Differential effects of CYP2E1 status on the metabolic activation of the colon carcinogens azoxymethane and methylazoxymethanol.

Methylazoxymethanol (MAM) and its chemical and metabolic precursor, azoxymethane (AOM), both strong colon carcinogens in rodents, can be metabolically activated by CYP2E1 in vitro. Using CYP2E1-null mice, we found that CYP2E1 deficiency differentially affects the activation of AOM and MAM, as reflected in DNA guanine alkylation in the colon and in the formation of colonic aberrant crypt foci (ACF). Male and female inbred 129/SV wild-type (WT) and CYP2E1-null (null) mice were treated with 189 micromol/kg of either AOM or methylazoxymethyl acetate (MAMAc), and 7-methylguanine (7-MeG) and O(6)-methylguanine (O(6)-MeG) were measured in the DNAs of various organs. The levels of O(6)-MeG (as pmol/nmol guanine) in the liver, colon, kidney, and lung of male null mice treated with AOM were 87, 48, 70, and 43% lower, respectively, than in AOM-treated WT mice. In null mice treated with MAMAc, the DNA O(6)-MeG levels were lower by 38% in the liver but were higher by 368, 146, and 194% in the colon, kidney, and lung, respectively, compared with the same organs of WT mice treated in the same way. Determination of ACF revealed that although AOM-induced ACF formation was significantly lower in the null group than in the WT group, MAMAc-induced ACF formation was significantly higher in the null group than in the WT group. These results demonstrate an important role for CYP2E1 in the in vivo activation of AOM and MAM and suggest that agents that modify CYP2E1 activity at the tumor initiation stage might either enhance or inhibit colon carcinogenesis, depending on whether AOM or MAMAc is used as the carcinogen. The mechanism of this effect is discussed.

Age-related changes in biotransformation of azoxymethane and methylazoxymethanol in vitro.

1. Age-related changes in hepatic hydroxylation of azoxymethane (AZO) to methylazoxymethanol (MAM), as well as colonic phase I metabolism of MAM by alcohol dehydrogenase (ADH) were examined in young (2-4 months), middle-aged (12-14 months), and old (22-24 months) male Fischer 344 rats. In addition, the possibility that colonic glucuronyltransferase might be involved in the biotransformation of MAM was also investigated. 2. A significant decrease in hepatic conversion of AZO to MAM was found in old vs young rats, concomitant with a decrease in hepatic cytochrome P-450 content, while no age-related difference was found in the colonic metabolism of MAM by ADH. MAM inhibition of colonic 4-methylumbelliferone glucuronyltransferase was non-competitive, suggesting indirectly that colonic glucuronyltransferase is not involved in conjugation of MAM. 3. It is concluded that ageing in the male Fischer 344 rat results in alternations of AZO and MAM biotransformation which indicate that AZO may be less carcinogenic in older rats.

A kinetic model of the cell culture cytotoxicity of metabolically activated agents: N-methyl-N-(acetoxymethyl)nitrosamine, methylnitrosourethane, and (methylazoxy)methanol acetate.

The activity of three metabolically activated methylating agents, N-methyl-N-(acetoxymethyl)nitrosamine (DMN-OAc), methylnitrosourethane (MNUT), and (methylazoxy)methanol acetate (MAM-Ac), were determined in cell culture by using a P388 cell growth rate inhibition assay. Experiments were conducted with normal P388 cells in Fischer's medium and under conditions in which the esterase-mediated activation was modified by pretreating cells with the irreversible esterase inhibitor paraoxon and by adding acetylcholinesterase to the medium. Inhibition of intracellular esterase had a much greater effect on activity than did addition of enzyme to medium. These experiments provided data that were used to assess the utility of kinetic models as a means to gain a more detailed understanding of the cytotoxicity process in cell culture. Growth rate inhibition was related to the amount of intracellular alkylation resulting from formation of metabolic intermediates and their subsequent chemical reaction to form methyldiazonium ion and methylation products by using kinetic rate laws and measured rate constants. The model is applicable to systems that form unstable metabolites that can, in part, partition between the cell volume and incubation medium. When growth rate inhibition effects were related to cumulative intracellular alkylation [P], the ED50 values were the same for all three agents and for three previously reported chemically activated methylating agents, N-methyl-N-nitrosourea, streptozotocin, and N-methyl-N'-nitro-N-nitrosoguanidine, which are also thought to act through the methyldiazonium ion. This observation is consistent with a growth rate inhibition effect of the methyldiazonium ion that reflects the intrinsic activity of this species that is independent of the precursor molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of chronic dietary ethanol on in vivo and in vitro metabolism of methylazoxymethanol and on methylazoxymethanol-induced DNA methylation in rat colon and liver.

We examined the metabolism of 14C-labeled methylazoxymethanol (MAM) in male F344 rats pair-fed for 21.0 days either a liquid control diet, an isocaloric liquid diet containing 6.6% ethanol by volume (continuous ethanol diet), or the ethanol diet for 20.5 days followed by the control diet for 0.5 day (interrupted ethanol diet). Compared to rats fed the control liquid diet, metabolism of [1,2-14C]MAM acetate to exhaled 14CO2 was inhibited by 25 to 42% in rats fed the continuous ethanol diet, but was initially stimulated by 90% in rats given the interrupted ethanol diet. MAM-induced DNA methylation, as reflected in 7-methylguanine and O6-methylguanine content 24 h after carcinogen administration, was inhibited in the colon mucosae of rats fed the interrupted ethanol diet by 52 to 54%, and an even greater inhibition (71 to 86%) of DNA methylation occurred in the colon mucosae of rats fed the continuous ethanol diet. Liver DNA methylation was significantly inhibited (by 32 to 42%) only in those rats fed the continuous ethanol diet. Liver microsomes isolated from rats fed the 3 diets metabolized MAM to formic acid and methanol in vitro, but liver microsomes from rats fed the continuous ethanol diet were 12 to 15 times more active than liver microsomes from rats fed the control diet. Liver microsomes isolated from rats fed the interrupted ethanol diet were only 3 to 5 times more active in MAM metabolism than liver microsomes from rats fed the control diet, indicating very rapid turnover of the ethanol-induced enzyme(s) catalyzing the oxidation of MAM. Although chronic ethanol feeding enhanced the activity of liver microsomes for MAM metabolism, ethanol was found to inhibit the reaction competitively. Hepatocytes isolated from rats fed the continuous ethanol diet were considerably more sensitive to MAM-induced unscheduled DNA synthesis than hepatocytes isolated from rats given the control liquid diet, indicating that the stimulation of MAM metabolism by dietary ethanol results in increased DNA damage, observable in an in vitro system. Thus, the increased metabolic activation of MAM, due to enzyme induction by ethanol which is observed in vitro, is not reflected in increased liver DNA methylation in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)