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, 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 defensive enzyme responsible for removal of toxic acetaldehyde and lipid peroxides, as we recently reviewed in Journal of Proteomics, on increased oxidative stress and their implications in our experimental models. In the past, we have developed a sensitive method of using biotin-N-maleimide as a specific probe to identify oxidatively-modified mitochondrial proteins and their inactivation in animal models of alcoholic fatty liver (AFLD). During this fiscal year, we continued our study to identify and characterize oxidatively-modified proteins in animal models of nonalcoholic acute liver disease to understand the mechanisms of mitochondrial dysfunction, apoptosis and tissue injury. In this effort, we identified and 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 published in Proteomics showed that many cytosolic proteins including anti-oxidant enzymes such as Cu-Zn-dependent cytosolic superoxide dismutase (SOD1) and peroxiredoxin were oxidized and inactivated after MDMA exposure. Consequently, we observed increased oxidative stress with elevated levels of lipid peroxides, stress-activated protein kinases such as JNK and p38 kinase, and phosphorylated (inactivated) Bcl-2 or Bcl-XL, all contributing to apoptosis of hepatocytes. As another model of nonalcoholic acute liver injury, we also studied the mechanism of hepatic damage caused by lipopolysaccharide (LPS). Our data published in Toxicol Letters revealed that LPS causes severe liver damage in mice deficient of peroxisomal proliferator-activated receptor alpha (PPAR-alpha) through activating the STAT-1 related inflammatory signaling pathways and increased oxidative/nitrosative stress. However, we observed a very low level of tissue damage in wild-type mice, strongly suggesting the protective role of PPAR-alpha in regulating STAT1 inflammatory signaling pathways and oxidative/nitrosative stress. In addition to oxidation, nitration of tyrosine (Tyr) residues of many proteins is also important in regulating the activities of cellular proteins. By using Cyp2e1-null mice, we have recently demonstrated that CYP2E1 is involved in promoting nitration and ubiquitin-dependent degradation of many proteins. To study the role of protein nitration in mitochondrial dysfunction and tissue damage, we have developed a method to purify nitrated proteins from mitochondrial fractions in liver tissues treated with a CYP2E1 substrate acetaminophen (APAP) or LPS. Increased protein nitration in mitochondria was observed within 1 and 2 h after treatment with APAP (or LPS) followed by disappearance of nitrated proteins at 4 h, possibly through ubiquitin-mediated degradation of nitrated proteins. Based on these results, nitrated proteins were purified by affinity purification with the antibody to nitrated proteins. Mass spectral analysis of the purified nitrated proteins revealed nitration of many mitochondrial proteins. Biochemical properties of nitrated mitochondrial proteins are being investigated to establish the effects of nitration of these proteins on their biological functions. We have recently 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 role of JNK in regulating mitochondrial function and cell/tissue damage through protein phosphorylation, we have initiated a study to identify and characterize JNK-mediated phosphorylation of many mitochondrial proteins. To specifically activate JNK without activating other mitogen-activated protein kinases, we chose a model of carbon tetrachloride (CCL4)-induced liver injury. We observed that activated JNK translocates to mitochondria and that many mitochondrial proteins are phosphorylated in a time-dependent manner after CCL4 exposure. To further characterize the phosphorylated mitochondrial proteins, we have purified phosphorylated mitochondrial proteins by affinity purification. We plan to determine the identities of the phosphorylated proteins by mass-spectral analysis followed by biochemical characterizations of some selected phospho-proteins to further establish the role of phosphorylation of mitochondrial proteins in promoting mitochondrial dysfunction and tissue injury. Although many animal models exist for studying the mechanisms of alcoholic and nonalcoholic fatty liver diseases (AFLD and NAFLD, respectively) without or with inflammation, the roles of PPAR-alpha and CYP2E1 in these areas have not been fully characterized. PPAR-alpha is a ligand-activated transcription factor involved in controlling the expression of many genes in the fatty acid transport, inflammatory reactions, and fat metabolism. Moreover, the expressed level of PPAR-alpha 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 various 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 published in J of Nutrition demonstrate that inhibition of PPAR-alpha 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. Based on our own data recently published and unpublished, we believe that Ppara-null or Cyp2e1-null mice are very useful in studying the mechanisms of AFLD and NAFLD treated with an ethanol-liquid diet alone or in combination with another potentially toxic agent including a HFD or nicotine, 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) against AFLD and NAFLD in Ppara-null or Cyp2e1-null mice compared to WT mice.

Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2011
Total Cost
$776,088
Indirect Cost
Name
National Institute on Alcohol Abuse and Alcoholism
Department
Type
DUNS #
City
State
Country
Zip Code
Cho, Young-Eun; Song, Byoung-Joon; Akbar, Mohammed et al. (2018) Extracellular vesicles as potential biomarkers for alcohol- and drug-induced liver injury and their therapeutic applications. Pharmacol Ther 187:180-194
Cho, Young-Eun; Song, Byoung-Joon (2018) Pomegranate prevents binge alcohol-induced gut leakiness and hepatic inflammation by suppressing oxidative and nitrative stress. Redox Biol 18:266-278
Lakshmi, Sreeja; Prakash, Parvathi; Essa, Musthafa M et al. (2018) Marine derived bioactive compounds for treatment of Alzheimer's disease. Front Biosci (Elite Ed) 10:537-548
Cho, Young-Eun; Yu, Li-Rong; Abdelmegeed, Mohamed A et al. (2018) Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury. J Hepatol 69:142-153
Mukhopadhyay, Partha; Eid, Nabil; Abdelmegeed, Mohamed A et al. (2018) Interplay of Oxidative Stress, Inflammation, and Autophagy: Their Role in Tissue Injury of the Heart, Liver, and Kidney. Oxid Med Cell Longev 2018:2090813
Choi, Youngshim; Abdelmegeed, Mohamed A; Song, Byoung-Joon (2018) Preventive effects of indole-3-carbinol against alcohol-induced liver injury in mice via antioxidant, anti-inflammatory, and anti-apoptotic mechanisms: Role of gut-liver-adipose tissue axis. J Nutr Biochem 55:12-25
Abdelmegeed, Mohamed A; Ha, Seung-Kwon; Choi, Youngshim et al. (2017) Role of CYP2E1 in Mitochondrial Dysfunction and Hepatic Injury by Alcohol and Non-Alcoholic Substances. Curr Mol Pharmacol 10:207-225
Guillemin, Gilles J; Essa, Musthafa Mohamed; Song, Byoung-Joon et al. (2017) Dietary Supplements/Antioxidants: Impact on Redox Status in Brain Diseases. Oxid Med Cell Longev 2017:5048432
Cho, Young-Eun; Mezey, Esteban; Hardwick, James P et al. (2017) Increased ethanol-inducible cytochrome P450-2E1 and cytochrome P450 isoforms in exosomes of alcohol-exposed rodents and patients with alcoholism through oxidative and endoplasmic reticulum stress. Hepatol Commun 1:675-690
Choi, Youngshim; Abdelmegeed, Mohamed A; Song, Byoung-Joon (2017) Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress. Food Chem Toxicol 103:111-121

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