Alcohol Metabolism, Functional Consequence And Signaling Mechanism

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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.