Protection of Oxidant Toxicity By GSTs

GST 保护氧化剂毒性

• 批准号: 7742093
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 44.3万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-21 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7742093
• 关键词: 4 hydroxynonenal; Affect; Animals; Antioxidants; Apoptosis; Apoptotic; Attenuated; Binding; Binding Sites; CD95 Antigens; CYP2E1 gene; Cell Line; Cell Nucleus; Cell model; Cells; Cessation of life; Chemicals; Clinical; Complex; Cytoplasm; Data; Degenerative Disorder; Doxorubicin; Drug Metabolic Detoxication; Drug toxicity; Embryo; Enzymes; Excision; Experimental Designs; Exposure to; Fibroblasts; Fourier transform ion cyclotron resonance; Funding; Gene Targeting; Glutathione; Glutathione S-Transferase; Hand; In Situ; In Vitro; Intraperitoneal Injections; Knockout Mice; Lipid Peroxidation; Lipid Peroxides; Liposomes; Liver; Mediating; Methods; Modeling; Mus; Nature; Necrosis; Nuclear; Oxidants; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Prevention; Prevention strategy; Process; Proteins; Recombinant Proteins; Recombinants; Recruitment Activity; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Stress; TP53 gene; Testing; Tissues; Toxic effect; Transcription Repressor/Corepressor; Transfection; Tumor Necrosis Factor Ligand Superfamily Member 6; Xenobiotics; base; impaired capacity; in vitro testing; in vivo; irradiation; novel; pro-caspase-8; proteoliposomes; public health relevance; reconstitution; trafficking

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.

描述（由申请人提供）：4-羟基壬烯醛（4-HNE）是脂质过氧化（LPO）的稳定终产物，可通过细胞凋亡和坏死引起毒性。在当前资助的年份中，我们已经确定 GSTA4-4 和 RLIP76 是 4-HNE 细胞内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，并通过一种新途径诱导 Fas 介导的细胞凋亡，该途径不同于 Fas 与 FADD 结合并招募 procaspase8 组装死亡诱导信号复合物 (DISC) 以最终执行细胞凋亡的经典途径。 4-HNE 还与转录抑制因子 Daxx 结合，导致其从细胞核易位至细胞质，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标。由于暴露于氧化剂会导致 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，并且 GSTA4-4（催化 4-HNE 与 GSH 的结合）和 RLIP76（介导 GS-HNE 结合物的转运）的过度表达应通过降低细胞内 4-HNE 水平。我们的初步研究支持了这一假设：a) Fas 和 p53 介导的细胞凋亡受到 GSTA4-4 过表达的抑制。 b) 将 RLIP76 脂质体递送至 RLIP76 (-/-) 小鼠（对氧化剂毒性更敏感）可保护这些小鼠免受毒性。在具体目标 1 中，该假设将通过拟议的体外研究进行测试，使用经 Cyp2E1 和两种模型氧化剂化合物 CCl4 和阿霉素 (DOX) 转染的 HepG2 细胞。在这些研究中，我们还将描述 4-HNE 诱导、Fas 介导的细胞凋亡的机制以及 Daxx 和 GST 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，因此在特定目标 2 中提出了研究来描述 4-HNE 与这些蛋白质之间相互作用的性质和意义。在具体目标 3 中，我们假设 4-HNE 解毒能力受损的 RLIP76 (-/-) 小鼠对这些药物的毒性更加敏感，并且 RLIP76 脂蛋白体的给药应减弱这种毒性。这将通过拟议的体内研究进行测试，通过比较这些氧化剂在 RLIP76 (-/-) 和 RLIP76 (+/+) 小鼠中的毒性，无论是否用 RLIP76 蛋白脂质体治疗。在这些研究中，我们还将比较不同组小鼠肝脏中的 4-HNE 水平和细胞凋亡程度。除了提供关于氧化剂毒性机制和 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，本申请中提出的研究还应有助于制定针对药物和异生物质毒性的预防策略，这是一个主要的临床和环境问题。公共健康相关性：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减轻环境氧化剂和药物的毒性以及预防和治疗氧化应激引起的退行性疾病的策略。