Oxygen/Nitrogen Stress in Acetaminophen Hepatotoxicity

对乙酰氨基酚肝毒性中的氧/氮应激

• 批准号: 7654947
• 负责人: Jack A. Hinson
• 金额: $ 34.8万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7654947
• 关键词: 3-nitrotyrosine; 7-nitroindazole; Acetaminophen; Acetylcysteine; Acute Liver Failure; Address; Affect; Analgesics; Antidotes; Binding; Cell Death; Cells; Clinical; Complex; Cyclosporine; Cytochrome P450; Data; Development; Event; Family; Glutathione; Goals; Hepatic; Hepatocyte; Hepatotoxicity; Hour; Human; Imines; In Vitro; Incubated; Injury; Knockout Mice; Laboratories; Measures; Mediating; Medical; Membrane; Metabolic; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mus; NOS1 gene; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrogen; Overdose; Oxidants; Oxidative Phosphorylation; Oxidative Stress; Oxygen; Pathway interactions; Patients; Permeability; Peroxonitrite; Pharmaceutical Preparations; Phase; Phase III Clinical Trials; Play; Poisoning; Production; Proteins; Proteomics; Research; Risk; Role; Screening procedure; Staging; Staining method; Stains; Stress; Superoxides; Testing; Time; Toxic effect; Transgenic Mice; Tyrosine; United States; Uric Acid; Work; acetaminophen overdose; clinical toxicology; drug candidate; early onset; ebselen; hepatic necrosis; hepatotoxin; in vivo; in vivo Model; inhibitor/antagonist; mitochondrial dysfunction; mouse model; nitration; novel strategies; omega-N-Methylarginine; oxidant stress; oxidation; p-Benzoquinones; para-benzoquinone; public health relevance; respiratory

DESCRIPTION (provided by applicant): Acetaminophen (APAP) poisoning is the most common cause of acute liver failure in the U.S. The early metabolic events in APAP-mediated toxicity are well described; however, the subsequent steps in development of toxicity are unknown. Using freshly isolated mouse hepatocytes treated with APAP, we recently showed that toxicity involves mitochondrial permeability transition (MPT), a mechanism mediated by oxidative stress and leads to a large increase in oxidative stress. We also demonstrated that protein tyrosine nitration, indicative of peroxynitrite, coincided with toxicity at later stages of treatment (2-5hr) following washing cells free of APAP. More recent studies have examined the mechanism leading to induction of MPT. Hence, addition of inhibitors of MPT, oxidant stress, and nitric oxide synthase (NOS1) inhibited mitochondrial injury, nitration, and development of APAP-mediated toxicity. Interestingly, GSH depletion was maximal by 0.5hr, but incubation of hepatocytes with APAP for 1hr followed by washing to remove APAP and reincubation did not result in toxicity. Therefore, we hypothesize that there are 3 stages of APAP toxicity: STAGE 1: the metabolic stage which occurs by 1hr and leads to STAGE 2: the oxidative stage which leads to mitochondrial injury and oxidant production prior to MPT; and finally STAGE 3: the toxicity stage which occurs with a large increase in protein nitration, MPT, and toxicity. Three specific aims are proposed. Specific Aim 1: Determine the early events (before 2 hr) important in the oxidative stage (Stage 2) of APAP-induced toxicity leading to MPT in hepatocytes. We hypothesize that oxidative stress develops in Stage 2 and is critical to the initiation of Stage 3. Specific Aim 2: Determine the molecular events which occur during and following MPT (Stage 3) in APAP-induced toxicity using in vitro and in vivo models. We hypothesize that mitochondrial superoxide and NOS1 activation play a fundamental role in MPT mediated toxicity. Specific Aim 3: Identify the hepatic proteins that are tyrosine nitrated in APAP toxicity. We hypothesize that a different subset of proteins are nitrated during the early onset of Stage 2 versus the later onset of Stage 3. The overall goal of the proposal is to test the hypothesis that mitochondrial protein nitration plays a fundamental role in induction and during MPT and toxicity of APAP. PUBLIC HEALTH RELEVANCE: Acetaminophen poisoning is the single most common cause of acute liver failure in the United States. An overdose of the analgesic can produce fatal liver necrosis. It is well recognized that metabolism to a reactive metabolite that depletes glutathione and covalently binds to protein is important. However, the subsequent events are unknown. Recent preliminary data in the PI's laboratory indicate that acetaminophen toxicity occurs with mitochondrial dysfunction and production of reactive oxygen/nitrogen species. The goal of the project will be to understand the role of reactive oxygen/nitrogen species and mitochondrial dysfunction in acetaminophen toxicity in mice. The ultimate goal is to develop new approaches to treat acetaminophen poisoning in humans.

描述（由申请人提供）：对乙酰氨基酚（APAP）中毒是美国最常见的急性肝衰竭原因。对APAP介导的毒性的早期代谢事件有很好的描述；然而，毒性发展的后续步骤是未知的。我们最近用APAP处理新鲜分离的小鼠肝细胞，发现其毒性涉及线粒体通透性转变（MPT），这是一种由氧化应激介导的机制，可导致氧化应激的大量增加。我们还证明，蛋白质酪氨酸硝化（指示过氧亚硝酸盐）与处理后期（2-5hr）无APAP洗涤细胞后的毒性一致。最近的研究已经检查了导致MPT诱导的机制。因此，添加MPT、氧化应激和一氧化氮合酶（NOS1）抑制剂可抑制线粒体损伤、硝化和apap介导的毒性发展。有趣的是，GSH耗竭在0.5小时时达到最大，但将肝细胞与APAP孵育1小时，然后清洗以去除APAP，再孵育并没有导致毒性。因此，我们假设APAP毒性有3个阶段：第1阶段：代谢阶段，发生在1hr，并导致第2阶段：氧化阶段，导致线粒体损伤和MPT之前的氧化剂产生；最后阶段3：毒性阶段，发生在蛋白质硝化、MPT和毒性大量增加的阶段。提出了三个具体目标。特异性目的1：确定apap诱导毒性导致肝细胞MPT的氧化阶段（第2阶段）重要的早期事件（2小时前）。我们假设氧化应激在第二阶段发展，对第三阶段的开始至关重要。具体目标2：使用体外和体内模型确定apap诱导毒性MPT（阶段3）期间和之后发生的分子事件。我们假设线粒体超氧化物和NOS1激活在MPT介导的毒性中起着重要作用。特异性目的3：鉴定在APAP毒性中酪氨酸硝化的肝脏蛋白。我们假设在第2阶段早期与第3阶段晚期发生时，不同的蛋白质亚群被硝化。该提案的总体目标是验证线粒体蛋白质硝化在APAP的诱导和MPT和毒性过程中起基本作用的假设。公共卫生相关性：在美国，对乙酰氨基酚中毒是导致急性肝衰竭的最常见原因。过量使用这种镇痛剂会导致致命的肝坏死。众所周知，代谢成一种消耗谷胱甘肽并与蛋白质共价结合的活性代谢物是很重要的。然而，随后的事件是未知的。PI实验室最近的初步数据表明，对乙酰氨基酚毒性与线粒体功能障碍和活性氧/氮物质的产生有关。该项目的目标是了解活性氧/氮物种和线粒体功能障碍在小鼠对乙酰氨基酚毒性中的作用。最终目标是开发治疗人类对乙酰氨基酚中毒的新方法。