Role of Glutathione Redox Status in Hepatotoxicity

描述（申请人提供）：肿瘤坏死因子（Tumor necrosis factor， TNF）在肝损伤中起重要作用。正常肝细胞对TNF细胞毒性具有抗性。然而，我们最近发现，线粒体外谷胱甘肽的选择性消耗对TNF诱导的凋亡敏感，这伴随着细胞质中持续的c-jun- n末端激酶（INK）激活和细胞核中NF-kB转激活受损。此外，我们发现抑制JNK对体内和体外对乙酰氨基酚（APAP）诱导的坏死有保护作用。规划的目标如下：1。确定GSH耗竭的速率、持续时间和程度对培养肝细胞JNK和NF-kappaB双氧化还原依赖性变化以及对tnf诱导的细胞凋亡的敏感性的影响。在这个目标中，我们将定义对TNF敏感的时间窗口以及与GSH/GSSG的关系，比较快速和渐进的GSH消耗，并确定GSH消耗对IkappaB亚型在TNF响应中的水平和区隔的影响。2. 在体外和体内研究JNK的作用及JNK抑制剂对apap诱导的坏死的保护机制。我们发现，APAP消耗GSH激活JNK， JNK抑制剂可以保护APAP诱导的体外和体内坏死，因此我们将使用其他方法来抑制JNK以及对bcl2家族的影响来研究JNK在APAP毒性中的作用。3. 测定体内APAP或AMAP对TNF致敏的影响。我们将比较用APAP和AMAP（无毒区域异构体）治疗后体内对内源性和外源性TNF的致敏性。4. 探讨GSH耗竭对NFkappaB转激活的影响机制。我们将评估GSH、GSSG和氧化还原调节蛋白对GSH干扰物和TNF的核区隔反应。染色质免疫沉淀（ChIP）试验将用于确定NF-kappaB与iNOS基因启动子的结合，并检查NF-kappaB的氧化还原依赖性翻译后修饰，包括二硫形成、谷胱甘肽化、磷酸化和乙酰化。总的来说，这项研究将增加我们对谷胱甘肽和氧化还原修饰在使肝脏对损伤敏感中的作用的理解。这一发现将广泛适用于药物、病毒、酒精和代谢性肝病

DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF) plays an important role in liver injury. Normal hepatocytes are resistant to TNF cytotoxicity. However, we have recently found that selective depletion of extramitochondrial GSH sensitizes to TNF induced apoptosis which is accompanied by sustained c-jun-N-terminal kinase (INK) activation in cytosol and impaired NF-kB transactivation in the nucleus. In addition, we have found that inhibition of JNK protects against acetaminophen (APAP)-induced necrosis in vitro and in vivo. The following aims are planned: 1. To determine the influence of rate, duration, and extent of GSH depletion on dual redox-dependent changes in JNK and NF-kappaB and sensitization to TNF-induced apoptosis in cultured hepatocytes. In this aim we will define the time window of sensitization to TNF and the relation to GSH/GSSG, compare rapid versus gradual GSH depletion, and determine the effect of GSH depletion on the levels and compartmentation of IkappaB isoforms in response to TNF. 2. To determine the role of JNK and the mechanism for protection by JNK inhibitor against APAP-induced necrosis in vitro and in vivo. We showed that GSH depletion by APAP activates JNK and JNK inhibitor protects against APAP-induced in vitro and in vivo necrosis, so we will examine the role of JNK in APAP toxicity using other approaches to inhibiting JNK and the effects on the Bcl2-family. 3. To determine the effect of APAP or AMAP in vivo on sensitization to TNF. We will compare sensitization to endogenous and exogenous TNF in vivo after treatment with APAP and AMAP (nontoxic regioisomer). 4. To determine the mechanism of the effect of GSH depletion on NFkappaB transactivation. We will assess nuclear compartmentalization of GSH, GSSG, and redox regulatory proteins in response to GSH perturbants and TNF. The chromatin immunoprecipitation (ChIP) assay will be employed to determine NF-kappaB binding to the iNOS gene promoter and redox-dependent, post-translational modifications of NF-kappaB, including disulfide formation, glutathionylation, phosphorylation, and acetylation, will be examined. Overall, this research will increase our understanding of the role of GSH and redox modifications in sensitizing the liver to injury. The findings will be broadly applicable to drug, viral, alcohol and metabolic liver diseases