A COMPARATIVE GENOTOXIC STUDY OF THE METABOLITES OF 2, 6-AND 3,5-DIMETHYLANILINE IN HUMAN LYMPHOBLASTOID TK6 CELLS

Objective: To characterize the genotoxicity of reactive metabolites of 2,6-dimethylaniline (2,6-DMA) and 3,5-DMA in the hypoxanthine‑guanine phosphoribosyl transferase (HPRT) gene of human lymphoblastoid TK6 cells.Methods: Cultures were exposed to N-hydroxylamine and aminophenol metabolites of 2,6-and 3,5-DMA for 1 h in serum-free medium. Cell survival 24 h after exposure was determined by trypan blue exclusion. Cells were then subcultured for 7-10 d to allow to the phenotypic expression of HPRT mutants. After the expression period, cells were plated in the presence of 2 µg/ml 6-thioguanine for selection of HPRT mutants. Plating efficiency was determined and mutant fraction calculated. Electron Paramagnetic Resonance (EPR) was also used to determine whether 3,5-DMAP-produced reactive oxygen species (ROS).Results: All of the metabolites tested were cytotoxic to these cells but exhibited considerable variation in potency. The aminophenol metabolites of 2,6-DMA and 3,5-DMA were considerably more toxic than the corresponding N-hydroxylamines. Also, each metabolite of 3,5-DMA was more toxic than its 2,6-DMA counterpart; N-OH-3,5-DMA and 3,5-DMAP were clearly mutagenic at a level of 50 µM. EPR studies showed intracellular oxidative stress induced under 3,5-DMAP treatment.Conclusion: Our findings suggest that genotoxic responses of 2,6-DMA and 3,5-DMA are mediated through the generation of ROS by hydroxylamine and/or aminophenol metabolites.