Enhancement of rat liver microsomal metabolism of azoxymethane to methylazoxymethanol by chronic ethanol administration: similarity to the microsomal metabolism of N-nitrosodimethylamine.

We compared the metabolism of azoxymethane (AOM) and of N-nitrosodimethylamine (NDMA) by liver microsomes obtained from male F344 rats pair-fed for 3 weeks either a control liquid diet or an isocaloric liquid diet containing ethanol at a concentration of 6.6% by volume. High-performance liquid chromatographic analysis of the products of the microsomal metabolism of AOM showed that methylazoxymethanol was the only primary metabolite. While the formation of small (less than 4% of methylazoxymethanol) quantities of methanol and formaldehyde could also be detected in this reaction, these products could be accounted for almost entirely by the spontaneous decomposition of methylazoxymethanol. With NDMA as the substrate in the incubation system, the formation of methylamine, formaldehyde, methanol, and an additional, as yet unidentified metabolite was detected. Liver microsomes obtained from rats fed the ethanol-containing diet up to the time of sacrifice were 12-18 times more active in the metabolism of both AOM and NDMA than liver microsomes obtained from rats fed the control, ethanol-free diet for the same period. When rats fed the ethanol diet for 20.5 days were fed the control diet for 0.5 days and then sacrificed, only a 2- to 3-fold increase in the metabolism of both AOM and NDMA by liver microsomes was observed, indicating that cessation of ethanol intake results in a rapid decrease of the ethanol-induced metabolic enzymes. Hepatocytes isolated from ethanol-fed rats showed a significantly enhanced sensitivity to AOM- as well as to NDMA-induced unscheduled DNA synthesis, indicating that the increased rate of microsomal metabolism induced by ethanol is associated with enhanced carcinogen activation in vitro. The metabolism of AOM and NDMA by liver microsomes was inhibited to similar extents by carbon monoxide, pyrazole, sodium azide, aminoacetonitrile, imidazole, and ethanol. In addition, both ethanol and NDMA were found to inhibit competitively the microsomal metabolism of AOM. These results suggest that AOM and NDMA are metabolized by very similar, indeed perhaps the same rat liver microsomal enzyme(s).

Differential effects of 4-iodopyrazole and 3-methylpyrazole on the metabolic activation of methylazoxymethanol to a DNA methylating species by rat liver and rat colon mucosa in vivo.

Using a hybrid ion-exchange reverse phase HPLC system, we found that F344 rat liver microsomes, in the presence of an NADPH-generating system, can metabolize methylazoxymethanol (MAM), a colon and liver carcinogen, to methanol and formic acid. This is in contrast to the spontaneous decomposition of MAM which yields methanol and formaldehyde. The metabolism of MAM by rat liver microsomes is sensitive to inhibition by carbon monoxide as well as to inhibition by 3-methylpyrazole (3-MeP) and 4-iodopyrazole (4-IP), with 4-IP being more potent in this respect than 3-MeP. Pretreatment of rats with 4-IP decreased the level of MAM acetate-induced DNA methylation in both the liver and the colon mucosa. In contrast, pretreatment with 3-MeP decreased MAM acetate-induced liver DNA methylation, but increased DNA methylation in the colon mucosa. This differential effect of the two compounds on DNA methylation in the liver and the colon suggests that different enzymes are responsible for activation of the carcinogen in the two organs.

Differential susceptibility of rat and guinea pig colon mucosa DNA to methylation by methylazoxymethyl acetate in vivo.

Methylazoxymethanol (MAM) and methylazoxymethyl acetate (MAMOAc) are powerful colon carcinogens in rats, mice and hamsters. In contrast, these agents are not carcinogenic to the colon of the guinea pig. To probe the mechanism responsible for this species difference, we determined the levels of DNA methylation in the livers and colon mucosae of F344 rats and strain-2 guinea pigs after the s.c. administration of 25 mg/kg MAMOAc. While no significant difference was observed between the two species with respect to the degree of liver DNA methylation, the level of O6-methylguanine in guinea pig colon mucosa DNA was 19 times lower than in rat colon mucosa DNA, and the level of 7-methylguanine was below detection limits. However, significant colon mucosa DNA methylation was observed in the guinea pig after the intrarectal administration of 1.25 mg methylnitrosourea. The methylation of colon mucosa DNA in response to MAMOAc in the two species correlated with the activity of alcohol dehydrogenase, an enzyme believed to be involved in the activation of MAM. Thus the resistance of the guinea pig colon to the carcinogenicity of MAM/MAMOAc may be ascribed to the lack of metabolic activation of MAM in this organ.