Unexpected hormonal activity of a catechol equine estrogen metabolite reveals reversible glutathione conjugation.
4-Hydroxyequilenin (4-OHEN) is a major phase I metabolite of the equine estrogens present in widely prescribed hormone replacement formulations. 4-OHEN is autoxidized to an electrophilic o-quinone that has been shown to redox cycle, generating ROS, and to covalently modify proteins and DNA and thus potentially to act as a chemical carcinogen. To establish the ability of 4-OHEN to act as a hormonal carcinogen at the estrogen receptor (ER), estrogen responsive gene expression and proliferation were studied in ER(+) breast cancer cells. Recruitment by 4-OHEN of ER to estrogen responsive elements (ERE) of DNA in MCF-7 cells was also studied and observed. 4-OHEN was a potent estrogen, with additional weak activity associated with binding to the arylhydrocarbon receptor (AhR). The potency of 4-OHEN toward classical ERalpha mediated activity was unexpected given the reported rapid autoxidation and trapping of the resultant quinone by GSH. Addition of thiols to cell cultures did not attenuate the estrogenic activity of 4-OHEN, and preformed thiol conjugates added to cell incubations only marginally reduced ERE-luciferase induction. On reaction of the 4OHEN-GSH conjugate with NADPH, 4-OHEN was observed to be regenerated at a rate dependent upon NADPH concentration, indicating that intracellular nonenzymatic and enzymatic regeneration of 4-OHEN accounts for the observed estrogenic activity of 4-OHEN. 4-OHEN is therefore capable of inducing chemical and hormonal pathways that may contribute to estrogen-dependent carcinogenesis, and trapping by cellular thiols does not provide a mechanism of termination of these pathways.
Peng
Kuan-Wei
KW
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612-7231, USA.
Chang
Minsun
M
Wang
Yue-Ting
YT
Wang
Zhican
Z
Qin
Zhihui
Z
Bolton
Judy L
JL
Thatcher
Gregory R J
GR
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D004273:DNA, Neoplasm / Q000187:drug effects; D004855:Equilenin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D006801:Humans; D008024:Ligands; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D011960:Receptors, Estrogen / Q000819:agonists / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D008024:Ligands; D011960:Receptors, Estrogen; D013438:Sulfhydryl Compounds; D009249:NADP; C065250:4-hydroxy-equilenin; D005978:Glutathione; D004855:Equilenin