Bagchi, M., C. Kuszynski, E. B. Patterson, L. Tang, D. Bagchi, T. Saani, and S.J. Stohs. In Vitro Free Radical Production in Human Oral Keratinocytes Induced by Smokeless Tobacco Extract. In Vitro Toxicol. 1997. 10(2): 263-274.

smokeless tobacco extract

Smokeless tobacco increases the risk of oral and other cancers. However, the mechanism by which smokeless tobacco constituents produce these effects is not clear. Therefore, the effects of a smokeless tobacco extract (STE) on protein kinase C activity, production of reactive oxygen species, DNA damage, and type IV collagen mRNA were determined on human keratinocytes in culture. Cells were obtained from the oral cavities of normal human subjects and grown to 100% confluency in keratinocyte growth medium (KGM), supplemented with pituitary extract. Approximately 70% confluent primary human oral keratinocytes grown in 60-mm Petri dishes were subcultured successfully in 6-well plates. These cells were treated with an aqueous extract of smokeless tobacco (STE) (0-300 ug/mL) and incubated for 24 hours at 37° C. The overall intracellular oxidized states of cells were measured by laser scanning confocal microscopy, using 2,7-dichlorofluorescein diacetate (2,7-DCF) as the fluorescent probe. The intensity of the 2,7-DCF fluorescence band increased with increasing concentration of the STE on confocal microscopy, indicating an increase in intracellular oxidation states of the keratinocytes. STE significantly increased protein kinase C (PKC) activity in the cells. The total PKC activity in cells exposed to STE was approximately 1.5 to 2.0 times that of control cells after 3 and 24 hours of incubation. Concentration-dependent increases occurred in the production of reductive oxygen species (ROS) and DNA damage in cultured keratinocytes. STE treated cells also exhibited a 2-3 fold increase in the steady state levels of mRNA for the a ₃, a ₄, a ₅ chains of type IV collagen. The data indicate that exposure of human keratinocytes to aqueous extracts of STE can result in the production of highly reactive forms of oxygen, DNA damage, and altered biosynthetic pathways of collagen synthesis that may lead to cancers of the oral cavity.