GENOTOXICITY OF N-HYDROXY AND AMINOPHENOL METABOLITES OF 2,6- AND 3,5-DIMETHYLANILINE AT THE HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE LOCUS IN TK6 CELLS

Abstract

Objective: The objective of this study as to characterize the genotoxicity of reactive metabolites of 2,6-dimethylaniline (2,6-DMA) and 3,5-DMA in the hypoxanthineguanine phosphoribosyltransferase (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 days to allow to phenotypic expression of HPRT mutants. After the expression period, cells were plated in the presence of 2 μg/ml 6-thioguanine for the selection of HPRT mutants. Plating efficiency was determined and mutant fraction calculated. Electron paramagnetic resonance (EPR) was also used to determine whether 3,5- dimethylaminophenol (DMAP) produced reactive oxygen species (ROS).Results: All of the metabolites tested were cytotoxic to these cells but exhibited a considerable variation in potency. The aminophenol metabolites of 2,6- and 3,5-DMA were considerably more toxic than the corresponding N-hydroxylamines. Furthermore, 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.Conclusions: Our findings suggest that genotoxic responses of 2,6- and 3,5-DMA are mediated through the generation of ROS by hydroxylamine and/ or aminophenol metabolites.