Oxygen/Nitrogen Stress in Acetaminophen Hepatotoxicity

对乙酰氨基酚肝毒性中的氧/氮应激

基本信息

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
项目类别：
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

项目摘要

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。但是，随后的毒性发展步骤尚不清楚。使用APAP处理的新鲜分离的小鼠肝细胞，我们最近表明毒性涉及线粒体通透性跃迁（MPT），这是一种由氧化应激介导的机制，并导致氧化应激大大增加。我们还证明了蛋白酪氨酸硝化作用，表明过氧亚硝酸盐，在治疗后期（2-5hr）的毒性是在洗涤细胞不含APAP后的毒性（2-5hr）。最近的研究检查了导致MPT诱导的机制。因此，添加MPT抑制剂，氧化剂应激和一氧化氮合酶（NOS1）抑制了线粒体损伤，硝化和APAP介导的毒性的发展。有趣的是，GSH的耗竭最大为0.5小时，但肝细胞与APAP孵育1小时，然后洗涤以去除APAP和再孵育并没有导致毒性。因此，我们假设有3个APAP毒性阶段：阶段1：由1小时发生的代谢阶段，导致2阶段：氧化阶段，导致MPT之前导致线粒体损伤和氧化剂的产生；最后，第3阶段：毒性阶段发生，蛋白质硝化，MPT和毒性大大增加。提出了三个具体目标。具体目标1：确定早期事件（2小时之前）在APAP诱导的毒性的氧化阶段（第2阶段）中很重要，从而导致肝细胞中MPT。我们假设氧化应激在第2阶段中发展，对于第3阶段的开始至关重要。特定目标2：确定在APAP使用体外和体内模型中APAP诱导的毒性中发生的分子事件（第3阶段）（第3阶段）。我们假设线粒体超氧化物和NOS1激活在MPT介导的毒性中起着基本作用。特定目的3：确定在APAP毒性中硝化酪氨酸的肝蛋白。我们假设在第2阶段的早期发作中与第3阶段的后期发作相比，蛋白质的不同子集被硝化。该提案的总体目标是检验以下假说，即线粒体蛋白硝化在诱导和MPT和APAP的MPT和毒性期间在诱导和毒性中起着基本作用。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。镇痛药过量会产生致命的肝坏死。众所周知，耗尽谷胱甘肽并共价结合蛋白质的反应性代谢产物的代谢很重要。但是，随后的事件未知。 PI实验室中的最新初步数据表明，对乙酰氨基酚的毒性发生在线粒体功能障碍和活性氧/氮种的产生中。该项目的目的是了解小鼠对乙酰氨基酚毒性中的活性氧/氮和线粒体功能障碍的作用。最终目标是开发新方法来治疗人类中毒。