Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase

Jiaqin Yao, Yan Li, Minsun Chang, Huaping Wu, Xiaofeng Yang, Julie E. Goodman, Xuemei Liu, Hong Liu, Andrew D. Mesecar, Richard B. Van Breemen, James D. Yager, Judy L. Bolton

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


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 Km of 2.4 μM and kcat 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 Ki of 26.0 μM and a k2 of 1.62 × 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.

Original languageEnglish (US)
Pages (from-to)668-675
Number of pages8
JournalChemical research in toxicology
Issue number5
StatePublished - May 1 2003

ASJC Scopus subject areas

  • Toxicology


Dive into the research topics of 'Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase'. Together they form a unique fingerprint.

Cite this