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. 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 enzyme, on increased oxidative stress and its physiological implications in our experimental models. Despite the well-established causal roles of ROS/RNS in alcohol-induced oxidative injury, the target proteins, that are oxidatively-modified by ROS/RNS, and their functional alterations are poorly understood. To solve these problems, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to identify oxidized and/or S-nitrosylated proteins. We continued our efforts to identify and characterize oxidatively-modified proteins in animal models of alcoholic and nonalcoholic fatty liver diseases without inflammation or with inflammation (AFLD and NAFLD, respectively) to understand the underlying mechanisms of mitochondrial dysfunction, apoptosis and tissue injury. By using our own method, we characterized oxidatively-modified cytosolic proteins in rat livers exposed to MDMA (3,4-methylenedioxymethamphetamine, ecstasy), which is often co-abused in alcoholic individuals. Our results showed that many cytosolic proteins including anti-oxidant enzymes such as superoxide dismutase (SOD1) and peroxiredoxin were oxidized and inactivated after MDMA exposure. Consistent with these results, we observed increased oxidative stress with elevated levels of lipid peroxides, stress-activated protein kinases, and phosphorylated (inactivated) Bcl-2 or Bcl-XL, leading to apoptosis. A manuscript about these results is now under revision in Proteomics. We also investigated the role of CYP2E1 in protein nitration and ubiquitin-dependent degradation during liver toxicity caused by a CYP2E1 substrate acetaminophen (APAP). Markedly increased centrilobular liver necrosis and 3-nitrotyrosine (3-NT) protein-adducts were only observed in APAP-exposed wild-type (WT) mice in a dose- and time-dependent manner but not in Cyp2e1-null mice, confirming a key role for CYP2E1 in causing APAP toxicity. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed WT mice, supporting the fact that nitrated proteins are more susceptible (than the native proteins) to ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic SOD1 was nitrated and ubiquitinated before it was degraded in APAP-exposed WT mice. These results were not observed in Cyp2e1-null mice, suggesting an important role of CYP2E1 in 3-NT formation, protein degradation, and liver damage. Our results also indicate that decreased levels of many proteins in the WT mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity. These results were published in Biochemical Pharmacology. In addition, we studied a mechanism of inhibition of mitochondrial ALDH2 in a model of carbon tetrachloride (CCL4)-induced liver injury. CCL4 administration caused marked liver necrosis, while it elevated oxidative stress and activated c-Jun-N-terminal protein kinase (JNK). However, hepatic ALDH2 activity was potently suppressed in a time-dependent manner after CCL4 injection. Little or no change in the ALDH2 protein level suggested that ALDH2 could be inhibited through covalent modifications such as JNK-mediated phosphorylation. To demonstrate phosphorylation, we compared the isoelectric points (pI) of ALDH2 in CCL4-exposed rats and untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCL4-exposed rats were shifted to acidic pI ranges on 2-D gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with concurrent alkaline pI shifts of ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria following CCL4 exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity with acidic pI shifts on 2-D gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser-Pro antibody revealed detection of one band in the CCL4-exposed rats but not in controls, suggesting phosphorylation of Ser residue(s) of ALDH2. These results collectively indicate a novel mechanism that CCL4 exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied with decreased cellular defense capacity and increased lipid peroxidation. These results were published in Free Radic Biol Med. Although many animal models exist for studying the mechanisms of AFLD and NAFLD, the roles of peroxisomal proliferator-activated receptor alpha (PPARalpha) and CYP2E1 in these areas have not been fully characterized. PPARalpha 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 PPARalpha in humans is much lower (less than one tenth) than that in rodents, suggesting Ppara-null mice can be used as a good model for studying the mechanisms of AFLD and NAFLD in human conditions. We hypothesized that Ppara-null mice are very sensitive to organ damage compared to WT mice while Cyp2e1-null mice are very resistant to tissue damage caused by ethanol and other potentially toxic agents or diets. Based on this hypothesis, we studied the mechanism of NAFLD in WT mice and Ppara-null mice fed a high fat diet (HFD). Age- and gender-matched WT and Ppara-null mice were fed a liquid HFD (70% energy derived from fat) or a standard liquid diet (STD, 35% energy derived from fat) ad libitum for 3 weeks in a 2 x 2 design. Ppara-null mice fed a HFD exhibited the highest levels of hepatocyte ballooning, steatosis, inflammation, and ultimately NASH activity score among the 4 groups. Elevated levels of CYP2E1, tumor necrosis factor-alpha, and lipid peroxides (e.g., malondialdehyde) were observed in HFD-fed Ppara-null mice. Consequently, protein nitration and oxidation were also increased in Ppara-null mice fed a HFD compared to their WT counterparts. Increased oxidative stress and inflammation were associated with activation of JNK and p38 kinase, contributing to increased hepatocyte apoptosis in Ppara-null mice fed a HFD compared with WT mice. These results demonstrate that inhibition of PPAR functions may increase susceptibility to nonalcoholic steatohepatitis (NASH) in the presence of a HFD. On the other hand, Cyp2e1-null mice fed a HFD were relatively resistant to the development of NAFLD or NASH. A manuscript for these results is now under revision in Journal of Nutrition. Based on our own data recently published and unpublished, we believe that Ppara-null or Cyp2e1-null mice are very useful for studying the mechanisms of AFLD and NAFLD treated with an ethanol-liquid diet or a combination of alcohol and another potentially toxic agent such as nicotine and lipopolysaccharide, simulating human conditions. Upon establishment of AFLD and NAFLD in these mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid (DHA) or S-adenosylmethionine against AFLD and NAFLD in Ppara-null or Cyp2e1-null mice compared to WT mice.
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