METABOLIC ACTIVATION OF 2,6-DIMETHYLANILINE: MUTATIONAL SPECIFICITY IN THE GPT GENE OF AS52 CELLS

Abstract

Objective: The purpose of the current work was to characterize the mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyl transferase (gpt) gene locus were assessed with doses, of which relative survival is 30% or more. Nested polymerase chain reaction-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction on treatment of 2,6-DMA and its metabolites but show a considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethylaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8-fold (13.2×10−5) greater than the spontaneous fraction of 1.62×10−5. Total deletion of the gpt gene sequences was found in 1 (4%) spontaneous and 2 (6%) the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusions: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo.