Toxicity of smokeless tobacco in human oral epithelium with emphasis on carcinogen metabolism and regulation of programmed cell death

Abstract: The oral mucosa is globally a common site for cancer development. Primary risk factors include tobacco smoking and alcohol consumption whereas the contribution from usage of smokeless tobacco remains debated. The susceptibility of the human oral epithelium to carcinogens in tobacco likely depends on the presence of biotransformation enzymes, capable of metabolically activating or detoxifying these agents as well opposing influences from oxidative stress. Induction of programmed cell death (PCD), including function of tumor suppressor p53, may also modulate smokeless tobacco toxicity. On this basis, the purpose of this study was to investigate the expression of biotransformation enzymes as well as the roles of PCD and p53 in smokeless tobacco toxicity in oral epithelium. Various qualitative and quantitative analyses of oral tissue specimens and normal, immortalized and malignant oral keratinocytes indicated presence of multiple biotransformation enzymes. Several cytochrome P450 (CYP) transcripts were demonstrated including ]AL 1A2, 2C, 2D6, 2E1, 3A4/7 and 3A5. 'typical CYP substrates, including ethoxyresorutin, methoxyresorufin and chlorzoxazone, were delectably oxidized in vitro and metabolism of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)1- (3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1) resulted in covalently bound adducts. Moreover, normal keratinocytes and SV40T antigen-immortalized keratinocytes (SVpgC2a) were shown to express enzymes catalyzing conjugation reactions and detoxification of reactive oxygen. Notably, SVpgC2a showed higher expression levels than normal keratinocytes of some enzymes, e.g. CYP1B1, By RT-PCR, the CYPs where generally shown to be expressed at levels <50 molecules, the conjugation enzymes at levels between 50-1000 molecules and the enzymes involved in detoxification of reactive oxygen at levels >1000 molecules, using 106 molecules of beta-actin as reference. Microarray analysis confirmed expression of these enzymes at levels >300 molecules per 1 06 molecules of beta-actin. The results indicated presence of several biotransformation enzymes in oral buccal mucosa in vivo and in vitro, indicating the usefulness of oral keratinocyte cell lines for studies of both single agents and complex mixtures in human oral epithelium. Studies of smokeless tobacco toxicity involved cultured oral keratinocyte cell lines and oral tissue specimens obtained from healthy controls, snuff users (SDL) and patients diagnosed for lichen planus (OLP). Assessments of net growth rates. apoptosis, necrosis and terminal differentiation in vitro showed that aqueous smokeless tobacco extract prepared from "Ettans snus" (STE) primarily caused necrotic death without substantial involvement of PCD. Carcinoma cells (SqCC/Y1) were more resistant to necrosis from STE as compared to normal cells. Extract prepared from "Kentucky standard reference tobacco" caused similar toxicity as STE. The latter extract induced increases in p53 content that did not associate to increased apoptosis, whereas in contrast. the DNA damaging agent mitomycin C (MMC) increased both p53-content and apoptosis. STE and nicotine separately. significantly inhibited apoptosis induced by various regimens. Slight increases in bcl-2 transcripts in STE-exposed keratinocytes indicated the involvement of this gene. Analysis of Jurkat cells implied that reactive smokeless tobacco chemicals might also block apoptosis by inhibiting caspase activity. Oral tissue analysis agreed with the concept that smokeless tobacco may inhibit apoptosis, i.e. increased mitosis in SDL (relative to normal controls) was not associated with increased apoptosis, whereas OLP exhibited increases in both mitosis and apoptosis. Finally, expression of the p53 and Bcl-2 proteins was noted in SDL whereas OLP expressed p53 but not bcl-2. In summary, the analysis of the expression of biotransformation enzymes and smokeless tobacco toxicity generally demonstrated similar results in tissue and cultured cell lines implying the usefulness of cell culture technology in the investigation of mechanisms underlying carcinogenesis and other oral disease processes. Thus. keratinocytes actively expressing multiple biotransformation enzymes were susceptible to smokeless tobacco toxicity. The toxicity mechanism of smokeless tobacco likely involves metabolism of carcinogenic agents, including N-nitrosamines. and inhibition of p53-mediated apoptosis. Thus, this study suggests several mechanisms whereby smokeless tobacco usage may contribute to adverse health effects including those associated with cancer development in the oral epithelium.