Methylazoxymethanol (MAM), methylazoxymethyl acetate (MAMAc), the stable form of MAM, and azoxymethane (AOM), the chemical and metabolic precursor of MAM, are potent colon carcinogens in rodents. AOM is activated by hydroxylation to MAM, which yields a DNA alkylating species spontaneously or by enzyme-catalyzed reactions. Both AOM and MAMAc, but most especially AOM, have been used extensively in rodent studies seeking to identify colon cancer chemopreventive agents. To make rational use of the data obtained from such studies, it is necessary that there exist an adequate information base on the in vivo activation of these carcinogens. The current absence of such information has often led to apparent paradoxes and the inability to optimally interpret the data for extrapolation to man. Recognizing these deficiencies, our goal here is to expand our knowledge of the enzyme systems responsible for the metabolic activation of AOM and MAM. We have shown previously that both MAM and AOM can be metabolically activated by CYP2E1 in vitro. Very recently, using cyp2e1-null and -wild type mice, we showed unambiguously that CYP2E1 also participates in the activation of AOM and MAM to DNA-reactive species in vivo. However, the same studies clearly showed that other enzymes, perhaps other members of the CYP family, were also involved. These studies also demonstrated that differences in CYP2E1 levels, as in the null- and wild-type mice, can profoundly influence the activation and tumorigenicity of AOM and MAMAc - relative to the wild-type mice, the tumorigenicity of AOM is decreased, and that of MAM is increased, in the null-type mice. The influences of chemopreventive agents which may either induce or inhibit CYP2E1 and other enzymes of AOM and MAM activation is expected to be similar.
As Specific Aim 1, we propose to identify CYP isozymes other than CYP2E1, which are active in the in vitro metabolism of 14C-MAM and 14C-AOM using insect cell microsomes expressing specific human CYP isozymes, with analysis of metabolites by a unique HPLC analytical system. As a logical consequence, in Specific Aim 2 we propose to use specific enzyme modifiers to obtain evidence that the isozymes identified in Specific Aim 1 are, in fact, involved in the activation of AOM and MAM in vivo, as reflected in DNA alkylation.
As Specific Aim 3, we test the relevance of the results obtained in the previous Aims by using specific modifiers of CYP isozymes in wild- and null-CYP2E1 mice and determining their effects on colonic aberrant crypt formation. The work under Specific Aim 4 will examine whether other enzymes, including alcohol dehydrogenase and prostaglandin synthase are additionally involved in the in vitro and in vivo metabolism of the carcinogens.
