Metabolic activation of 2,4-diaminoanisole, a hair-dye component--II. Role of cytochrome P-450 metabolism in irreversible binding in vitro.

Incubation of rat liver microsomes with radiolabeled 2,4-diaminoanisole (2,4-DAA) in the presence of NADPH and oxygen led to the formation of irreversibly bound products to microsomal protein. The binding was inhibited by a CO:O₂ atmosphere and by an antibody against NADPH cytochrome c reductase. In vivo and in vitro inhibitors of cytochrome P-450 decreased the binding and phenobarbital-pretreatment increased binding, whereas β-napthoflavone-pretreatment was without effect. Binding of ring-labeled 2,4-DAA was much higher than with methyl-labeled-2,4-DAA. Experiments with [³H]-ring-and [¹⁴C]-ring-labeled-2,4-DAA indicated some loss of tritium; this was confirmed by isolation of labile tritium. Substitution of the hydrogens in the methyl group with deuterium led to increases in both binding and mutagenicity of 2,4-DAA. Formation of formaldehyde and a small amount of methanol could be demonstrated during the oxidative metabolism of methyl-labeled-2,4-DAA. Addition of superoxide dismutase and ascorbic acid inhibited binding, and a small amount of irreversible binding could be demonstrated when NADPH was replaced by a xanthine-xanthine oxidase system. Microsomes from rat kidneys also activated 2.4-DAA in the presence of NADPH. Thin-layer chromatography revealed that 30–40 per cent of 2.4-DAA was oxidized during 10 min of incubation with liver microsomes. And a tentative scheme involving aromatic hydroxylation, oxidative demethylation and N-hydroxylation for the microsomal metabolism of 2,4-DAA is presented. Irreversible binding could also be shown with liver microsomal RNA in vitro, whereas no binding to exogenously added DNA could be found.