No 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.

Oxidation of diethylstilbestrol (I) by peroxidases from horseradish or mouse uterus in the presence of H2O2 in vitro leads to Z,Z-dienestrol (II) and to a number of cleavage products, five of which were identified by g.l.c.-mass spectrometry and comparison with authentic reference compounds as 4-hydroxybenzoic acid (III), 4'-hydroxypropiophenone (IV), 1'(4'-hydroxyphenyl)-propan-1-on-2-ol (V), 1-(4'-hydroxyphenyl)-propan-1,2-dione (VI) and 3-(4'-hydroxyphenyl)-hex-2-en-4-one (VII). The formation of 3-(4'-hydroxyphenyl)-hex-2-en-4-one (VII) from diethylstilbestrol is the first reported example of a metabolic dearylation reaction. The amount of cleavage products depends on the excess of H2O2 used. The amount of H2O2 does not affect the extent of binding of diethylstilbestrol to DNA as mediated by peroxidases. The syntheses of V, VI and VII are described.

No abstract

[14C]Diethylstilbestrol (DES) was incubated in vitro with liver and kidney microsomes from male and female hamsters and rats, and the extent of non-extractable binding of radioactivity to microsomal protein was determined. Binding to microsomes from male hamster kidney, which is a target organ for DES carcinogenicity in vivo, was found to be 5-10 times higher than binding to microsomes from non-target tissues. Pretreatment with phenobarbital led to a marked increase in binding of DES to kidney microsomes but not to liver microsomes from female hamsters and male and female rats. The correlations of in vitro covalent binding with organ susceptibility implies a role for metabolic activation of DES in the mechanism of its carcinogenicity.