Protection of Oxidant Toxicity by Glutathione S Transferases

描述（由申请人提供）：许多外源药物的毒性与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（ggpxs）通过减少脂质氢过氧化物来提供抗LPO的保护。的吗?-类谷胱甘肽s转移酶(？-GSTs)也显示GPx对脂质氢过氧化物的活性，但这种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活性原位还原膜磷脂氢过氧化物（PL-OOH）。这些酶在K562细胞中的过度表达可减弱氧化剂（H202或外源）诱导的LPO和细胞毒性。另一个人的过度表达？- gst， hGSTA4-4通过与谷胱甘肽偶联来解毒LPO的最终产物，4-羟基壬烯醛（4- hne），也可以防止氧化毒性。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来保护细胞免受氧化剂/异种生物的毒性。为了验证这一假设，提出以下具体目标：的生理意义？-GSTs， GSTA1-1和GSTA2-2将通过测定它们对LPO产物，PL-OOH和4-HNE的动力学性质来研究。他们在大鼠和小鼠肝脏中减少PL-OOH的贡献将被确定并与硒GPxs进行比较。我们将研究过度表达这些酶的细胞是否能保护其免受H202或氧化外源药物（如阿霉素、CCI4）诱导的c-Jun n -末端激酶（JNK）、半胱天冬酶3的激活和随后的凋亡。2. 细胞将被转染？在转染细胞和对照细胞中比较-GST同工酶、解毒4-HNE的GSTA4-4以及诱导LPO的H202和氧化外源物的毒性。我们将在对照和转染细胞中比较外源药物、H202、黄嘌呤/氧化酶、DOX诱导的毒性和凋亡，以阐明4-HNE和GSTA4-4在氧化应激介导的信号传导中的作用。3. 我们将研究GSTA4-4敲除小鼠中氧化剂的毒性是否因为它们无法解毒4-HNE而增强。由于gst可由无毒微量营养素诱导，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，后者导致与年龄有关的退行性疾病。

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.