Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones.

Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen ortho-quinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO1) stems from earlier reports. In this investigation, we were successful in circumventing the problem of nonenzymatic reduction of estrogen quinone by NAD(P)H, which led to the above conclusion, and for the first time we show that NQO1 catalyzes the reduction of estrogen quinones. Mass spectrometric binding studies involving estradiol-3,4-quinone or menadione with NQO1 clearly support the formation of an enzyme-substrate physical complex. However, the NQO1 mass spectrum did not alter after addition of cholesterol, the control. Two different strategies were employed to ascertain the NQO1 activity in estrogen quinone reduction. First, the ping-pong mechanism of NQO1 catalysis was utilized to overcome the problem of nonenzymatic reduction of the substrate by NADH. Second, tetrahydrofolic acid, which has a lower reducing potential, was used as an alternate cofactor. Both of these methods confirmed the reduction of estradiol-3,4-quinone by NQO1, when the assay mixtures were analyzed by UV or liquid chromatography-mass spectrometry. Furthermore, reduction of 9,10-phenanthrene quinone or menadione was observed using the reported assay conditions. Thus, clear evidence for the catalytic reduction of estrogen ortho-quinones by NQO1 has been obtained; its mechanism and implications are discussed.