Protection of Oxidant Toxicity by Glutathione S Transferases

描述（由申请方提供）：许多外源性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，已知仅谷胱甘肽过氧化物酶（GPx）通过减少脂质氢过氧化物来提供针对LPO的保护。什么？-类谷胱甘肽S-转移酶（？- GST）也显示出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hGSTA 1 -1和hGSTA 2 -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细胞中的过表达减弱了氧化剂（H2 O2或异生素）诱导的LPO和细胞毒性。另一个人的过度表达？GST，hGSTA 4 -4，其通过将LPO的终产物4-羟基壬烯醛（4-HNE）缀合至谷胱甘肽而使其解毒，也保护免受氧化剂毒性。我们假设GST提供了对抗ROS的第二道防线，并作为抗氧化酶，通过减弱LPO保护细胞免受氧化剂/外源性物质的毒性。提出了以下具体目标来检验这一假设：1。的生理意义？-将通过测定GST、GSTA 1 -1和GSTA 2 -2对LPO产物、PL-OOH和4-HNE的动力学性质来研究它们。将测定它们在大鼠和小鼠肝脏中PL-OOH减少中的贡献，并与硒GPx的贡献进行比较。我们将检查过表达这些酶的细胞是否被保护免受H2 O2或氧化剂异生物质（例如，多柔比星（doxorubucin，CCI 4）诱导c-Jun N-末端激酶（JNK）、半胱天冬酶3的活化和随后的细胞凋亡。2.细胞将被转染？-将在转染细胞和对照细胞中比较GST同工酶GSTA 4 -4（其使4-HNE解毒）和H2 O2和氧化剂异生物质（其诱导LPO）的毒性。将在对照和转染的细胞中比较外源性物质、H2 O2、黄嘌呤/氧化酶、DOX诱导的毒性和细胞凋亡，以描述4-HNE和GSTA 4 -4在氧化应激介导的信号传导中的作用。3.我们将检查是否氧化剂的毒性在GSTA 4 -4基因敲除小鼠中增强，因为它们不能解毒4-HNE。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，以消除环境化学品和慢性氧化应激的毒性，从而导致与年龄相关的退行性疾病。

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.