Covalent binding of estrogen metabolites to hamster liver microsomal proteins: inhibition by ascorbic acid and catechol-O-methyl transferase.

Abstract

We have studied the irreversible binding of [14C]estradiol to hamster liver and kidney microsomes of castrated hamsters. The binding of estradiol metabolites to kidney microsomes was approximately 25 times lower than seen for liver microsomes, and was not affected by increases in protein or substrate concentration. Our results indicate that this irreversible binding, covalent in nature, is dependent on the metabolism of estrogens to catechols since the absence of NADPH markedly reduces this binding. The irreversible binding was inhibited nearly 70% by addition of catechol-O-methyl transferase (COMT) and S-adenosylmethionine (SAM). Ascorbic acid also inhibited the binding to 85% in a dose-dependent manner. Utilizing a displacement assay to assess the relative covalent binding of different stilbene and steroidal estrogens with homologous radiolabeled hormones, we found that only indenestrol B exhibited greater ability than diethylstilbestrol (DES) to displace [14C]DES from hepatic microsomal proteins. Except for hydroxypropiophenone and beta-dienestrol, all of the stilbene estrogens studied displaced the radioactive DES binding from these liver proteins to a greater extent than estradiol at comparable concentrations. A marked difference was observed in the ability of alpha- and beta-dienestrol to displace [14C]DES. Using radioinert steroidal estrogens to displace [14C]estradiol, we observed that both estriol and deoxoestrone were significantly less effective in displacing radiolabeled estradiol from liver microsomal proteins. Only ethinyl estradiol and 2-hydroxyestradiol displaced greater than 50% of the radioactive hormone at 1-fold excess concentrations. Interestingly, 11 beta-methoxyethinyl estradiol (Moxestrol) exhibited essentially the same ability to bind liver microsomal proteins as estradiol. When estrone and 2,4-dibromoestradiol were used as substrates together, we found the latter compound to be inactive as a substrate for estrogen hydroxylase (ESH) and additionally inhibited the metabolism of estrone to form the catechol metabolite. Data presented herein suggest that the chemically reactive estrogen metabolites responsible for covalent binding of both stilbene and steroidal estrogens are quinoids derived from catechols formed earlier in metabolism. Except for ethinyl estradiol which is a good substrate for liver, but not kidney microsomal ESH, the carcinogenicity data for the hamster kidney with respect to these estrogens is consistent with the covalent binding data presented.