Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17 alpha- and 17 beta -dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescrib ed estrogen formulation in estrogen replacement therapy. Despite the wide c linical 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 s tudy, we investigated the metabolism of equilenin in two lines of human bre ast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment wi th Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whe reas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of syn thetic metabolite standards and deuterium-labeled analogues as internal sta ndards. In the two cell lines, the same pathways of equilenin metabolism we re observed. Equilenin was reduced at C-17 to the 17 beta -dihydro form, wi th minimal production of the 17 alpha -dihydro isomer. Both equilenin and 1 7 beta -dihydroequilenin were hydroxylated at the C-4 position, and the res ultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17 beta -dihydroequilenin. Rates of equilenin metabolism were mar kedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tet rachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation path ways of equilenin and 17 beta -dihydroequilenin metabolism were not observe d. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A l and 1B1 catalyzed the 4-hydroxylation of 17 beta -dihydroequilenin, where as with 17 beta -estradiol as substrate P450 1A1 catalyzes predominantly 2- hydroxylation and P450 1B1 predominantly 4-hydroxylation, Since F450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indica te the potential for 4-hydroxylation of equilenin and 17 beta -dihydroequil enin in extrahepatic, estrogen-responsive tissues.