Lipid Hydroperoxide Cytotoxicity and Detoxification

描述(申请人提供)：面对物理或化学氧化损伤的细胞中的不饱和膜脂可能会发生过氧化损伤，通常会造成有害的后果。脂质过氧化氢(LOOHs)，包括磷脂(PL)和胆固醇(CH)衍生的物种，是这类反应中重要的非自由基中间体。一旦形成，LOOHs可以有多种命运，这些命运影响靶细胞的生存能力。新生的LOOH可能会发生铁催化的单电子还原为自由基物种，从而引发破坏性(毒性增强)的链过氧化反应。或者，也可以发生硒过氧化物酶(Gpx4)催化的LOOHs解毒，即两电子还原为相对无害的醇。在研究这些过程时，我们发现了一条新的途径，它扩展了LOOH动力学，即。膜间易位。这项拟议的研究将在考虑到两个假设的情况下更深入地研究这一现象：(I)LOOHs在膜之间的移动速度比亲体脂更快，无论是在细胞内还是在细胞间，这一点都得到了转移蛋白的增强。(Ii)氧化还原铁或Gpx4在受体部位的可用性将决定易位是扩大还是减弱LOOH细胞毒性。这些研究将利用Gpx4缺失的COH-BR1乳腺肿瘤细胞，其过表达线粒体或非线粒体Gpx4的转染体，以及过表达非特异性脂转移蛋白(SCP-2)的转染体。将采用检测LOOHs和其他氧化产物的高灵敏度/特异性技术，即。高效液相色谱-EC(汞)和HPTLC-PI，均为本实验室研制。这些假说将通过研究(I)从肿瘤细胞分离的质膜(PM)和线粒体(MITO)之间的自发和SCP-2促进的LOOH转移；(Ii)转移获得的PM和MITO中LOOH的单电子和双电子反应性；(Iii)SCP-2过表达对亚细胞LOOH运输和细胞毒性的影响；以及(Iv)SCP-2和Gpx4过表达在LOOH解毒方面的可能协同作用。通过探索LOOH生物反应性的这一新维度，这些研究将为这些物种的细胞病理效应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.