Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones
Evidence from ESI-MS for NQO1-catalyzed reduction of estrogen ortho-quinones
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DOI：
10.1016/j.freeradbiomed.2007.07.021
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Cavalieri, Ercole L.
中科院分区：
文献类型：
--
作者：
Gaikwad, Nilesh W.;Rogan, Eleanor G.;Cavalieri, Ercole L.
雌激素邻醌类被认为是雌激素的最终致癌代谢物。雌激素原醌类不是NAD（P）H：醌氧化还原酶（NQO 1）的底物的结论源于早期的报道。在这项研究中，我们成功地规避了NAD（P）H非酶还原雌激素醌的问题，从而得出上述结论，并首次表明NQO 1催化雌激素醌的还原。涉及雌二醇-3，4-醌或甲萘醌与NQO 1的质谱结合研究清楚地支持酶-底物物理复合物的形成。然而，NQO 1质谱在添加胆固醇（对照）后没有改变。采用两种不同的策略来确定NQO 1在雌激素醌还原中的活性。首先，利用NQO 1催化的乒乓机制来克服底物被NADH非酶还原的问题。其次，四氢叶酸，具有较低的还原潜力，被用作替代辅因子。这两种方法证实了减少雌二醇-3，4-醌的NQO 1，当测定混合物进行了分析，通过UV或液相色谱-质谱法。此外，使用报告的测定条件观察到9，10-菲醌或甲萘醌的还原。因此，明确的证据表明，雌激素邻醌的催化还原NQO 1已获得，其机制和影响进行了讨论。(C)2007爱思唯尔公司All rights reserved.
Estrogen ortho-quinones have been implicated as ultimate carcinogenic metabolites of estrogens. The present conclusion that estrogen orthoquinones are not substrates for NAD(P)H:quinone oxidoreductase (NQO 1) 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. (C) 2007 Elsevier Inc. All rights reserved.