The carcinogen methylazoxymethanol acetate induces a high incidence of tumors in select tissues of the rat. It has been shown that methylazoxymethanol is a substrate for the enzyme horse liver alcohol dehydrogenase, and it has been suggested that the organotropic effects are related to the metabolism of methylazoxymethanol within the sensitive tissues by β-nicotinamide adenine dinucleotide (NAD+)- or β-nicotinamide adenine dinucleotide phosphate-dependent dehydrogenase reactions. The fate of the product of the enzyme reaction, presumably the aldehydic form of methylazoxymethanol, and its mechanism of reaction with cellular macromolecules have heretofore not been investigated. We designed an assay to monitor the formation of carbonium ions using sodium-[14C]acetate as the trapping agent. This nucleophile was added to an incubation mixture containing methylazoxymethanol, NAD+, and alcohol dehydrogenase. The amount of methylazoxymethanol converted was determined by measuring the amount of reduced β-nicotinamide adenine dinucleotide formed. Significantly more methyl [14C]acetate formed when methylazoxymethanol was incubated with enzyme and NAD+ as compared to when it was incubated alone. Analysis of the data comparing the amounts of reduced β-nicotinamide adenine dinucleotide and of methyl[14C]acetate formed indicates that the aldehydic form of methylazoxymethanol is a very unstable compound which rapidly releases carbonium ions. Experiments with N-methyl-N-nitrosourea, a methylating agent not requiring metabolic activation, showed no increase in the amount of methyl[14C]acetate obtained when enzyme and NAD+ were added as opposed to when enzyme was omitted. The significance of these findings is discussed in relation to the organotropism exhibited by methylazoxymethanol.
Supported by USPHS Grants CA 08748 from the National Cancer Institute and CA 15637 from the National Cancer Institute through the National Large Bowel Cancer Project and by the Elsa U. Pardee Foundation.