Protection of Oxidant Toxicity By GSTs

描述(申请人提供)：4-羟基壬烯醛(4-HNE)，脂质过氧化(LPO)的稳定最终产物，可通过细胞凋亡和坏死引起毒性。在当前的资助年中，我们已经确定GSTA4-4和RLIP76是细胞内4-HNE浓度的主要决定因素。我们的初步研究表明，4-HNE与死亡受体Fas结合，通过一种新的途径诱导Fas介导的细胞凋亡，不同于Fas与FADD结合并招募proaspase8组装死亡诱导信号复合体(DISC)以最终执行细胞凋亡的典型途径。4-HNE还与转录抑制因子Daxx结合，使其从细胞核转位到细胞质，从而影响Fas介导的细胞凋亡。此外，4-HNE促进P53的磷酸化和易位，并激活其促凋亡基因靶点。由于接触氧化剂会导致LPO，我们假设氧化剂的部分毒性是由4-HNE的促凋亡作用造成的，GSTA4-4(催化4-HNE与GSH的结合)和RLIP76(介导GS-HNE结合物的运输)的过度表达应该通过降低细胞内4-HNE的水平来减轻氧化剂诱导的毒性。我们的初步研究表明：a)GSTA4-4的过表达抑制了Fas和P53介导的细胞凋亡。B)RLIP76脂质体给RLIP76(-/-)小鼠(对氧化毒性更敏感)可保护这些小鼠免受毒性。在具体目标1中，这一假设将通过建议的体外研究来验证，该研究使用了转染了CYP2E1和两种模型氧化剂化合物CCL4和阿霉素(DOX)的HepG2细胞。在这些研究中，我们还将阐明4-HNE诱导、Fas介导的细胞凋亡的机制以及Daxx和GSTs的调节作用。由于我们的初步研究表明，4-HNE与Fas、Daxx和P53结合，并影响它们的功能和/或细胞定位，因此建议在特定目标2中进行研究，以描述4-HNE与这些蛋白相互作用的性质和意义。在具体目标3中，我们假设对4-HNE解毒能力受损的RLIP76(-/-)小鼠对这些药物的毒性更敏感，给予RLIP76蛋白脂质体应可减轻这种毒性。这一点将通过拟议的体内研究进行验证，方法是比较这些氧化剂在RLIP76(-/-)和RLIP76(+/+)小鼠中的毒性，并分别使用和不使用RLIP76蛋白脂体进行治疗。在这些研究中，我们还将比较不同组小鼠肝脏中4-HNE水平和细胞凋亡程度。除了提供有关氧化剂毒性机制和4-HNE在氧化应激相关退行性疾病中的作用的基本新信息外，这项申请中提出的研究还应有助于制定预防药物和异生物质毒性的策略，这是一个主要的临床和环境问题。公共卫生相关性：关于谷胱甘肽转移酶和RLIP76对氧化剂毒性的保护作用的拟议研究应有助于制定战略，以减轻环境氧化剂和药物的毒性，并预防和治疗氧化应激诱导的退行性疾病。

DESCRIPTION (provided by applicant): 4-Hydroxynonenal (4-HNE), a stable end-product of lipid peroxidation (LPO) can cause toxicity through apoptosis and necrosis. During current funded years, we have established that GSTA4-4 and RLIP76 are the major determinants of the intracellular concentrations of 4-HNE. Our preliminary studies show that 4-HNE binds to the death receptor Fas and induces Fas-mediated apoptosis through a novel pathway that is distinct from the canonical pathway in which Fas binds to FADD and recruits procaspase8 to assemble the death inducing signaling complex (DISC) for eventual execution of apoptosis. 4-HNE also binds to Daxx, a transcription repressor and causes its translocation from nucleus to cytoplasm to affect Fas-mediated apoptosis. In addition, 4-HNE promotes phosphorylation and translocation of p53 and activates its pro-apoptotic gene targets. Since exposure to oxidants causes LPO, we hypothesize that part of toxicity of oxidants is contributed by the pro-apoptotic effects of 4-HNE and that the over expression of GSTA4-4 (which catalyzes conjugation of 4-HNE to GSH) and RLIP76 (which mediates transport of GS- HNE conjugate) should attenuate the oxidant-induced toxicity by lowering the intracellular levels of 4-HNE. This hypothesis is supported by our preliminary studies showing that: a) Fas- and p53-mediated apoptosis is inhibited by the over-expression of GSTA4-4. b) Liposomal delivery of RLIP76 to RLIP76 (-/-) mice (which are more sensitive to oxidant toxicity) protects these mice from toxicity. In Specific Aim 1 this hypothesis will be tested through proposed in-vitro studies using HepG2 cells transfected with Cyp2E1 and two model oxidant compounds, CCl4, and doxorubicin (DOX). In these studies we will also delineate the mechanisms of 4-HNE induced, Fas- mediated apoptosis and the regulatory role of Daxx and GSTs. Since our preliminary studies show that 4-HNE binds to Fas, Daxx, and p53, and affects their functions and/or cellular localization, studies are proposed in Specific Aim 2 to delineate the nature and significance of the interactions between 4-HNE and these proteins. In Specific Aim 3, we hypothesize that RLIP76 (-/-) mice with impaired capacity to detoxify 4-HNE would be more sensitive to the toxicity of these agents and administration of RLIP76 proteoliposomes should attenuate this toxicity. This will be tested through proposed in vivo studies by comparing the toxicity of these oxidants in RLIP76 (-/-) and RLIP76 (+/+) mice, with and without treatment with RLIP76 proteolipsomes. In these studies we will also compare the 4-HNE levels and the extent of apoptosis in the liver of different groups of mice. Besides providing fundamental novel information on the mechanisms of oxidant toxicity and the role of 4-HNE in oxidative stress associated degenerative disorders, studies proposed in this application should also help to develop prevention strategies against the toxicity of drugs and xenobiotics which is a major clinical and environmental problem. PUBLIC HEALTH RELEVANCE: Proposed studies on the protective role of glutathione transferases and RLIP76 against oxidant toxicity should help in developing strategies for alleviating the toxicity of environmental oxidants and drugs and also for the prevention and treatment of oxidative stress-induced degenerative diseases.