Glutamate-L-cysteine ligase expression and liver injury

描述（由申请人提供）：氧化应激涉及大量疾病的病理生理。这种应激不仅来自于正常的有氧代谢，也来自于外来化合物的代谢，并且是某些细胞类型释放活性氧的直接结果。生物体已经进化出抗氧化防御，包括抗氧化酶和抗氧化化合物的消耗。参与抗氧化防御的一个非常重要的酶是谷氨酸-半胱氨酸连接酶（GCL），它是细胞抗氧化剂谷胱甘肽（GSH）合成的限速酶。这个项目的主要目标是研究GCL在抵抗引起肝脏氧化损伤的物质和条件中的作用。我们拟建立GCL过表达转基因小鼠模型，并评估调节GCL表达对肝脏氧化损伤易感性的影响。我们建议使用三种已知引起氧化性肝损伤的药物，即对乙酰氨基酚、四氯化碳和肿瘤坏死因子- α。转基因和野生型（正常）幼崽将暴露于非致死剂量的这些制剂，并在6至48小时后牺牲。肝组织将被切除并检查氧化损伤、细胞坏死和凋亡的迹象，并对细胞活力和功能进行生化和细胞测量。这些信息将有助于确定GCL在防御活性氧和诱导氧化应激的异种生物中的功能意义，并有助于更好地了解人类GCL表达的变化意义。

DESCRIPTION (provided by applicant): Oxidative stress is involved the pathophysiology of a large number of diseases. This stress originates not only from normal aerobic metabolism, but also from the metabolism of foreign compounds, and as a direct result of the release of reactive oxygen species by certain cell types. Organisms have evolved antioxidant defenses against oxidative insults, which include antioxidant enzymes and through the consumption of antioxidant compounds. A very important enzyme involved in antioxidant defense is glutamate-cysteine ligase (GCL), the rate limiting enzyme for the synthesis of the cellular antioxidant glutathione (GSH). The primary goal of this project will be to investigate the role of GCL in defense against substances and conditions which induce oxidative damage to the liver. We propose to characterize a transgenic mouse model of GCL overexpression, and to assess the effects of modulating GCL expression on susceptibility to oxidant-induced damage to the liver. We propose to use three agents known to cause oxidative liver injury, namely acetaminophen, carbon tetrachioride, and tumor necrosis factor-alpha. Transgenic and wild-type (normal) littermates will be exposed to non-lethal doses of these agents, and sacrificed from 6 to 48 hours later. Liver tissue will be excised and examined for signs of oxidative damage, cellular necrosis and apoptosis, and biochemical and cellular measures of cell viability and function will be made. Such information will be useful in determining the functional significance of GCL in defense against reactive oxygen species and xenobiotics which induce oxidative stress, and lead to a better understanding of the significance of variable GCL expression in humans.