In vitro metabolism of o-aminoazotoluene and mutagenesis of Salmonella by the metabolites

Incubation of hepatocarcinogenic aminoazo dye, o-aminoazotoluene (OAT) with rat liver microsomes together with NADPH and NADH yielded N-hydroxy-OAT (I), 4′ -hydroxy-OAT (II) and a smaller amount of 2′ -hydroxymethyl-3-methyl-4-aminoazobenzene (III). As an artifact 4, 4′-bis(otolylazo)-2, 2′ -dimethylazoxybenzene (IV) was also detected in a small quantity. The mutagenicities of these metabolites were assayed by using Salmonella typhimurium TA98 and TA100 together with S-9 prepared from the livers of rats treated with polychlorinated biphenyl mixture (PCB). OAT and III were strongly mutagenic, but only when S-9 was present. In contrast, I was a strong mutagen regardless of the presence or absence of S-9. II and IV were non-mutagenic. The yields of I, II and III from OAT were pronouncedly reduced by addition of cytochrome P-450 inhibitors, especially by a cytochrome P-448 inhibitor 7,8-benzoflavone. Mutagenesis by OAT was also inhibited by addition of 7,8-benzoflavone. Activation of OAT for mutagenesis was enhanced by pretreatment of the donor rats with PCB or 3-methyl-cholanthrene and to a much lesser extent by phenobarbital. These findings suggest that N-hydroxylation of OAT mainly proceeds via catalysis by cytochrome P-448 and that this process is an obligatory step for the activation of OAT. Synthetic methods for the preparation of new azo compounds such as I, IV and 2′, 3-dimethyl-4-nitrosoazobenzene are described.