Rat Liver Microsome-mediated N-Demethylation and Mutagenicity of Azoxymethane¹

The in vitro metabolism of azoxymethane (AOM), a highly specific intestinal carcinogen, was studied. Liver microsomes from male Sprague-Dawley rats in the presence of NADPH metabolize AOM to a mutagen detected by Ames' Salmonella typhimurium tester strains HisG46, TA1530, TA1535, and TA100 in liquid incubation. Mutagen formation was dependent on the length of incubation, microsomal protein, and AOM concentration, but its detection was most sensitive with strain TA100. The exact chemical nature of the mutagen was not determined.

An assay for the N-demethylation of AOM to formaldehyde by liver microsomes was also established. The reaction required reduced nicotinamide adenine dinucleotide phosphate and O₂ thus exhibiting mixed-function oxidase characteristics. Pretreatment of animals with sodium phenobarbital or 3-methylcholanthrene did not significantly alter the specific activity of microsomal AOM N-demethylation but did increase microsomal cytochrome P-450 levels. Cobaltous chloride (60 mg/kg s.c.) reduced AOM N-demethylation, 49%; and cytochrome P-450, 69%. SKF-525A in vitro caused inhibition of AOM N-demethylation (43% initial activity at 3 mm), but 7,8-benzoflavone did not. Disulfiram, an inhibitor of 1,2-dimethylhydrazine and AOM carcinogenesis, reduced microsomal AOM N-demethylation by 70% when given at 0.25% (w/w) in Purina rat chow but did not alter cytochrome P-450 levels. The specific activity of microsomal AOM N-demethylation was significantly greater (+86%) when animals were fed a Purina rat chow diet than when fed a semisynthetic diet containing 56% dextrose, 25% casein, 7% salt mix, 5% corn oil, 5% bran, and 2% vitamin supplement. Compared to a semisynthetic diet control, AOM N-demethylationwas significantly increased by an 18-hr starvation period from the semisynthetic diet (+55%) or from the Purina rat chow diet (+ 175%). The addition of 30% beef fat, however, did not alter the specific activity of AOM N-demethylation.