title: Bioreductive activation of catechol estrogen-ortho-quinones: aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity.
abstract: There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 60612-7231, USA.
authors.forename: L
authors.initials: L
authors.lastname: Shen
authors.forename: E
authors.initials: E
authors.lastname: Pisha
authors.forename: Z
authors.initials: Z
authors.lastname: Huang
authors.forename: J M
authors.initials: JM
authors.lastname: Pezzuto
authors.forename: E
authors.initials: E
authors.lastname: Krol
authors.forename: Z
authors.initials: Z
authors.lastname: Alam
authors.forename: R B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: J L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D000818:Animals; D004855:Equilenin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002393:Estrogens, Catechol / Q000633:toxicity*; D006801:Humans; D007700:Kinetics; D008545:Melanoma / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011809:Quinones / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D002393:Estrogens, Catechol; D011809:Quinones; D009249:NADP; C065250:4-hydroxy-equilenin; D016660:NAD(P)H Dehydrogenase (Quinone); D004855:Equilenin