Alcohol Metabolism, Functional Consequence and Apoptosis Signaling Mechanism

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。在建立了一种敏感的方法后，我们扩展了我们的方法，在酒精性和非酒精性脂肪性肝病伴炎症性损伤（分别为AFLD和NAFLD）的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AFLD和NAFLD的保护剂。

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 being 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 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 non-alcoholic fatty liver diseases with inflammatory injury (AFLD and NAFLD, respectively) to investigate the underlying mechanisms of mitochondrial dysfunction and apoptosis. Furthermore, our method allows us to find protective agents against AFLD and NAFLD. During this fiscal year, we collaborated with Dr. Pal Pacher, LPS, NIAAA, to identify the oxidized proteins to identify early biochemical changes and study the mechanism of mitochondrial dysfunction (at 2-h) long before visible signs of tissue injury (observed at 10- or 24-h reperfusion) following hepatic ischemia-reperfusion (I/R) as a mouse model of NAFLD with or without 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 10-, or 24-h) without MnTMPyP. These changes were accompanied with elevated levels of nitrite, 3-nitrotyrosine (3-NT), and iNOS compared to those in sham-operated controls. Pretreatment with MnTMTyP significantly protected against liver damage and with normalized levels of plasma transminases, nitrite, 3-NT, and iNOS. Comparative 2-D gel analysis revealed that the number and intensity of oxidized and S-nitrosylated mitochondrial proteins were markedly increased 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 the suppressed activities of mitochondrial ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase following the I/R procedure. 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, which ultimately contributes to necrotic tissue damage. In collaboration with Drs. Natalie D. Eddington and James Lee at University of Maryland, we also studied the mechanism of mitochondrial dysfunction and non-alcoholic liver damage caused by acute exposure to MDMA (3,4-methylenedioxymethamphetamine, ecstasy). MDMA-treated rats showed abnormal liver histology with significant elevations of plasma transaminases, iNOS, and the increased production of hydrogen peroxide. Comparative 2-D gel analysis revealed markedly increased levels of biotin-NM labeled, oxidatively-modified proteins in MDMA-exposed rats compared to control rats. Mass spectrometric analysis revealed the identities of oxidatively-modified mitochondrial proteins. . Among these, the activities of ALDH2, 3-ketoacyl-CoA thiolases, and ATP synthase involved in antioxidant defense, fat metabolism, and energy supply, respectively, were significantly inhibited through oxidative modifications (e.g., S-nitrosylation and nitration of active site Cys and Tyr residues, respectively) following MDMA exposure. In addition, we extended our study by concurrent administration of MDMA and alcohol to determine whether these two widely-abused substances can synergistically work toward tissue damage, as frequently observed in young adults. Our data showed elevated levels of acetaldehyde and malondialdehyde with more severe liver damage as assessed by increased plasma transaminase activities and liver histology. These data not only confirmed our earlier data of ALDH2 inhibition by MDMA but also indicate that these two agents synergistically work (potentiation) toward acute liver damage. Although many animal models exist for studying the mechanisms of AFLD and NAFLD, the roles of peroxisomal proliferator-activated receptor (PPAR) and CYP2E1 in these areas have not been fully characterized. PPAR is a transcription factor involved in controlling the expression of many genes in the fatty acid transport, inflammatory reactions, peroxisomal and mitochondrial fat metabolism. Moreover, the expressed level of PPAR in human is much lower than that in rodents, suggesting Ppara-null mice can be used as a good model for studying the mechanisms of AFLD and NAFLD simulating human conditions. At first, we studied the role of PPAR in hepatosteatosis and oxidative stress during fasting. Fasted Ppara-null mice exhibited marked hepatosteatosis, which was associated with elevated levels of lipid peroxidation, NOS activity, and hydrogen peroxide production. Total glutathione (GSH), mitochondrial GSH, and the activities of major anti-oxidant enzymes were also lower in the fasted Ppara-null mice. As expected, oxidatively-modified proteins were only found in the fasted Ppara-null mice. These results with increased oxidative stress observed in the fasted Ppara-null mice compared with other groups demonstrate a role for PPAR in fasting-mediated oxidative stress and that inhibition of PPAR functions may increase the susceptibility to oxidative damage in the presence of another toxic agent. In addition, we studied the role of CYP2E1 in protein nitration and ubiquitin-mediated degradation during acetaminophen (APAP) toxicity in wild-type and Cyp2e1-null mice exposed to APAP (200 and 400 mg/kg) for 4 and 24 h. Markedly increased centrilobular liver necrosis and 3-NT formation were only observed in APAP-exposed wild-type mice in a dose- and time-dependent manner, confirming an important role for CYP2E1 in APAP biotransformation and toxicity. However, the pattern of 3-NT protein adducts, not accompanied by concurrent activation of NOS, was similar to that of protein ubiquitination. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed wild-type mice, confirming the fact that nitrated proteins are more susceptible (than the native proteins) to ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic superoxide dismutase (SOD1) levels were clearly decreased and immunoprecipitated SOD1 was nitrated and ubiquitinated, likely leading to its accelerated degradation in APAP-exposed wild-type mice. These data suggest that CYP2E1 appears to play a key role in 3-NT formation, protein degradation, and liver damage, which is independent of NOS, and that decreased levels of many proteins in the wild-type mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity. Based on our own data recently published, we believe that Ppara-null or Cyp2e1-null mice are suitable for studying the mechanisms of AFLD and NAFLD treated with an ethanol-liquid diet or a high fat diet. Therefore, we plan to test the beneficial effects of various anti-oxidants including the DHA-containing diet against AFLD and NAFLD in Ppara-null or Cyp2e1-null mice compared to wild-type mice.