title: Effect of halogenated substituents on the metabolism and estrogenic effects of the equine estrogen, equilenin.
abstract: Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors alpha and beta, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17beta-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17beta-equilenin. Because 17beta-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated equilenin derivatives in vitro, we studied the relative ability of the 17beta-hydroxy-4-halogenated equilenin derivatives to induce estrogenic effects in the ovariectomized rat model. The 4-fluoro derivative showed higher activity than 4-chloro and 4-bromo derivatives as demonstrated by inducing higher vaginal cellular differentiation, uterine growth, and mammary gland branching. However, 17beta-hydroxy-4-fluoroequilenin showed a lower estrogenic activity than 17beta-hydroxyequilenin and estradiol, which could be due to alternative pharmacokinetic properties for these compounds. These data suggest that the 4-fluoroequilenin derivatives have promise as alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street (M/C 877), Chicago, Illinois 60612-7231, USA.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Fagen
authors.initials: F
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Joanna E
authors.initials: JE
authors.lastname: Burdette
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Cassia R
authors.initials: CR
authors.lastname: Overk
authors.forename: Emily
authors.initials: E
authors.lastname: Pisha
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Yao
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: Steven M
authors.initials: SM
authors.lastname: Swanson
authors.forename: Judy L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004855:Equilenin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000633:toxicity*; D030762:Estrous Cycle / Q000187:drug effects; D005260:Female; D006219:Halogens / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000473:pathology; D009929:Organ Size / Q000187:drug effects; D010052:Ovariectomy; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011960:Receptors, Estrogen / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014599:Uterus / Q000187:drug effects / Q000473:pathology; D014621:Vagina / Q000187:drug effects / Q000473:pathology
chemical_list: D006219:Halogens; D012333:RNA, Messenger; D011960:Receptors, Estrogen; D000469:Alkaline Phosphatase; D004855:Equilenin