Alcohol Metabolism, Functional Consequence And Signaling Mechanism

酒精代谢、功能后果和信号机制

• 批准号: 7732090
• 负责人: BYOUNG-JOON SONG
• 金额: $ 71.82万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732090
• 关键词: 3-nitrotyrosine; Acetaldehyde; Acetaminophen; Acetyl-CoA C-Acetyltransferase; Acids; Acute; Alcoholic Fatty Liver; Alcohols; Aldehyde dehydrogenase (NAD+); American Society of Hematology; Animal Feed; Animal Model; Animals; Antioxidants; Apoptosis; Area; Asians; Biochemical; Biotin; Brain; Carbon Tetrachloride; Cell Death; Cells; Chemicals; Chronic; Clinic; Coenzyme A; Collaborations; Condition; Coupled; Cultured Cells; Cytochrome P-450 CYP2E1; DNA; Data; Diabetes Mellitus; Diet; Dietary intake; Docosahexaenoic Acids; Dominant-Negative Mutation; Dose; Elevation; Energy Supply; Enzymes; Ethanol; Ethanol Metabolism; Evaluation; Exhibits; Exposure to; Fatty Liver; Fatty acid glycerol esters; Gel; Genes; Glutathione; Goals; Heart; Hepatic; Histology; Human; Hydrogen Peroxide; Inflammatory; Injury; Ischemia; Isoenzymes; Kidney; Knockout Mice; Label; Linolenic Acids; Liquid substance; Liver; Liver Dysfunction; Long-Evans Rats; Lung; MAPK14 gene; MAPK8 gene; Maleimides; Malnutrition; Maryland; Measurement; Mediating; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Chaperones; Mus; Mutation; NADPH Oxidase; Necrosis; Nicotine; Nitrites; Nitrogen; Numbers; Obesity; Omega-3 Fatty Acids; Oral; Organ; Oxidative Stress; Oxygen; Peroxonitrite; Pharmaceutical Preparations; Phosphotransferases; Physiological reperfusion; Plasma; Poison; Polyunsaturated Fatty Acids; Problem Solving; Procedures; Protective Agents; Proteins; Rattus; Reperfusion Injury; Reperfusion Therapy; Respiratory Chain; Rodent; Role; Serum; Signal Pathway; Signal Transduction; Smoking; Stress; Time; Tissues; Transaminases; Transcriptional Activation; Universities; Up-Regulation; Virus Diseases; Wild Type Mouse; Xanthine Oxidase; alcohol exposure; aldehyde dehydrogenase 1; aldehyde dehydrogenases; animal tissue; base; carcinogenesis; comparative; dietary supplements; drug of abuse; ecstasy; feeding; hepatoma cell; human NOS2A protein; inhibitor/antagonist; interest; lipid metabolism; mitochondrial dysfunction; mouse model; nitration; non-alcoholic; non-alcoholic fatty liver; nonalcoholic steatohepatitis; prevent; problem drinker; streptavidin-agarose; stress activated protein kinase

Oxidative stress is one of the major contributing factors in ethanol (alcohol)-mediated cell and tissue damage. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells/tissues are produced through direct inhibition of the mitochondrial respiratory chain and induction/activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), NADPH-oxidase, and xanthine oxidase. Despite the well-established causal roles of ROS/RNS in alcohol-induced mitochondrial dysfunction and injury, the target proteins, that are oxidatively-modified by elevated ROS/RNS, and their functional alterations are poorly understood. To solve these problems, we have recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to positively identify oxidized and/or S-nitrosylated proteins in ethanol-exposed hepatoma cells or animal tissues. Having established a sensitive method, we extended our approaches to identify oxidatively-modified proteins in animal models of alcoholic and nonalcoholic fatty liver and inflammatory injury (ASH and NASH) to investigate the underlying mechanisms of mitochondrial dysfunction and apoptosis. Furthermore, our method allows us to find protective agents against ASH and NASH. During this fiscal year, we have collaborated with Dr. Norman Salem Jr, LMBB, to study the beneficial effects of dietary intake of polyunsaturated fatty acids (PUFA) such as arachidonic (AA,20:4n6,omega-6) and docosahexaenoic (DHA,22:6n3, omega-3) acids against alcoholic fatty liver and mitochondrial dysfunction in Long Evans rats. Our result showed that chronic alcohol administration with an ethanol liquid diet containing low but adequate levels of linoleic and linolenic acids without AA and DHA promotes fatty liver. Fat accumulation was accompanied by increased oxidative/nitrosative stress through elevated levels of ethanol-inducible CYP2E1, iNOS, nitrite and mitochondrial hydrogen peroxide. However, these increments and alcoholic fatty liver were normalized in rats fed the alcohol-DHA/AA-supplemented diet. The number of oxidatively-modified mitochondrial proteins displayed on 2-D gels was markedly increased following alcohol exposure but significantly reduced in rats fed the alcohol-DHA/AA-supplemented diet. The suppressed activities of mitochondrial aldehyde dehydrogenase (ALDH2), ATP synthase, and 3-ketoacyl-CoA thiolase in ethanol-exposed rats were also recovered in animals fed the ethanol-DHA/AA-supplemented diet. These results suggest that physiologically relevant levels of DHA-containing PUFA are protective against alcohol-mediated mitochondrial dysfunction and fatty liver through decreasing the levels of ROS/RNS. We expect that DHA-supplemented PUFA diet may also provide beneficial effects against NASH caused by obesity, diabetes, and many drugs being used in clinics. We also collaborated with Dr. Pal Pacher, LPS, to identify the oxidized proteins with an aim to understand the mechanism of mitochondrial dysfunction and injury following hepatic ischemia-reperfusion (I/R) as a mouse model of NASH in the absence and presence of a peroxynitrite scavenger MnTMPyP. Liver histology and plasma transaminase activity results showed that mouse livers were severely damaged following the I/R procedure (1-h ischemia followed by reperfusion for 2-h, 10-h, or 24-h) in the absence of MnTMPyP. These changes were accompanied by elevated levels of nitrite, 3-nitrotyrosine (3-NT), and iNOS compared to those in sham-operated controls. Pretreatment with MnTMTyP fully restored liver histology with normalized levels of plasma transminases, nitrite, 3-NT, and iNOS. Comparative 2-D gel analysis revealed markedly increased numbers of oxidized and S-nitrosylated mitochondrial proteins following hepatic I/R injury. Many key mitochondrial enzymes involved in cellular defense, fat metabolism, energy supply, and chaperones were oxidatively-modified. MnTMPyP pretreatment decreased the number of oxidatively-modified proteins and restored I/R-induced suppressed activities of mitochondrial ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase. These results strongly suggest that increased nitrosative stress is critically important in promoting S-nitrosylation and nitration of various mitochondrial proteins, leading to mitochondrial dysfunction with decreased energy supply and increased hepatic injury. Based on these results, we believe that this approach can be used to investigate the mechanism of tissue damage and to identify another beneficial agent against oxidative tissue injury in many other organs such as brain, heart, lung and kidney. In collaboration with Drs. Natalie D. Eddington and James Lee from the University of Maryland, we also studied the mechanism of mitochondrial dysfunction and nonalcoholic liver damage caused by acute exposure to MDMA (3,4-methylenedioxymethamphetamine, ecstasy). We hypothesized that key mitochondrial proteins are oxidatively-modified and inactivated, contributing to liver damage in MDMA-exposed tissues. MDMA-treated rats showed abnormal liver histology with significant elevations of plasma transaminases, iNOS, and the level of hydrogen peroxide. Oxidatively-modified mitochondrial proteins in control and MDMA-exposed rats were labeled with the biotin-NM probe, purified with streptavidin-agarose, and resolved using 2-D PAGE. Comparative 2-D gel analysis revealed markedly increased levels of oxidatively-modified proteins following MDMA exposure. Mass spectrometric analysis identified oxidatively-modified mitochondrial proteins involved in energy supply, fat metabolism, antioxidant defense, and chaperone activities. Among these, the activities of ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase were significantly inhibited following MDMA exposure. Our data show for the first time that MDMA causes oxidative inactivation of key mitochondrial enzymes which most likely contributes to mitochondrial dysfunction and subsequent liver damage in MDMA-exposed animals. Accumulation of toxic acetaldehyde, produced from ethanol metabolism and through the inhibition of mitochondrial ALDH2 enzyme by chemical inhibitors or dominant negative mutation of ALDH2 gene, can interact with many cellular proteins and DNA while it increases oxidative stress by depleting cellular antioxidants such as glutathione. In fact, people with a defective ALDH2 gene (frequently observed in East Asians) are significantly much more susceptible to alcohol-mediated tissue injury and carcinogenesis than normal people without the mutation. Therefore, we hypothesized that mice deficient of mouse ADLH2 gene may be more susceptible to alcohol-mediated tissue injury. Based on this hypothesis, we have extensively evaluated potential liver damage in ALDH2 knockout (KO) mice compared to wild type (WT) mice following binge ethanol exposure. However, our results did not show any severe liver damage (histological evaluation, serum transaminase levels and other biochemical measurements) in ALDH2 KO mice treated with large doses of ethanol (oral, 3 doses of 4 g/kg/dose at 12-h intervals) compared to WT mice. The low levels of hepatic damage may result from up-regulation of some compensatory proteins (or factors) in the ethanol-exposed ALDH2 KO mice. Alternatively, the negative result could be due to the presence of another ALDH isozyme such as cytosolic ALDH1, which exhibits a relatively low Km value for acetaldehyde (Km=14-15 uM in rodents compared to >190 uM in humans), preventing acetaldehyde accumulation in ALDH2 KO mice. Based on the negative results, we stopped using ALDH2 KO mice but plan to study the mechanism of mitochondrial dysfunction and liver damage (necroinflammation) in CYP2E1 KO mice fed an ethanol liquid diet and a high fat diet, as models of ASH and NASH, respectively.

氧化应激是乙醇介导的细胞和组织损伤的主要因素之一。在酒精暴露的细胞/组织中，大多数活性氧和活性氮（ROS/RNS）是通过直接抑制线粒体呼吸链和诱导/激活乙醇诱导的细胞色素P450 2E1 （CYP2E1）、诱导型一氧化氮合酶（iNOS）、nadph氧化酶和黄嘌呤氧化酶产生的。尽管已经确定ROS/RNS在酒精诱导的线粒体功能障碍和损伤中的因果作用，但被ROS/RNS升高氧化修饰的靶蛋白及其功能改变尚不清楚。为了解决这些问题，我们最近开发了一种使用生物素-n -马来酰亚胺（生物素- nm）作为特异性探针的敏感方法，以阳性识别乙醇暴露的肝癌细胞或动物组织中的氧化和/或s -亚硝基化蛋白质。在建立了一种灵敏的方法后，我们扩展了我们的方法，在酒精性和非酒精性脂肪肝和炎症性损伤（ASH和NASH）动物模型中鉴定氧化修饰蛋白，以研究线粒体功能障碍和细胞凋亡的潜在机制。此外，我们的方法使我们能够找到针对ASH和NASH的保护剂。

项目成果

Acetaminophen induces apoptosis of C6 glioma cells by activating the c-Jun NH(2)-terminal protein kinase-related cell death pathway.

• 发表时间: 2001-10
• 期刊: Molecular pharmacology
• 影响因子: 3.6
• 作者: Myung-Ae Bae;J. Pie;Byoung Joon Song

Molecular cloning of cytochrome P4501A cDNA of medaka (Oryzias latipes) and messenger ribonucleic acid regulation by environmental pollutants.

青鳉细胞色素 P4501A cDNA 的分子克隆和环境污染物对信使核糖核酸的调节。

• DOI: 10.1897/03-27
• 发表时间: 2004
• 期刊: Environmental toxicology and chemistry / SETAC
• 作者: Ryu,Jisung;Lee,Moon-Soon;Na,JinGyun;Chung,Kyuhyuck;Song,Byoung-Joon;Park,Kwangsik
• 通讯作者: Park,Kwangsik

Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats.

禁食的 Sprague-Dawley 大鼠中氯唑沙宗代谢增加。

• DOI: 10.1211/jpp.58.1.0007
• 发表时间: 2006
• 期刊: The Journal of pharmacy and pharmacology
• 作者: Wan,Jie;Ernstgård,Lena;Song,ByoungJ;Shoaf,SusanE
• 通讯作者: Shoaf,SusanE