Protection of Oxidant Toxicity by Glutathione S Transferases

谷胱甘肽 S 转移酶对氧化剂毒性的保护

• 批准号: 7337306
• 负责人: YOGESH Chandra AWASTHI
• 金额: $ 25.45万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2009-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7337306
• 关键词: 4 hydroxynonenal; Acrolein; Antibodies; Antioxidants; Apoptosis; Apoptotic; Attenuated; Biological; Biological Assay; CYP2E1 gene; Cell Line; Cells; Chronic; Class; Condition; DNA; DNA Binding; DNA Damage; Degenerative Disorder; Doxorubicin; Enzymes; Ephrin-A5; Erythrocyte Membrane; Fibroblasts; Glutathione; Glutathione S-Transferase; HL-60 Cells; Human; Human Cell Line; Hydrogen Peroxide; In Situ; In Situ Nick-End Labeling; Injury; Iron Overload; Isoenzymes; JUN gene; K-562; K562 Cells; Kinetics; Knock-out; Knockout Mice; Laboratories; Lipid Peroxidation; Lipid Peroxides; Liver; Measures; Mediating; Membrane; Metabolism; Methods; Micronutrients; Modeling; Mus; Naphthalene; Naphthalenes; Necrosis; Orthologous Gene; Oxidants; Oxidative Stress; Pathology; Phospholipids; Phosphorylation; Physiological; Property; Protein Overexpression; Proteins; Rat Cell Line; Rattus; Reactive Oxygen Species; Recombinants; Relative (related person); Rodent; Role; Signal Pathway; Signal Transduction; Specific qualifier value; Subgroup; Superoxide Dismutase; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Testis; Tissues; Toxic effect; Transcription Factor AP-1; Transfection; Xanthine Oxidase; Xanthines; Xenobiotics; adduct; age related; caspase-3; cytotoxicity; environmental chemical; glutathione peroxidase; hGSTA4; in vivo; mGSTA4-4; research study; stress-activated protein kinase 1; xanthine

DESCRIPTION (provided by applicant): Toxicity of many xenobiotics has been associated with lipid peroxidation (LPO) caused by reactive oxygen species (ROS) generated during their metabolism. Among the antioxidant enzymes, only glutathione peroxidases (GPxs) are known to provide protection against LPO by reducing lipid hydroperoxides. The ?-class glutathione S-transferases (?-GSTs), also show GPx activity towards lipid hydroperoxides, but the physiological role of this activity is not understood. Our preliminary studies show that human hGSTA1-1 and hGSTA2-2 can reduce membrane phospholipid hydroperoxides (PL-OOH) in situ by their GPx activity. Over expression of these enzymes in K562 cells attenuates oxidant (H202 or xenobiotic) induced LPO and cytotoxicity. The over expression of another ?-GST, hGSTA4-4 which detoxifies the end- product of LPO, 4- hydroxynonenal (4-HNE), by conjugating it to glutathione, also protects against oxidant toxicity. We hypothesize that GSTs provide a second line of defense against ROS and act as antioxidant enzymes, which protect cells against toxicity of oxidants/xenobiotics by attenuating LPO. The following specific aims are proposed to test this hypothesis: 1. The physiological significance of the ?-GSTs, GSTA1-1 and GSTA2-2 will be studied by determining their kinetic properties towards LPO products, PL-OOH and 4-HNE. Their contributions in the reduction of PL-OOH in rat and mouse liver will be determined and compared with those of the seleno GPxs. We will examine if the cells over expressing these enzymes are protected against H202 or oxidant xenobiotics (e.g., doxorubucin, CCI4) induced activation of c-Jun N-terminal kinase (JNK), caspase 3, and subsequent apoptosis. 2. Cells will be transfected with the ?-GST isozyme, GSTA4-4, which detoxifies 4-HNE and the toxicity of H202 and oxidant xenobiotics which induce LPO will be compared in the transfected and control cells. Xenobiotics, H202, xanthine/oxidase, DOX induced toxicity and apoptosis will be compared in the control and transfected cells to delineate the role of 4-HNE and GSTA4-4 in oxidative stress mediated signaling. 3. We will examine whether the toxicity of oxidants is enhanced in GSTA4-4 knock out mice because of their inability to detoxify 4-HNE. Since GSTs can be induced by non-toxic micronutrients, these studies will help in devising strategies for negating the toxicity of environmental chemicals and chronic oxidative stress, which leads to age, related degenerative disorders.

描述（由申请人提供）：许多外源物质的毒性与其代谢过程中产生的活性氧（ROS）引起的脂质过氧化（LPO）有关。在抗氧化酶中，已知只有谷胱甘肽过氧化物酶 (GPx) 可以通过减少脂质氢过氧化物来提供针对 LPO 的保护。 β-类谷胱甘肽 S-转移酶 (β-GST) 也显示出针对脂质氢过氧化物的 GPx 活性，但该活性的生理作用尚不清楚。我们的初步研究表明，人 hGSTA1-1 和 hGSTA2-2 可以通过其 GPx 活性原位减少膜磷脂氢过氧化物 (PL-OOH)。这些酶在 K562 细胞中的过度表达可减弱氧化剂（H2O2 或异生素）诱导的 LPO 和细胞毒性。另一种α-GST，即hGSTA4-4的过度表达，通过将LPO的终产物4-羟基壬烯醛(4-HNE)与谷胱甘肽缀合来解毒，也可以防止氧化剂毒性。我们假设 GST 提供了针对 ROS 的第二道防线，并充当抗氧化酶，通过减弱 LPO 来保护细胞免受氧化剂/外源物质的毒性。提出以下具体目标来检验这一假设： 1. 将通过确定 α-GST、GSTA1-1 和 GSTA2-2 对 LPO 产物、PL-OOH 和 4-HNE 的动力学特性来研究它们的生理意义。将确定它们对减少大鼠和小鼠肝脏中 PL-OOH 的贡献，并与硒 GPx 进行比较。我们将检查过度表达这些酶的细胞是否受到保护，免受 H2O2 或氧化剂异生物质（例如阿霉素、CCI4）诱导的 c-Jun N 末端激酶 (JNK)、半胱天冬酶 3 的激活以及随后的细胞凋亡。 2.用α-GST同工酶GSTA4-4转染细胞，其解毒4-HNE，并且将在转染细胞和对照细胞中比较H 2 O 2 和诱导LPO的氧化剂异生物质的毒性。将在对照细胞和转染细胞中比较异生素、H2O2、黄嘌呤/氧化酶、DOX 诱导的毒性和细胞凋亡，以描绘 4-HNE 和 GSTA4-4 在氧化应激介导的信号传导中的作用。 3. 我们将检查GSTA4-4敲除小鼠中氧化剂的毒性是否因它们无法解毒4-HNE而增强。由于GST可以由无毒的微量营养素诱导，这些研究将有助于制定策略来消除环境化学物质的毒性和慢性氧化应激，从而导致与年龄相关的退行性疾病。