Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats.

Abstract

Primary cultures of hepatocytes from adult male F344 rats were used to investigate the activation of the hepatocarcinogen 2,4-diaminotoluene (2,4-DAT) to metabolites which bound covalently to DNA. Covalent binding of 2,4-DAT to DNA was significantly greater than that of a non-carcinogenic isomer, 2,6-DAT. Treatment of male rats with 5,6-benzoflavone (BNF), an inducer of cytochrome P-450c and P-450d, had no effect on the binding of 2,4-DAT to DNA of hepatocytes from these animals. However, treatment of hepatocytes in vitro with metyrapone or piperonyl butoxide, two general inhibitors of P-450 enzymes, inhibited the binding of 2,4-DAT to DNA by approximately 80-85%. Two inhibitors of sulfation, pentachlorophenol and 2,5-dichloro-4-nitrophenol, also inhibited DNA binding in hepatocytes from both BNF-induced (91 and 85% respectively) and control rats (82 and 41% respectively), indicating that sulfation may also be required. 2,4-DAT was a more potent mutagen than 2,5- or 2,6-DAT in the Ames Salmonella mutagenesis assay using hepatic S9 fractions from F344 rats as an activating system. In contrast to DNA binding, activation of 2,4-DAT to mutagens by S9 fractions from BNF-treated rats was greater than that by S9 from control rats. The present study shows that 2,4-DAT is activated by hepatocytes of F344 rats to products which bind covalently to DNA. Both cytochrome P-450 and sulfation appear to be involved in the activation.