Sulfate conjugates of the B-ring unsaturated
estrogens,
equilin,
equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of
Premarin (Wyeth-Ayerst), the most commonly prescribed
estrogen formulation in
estrogen replacement therapy. Despite the wide clinical use of
Premarin, there have been very few studies on the metabolism of the B-ring unsaturated
estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or
tumors. In this study, we investigated the metabolism of
equilenin in two lines of human
breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with
Ah-receptor agonists with induction of
cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of
equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and
deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of
equilenin metabolism were observed.
Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both
equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant
catechol metabolites were methylated to form
4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of
equilenin metabolism were markedly elevated in cultures exposed to the
Ah-receptor agonists,
2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of
equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with
cDNA-expressed human
enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of
equilenin and 17beta-dihydroequilenin in extrahepatic,
estrogen-responsive tissues.