Objective: Having recently shown that the use of smokeless tobacco (ST) results in the production of nitric oxide radicals (•NO), we wanted to assess the potential of ST and some of it's constituent compounds to induce protein nitrosation in human oral keratinocytes in vitro. Methods: Human keratinocytes, grown on chamber slides, were treated with a range of doses of either ST, nicotine, nitroso-nornicotine (NNN), 4-(N-methyl-N-nitrosoamino)- 1-(3-pyrdyl)-1-butanone (NNK) or peroxynitrite for a 60 minute period. Following treatment, the cells were probed using an antibody against nitrotyrosine residues. Visualization of the extent of protein nitrosation was achieved through the use of a CY-3 conjugated secondary antibody and a fluorescent microscope. Results: Dose-dependent increases in cellular nitrotyrosine residues were observed for all compounds as they approached their LC50 value. Peroxynitrite, the reaction product of •NO and superoxide (O2•) was used as a control and yielded the highest levels of protein nitrosation, followed by nicotine, NNK, NNN with the lowest levels seen in ST. Conclusion: These results suggest that ST as well as many of it's constituent compounds may yield •NO, that under physiologic conditions is converted into peroxynitrite which acts as a potent agent in protein nitrosation. This work was supported by the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research.

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