Morphologic and growth effects of tobacco-associated chemical carcinogens and smokeless tobacco extracts on human oral epithelial cells in culture

Epidemiologic studies show an increase in the use of smokeless tobacco but few in vitro studies have directly assessed the potential for smokeless tobacco-induced oral carcinogenesis. Oral keratinocytes were grown to 90% confluence from explants of human labial and gingival mucosa at 34° C, 5% CO₂ in defined media. Epithelial monolayers were subsequently subcultured and then treated for 1 hour with aqueous extracts of moist or leaf smokeless tobacco, or with 0.25 to 1.0 ng/ml of three common smokeless tobacco carcinogens: 4-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-1-butanone; N-nitrosonornicotine; and benzo(a)pyrene. Even though the controls and most treatment groups terminally differentiated, cells exposed to 4-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-1-butanone, N-nitrosonornicotine, and moist and dry extract continued to divide, maintained a differentiated phenotype for 8 1 2 to 10 weeks in culture, and displayed focal growth and morphologic changes suggestive of early stages in cell transformation.