The major metabolite of equilin, 4-hydroxyequilin, autoxidizes to an o-quinone which isomerizes to the potent cytotoxin 4-hydroxyequilenin-o-quinone

The risk factors for women developing breast and endometrial cancers are al l associated with a lifetime of estrogen exposure. Estrogen replacement the rapy in particular has been correlated with a slight increased cancer risk. Previously, we showed that equilenin, a minor component of Premarin (Wyeth -Ayerst), was metabolized to highly cytotoxic quinoids which caused oxidati ve stress and alkylation of DNA in vitro [Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113-1127]. In this study, w e have compared the chemistry of the major catechol metabolite of equilin ( 4-hydroxyequilin), which is found in several estrogen replacement formulati ons, to the equilenin catechol (4-hydroxyequilenin). Unlike endogenous cate chol estrogens, both equilin and equilenin were primarily converted by rat liver microsomes to 4-hydroxylated rather than 2-hydroxylated o-quinone GSH conjugates. With equilin, a small amount of 2-hydroxyequilin GSH quinoids were detected (4-hydroxyequilin:2-hydroxyequilin ratio of 6:1); however, no peaks corresponding to 2-hydroxyequilenin were observed in incubations wit h equilenin. These data suggest that unsaturation in the B ring alters the regiochemistry of P450-catalyzed hydroxylation from primarily 2-hydroxylati on for endogenous estrogens to 4-hydroxylation for equine estrogens. 4-Hydr oxyequilenin-o-quinone reacts with GSH to give two mono-G;SH conjugates and one di-adduct. The behavior of 4-hydroxyequilin was found to be more compl ex than 4-hydroxyequilenin as conjugates resulting from 4-hydroxyequilenin were detected in addition to the 4-hydroxyequilin-GSH adducts. The mechanis m of decomposition of 4-hydroxyequilin likely involves isomerization to a q uinone methide which readily aromatizes to 4-hydroxyequilenin followed by a utoxidation to 4-hydroxyequilenin-o-quinone. Similar results were obtained with 2-hydroxyequilin, although, in contrast to 4-hydroxyequilenin, 2-hydro xyequilenin does not autoxidize and the reaction stops at the catechol. Sin ce 4-hydroxyequilin is converted to 4-hydroxyequilenin and 4-hydroxyequilen in-o-quinone, similar effects were observed for this equine catechol, inclu ding consumption of NAD(P)H likely by the 4-hydroxyequilenin-o-quinone, dep letion of molecular oxygen by 4-hydroxyequilenin or its semiquinone radical , and alkylation of deoxynucleosides and DNA by 4-hydroxyequilenin quinoids . Finally, preliminary studies conducted with the human breast tumor cell l ine MCF-7 demonstrated that the cytotoxic effects of the catechol estrogens from estrone, equilin, and 2-hydroxyequilenin were similar, whereas 4-hydr oxyequilenin was a much more potent cytotoxin (similar to 30-fold). These r esults suggest that the catechol metabolites of equine estrogens have the a bility to cause alkylation/redox damage in vivo primarily through formation of 8-hydroxyequilenin quinoids.