Studies on Carcinogenic Azo Dyes. V. The NIH Shift during the Aryl Hydroxylation of 3'-Methyl-4-(dimethylamino) azobenzene and 3-Methylacetanilide by the Rat

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The NIH shift during the aryl hydroxylations of the specifically deuterated or tritiated 3'-methyl-4-(dimethylamino) azobenzene (3'-Me-DAB) and 3-methylacetanilide by a rat in vivo or in vitro has been investigated. 3'-Me-4'-OH-DAB was excreted in the bile and 3-methyl-4-hydroxyacetanilide in urine when 3'-Me-DAB [4'-²H] was administered orally and 3-methylacetanilide [4-²H, 4-³H, or 5-²H] intraperitoneally to rats. In in vitro experiments, 3'-Me-DAB [4'-²H or 4'-³H] was incubated at 37° in an aerobic conditions for 1 hr with liver homogenates and 3-methylacetanilide [4-²H, 4-³H, or 5-²H] with liver microsomes. Retention of heavy hydrogen in the 4'-or 4-hydroxylated metabolite was determined by mass spectrometry or radioactivity counting. The NIH shift was observed also during these hydroxylations. In in vitro hydroxylation of 4'-labeled 3'-Me-DAB, tritium was retained 94.1±2.7% and deuterium 43.7±3.4%. Therefore, isotope effect in retention was great in this reaction. In the case of 3-methylacetanilide, such isotope effect was not observed, and heavy hydrogen was retained 19-23% in the 4-hydroxylated metabolite in vivo or in vitro. Isotopic hydrogens at the position adjacent to hydroxylation in both substrates were stable under the employed conditions. It was demonstrated that 3-methylacetanilide belongs to class I substrate and 3'-Me-DAB to class II because of the effect of substituent on their retention of isotopic hydrogen.