17-BETA-ESTRADIOL METABOLISM BY HAMSTER HEPATIC MICROSOMES - IMPLICATIONS FOR THE CATECHOL-O-METHYL TRANSFERASE-MEDIATED DETOXICATION OF CATECHOL ESTROGENS

We have shown that the metabolism of 17 beta-estradiol in hamster live r microsomes is concentration-dependent. At low (<25 mu M) concentrati ons of 17 beta-estradiol, 16 alpha-hydroxylase activity predominated, and estriol was the major metabolite, At higher concentrations (25-75 mu M), 16 alpha-hydroxylation and aromatic hydroxylation at C2 contrib uted equally to 17 beta-estradiol metabolism. Aromatic C4-hydroxylatio n was maximal at 75 mu M of 17 beta-estradiol and was always less than C2-hydroxylation. Dehydrogenation of the 17 beta-hydroxyl group to th e ketone (estrone) was also observed, but both estrone and 2-hydroxyes trone were minor (similar to 3%) metabolites of 17 beta-estradiol, onl y detectable at concentrations of 50 mu M and above, Catechol-O-methyl transferase (COMT) effectively converted both 2- and 4-hydroxy-17 bet a-estradiol to their corresponding monomethoxy metabolites. Effective reducing conditions are required for COMT activity, because catechol e strogens are readily oxidized to their corresponding ortho-quinones, a nd ascorbic acid is routinely added to assays of COMT activity, Intere stingly, although ascorbic acid (1 mM) increased the recovery of 2- an d 4-hydroxy-17 beta-estradiol from microsomal incubations, it decrease d the recovery of the methoxy metabolites (similar to 40%). Since the enediol function of ascorbate resembles that of a catechol group, asco rbate is a substrate for COMT and probably competes with the catechol estrogens for methylation, Because previous studies describing the abi lity of COMT to inhibit the covalent binding of electrophilic reactive metabolites of [4-C-14]17 beta-estradiol to microsomal protein were p erformed in the presence of high (100 mM) Mg2+ concentrations, we also investigated the effects of Mg2+ on 17 beta-estradiol metabolism. Con centrations of Mg2+ > 10 mM inhibited the metabolism of 17 beta-estrad iol, as evidenced by i) the increased recovery of substrate; ii) a dec rease in the formation of estriol, estrone, and 2-, and 4-hydroxy-17 b eta-estradiol; iii) a decrease in the recovery of water-soluble metabo lites when incubations were performed in the presence of glutathione ( GSH) to trap the reactive electrophilic metabolites; and iv) a decreas e in the amount of reactive electrophilic metabolites bound to microso mal protein. GSH also decreased the covalent binding of electrophilic metabolites of [4-C-14]17 beta-estradiol to microsomal protein, with t he concomitant formation of water-soluble metabolites, Thus, both COMT and GSH combine to limit the formation of electrophilic metabolites f rom 17 beta-estradiol. The relative importance of each of these pathwa ys to the disposition of the catechol estrogens remains to be determin ed.