Ethanol (alcohol)-mediated cell and tissue damage is partly caused by increased oxidative and nitrosative stress. 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, xanthine oxidase, etc. We are particularly interested in studying the combined effects of activated CYP2E1, a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an anti-oxidant defense enzyme responsible for removal of toxic acetaldehyde and lipid peroxides, on increased oxidative stress and their implications in our experimental models. In the past, we developed a sensitive method to identify oxidatively-modified mitochondrial proteins and demonstrated causal relationship between oxidative modifications and their inactivation in mouse models of alcoholic fatty liver (AFLD) and nonalcoholic fatty liver (NAFLD). During fiscal year 2012, we have established sensitive methods to identify other post-translational modifications such as nitration and phosphorylation of various proteins to investigate their roles in mitochondrial dysfunction and acute liver injury. For studying the functional role of protein nitration, we used a mouse model of acetaminophen (APAP)-induced liver injury because of the well-established role of nitration in liver injury. For evaluating the roles of protein phosphorylation in causing cell/tissue injury, we used a model of acute liver injury by carbon tetrachloride (CCL4). In collaboration with Dr. Bong-Hee Lee at Gachon University Medical School in Korea, we also developed a method to identify the glycosylated proteins and studied their roles in promoting NAFLD and neuronal damage in rats. Furthermore, we collaborated with Dr. Ali Keshavarzian at Rush Medical Center in Chicago to investigate the role of CYP2E1 in exacerbating AFLD through increasing gut leakiness. Many investigators reported pathophysiological implications of protein nitration in promoting various types of cell/tissue injury. Protein nitration is a contributing factor in alcohol- or drug-induced liver injury since iNOS-null mice were markedly protected from these types of liver injury. However, the mechanisms of alcohol- or drug-induced liver injury are poorly understood. In fact, it is largely unknown which cellular (including mitochondrial) proteins are nitrated and how the functions of nitrated proteins are altered and thus contribute to alcohol- or APAP-mediated mitochondrial dysfunction and hepatotoxicity. By using Cyp2e1-null mice, we recently showed that CYP2E1 is involved in promoting protein nitration and ubiquitin-dependent degradation of many proteins (Abdelmegeed et al., 2010). Based on the numerous spots of nitrated proteins displayed on 2-D gels, we hypothesized that many more proteins could be nitrated and that nitrated proteins then contribute to liver injury caused by a single dose (350 mg/kg, ip) of APAP, a CYP2E1 substrate. Thus, we aimed to systematically identify nitrated proteins and investigate their functional roles in APAP-induced liver injury. During this study, we showed that many cytosolic and mitochondrial proteins were rapidly nitrated at 1 or 2 h following APAP exposure. We thus antibody-based affinity-purified nitrated proteins from the mouse livers exposed to APAP for 2 h when liver damage was minimal and determined their identities by mass-spectral analysis. Our data revealed that more than 30 cytosolic and 65 mitochondrial proteins involved in anti-oxidant defense, energy supply, amino acid and fat metabolic pathways were nitrated by APAP exposure. The enzyme activities of a few selected nitrated proteins such as ALDH2, ATP synthase, and 3-ketoacyl-CoA thiolase were suppressed in APAP-exposed mice but restored by co-treatment with a peroxynitrite scavenger N-acetylcysteine, which also prevented APAP-induced protein nitration and liver injury. These results established the causal role of protein nitration in APAP-induced liver injury. We previously reported the critical role of the activated JNK in promoting cell death by phosphorylating critical proteins including pro-apoptotic Bax and mitochondrial ALDH2. To better understand the roles of JNK and its target proteins in regulating mitochondrial function and cell/tissue damage, we have initiated a study to identify and characterize JNK-mediated phosphorylation of many mitochondrial proteins. For specific activation of JNK without activating other mitogen-activated protein kinases (MAPKs), we chose a model of liver injury caused by a single dose (50 mg/kg, ip) of carbon tetrachloride (CCL4), another substrate of CYP2E1. We observed that JNK, activated within 30 min, translocated to mitochondria and that many mitochondrial proteins were rapidly phosphorylated between 1 and 8 h after CCL4 exposure where liver injury was minimal. However, these events were not observed in the corresponding Cyp2e1-null mice. To further characterize their functions, we purified phosphorylated mitochondrial proteins from WT mouse livers exposed to CCL4 for 2 h by using metal-affinity columns. Mass-spectral analysis of purified phospho-proteins revealed that more than 100 mitochondrial proteins were phosphorylated by activated JNK. These proteins include pyruvate dehydrogenase, ATP synthase, ALDH2, etc involved in energy supply and cellular defense, respectively. The activities of these phospho-proteins were markedly suppressed in CCL4-exposed mice but significantly restored in CCL4-exposed mice pretreated with a selective JNK inhibitor SU-3327, which blocked the JNK-mediated protein phosphorylation and prevented liver damage. These results provide direct evidence for critical roles of JNK and protein phosphorylation in promoting CCL4-mediated mitochondrial dysfunction and acute liver injury. We also studied the role of CYP2E1 in the development of inflammatory liver disease e.g., alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) induced by binge alcohol (6 g/kg oral gavage for 3 times at 12 h intervals) and a high fat diet (HFD), respectively. Histological signs of inflammatory liver injury were easily observed in WT mice while the age- and gender-matched Cyp2e1-null mice were protected from alcohol-induced ASH and HFD-mediated NASH (60% energy derived from fat compared to a low fat diet with 10% fat-derived energy for 10 weeks in a 2 x 2 design), respectively. Elevated inflammation scores with increased levels of CYP2E1, tumor necrosis factor-alpha, and lipid peroxides (e.g., malondialdehyde+4-hydroxyalkenals) with suppressed ALDH2 activity were observed in binge alcohol-exposed and HFD-fed WT mice. Consequently, protein nitration, oxidation, and glycosylation were also increased in WT mice fed a HFD or exposed to binge alcohol. Our data also showed that CYP2E1 is important promoting gut leakiness with increased levels of endotoxin and translocation of enterobacteria into the liver in binge alcohol-exposed WT mice. In contrast, all these parameters were significantly reduced or absent in the corresponding Cyp2e1-null mice, suggesting an important role of CYP2E1 in promoting gut leakiness and the development of ASH and NASH. Based on the establishment of AFLD/ASH and NAFLD/NASH in our mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid (DHA) against AFLD/ASH and NAFLD/NASH in Ppara-null or Cyp2e1-null mice compared to the WT mice.
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