Carcinogenic Azo Dyes. XII. Detection of New Metabolites of Aminoazo Dyes by Rat Liver

Metabolism of 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB) and 3'-methyl-4-(methylamino) azobenzene (3'-Me-MAB) by the rat liver was investigated by the use of a tracer technique. 3'-Me-DAB [5'-3H] or 3'-Me-MAB [5'-3H] was incubated with a 9000 g supernatant fraction at 37°in an aerobic condition for 30 min. The metabolites were extracted with benzene-acetone. The residual aqueous solution was applied to an Amberlite XAD-2 column and the metabolites were eluted with methanol. The metabolites in the extract or eluate were determined by the reverse isotope dilution analysis after separation by thin-layer chromatography. The recovery of radioactivity in benzene-acetone extract after incubation of 3'-Me-DAB [5'-3H] was 70.8±7.5%. About 99.5% of the radioactivity extracted was identified with the N-demethylated, oxidized at 4' position and ring methyl group, and azo-reduced metabolites and the substrate. On the other hand, 79.4% or 83.8% of radioactivity recovered in each eluate was identified with 3-aminobenzoic acid and 3-acetaminobenzoic acid. Accordingly, it was proved that the metabolites oxidized at the ring methyl group was also detected in the in vitro metabolism of 3'-Me-DAB or 3'-Me-MAB.