Protection of Oxidant Toxicity by Glutathione S Transferases

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物种(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)通过减少脂质过氧化氢来保护LPO。谷胱甘肽S转移酶(？-GSTs)对脂质过氧化也具有GPx活性，但其生理作用尚不清楚。我们的初步研究表明，hGSTA1-1和hGSTA2-2可通过其GPx活性原位降低膜磷脂过氧化产物(PL-OHO)。这些酶在K562细胞中的过表达可以减轻氧化剂(H202或异种生物)诱导的LPO和细胞毒性。另一种？-GST，hGSTA4-4的过度表达，通过将其与谷胱甘肽结合来解毒LPO的最终产物4-羟基壬烯醛(4-HNE)，也可以保护其免受氧化毒性。我们假设GSTs提供了抵抗ROS的第二道防线，并作为抗氧化酶发挥作用，通过减弱LPO来保护细胞免受氧化剂/外源物质的毒性。1.通过测定β-GSTs、GSTA1-1和GSTA2-2对LPO产物PL-OOH和4-HNE的动力学性质，研究它们的生理意义。将测定它们在降低大鼠和小鼠肝脏中PL-OH值方面的贡献，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H202或氧化剂外源物质(如多柔比星，CCI4)诱导的c-jun氨基末端激酶(JNK)、半胱氨酸天冬氨酸蛋白酶3(Caspase 3)激活和随后的凋亡的保护。2.将解毒4-HNE的β-GST同工酶GSTA4-4导入细胞，比较H202和氧化性外源物质对细胞的毒性。通过比较外源物质、H202、黄嘌呤/氧化酶、DOX诱导的细胞毒性和细胞凋亡来阐明4-HNE和GSTA4-4在氧化应激介导的信号转导中的作用。3.我们将检查GSTA4-4基因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，慢性氧化应激会导致年龄和相关的退行性疾病。

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.