysplasia and other histomorphologic changes (18). In respect to this and other studies in which animals are infected with herpes virus in addition to treatment with snuff extracts, it should be noted that 20 to 40 percent of the U.S. population have periodic occur- rences of labial herpes (19). Male F344 rats were treated for up to 30 months by swabbing the oral cavity with either a concentrated water extract of snuff (group 1; 13.2 vg NNN and 2.8 ug NNK per milliliter snuff extract solution), snuff ex- tract enriched with the tobaccc~specific nitrosamines NNN and NNK (group 2; 148 ug NNN and 30 ug NNK per milliliter snuff extract solu- tion), NNN and NNK alone in concentrations corresponding to those applied in group 2 (group 3; 135 pg NNN and 27.6 ug NNK per milliliter test solution), or with water alone (group 4). Groups 1,2, and 3 consisted of 30 male rats each and group 4 (control) of 21 rats. The incidence of tumors in groups 1 and 2 was not significantly increased over that in the control group. In the group of 30 rats treated with NNN and NNK alone, 8 animals had oral tumors (6 papillomas in the cheek, 4 papillomas in the hard palate, and 1 papihoma of the tongue), and 4 animals had lung carcinoma. This study indicates that snuff contains czucinogenic N-nitrosamines; however, when they are being tested in an admixture with other components in the water extract of snuff, their carcinogenic activity may be suppressed (20). A group of 21 male and 21 female SpragueDawley rats were treated with snuff placed in a surgically created canal in the lower lip. Approxi- mately 0.2 g of a standard Swedish snuff (pH 8.3) was given twice daily 5 days per week for 9 to 22 months. The mean retention time of the snuff in the canal was 6 hours, and the estimated daily dose was 1 g of snuff/kg b.w. Using the same methodology, another group of 5 male and 5 female rats was treated with alkaline snuff in the surgically created canal (pH 9.3). One of the 42 rats treated with regular snuff developed a squamous carcinoma in the oral cavity after 8.5 months. The exposure to the regu- lar snuff resulted in mild to moderate hyperplasia of the epithelium, 84 hyperorthokeratosis, and acanthosis. Among rats exposed to snuff for 18 to 22 months, 16 of 42 showed vacuolated cells penetrating deeper into the epithelium with hyperplastic and atropic lesions. Rats exposed to alkaline snuff differed little from those in the group treated with regular snuff. Outside the area of treatment, squamous cell hyperplasia of the forestomach was found in rats exposed to snuff for 18 to 22 months (21). In another bioassay using the same methodology as described by Hirsch and Johansson (21), the surgically created canal in the lower lip of F344 rats was filled five times each week over 28 months with either U.S. snuff (average 0.2 g per application; n=30), snuff enriched with its own water extract (n=30), or the extracted residue of snuff (n=21). Ten rats with the surgically created lip canal, and otherwise untreated served as controls. The incidence of nonspontaneous tumors in each group was the following rats treated with snuff had one squamous carcinoma of the oral cavity, one squamous cell papilloma of the hard palate, and one meningioma; treatment with enriched snuff led to one squamous cell papilloma of the floor of the mouth and one nasal olfactory tumor; treat- ment with extracted snuff induced one squamous cell papilloma of the hard palate. There were no tumors in the control group (20). Four groups of female SpragueDawley rats with surgically created canals in the lower lip, received the following treatments beginning at 3 months of age: group 1 was infected with herpes simplex virus type 1 (HSV-1) by scarification and topical application followed 10 days later by administration of snuff into the canal morning and night on 5 days per week; group 2 was infected with virus and received no other treatment; group 3 was sham-infected with sterile saline followed by snuff treat- ment; and group 4, not given virus or snuff, served as controls, The HSV-1 infection was repeated once after a l-month interval, and snuff treatment was continued for 18 months after which time all animals were killed. Three animals in each of groups 1 and 2 died from encephalitis shortly after the second infection with HSV-1. Squamouscell carcinomas of the oral cavity developed in two of seven rats, and a retroperitoneal sarcoma was seen in one of seven rats exposed to HSV-1 plus snuff. In the group exposed to snuff alone, 1 of 10 animals developed a squamous carcinoma of the anus and 1 of 10 a retroperitoneal sarcoma (22). In several studies, various forms of snuff were installed in the cheek pouches of Syrian golden hamsters for up to 20 months. The application of snuff did not lead to the induction of tumors in the cheek pouches nor at any other site of the oral cavity in any of these studies even though malignant tumors were induced in the oral cavity with high doses of 7, 12dimethylbenz(a) anthracene and 3-methylcholanthrene (z23-26). In an assay for the joint action of HSV and snuff, the buccal pouches of 125 Syrian hamsters were inoculated with HSV-1, HSV-2, or culture medium. The control and HSV inoculations were done once a month for 6 consecutive months. Then 25 hamsters with HSV-inoculated pouches received installations of commercial snuff twice daily into both the right 85 and left pouches. One month after the last HSV inoculation and 6 months after continuous snuff application, the assay was terminated. The buccal pouches were removed for histopathologic examination. Neither the application of snuff to the cheek pouches nor HSV infection alone induced neoplastic changes in hamster buccal pouches. However, HSV infection in combination with snuff resulted in epitheliaI dysplasia and in squamous carcinoma of the buccal pouches in 11 out of 25 ham- sters (27). This investigation provides the strongest evidence to date that snuff may increase cancer risk in animals; however, full evaluation is pre eluded since the findings have been published only in abstract form. Subcutaueous Administration A Swedish snuff was extracted with 6Opercent alcohol and resulted in 18percent dry extract, which was injected subcutaneously into rats with 7Opercent ethanol and tri-n-caprylin (1:ll as vehicle. The rats received a total dose of 4.2 g of extract with 84 weekly doses of 50 mg of extract. No tumors were observed at the area of injection (28). This result is quite different from an earlier one by the same investigators in which an alcohol extract from cigarette tobacco (20percent yield) was injected into 75 rats with 70percent alcohol and glycerol as solvent (1:3). Per week, 45 mg extracts were injected until the total dose amounted to 3.2 g/rat. After 25 months, 18 of 75 rats had developed malignant tumors, primarily sarcomas at the injection site (29). The bioassay data with snuff are summarized in table 2. Bioassays With Constituents of Smokeless Tobacco At least three types of carcinogens occur in smokeless tobacco: poly- nuclear aromatic hydrocarbons (PAH), polonium-210 (21oPo), and N-nitrosamines. One of the PAH identified in smokeless tobacco, benzo(a)pyrene (up to 72 ppb), has long been recognized as an animal carcinogen (18,24,30). Levels of ZloPo in processed tobacco amount to 0.1-1.0 pCi per gram and to 0.18-1.22 pCi/g in commercial U.S. snuff products. Ionizing radiation can cause multiple types of cancer in ani- mals and humans raising the possibility that the alpha-radiation of 2loPo may contribute to the carcinogenic potential of smokeless tobacco and especially snuff f31,31). Three groups of N-nitrosamines have been identified in smokeless tobacco. All of the 4 volatile nitrosamines thus far identified are carcino- genic in animals (3). These are nitrosodimethylamine (0 to 215 ppb), nitrosopyrrolidine (0 to 291 ppb), nitrosopiperidine (0 to 107 ppb), and ni- trosomorpholine (0 to 690 ppb). Seven nonvolatile nitrosamines have also been identified in smokeless tobacco. Of these, only nitrosodiethano lamine (30 to 6,800 ppb) is a known carcinogen in mice, rats, and hamsters (33) Swabbing of the oral cavity of 20 male and 20 female hamsters with solutions of these agents three times weekly for 45 weeks 86 (20 mg per application) induced tumors of the nasal cavity in 17 animals, tumors of the trachea in 6, and a tumor of the larynx in 1 of the hamsters (34). The most abundant carcinogens in smokeless tobacco yet identified are the tobaccospecific nitrosamines (TSNA). These are formed during the processing of tobacco from its alkaloids. So far, seven TSNA have been identified in smokeless tobacco. Of these, N `-nitrosonornicotine (NNN; 470-135,000 ppb) and 4-(methylnitrosamino)-1-(3-pyridyl)-l- butanone (NNK; 30-13,600 ppb) are powerful carcinogens in mice, rats, and hamsters (table 1; 1,9). Table 3 summarizes results from bioassays administering TSNA to test animals. A variety of tumors were produced, particularly in the esophagus, nasal cavity, and lung. In a recently com- pleted investigation, daily swabbing for up to 30 months of the oral cavity of F344 rats with a saline solution containing 135 ppm NNN and 28 ppm NNK led to the development of benign oral tumors in 8 and lung carcinoma in 4 of 30 rats. Neither oral tumors nor tumors of the lung were observed in the negative control group (20). This study sug- gests that NNN and NNK may be tumor&&c at the site of exposure as well as systemically. Full evaluations of these results are precluded, however, since the original manuscript is now under journal review and not published. It is noteworthy that some of the bioassays indicated that relatively low doses of the TSNA could induce tumors. In hamsters, a total dose of only 0.2 mmol/kg of NNK induced a significant incidence of tumors (3s), whereas in F344 rats, 60 subcutaneous injections of a total dose of 20 mg (0.33 mmokkg) of NNK induced tumors of the liver in 10, tumors of the lung in 13, and tumors of the nasal cavity in 6 of 30 rats. Subcu- taneous applications to 27 rats of the same molar dose (0.33 mmokkg) of nitrosodimethylamine resulted in 6 animals with tumors of the liver and 1 rat with a tumor of the nasal cavity (36). For NNN, high tumor inci- dences were produced in F344 rats by a total dose of 1.0 mmovkg (37). Based on daily use for 30 years of 10 g of snuff containing 3.1 ppm of NNK, the estimated NNK exposure of a snuff dipper would be approxi- mately 0.02 mmollkg. Exposure to NNN from the same brand would be 0.4 mmol/kg (figure 3, chapter 2). Hence, the bioassays indicate that exposures in the dose range actually experienced by long-term snuff dippers induce tumors in animals. This is a distinctive and potentially important finding, since for most chemical carcinogens their carcino genicity was detected following exposure at doses much higher than usually received by humans. Of the other five TSNA, besides NNN and NNK, N `nitrosoanabasine (NAB; lo-6,700 ppb) and 4(methylnitrosamino)-1-(&pyridyl)-1-butanol (NNAL; 140-300 ppb) were moderately active carcinogens, and N-nitro soanatabine (NAT; 300-338,000 ppb) was inactive when tested at the low dose level of 9 n-mol./kg (9,38). 87 Recently, two additional TSNA have been identified in snuff: 4 (methyhritrosamino)-4-(3.pyridyl)-1-butanone (1,300-1,800 ppb) and 4-(methyhritrosamino)-(3-pyridyl)butenel (10 ppb; 6). These two nitro w-nines have not yet been tested for carcinogenicity. Mutagenicity Assays and Other Short-Term Tests Chewing Tobacco Nicotinu rusticu is a tobacco variety that is widely cultivated and used throughout India. Its ethanol extracts induced mutations in Sahwn~lla typhimurium TA98 and in V79 cells of Chinese hamsters. The addition of S9 liver homogenate from Aroclor-pretreated rats enhanced the mutagenic effect. No mutations were induced in TAlOO, TA1535, or TAX38 in the presence of the S9 homogenate. This ethanol extract of tobacco also induced micronuclei in bone marrow cells of Swiss mice (1,39,40). An ethyl acetate extract of Indian chewing tobacco induced sister chromatid exchange (SCE) in human lymphocytes and in a human lymphoblastoid cell line. In the latter system, S9 rat liver homogenate enhanced the effect. When the tobacco extract was tested in the absence of the S9 homogenate it did not induce ouabain-resistance in Chinese hamster V79 cells. The same extract, another ethyl acetate extract, and an ethanol extract of tobacco induced cell transformation in Syrian hamster embryo cells (41&f). The incidence of micronucleated oral mucosa cells in 27 Indians using khani chewing tobacco was 2.2 percent (0.8-4.9 percent). The incidence of micronuclei in exfoliated cells of nonchewers of similar ethnic back- grounds and dietary habits was 0.47 percent (0.0-0.9 percent) (43). Snuff The residue of organic solvent extracts from a U.S. commercial snuff was dissolved in DMSO and tested for the induction of SCE's in human peripheral lymphocytes. The organic snuff extract induced significant SCE's with a 0.05 percent concentration in lymphocytes of one of three donors, with a 0.15 percent concentration in lymphocytes in two of three donors, and with a 0.5 percent concentration in lymphocytes of all three donors (44). Tobacco-Specific N-Nitrosammes (`I'SNA) Of the seven TSNA so far identified in smokeless tobacco, only NNN and NNK were also tested for genotoxicity in short-term tests. In the presence of a liver microsomal preparation from Aroclor-induced rats, NNN and NNK caused dose-dependent mutations in SuZmoneUu typhimurium TAlOO and TA1535. Increased mutation frequencies were observed in the case of NNN at 2.5 umol and at 5.65 timoUplate and in the case of NNK at 1.4 wokplate (45-47). 88 NNN and NNK at 10-3 and 10-2 molar concentration each induced unscheduled DNA synthesis in freshly isolated hepatocytes from adult rats (48). Summary Chewing tobacco and extracts from various chewing tobaccos have been tested by oral administration in mice, topical application to the oral mucosa of mice, rats, and hamsters, and by subcutaneous adrnin- istration and shin application to mice. The investigations failed to demonstrate significantly increased tumor production. Short applica- tion times and low-dose exposures, however, limit the evaluation of the carcinogenicity of chewing tobacco or its extracts. Bioassays of snuff have likewise generally shown no excess cancer, although some experi- ments suggest that it may cause oral tumors in rats and hamsters that are infected with herpes simplex virus. Among the chemical com- ponents of snuff, the tobaccospecific nitrosamines NNN and NNK are powerful carcinogens. The doses of NNN and NNK that produce tumors in experimental animals are close to the doses estimated from lifetime exposure among human snuff dippers. References (1) Shah, A.S., Sarode, A.V., and Bhide, S.V. Experimental studies on mutagenic and carcinogenic effects of tobacco chewing. J. Cancer Res. Clin. OncoL 109: 203-207,1985. (2) Mody, J.K., and Ranadive, J.K. Biological study of tobacco in relation to oral cancer. Ind. J. Med. Sci. 13: 1023-1037, 1959. (3) Gothoskar, S.V., Sant, S.M., and Ranadive, K.J. Effect of tobacco and Iime on oral mucosa of rats fed on vitamin B deficient diet. Int. J. cancer 12 424-429,1975. (4) Suri, K., Goldman, H.M., and Wells, H. Carcinogenic effect of a dimethylsulphoxide extract of betel nut on the mucosa of the hamster buccaI pouch. Nature 2%? 383-384, 1971. (5) Ranadive, K.J., and Gothoskar, S.V. Betel quid chewing and oral cancer: Experimental studies. In: H.E. Nieburgs ted.). Prevention and Detection of Cancer, Part I, VoL 2. New York, Marcel Dekker, 1976, pp. 1745-1766. (6) Rao, A.R. Modifying influences of betel quid ingredients on B(a)P- induced carcinogenesis in the buccaI pouch of hamster. Int. J. Cancer 3.5: 581-586, 1984. (71 Peacock, E.E., Jr., and Brawley, B.W. An evaluation of snuff and tobacco in the production of mouth cancer. Plast. Reconstr. Surg. 23: 628-635,1959. (8) Ftanadive, K.J., Gothoskar, S.V., and Khanolka, V.R. Experimental studies on the etiology of cancer types specific to India. A. Oral cancer, B. Kangri cancer. Acta Unio. IntematL Contra Cancrum 19: 634639, 1963. 89 (9) International Agency for Research on Cancer. Tobacco habits other than smoking: Betelquid and areca-nut chewing and some related nitrosamines. IARC Monogr. EvaI. Carcinog. Risk Chem. Hum. 37: 291, 1985. (10) Wynder, E.L., and Hoffmann. D. Tobacco and Tobacco Smoke. Studies in Experimental Carcinogenesis. New York, Academic Press, 1967, pp. 198-202. (11) Hoffmann, D., Hecht, S.S., Omaf, R.M., and Wynder, E.L. Nitrosonor- nicotine: Presence in tobacco, formation and carcinogenicity. IARC Sci. Publ. 14: 307-320, 1976. (12) Wynder, E.L., and Hoffmann, D. A study of tobacco carcinogenesis, X. `Rimor promoting activity. Cancer 24: 289-301, 1969. (13) Wynder, E.L., and Wright, G.A. A study of tobacco carcinogenesis, I. The primary fractions. Cancer 10: 255-271, 1957. (14) Bock, F.G., Moore, G.E., and Crouch, S.K. Tumor promoting activity of extracts of unburned tobacco. Science 145: 831-833.1964. (15) Bock, F.G., Shamberger, R.J., and Meyer, H.K. `Armor promoting agents in unburned cigarette tobacco. Nature 208: 584-585, 1965. (16) Van Duuren. B.L., Sivak, A., SegaI, A., Orris, L., and Langseth, L. The tumor-promoting agents of tobacco leaf and tobacco smoke conden- sate. J. Natl. Cancer Inst. 37: 519-526, 1966. (17) Homburger, F., Hsueh, S.S., Russfield, A.B., Laird, C.W., and Van Dongen, C.G. Absence of carcinogenic effects of chronic feeding of snuff in inbred Syrian hamsters. `Ibxicol. Pharmacol. 35: 515-521, 1976. (18) Park, N.H., Herbosa, E.G., Ninkian, K., and Shklar, G. Combined effect of herpes simplex virus and tobacco on the histopathologic changes in lips of mice. Oral Surg. 59: 154-158.1985. (19) Barker, R., Burke, J., and Zieve, P. (eds.). Principles of Ambulatory Medicine. Baltimore, Wilhams and Wilkins, 1983, p. 1074. (20) Hecht, S.S., Rivenson, A., Braley, J., DiBeIlo, J., Adams, J.D., and Hoffmann, D. Induction of oral cavity tumors in F344 rats by tobacco specific nitrosamines and snuff. Submitted (1986). (21) Hirsch, J.M., and Johansson, S.L. Effect of long-term application of snuff on the oral mucosa: An experimental study in the rat. J. Oral Pathol. 12: 187-198, 1983. (22) Hirsch, J.M.. Johansson, S.L., and Vahlne, A. Effect of snuff and herpes simplex virus-l on rat oral mucosa: Possible associations with the development of squamous cell carcinoma. J. Oral Pathol. 13: 52-62, 1984. (23) Dunham, L.J., and Herrold, K.M. Failure to produce tumors in the cheek pouch by exposure to ingredients of betel quid; histopathological changes in the pouch and other organs by exposure to known carcino gens. J. Natl. Cancer Inst. 29: 1047-1067, 1962. (24) Dunham L.J., Muir, C.S., and Hamner, J.E., III. Epithehal atypia in hamster cheek pouches treated repeatedly with calcium hydroxide. Br. J. Cancer 20: 588-593, 1966. 90 (2.5) Dunham, L. J ., Snell, K.C., and Stewart, H.L. Argyrophihc carcinoids in two Syrian hamsters (Mesocricetis uumtus). J. Natl. Cancer Inst. 54: 507-513, 1975. (2s Homburger, F. Mechanical irritation, polycyclic hydrocarbons, and snuff. Effects on facial skin, cheek pouch, and oral mucosa in Syrian hamsters. Arch. PathoL 91: 411-417, 1971. (27) Park, N.H., Herbosa, E.G., and Sapp, J.P. Oral cancer induced in hamsters with herpes simplex infection combined with simulated snuff- dipping (Abstract 10). International Herpes Virus Workshop, Ann Ar- bor, August 11-16, 1985, p. 297. (28) Schm%hl. D. Priifung von Kautabakextract auf cancerogene Wirkung bei Batten. Anneimittel-Forsch. 15: 704-705, 1965. (29) Dmckrey, H., S&m&I, D., Beuthner, H., and Muth, F. Vergleichende Priifung von Tabakrauch-Kondensatn, Benzopyren und Tabakextract auf carcinogene Wirkung bei Batten. Naturwissenschaften 47: 605-606, 1960. (30) Campbell, J.M., and Lindsey, A.J. PolycycIic aromatic hydrocarbons in snuff. Chem. Ind. London 951, July 6,1957. (31) Harley, N.H., Cohen, B.S., and no, T.C. Polonium-210. A questionable risk factor in smoking-related carcinogenesis. Banbury Report 3: 93-104, 1980. (32) Hoffmann, D., Harley, N.H., Fisenne, I., Adams, J.D., and Brunnemann, K.D. Carcinogenic agents in snuff. J. Natl. Cancer Inst. (in press). (33) International Agency for Cancer Research. Monograph on the evahra- tion of the carcinogenic risk of chemicals to humans, VoL 17. Some N-nitroso compounds. Lyon, France, International Agency for Cancer Fitsearch. 1978, p. 365. (34) Hoffmarm, D., Rivenson, A., Adams, J.D.. Juchatz, A., Vinchkoski. N.. and Hecht, S.S. Effects of route of administration and dose on the car- cinogenicity of ZV-nitrosodiethanolamine in the Syrian golden hamster. Cancer Res. 4.X: 2521-2524, 1983. (35) Hecht, S.S., Adams, J.D., Numoto, S., and Hoffmann, D. Induction of respiratory tract tumors in Syrian golden hamsters by a single dose of 4-(methyInitrosamino)-l-(3-pyridylbl-butanone (NNK) and the effect of smoke inhalation. Carcinogenesis 4: 1287-1290,1983. (36) Hecht, S.S.. `B-u&in, N., Castonguay, A., and Rivenson. A. Compara- tive tumorigenicity and DNA methylation in F344 rata by 4-(methyi- nitrosamino)-l-(3-pyridyl)-1-butanone and N-nitrosodimethylamine. Cancer R.-es. 46: 498-502.1986. (37) Hoffmann, D., Rivenson, A., Amins, S., and Hecht, S.S. Dose-response study of the carcinogenicity of tobacco-specific N-nitrosamines in F344 rats. J. Cancer Res. CIin. Oncol. 108: 81-86, 1984. (38) Hoffmann, D., and Hecht, S.S. Perspectives in cancer research. Nicotine-derived N-nitrosamines and tobaccorelated cancer: Current status and future directions. Cancer Res. 4.5: 935-944,1985. 91 fW Bhide, S.V., Shah, A.S., Nair, J., and Nagarajrow, D. Epidemiological and experimental studies on tobacco related oral cancer in India. IARC Sci. Publ. 57: 851-857, 1984. f4OJ Sbimame, L.P., Monon, M.M., and Bhide, S.V. Mutagenicity of betel quid and its ingredients using " test systems. Carcinogens sis 5: 501-503, 1984. (41) Umezawa, K., Fujie, S., Sawamur, M., Matsushima, T., Koath, Y, Tanaka M., and Takayama, S. Morphological transformation, sister cbromatid exchange and mutagenicity assay of betel constituents. `Ibx- icol. Lett. 8: 17-22, 1981. (421 Umezawa, K., `Ibkayama, S., Fujie, S., Matsushima, T., and Sugimura, T. In vitro transformation of hamster embryo cells by betel tobacco ex- tracts. !IbxicoL Lett. 2: 243-246,1978. (4.3) Stich, H.F., Curtis, J.R., and Parida, B.B. Application of the micro- nucleus test to exfoliated cells of high cancer risk groups: Tobacco chewers. Int. J. Cancer 30: 553-559, 1982. (44) Tucker, J.D., and Ong, T. Induction of sister chromatic exchanges by coal dust and tobacco snuff extracts in human peripherai lymphocytes. Environ. Mutagen. 7: 313-324. 1985. 145) Andrews, A.W., Thibault, L.H., and Lijinsky, W. The relationship be- tween mutagenicity and carcinogenicity of some nitrosamines. Mutat. Res. 51: 319-326. 1978. (46) Bartsch, H., MaIaveiIIe, C., Camus, A.M., Mart.&PIanche, G., Brun, G., Hautefeuille, A., Sabadie, N., Berlin, A., Kuroki, T., Drevon, C., PiccoIi, C., and Montesano, R. Validation and comparative studies on 180 chemicals with S. typhimurium and V79 Chinese hamster cells in the presence of metabolizing systems. Mutat. Res. 76: l-50, 1980. (47) Hecht, S.S., Lin, D., and Castonguay, A. Effects of alphadeuterium substitution on the mutagenicity of 4-(metbylnitmsamino)-l-(3-pyridyl)- 1-butanone. Carcinogenesis 4: 305-310, 1983. (48) Williams, G.M., and Laspia, M.F. The detection of various nitre samines in the hepatocyte primary culture/DNA repair test. Cancer L&t. 6: 199206, 1979. CONCLUSIONS 1. The scientific evidence is strong that the use of smokeless toham can cause cancer in humans. The association between smokeless tobacco use and cancer is strongest for cancers of the oral cavity. 2. Oral cancer has heen shown to occur several times more fre quently among snuff dippers than among nontobacco users, and the excess risk of cancers of the cheek and gum may reach nearly fiftyfold among long-term snuff users. 3. Some investigations suggest that the use of chewing tobacco also may increase the risk of oral cancer. 92 4. Evidence for an association between smokeless tobacco use and cancers outside of the oral cavity in humans is sparse. Some investigations suggest that smokeless tobacco users may face in- creased risks of tumors of the upper aerodigestive tract, but results are currently inconclusive. 5. Experimental investigations have revealed potent carcinogens in snuff and chewing tobacco. These include nitrosamines, poly- cyclic aromatic hydrocarbons, and radiation-emitting polonium. The tobacco-specific nitrosamines N-nitrosonornicotine and 4-(methylnitro samino)-1-(3-pyridyl)-1-butanone have been de t.ect.ed in smokeless tobacco at levels 100 times higher than the regulated levels of other nitrosamines found in bacon, beer, and other foods. Animals exposed to these tobaccospecific nitro samines, at levels approximating those thought to be accumu- lated during a human lifetime by daily smokeless tobacco users, have developed an excess of a variety of tumors. The nitro- samines can be metabolized by target tissues to compounds that can modify cellular genetic material. 6. Bioassays exposing animals to smokeless tobacco, however, have generally shown little or no increased tumor production, although some bioassays suggest that snuff may cause oral tumors when tested in animals that are infected with herpes simplex virus. RESEARCHNEEDS It has been established beyond reasonable doubt that smokeless tobacco use can in- the risk of cancer. The experimental and epi- demiologic evidence is strongest for the association between oral cancer and the chronic use of snuff. Additional studies are needed to determine whether the patterns of risk differ according to the form of smokeless tobacco, including reseamh evaluating cancer risks that are associati with chewing tobacco and dry versus moist snuff, and to quantify fur- ther the levels of risk in relation to differing levels of smokeless tobacco expose. The influence of smoking, alcohol, and other factors (including viral expmues) on the smokeless tobaccoassociated risk of oral cancer also should be explored further with an emphasis on detecting possible inter- actions between these factors and smokeless tobacco. Inhaled snuff may increase the risk of nasal carcinoma. The feasibil- ity of initiating studies in areas where snuff sniffing is common should be ascertained, and studies should be launched to confirm and quanti- tate this possible relationship. There have been few studies of smokeless tobacco and esophageal, laryngeal, and gastric cancers. These investigations have provided equivocal results, but in the aggregate, their findings raise the possibil- 93 ity of some increase in risk among smokeless tobacco users. Additional case-control studies of these neoplasms should be encouraged. These studies should be large enough to assess the risks that are associated with smokeless tobacco use while controlling for the potential con- founding effects of smoking, alcohol, and other risk factors. Isolated reports have associated smokeless tobacco with cancers of the cervix, pancreas, and other anatomic sites. Investigators with exist- ing data from case-control studies of these neoplasms should be encour- aged to perform analyses to determine whether associations with smokeless tobacco exist. Similarly, existing data from cohort studies with information on smokeless tobacco use should be analyzed. Reports from two relatively large cohort studies have been published only as ab- stracts. These should be expanded with detailed descriptions of both the methods used and the findings for various cancers and should be up dated to include followup into the 1980's. Recommendations for addi- tional studies of the role, if any, of smokeless tobacco in the etiology of cancers outside of the upper aerodigestive tract should await the results of these analyses. On the basis of current knowledge, it can be assumed that chewing tobacco and snuff contain several unknown nitroso compounds that may be contributors to the carcinogenic potential of these products. In- depth analytical studies are needed for the identification of these unknown compounds. Furthermore, mechanisms of their in vitro and endogenous formation should be studied together with those of the ni- troso compounds that are already known to occur in smokeless tobac- cos. For the validation of the uptake of the major carcinogens by to bacco chewers and snuff dippers, markers should be measured in the target tissues and in physiological fluids. Major emphasis should be placed on the identification and assays of DNA-adducts with tobacco specific compounds in tissues of the oral cavity. Finally, trends over time in agespecific oral cancer incidence and mortality rates should be monitored to determine whether the increas- ing use of smokeless tobacco by Americans is influencing national or regional cancer patterns. Changes in the prevalence of use and in the characteristics of smokeless tobacco products should also be docu- mented. Such monitoring will provide a base upon which future investi- gations of associations between smokeless tobacco and cancer can be built. 94 Chapter 3. NONCANCEROUSAND PRECANCEROUS ORAL HEALTH EFFECTS ASSOCIATED WiTH SMOKELESS TOBACCO USE CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...99 The Effects of Smokeless Tobacco Use on Oral LeukopIakia/MucosaI Pathology and the %ansformation of Oral Soft Tissues ,107 Oral LeukopIakia/Mucosal Pathology . ,107 %ansformationofOraISoftTissues.. . . ,113 The Effects of Smokeless Ibbacco Use on the Gingiva, Periodontal Tissue, and salivary Glands ........ ,123 Background and Definitions ...................... ,123 Gingival and Periodontal Tissue ................... .123 SalivaryGlands.. ............................ ..12 6 The Effects of Smokeless `Ibbacco Use on lb&h .......... ,128 Background and Definitions ...................... ,128 DentaICaries ................................ ..12 9 OtherHardTissueEffects ....................... ,130 C0ncIusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...130 ResearchNeeds....................................131 References.............. . . . . . . . . . . . . . . . . . . . . . . . . ..132 97 This chapter addresses the health effects of smokeless tobacco use on the oral tissues through a systematic review of the relevant scientific literature of animal and human studies. The major areas addressed are the effects of smokeless tobacco use on the oral soft tissues, the periodontium, and the teeth. This chapter also reviews information regarding the potential of oral tissue altered by smokeless tobacco use to transform to dysplasia and malignancy. Within each area, except for the section on the transformation of oral soft tissues, those tissues or conditions that are suspected to be most af- fected by smokeless tobacco use, or that hold the greatest potential for health effects, are considered initially. Where contradictory evidence exists, these date are also presented. Studies that were judged to meet stringent selection criteria* are presented first, followed by data from less rigorous study designs and case reports. Within the section on the transformation of oral soft tissues, the pre sent&ion of the evidence is grouped according to clinical reports, cohort studies, and case-control studies. This was done so as to be consistent with the format used in the chapter on Csrcinogenesis Associated With Smokeless `Ibbacco Use (chapter 2). In some cases, studies referenced in this chapter are the same as those used in chapter 2. The reader should review both chapters to obtain all pertinent information contained in these studies. Only studies from the United States and Scandinavia are included for the sections on oral leukoplakia/mucosal pathology, gingival and perio dontal tissues, and salivary glands. This assures that studies dealing with similar types of smokeless tobacco are used for comparison pur- poses. However, the section on the transformation of oral soft tissues includes a fuller range of studies that have reviewed the histopathologic changes associated with smokeless tobacc&nduced lesions. Studies in- vestigating the histopathologic transformation of nonsmokeless tobaccuinduced lesions have not been included. A summary of selected studies that addresses study sample, methods, and observations is provided in table 1 as a ready alphabetical reference to the text. In addition, a summary of selected case reports is provided in table 2. Emphasis has been placed on the issues of preva- lence of oral tissue changes, types of changes, sitespecificity of changes, and the effects of doseresponse. o See Introduction. Overview. and Conclusions for discussion of criteria for causality 99 TABLE I.-Selected Study Summaries for the Noncancerous Oral Health Effects From the Use of Smokeless Tobacco Study Sample Methods Observations cmnmenta AxklL 1976 o 20,333 Swedes: o ????????????*?o 51% females, design. 49% males. o Data collected o Ages 15 years on tobacco and older. habits, medications taken. oral hygiene status, and prosthetic status. o ?o?*???o examinations utilized diagnosis based on specific clinical criteria. . Photographic documentation of all lesions diagnosed as leukoplakia or lichen planus. o Tissue speck- n-lens taken of selected ca3es. o Statistical analysis con- ducted: t-tests, chi square teats. and. if appropri- ate, Fisher's exact test. o Of 1,444 snuff users, 116 (8%) had "snuff dip per's lesion" (oral leuko plakia). o The prevalence of oral leukoplakia was 3.6% among the total population examimd o It is not clear how many of the snuff users were also tobacco smokers. o snuff dipper's lesion implies mumsal tissue changeaatthe site of snuff placement. Greer and Paulson. 1983 o 1.119 teenagers in grades 9-12. o 117 110.5%) smokeless tobacco users: 113 males. 4 females. o Denver. Colorado. o Cross-sectional design. o Questionnaire administered to determine years of use, frequency of use. brand of tobacco used. site of applica- tion, use of other confounding agents, and dental care history. LA?ukopl&i% Mucosal Pathology o A suggested association between level and duration of smokeless tobacco use and mucosal lesions (42.7% of smoke less tobacco users had oral mucosal lesions). o An analysis of the influence of cofactors was not conducted. o No statistical analyses reported. . Examiners blind to responses on questionnaire. o No comparisons reported between users of smokeless tobacco and nonusers. 100