Lipid Hydroperoxide Cytotoxicity and Detoxification

描述（由申请人提供）：细胞中的不饱和膜脂面临物理或化学氧化损伤可能发生过氧化损伤，通常具有有害后果。脂质氢过氧化物（LOOHs），包括磷脂(PL)-和胆固醇(Ch)-衍生物，是此类反应中重要的非自由基中间体。一旦形成，looh可能会有各种各样的命运，影响目标细胞的生存能力。新生的LOOH可能经过铁催化的单电子还原成自由基，从而引发破坏性（增强毒性）的链过氧化反应。或者，硒过氧化物酶（GPX4）催化的looh解毒可能发生，即双电子还原为相对无害的醇。在研究这些过程的过程中，我们发现了一个新的途径，扩大了LOOH的动态，即膜间易位。拟议的研究将更深入地研究这一现象，并考虑到两个假设：(i)在细胞内和细胞间，looh在膜之间的移动速度比亲本脂质更快，并且这种移动被转移蛋白增强。（ii）受体位点上氧化还原铁或GPX4的可用性将决定易位是扩大还是减弱LOOH细胞毒性。该研究将利用GPX4缺失的COH-BR1乳腺肿瘤细胞，其转染物过表达线粒体或非线粒体GPX4，转染物过表达非特异性脂质转移蛋白（SCP-2）。将采用高灵敏度/特异性的检测LOOHs和其他氧化产物的技术，即HPLC-EC（Hg）和HPTLC-PI，这两种技术都是在本实验室开发的。这些假设将通过研究(i)从肿瘤细胞中分离的质膜（PM）和线粒体（Mito）之间自发的和scp -2促进的LOOH转移来验证；（ii） PM和Mito中转移获得的LOOHs的单电子与双电子反应性；（iii） SCP-2过表达对亚细胞LOOH运输和细胞毒性的影响；（iv） SCP-2和GPX4共同过表达对LOOH解毒的可能协同作用。通过探索LOOH生物活性的新维度，这些研究将为这些物种的细胞病理学作用提供有价值的新见解。

DESCRIPTION (provided by applicant): Unsaturated membrane lipids in cells confronted with physical or chemical oxidative insults may undergo peroxidative damage, often with deleterious consequences. Lipid hydroperoxides (LOOHs), including phospholipid (PL)- and cholesterol (Ch)-derived species, are important non-radical intermediates in such reactions. Once formed, LOOHs can have a variety of fates which impact on the viability of a targeted cell. A nascent LOOH may undergo iron-catalyzed one-electron reduction to free radical species, which trigger damaging (toxicity-enhancing) chain peroxidation reactions. Alternatively, selenoperoxidase (GPX4)-catalyzed detoxification of LOOHs may occur, i.e. two-electron reduction to relatively innocuous alcohols. While studying these processes, we have identified a new pathway, which expands the LOOH dynamic, viz. intermembrane translocation. The proposed research will delve more deeply into this phenomenon with two hypotheses in mind: (i) LOOHs move between membranes more rapidly than parent lipids, both intra- and intercellularly, and this is enhanced by transfer proteins. (ii) Availability of redox iron or GPX4 at acceptor sites will determine whether translocation expands or attenuates LOOH cytotoxicity. The studies will utilize GPX4-null COH-BR1 breast tumor cells, transfectants thereof overexpressing either mitochondrial or non-mitochondrial GPX4, and transfectants overexpressing non-specific lipid transfer protein (SCP-2). High sensitivity/specificity techniques for detecting LOOHs and other oxidation products will be employed, viz. HPLC-EC(Hg) and HPTLC-PI, both of which were developed in this laboratory. The hypotheses will be tested by studying (i) spontaneous and SCP-2-facilitated LOOH transfer between plasma membranes (PM) and mitochondria (Mito) isolated from tumor cells; (ii) one-electron vs. two-electron reactivity of transfer-acquired LOOHs in PM and Mito; (iii) effects of SCP-2 overexpression on subcellular LOOH trafficking and cytotoxicity; and (iv) possible cooperative effects of SCP-2 and GPX4 co-overexpression on LOOH detoxification. By exploring this new dimension in LOOH bioreactivity, these studies will provide valuable new insights into the cytopathological effects of these species.