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UBC Theses and Dissertations

Modulation of the risk to oral cancer Hornby, Antony Paul


Millions of people use smokeless tobacco, such as snuff, chewing tobacco, nass or nasswar (a mixture of tobacco, slaked lime, ash and oil), Khaini tobacco (tobacco and slaked lime), or as part of a simple betel quid (areca nut, tobacco, lime, betel leaf) or a complex "pan" (betel quid with catechu, seeds, perfumes and silver foils). These habits which are involved in the etiology of oral cancer, have been of concern since snuff dipping is becoming popular among teenagers of Canada and the United States. To prevent the development of oral cancer among snuff dippers, it appeared necessary to trace the etiological factors, to develop markers which would identify individuals at elevated risk for oral cancer, and to test the usefulness of these "intermediate endpoints" in following the response of the oral mucosa to the administration of chemopreventive agents. The population groups chosen for this study included Inuit, Canadian Indians and East Indians. Approximately 57.0% of Inuit males from Gjoa Haven (Northwest Territories) used snuff, 62.6% of native Indians of La Loche (Saskatchewan) dipped snuff daily, and 54.0% of East Indian fisherman from along the coast of Kerala (India) chewed tobacco in the form of a betel quid. The chewing patterns were as follows: number of dips per day for the Inuit were 8.03 at 25.2 min per dip, for the native Indians 9.1 dips per day at 20.3 min per dip and for the East Inians 17.2 chews per day at 15.2 min per chew. N-Nitroso compounds were found in the saliva of snuff dippers and betel quid chewers. They are considered to be the most probable etiological factors in the development of oral cancers, since they are the only known carcinogens present in mg/kg quantities in the various tobacco mixtures. High levels of nitrite, a precursor to nitrosamines, were found in tobaccos used by the Canadian natives. Up to 1040 mg/kg nitrite was detected in tobacco samples used by this population. High levels of nitrite were also detected in the saliva of Inuit and Canadian Indian snuff dippers: up to 0.25 mg/ml of nitrite appeared in the saliva within 5 to 10 min of a snuff dipping session. Nitrite was also detected in the saliva of East Indian betel quid chewers, averaging 36.27 μg/ml. Since nitrite can serve as a precursor to nitroamine reactions, the in vitro nitrosation capacity of saliva from a snuff dipper was tested. After the addition of 200 mg proline to the saliva of a snuff dipper at pH 2.5, an 18fold increase in nitrosoproline (NPRO) was observed over control levels. Moreover, NPROwas observed in the urine of chewers at a fivefold increased level over non-chewers. These results indicate an elevated level of nitrosation within individuals who dip snuff. This increased endogenous nitrosation reaction in snuff dippers can lead to the formation of carcinogenic N-nitrosamines. Snuff dippers and tobacco chewers are also exposed to tobacco-specific nitrosamines (TSNA). Levels from 3200 ppb for N-nitrosonornicotine (NNK) to 170,000 ppb for N-nitrosoanatabine (NAT) were found in commonly used brands of snuff which were commercially available in the Northwest Territories. In addition, relatively high levels of these carcinogenic nitrosamines were detected in the saliva of chewers (up to 980 μg/ml of saliva). The second objective of this study was the development of markers which indicate early changes in a human tissue exposed to carcinogens. Two markers were used to detail the damage occurring in the oral mucosa of users of smokeless tobacco. The first was micronuclei which was applied to exfoliated cells from the oral mucosa. This assay is a quantitative indicator of chromosomal breakage. An elevated frequency of micronucleated cells was observed in the oral mucosa of Inuit snuff dippers, native Canadian Indians and East Indian chewers of tobacco-containing betel quids, as compared to corresponding individuals who did not use smokeless tobacco. The second marker was oral leukoplakia, a preneoplastic lesion commonly found in the oral mucosa of betel quid chewers. By using these two markers to quantify carcinogen-induced damage during the preneoplastic stage, it appeared feasible to identify individuals at elevated risk for developing oral cancer. The study explored the possibility of using the above-mentioned markers to follow the response of smokeless tobacco users to the administration of beta-carotene and vitamin A. The administration of beta-carotene (180 mg/week) for ten weeks significantly reduced the level of micronucleated cells in the oral mucosa of a group of Inuit snuff dippers who continued to use their usual amount of snuff during the trial period. Similarly, the levels of micronucleated cells in the oral mucosa of chewers of tobacco-containing betel quids (East Indians) were significantly reduced after three months on a regime of either 180 mg/week of beta-carotene or 180 mg/week of beta-carotene plus 100,000 IU of vitamin A. The reduction of micronucleated oral mucosal cells occurred more rapidly than the remission of leukoplakia in the East Indian group as did the inhibition of newly formed leukoplakia following the administration of the two chemopreventive agents. The remission of established oral leukoplakia was significant (P = 0.004) only after six months in the betel quid chewers receiving beta-carotene plus vitamin A compared to the group receiving a placebo. The treated group also showed a significant reduction in the appearance of new oral leukoplakia (P = 0.08). The administration of vitamin A alone (200,000 IU/week) to betel quid chewers produced a highly significant remission of established leukoplakia (P = 0.0000089) plus an inhibition of the formation of new leukoplakia (P = 0.024) as compared to the placebo group. The East Indians continued to chew betel quids throughout the administration of chemopreventive agents. Many different scientific disciplines are necessary to obtain understanding of the etiological factors involved in the development of a particular cancer, and to recognize early changes in the target tissue to carcinogens. Only a profound understanding of these events will help in the early identification of individuals at elevated risk to cancer, the design of largescale chemopreventive trials, and the selection of the most effective chemopreventive agents.

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