REDOX INTERACTIONS OF DT DIAPHORASE AND VITAMIN E

NAD(P)H：苯醌氧化还原酶(NQO1，EC 1.699.2，DT-黄递酶)是一种普遍存在的酶，它对多种苯醌底物进行2电子还原，并在抗氧化反应中被诱导。尽管NQO1的“生理”底物尚不清楚，但我们最近观察到，NQO1将内源性α-生育酚(AlphaTH，维生素E)氧化产物α-生育酚(TQ)还原为α-生育酚氢醌(TQH2)，后者又能从α-生育酚自由基中再生出α-生育酚。AlphTH再生系统被认为是细胞抗氧化保护的重要因素。我们假设NQO1的一个基本功能是为αTh的氧化还原循环提供还原等价物，从而维持细胞抗氧化保护，防止膜中氧化损伤的传播。这种作用可能涉及NQO1本身或TQH2对单电子氧化生育氧自由基或两电子氧化生育酚的还原。为了验证这些假设，我们提出：目的1)表征TQH2和NQO1在单电子αTH氧化还原循环中的参与。生育氧自由基经NQO1或TQH2直接还原为αTH的研究将在脂质体和膜系统中进行。目的2)研究TQH2和NQO1在双电子αTh氧化还原循环中的作用。直接或通过TQH2将8a-取代生育酚还原为α-TH将在脂质体和膜模型中进行研究。目的3)研究NQO1对正常细胞αTh氧化还原状态和抗氧化剂代谢的影响。这些研究将使用表达NQO1多态的CHO细胞和人类细胞系。αTH及其氧化产物的水平和分布将作为NQO1活性的函数在受到氧化应激的细胞中进行测量。这些研究将阐明NQO1的内源性抗氧化作用，并确定自然界最普遍的膜抗氧化剂AlphaTH的抗氧化化学是如何与细胞代谢氧化还原平衡联系在一起的。对这些问题的回答对于提高我们对细胞抗氧化剂防御之间的相互作用及其在人类健康和疾病中的作用的理解至关重要。

NAD(P)H:quinone oxidoreductase (NQO1, EC 1.699.2, DT-diaphorase) is a ubiquitous enzyme that carries out 2-electron reduction of numerous quinone substrates and is induced in antioxidant responses. Although a "physiologic" substrate for NQO1 had been obscure, we observed recently that NQO1 reduces the endogenous alpha-tocopherol (alphaTH, vitamin E) oxidation product alpha- tocopherolquinone (TQ) to alpha-tocopherolhydroquinone (TQH2), which can, in turn, regenerate alphaTH from the alpha- tocopheroxyl radical. An alphTH regenerating system is thought to be an essential contributor to cellular antioxidant protection. We hypothesize that an essential function of NQO1 is to supply reducing equivalents to redox cycles of alphaTh, thus sustaining cellular antioxidant protection against propagation of oxidative damage in membranes. This action may involve either reduction of one-electron oxidized tocopheroxyl radicals or 2- electron oxidized tocopherones either by NQO1 itself or by TQH2. To test these hypotheses, we propose: Aim 1) To characterize the participation of TQH2 and NQO1 in a one-electron alphaTH redox cycle. Reduction of the tocopheroxyl radical to alphaTH either directly by NQO1 or via TQH2 will be studied in liposome and membrane systems. Aim 2) To characterize the participation of TQH2 and NQO1 in a two-electron alphaTh redox cycle. Reduction of 8a-substituted tocopherones to alphaTH either directly or via TQH2 will be studied in liposome and membrane models. Aim 3) To characterize the impact of NQO1 on alphaTh redox status and antioxidant turnover in intact cells. These studies will employ CHO cells and human cell lines expressing NQO1 polymorphisms. The levels and distribution of alphaTH and its oxidation products will be measured as a function of NQO1 activity in cells subjected to oxidative stress. These studies will clarify the endogenous antioxidant role of NQO1 and determine how the antioxidant chemistry of alphaTH, Nature's most ubiquitous membrane antioxidant, is tied to cellular metabolic redox balance. Answers to these questions are essential to improving our understanding of the interplay between cellular antioxidant defenses and their roles in human health and disease.