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

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 μM 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 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.