The tobacco alkaloid nicotine demonstrates genotoxicity in human tonsillar tissue and lymphocytes.

Recent studies suggest a direct contribution of nicotine, the addictive component of tobacco and tobacco smoke, to human carcinogenesis. To assess the genotoxicity of nicotine, the DNA-damaging effect on human lymphocytes and target cells from lymphatic tissue of the palatine tonsils from 10 healthy patients was tested with the alkaline single-cell microgel electrophoresis (Comet) assay. The degree of DNA migration, a measure of possible DNA single strand breaks, alkali labile sites, and incomplete excision repair sites, was expressed as the Olive tail moment, the percentage of DNA in the tail, and the tail length. One hour exposure to nicotine at 0.125, 0.25, 0.5, 1, 2, and 4 mM induced a statistically significant dose-dependent increase of DNA migration up to 3.8-fold and 3.2-fold in tonsillar cells and lymphocytes, respectively. The lowest concentration eliciting significant DNA damage was 0.5 mM nicotine. The genotoxic effect was confirmed in a second series of experiments using nicotine of high purity from two different suppliers. There were no significant differences between the two series, excluding artifacts from the source of nicotine. Finally, DNA damage by nicotine was compared in cells incubated in medium strictly adjusted to neutral pH, with non-adjusted medium becoming alkaline with increasing nicotine concentrations. Again no differences in DNA migration were observed. The data indicate that nicotine expresses significant direct genotoxic effects in human target cells in vitro. However, no differences in DNA damage were observed in cells from smokers and nonsmokers incubated without nicotine. The lack of higher DNA damage in smokers compared to nonsmokers could be a question of nicotine dose, rapid DNA repair, or interactions with other smoke constituents. These results require further investigations on the contribution of nicotine to tobacco carcinogenesis.

Genotoxicity of nicotine in mini-organ cultures of human upper aerodigestive tract epithelia.

The direct role of nicotine in tobacco carcinogenesis is still controversial. Recently, DNA damage by nicotine has been demonstrated in isolated human tonsillar tissue cells. Presently, these effects were investigated using mini-organ cultures (MOC) of human nasal epithelia. Intact MOC were repeatedly exposed to 2 and 4 mM nicotine for 1 h on culture days 7, 9, and 11. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. DNA damage was examined by Comet assay either directly after exposure or following a 24-h recovery period. Cell viability was not reduced by any treatment. On day 7, 1 h exposure to 2 and 4 mM nicotine caused a significant dose-dependent 3.3- and 5.6-fold increase in DNA damage compared to solvent controls. Although there was no evidence of significant repair within 24 h recovery, DNA damage was not further increased by nicotine on days 9 and 11. After double and triple exposure to 4 mM nicotine a significant reduction in DNA damage following 24 h recovery was observed. In contrast, treatment with MNNG resulted in a highly significant and cumulative increase in DNA migration up to 110-fold compared to controls. During recovery periods, MNNG-induced DNA damage was significantly repaired, leading to a 1.5- to 1.8-fold reduction in DNA migration within 24 h. These results confirm genotoxic effects of nicotine on human nasal epithelia. Further studies are needed to explain the lack of cumulative DNA-damaging effects of nicotine and the absence of significant DNA repair. These studies should include a battery of assays with multiple end points.