title: Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase.
abstract: Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a K(m) of 2.4 microM and k(cat) of 6.0 min(-)(1) but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous catechol estrogen 4-hydroxyestradiol with a K(i) of 26.0 microM and a k(2) of 1.62 x 10(-)(2) s(-)(1). 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous catechol estrogen clearance in vivo and further enhance toxicity.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy (M/C 781), College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, 60612, USA.
authors.forename: Jiaqin
authors.initials: J
authors.lastname: Yao
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Minsun
authors.initials: M
authors.lastname: Chang
authors.forename: Huaping
authors.initials: H
authors.lastname: Wu
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Yang
authors.forename: Julie E
authors.initials: JE
authors.lastname: Goodman
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Mesecar
authors.forename: Richard B
authors.initials: RB
authors.lastname: Van Breemen
authors.forename: James D
authors.initials: JD
authors.lastname: Yager
authors.forename: Judy L
authors.initials: JL
authors.lastname: Bolton
mesh_terms: D002394:Catechol O-Methyltransferase / Q000737:chemistry; D065098:Catechol O-Methyltransferase Inhibitors; D002851:Chromatography, High Pressure Liquid; D004855:Equilenin / Q000031:analogs & derivatives* / Q000737:chemistry*; D045166:Estradiol Congeners / Q000737:chemistry*; D002393:Estrogens, Catechol / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008745:Methylation; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D065098:Catechol O-Methyltransferase Inhibitors; D045166:Estradiol Congeners; D002393:Estrogens, Catechol; D011994:Recombinant Proteins; C065250:4-hydroxy-equilenin; D002394:Catechol O-Methyltransferase; D004855:Equilenin