Oxygen/Nitrogen Stress in Acetaminophen Hepatotoxicity

对乙酰氨基酚肝毒性中的氧/氮应激

• 批准号: 8252203
• 负责人: Jack A. Hinson
• 金额: $ 30.91万
• 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/8252203
• 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）中毒是美国急性肝衰竭的最常见原因。APAP介导的毒性中的早期代谢事件已得到充分描述;然而，毒性发展的后续步骤尚不清楚。使用新鲜分离的小鼠肝细胞与APAP处理，我们最近表明，毒性涉及线粒体通透性转换（MPT），氧化应激介导的机制，并导致氧化应激大幅增加。我们还证明，蛋白酪氨酸硝化，指示过氧亚硝酸盐，符合毒性在后期阶段的治疗（2- 5小时）后，洗涤细胞的APAP。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（NOS 1）抑制剂可抑制线粒体损伤、硝化和APAP介导的毒性的发展。有趣的是，GSH消耗在0.5小时时达到最大值，但将肝细胞与APAP孵育1小时，然后洗涤以去除APAP并重新孵育不会导致毒性。因此，我们假设APAP毒性有3个阶段：第1阶段：代谢阶段，在1小时内发生，并导致第2阶段：氧化阶段，在MPT之前导致线粒体损伤和氧化剂产生;最后第3阶段：毒性阶段，蛋白质硝化，MPT和毒性大幅增加。提出了三个具体目标。具体目标1：确定导致肝细胞MPT的APAP诱导毒性氧化阶段（第2阶段）的重要早期事件（2小时前）。我们假设氧化应激发生在第2阶段，对第3阶段的开始至关重要。具体目标二：使用体外和体内模型确定APAP诱导毒性中MPT（第3阶段）期间和之后发生的分子事件。我们推测，线粒体超氧化物和NOS 1的激活在MPT介导的毒性中起着重要作用。具体目标3：确定在APAP毒性中酪氨酸硝化的肝蛋白。我们假设，不同的蛋白质亚组在第2阶段的早期发作与第3阶段的晚期发作期间被硝化。该提案的总体目标是检验线粒体蛋白硝化在APAP的诱导和MPT和毒性过程中发挥重要作用的假设。公共卫生相关性：对乙酰氨基酚中毒是美国急性肝衰竭的最常见原因。过量的止痛药会导致致命的肝坏死。众所周知，代谢为消耗谷胱甘肽并与蛋白质共价结合的反应性代谢物是重要的。然而，随后发生的事件不详。PI实验室的最新初步数据表明，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/氮物质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。