Genotoxicity and mutagenicity of Arecoline to Chinese hamster ovary cells

Objectives: Betel quid (BQ) chewing, a popular oral habit, is closely associated with the high incidence of oral cancer. Areca nut (AN), the major component of the BQ, has been demonstrated to be carcinogenic. AN contains many polyphenols and alkaloids. Arecoline, the most abundant areca alkaloid, has been suggested as a possible carcinogen. Therefore, it is necessary to further examine the cytoxicity and genotoxicity of arecoline and clarify the underlying mechanisms. Methods: We used trypan blue dye exclusion assay, clonogenic cell survival assay, cytokinesis-block micronucleus assay, hypoxanthine-guanine phosphoribosyltransferase (hprt) gene mutation assay and flow cytometric analysis to test these events using Chinese hamster ovary (CHO-K1) cells. Results: we found that arecoline inhibited the clonal growth and proliferation of CHO-K1 cells in a concentration- and time-dependent manner. After 24-h of exposure, arecoline increased the intracellular H2O2 production at 0.8 mM. In cytokinesis-block micronucleus assay, a dose-dependent increase in the micronuclei frequency by arecoline was found. Moreover, arecoline itself increased the mutation frequency in hprt gene mutation assay. Treatment with arecoline (0.8mM) for 24-h caused cell cycle arrest and subsequently prolonged exposure induced cell death. Co-incubation of cells to catalase (2000 U/ml) and arecoline (0.8mM) reduced the micronuclei frequency, indicating that arecoline-induced toxicity was associated with ROS production. Conclusion: our results suggest that arecoline itself posseses the cytotoxicity and genotoxicity to CHO-K1 cells. An increase in micronuclei frequency and mutation frequency are possibly associated with ROS production by areoline. BQ chewing can be a risk factor of oral cancer.