OXIDANT MECHANISMS IN DRUG INDUCED HEPATIC NECROSIS

活性氧物种和自由基被认为是导致 与许多人类疾病相关的许多不良反应 和毒物。细胞或组织暴露在活性氧物种中 通常以谷胱甘肽二硫化物的形成为特征 蛋白质混合二硫化物，但氧化细胞损伤似乎更多 与化学催化的氧化类型密切相关 活性铁络合物。尽管铁对人体健康是绝对必要的 一系列生物功能，获得这种铁是可用的 受酶或贮藏蛋白质的限制。损失 因此，铁有效性的动态平衡控制可能是一个关键 参与了由活性氧引起的组织损伤。近期 研究表明，活性氧介导的肝坏死导致 通过对Fischer-344大鼠的敌草快和Spraogue-Dawley大鼠的抵抗力， 可能是铁代谢控制不同的结果。这个 Fischer-344大鼠比Spraogue-Dawley大鼠对 高氧性肺损伤与肺内非血红素铁的再分布 高氧暴露的Fischer大鼠进一步表明， 这些动物模型揭示了活性氧的基本机制-- 介导性组织损伤。目前所描述的实验 应用程序还将检查谷胱甘肽还原酶的贡献 通过对细胞中的抗氧化防御机制的研究 反义靶向转染法抑制还原酶活性 人类谷胱甘肽还原酶基因。拟议的研究还包括 将检验硫醇/二硫化班次被隔间的假设 在体内线粒体导致氧化细胞损伤，采用 辅酶A及其与谷胱甘肽混合二硫化物的测定 (CoASSG)在冷冻夹住的组织中在酸中处理以最大限度地减少 在锻炼过程中人为的氧化还原转移。因为谷胱甘肽- 阻止蛋白质和肝脏烷基化的依赖剂量阈值 治疗剂量的对乙酰氨基酚造成的伤害可能与 对乙酰氨基酚体内与DNA的共价结合--DNA的烷基化 将检查低剂量给药，以及化学相互作用 与N-乙酰-对苯二酚亚胺的DNA，许多研究人员认为 是扑热息痛的代谢物，对细胞损伤负责 通过烷基化，也可能通过氧化。了解 对生物分子负责的因素，以及生化和 各自变化的病理生理学后果代表 生物医学研究的重要基本目标。这些是宽阔的， 本申请中描述的研究的长期目标。

Reactive oxygen species and free radicals are thought to contribute to many of the adverse effects associated with a number of human diseases and toxicities. Exposure of cells or tissues to reactive oxygen species often is characterized by the formation of glutathione disulfide and protein mixed disulfides, but oxidant cell damage appears to be more closely associated with the types of oxidations catalyzed by chemically reactive iron chelates. Although iron is absolutely essential for a wide range of biological functions, access to this iron is availability restricted by containment in enzymes or in storage proteins. Loss of homeostatic control of iron availability thus may represent a pivotal step in the initiation of tissue damage by reactive oxygen. Recent studies suggest that the reactive oxygen-mediated hepatic necrosis caused by diquat in Fischer-344 rats, and the resistance of Sprague-Dawley rats, may be a result of differences in control of iron metabolism. The greater sensitivity of Fischer-344 rats than of Sprague-Dawley rats to hyperoxic lung injury and the redistribution of lung nonheme iron in the hyperoxia-exposed Fischer rats further suggests the broader relevance of these animal models to the fundamental mechanisms of reactive oxygen- mediated tissue injury. The experiments described in the present application also will examine the contributions of glutathione reductase to antioxidant defense mechanisms through studies in cells in which reductase activity has been inhibited by antisense-oriented transfection of the human gene for glutathione reductase. The proposed studies also will test the hypothesis that thiol/disulfied shifts compartmentalized to the mitochondria contribute to oxidant cell injury in vivo, employing measurements of coenzyme A and its mixed disulfide with glutathione (CoASSG) in freeze-clamped tissues worked up in acid to minimize artifactual redox shifts during workup. Because the glutathione- dependent dose-threshold that prevents alkylation of protein and hepatic injury by therapeutic doses of acetaminophen may not be relevant to alkylation of DNA, the covalent binding to DNA in vivo of acetaminophen given at low doses will be examined, as will the chemical interactions with DNA of N-acetyl-p-benzoquinone imine, thought by many investigators to be a metabolite of acetaminophen responsible for cellular damage through alkylation and possibly through oxidation. Understanding the factors responsible for biological molecules, and the biochemical and pathophysiological consequences of the respective alterations represent important fundamental goal of biomedical research. These are the broad, long-term goals of the research described in the present application.