Alcohol steatosis and cytotoxicity: The role of cytochrome P4502E1 and autophagy.
ABSTRACT The goal of the current study was to evaluate whether CYP2E1 plays a role in binge-ethanol induced steatosis and if autophagy impacts CYP2E1-mediated hepatotoxicity, oxidative stress and fatty liver formation produced by ethanol. Wild type (WT), CYP2E1 knockin (KI) and CYP2E1 knockout (KO) mice were gavaged with 3g/kg body wt ethanol twice a day for four days. This treatment caused fatty liver, elevation of CYP2E1 and oxidative stress in WT and KI mice but not KO mice. Autophagy was impaired in ethanol-treated KI mice compared to KO mice as reflected by a decline in the LC3-II/LC3-I ratio and lower total LC-3 and Beclin-1 levels coupled to increases in P62, pAKT/AKT and mTOR. Inhibition of macroautophagy by administration of 3-methyladenine enhanced the binge ethanol hepatotoxicity, steatosis and oxidant stress in CYP2E1 KI, but not CYP2E1 KO mice. Stimulation of autophagy by rapamycin blunted the elevated steatosis produced by binge ethanol. Treatment of HepG2 E47 cells which express CYP2E1 with 100mM ethanol for 8 days increased fat accumulation and oxidant stress but decreased autophagy. Ethanol had no effect on these reactions in HepG2 C34 cells which do not express CYP2E1. Inhibition of autophagy elevated ethanol toxicity, lipid accumulation and oxidant stress in the E47, but not C34 cells. The antioxidant N-acetylcysteine, and CYP2E1 inhibitor chlormethiazole blunted these effects of ethanol. These results indicate that CYP2E1 plays an important role in binge ethanol-induced fatty liver. We propose that CYP2E1-derived reactive oxygen species inhibit autophagy, which subsequently causes accumulation of lipid droplets. Inhibition of autophagy promotes binge ethanol induced hepatotoxicity, steatosis and oxidant stress via CYP2E1.
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ABSTRACT: In this review, we describe research findings on the effects of alcohol exposure on two major catabolic systems in liver cells: the ubiquitin–proteasome system (UPS) and autophagy. These hydrolytic systems are not unique to liver cells; they exist in all eukaryotic tissues and cells. However, because the liver is the principal site of ethanol metabolism, it sustains the greatest damage from heavy drinking. Thus, the focus of this review is to specifically describe how ethanol oxidation modulates the activities of the UPS and autophagy and the mechanisms by which these changes contribute to the pathogenesis of alcohol-induced liver injury. Here, we describe the history and the importance of cellular hydrolytic systems, followed by a description of each catabolic pathway and the differential modulation of each by ethanol exposure. Overall, the evidence for an involvement of these catabolic systems in the pathogenesis of alcoholic liver disease is quite strong. It underscores their importance, not only as effective means of cellular recycling and eventual energy generation, but also as essential components of cellular defense.12/2014; 3. DOI:10.1016/j.redox.2014.10.006
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ABSTRACT: We have shown that alcohol increases Caco-2 intestinal epithelial cell monolayer permeability in vitro by inducing the expression of redox sensitive circadian clock proteins CLOCK and PER2 and that these proteins are necessary for alcohol-induced hyperpermeability. We hypothesized that alcohol metabolism by intestinal Cytochrome-P450 2E1 (CYP2E1) could alter circadian gene expression (Clock and Per2) resulting in alcohol-induced hyperpermeability. Methods. In vitro Caco-2 intestinal epithelial cells were exposed to alcohol and CYP2E1 protein, activity, and mRNA were measured. CYP2E1 expression was knocked down via siRNA and alcohol-induced hyperpermeability and CLOCK and PER2 protein expression were measured. Caco-2 cells were also treated with alcohol or H2O2 ± N-acetylcysteine (NAC) anti-oxidant and CLOCK and PER2 proteins were measured at 4 or 2 hours. In vivo Cyp2e1 protein and mRNA were also measured in colon tissue from alcohol-fed mice. Results. 1) Alcohol increased CYP2E1 protein by 93% and enzyme activity by 69% in intestinal cells in vitro; 2) Alcohol feeding also increased mouse colonic Cyp2e1 protein by 73%. 3) mRNA levels of Cyp2e1 were not changed by alcohol in vitro or in mouse intestine; 4) siRNA knockdown of CYP2E1 in Caco-2 cells prevented alcohol-induced hyperpermeability and induction of CLOCK and PER2 proteins; 5) Alcohol-induced and H2O2-induced increases in intestinal cell CLOCK and PER2 were significantly inhibited by treatment with NAC. 6) Conclusion. Our data support a novel role for intestinal CYP2E1 in alcohol-induced intestinal hyperpermeability via a mechanism involving CYP2E1-dependent induction of oxidative stress and upregulation of circadian clock proteins CLOCK and PER2.AJP Gastrointestinal and Liver Physiology 05/2013; DOI:10.1152/ajpgi.00354.2012
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ABSTRACT: Puerarin (PR) has been utilized as a phytomedicine to managing liver disease in China. Thus, this study aimed to evaluate the potential PR-mediated hepatoprotective role against chronic alcohol-induced liver injury in rats. The results indicated that serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and pro-inflammatory cytokines were significantly reduced following PR treatment, while the albumin (ALB) level was increased. Meanwhile, intrahepatic contents of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) were elevated. Pathological examination showed that alcohol-lesioned hepatocytes were mitigated through the PR treatment. In addition, the endogenous levels of glycogen synthase kinase-3β (GSK-3β) at the protein level and β-catenin expression at the mRNA level were notably down-regulated, whereas the tumor necrosis factor alpha (TNF-α) and nuclear factor-kappa B (NF-κB) proteins in the liver tissue were effectively decreased following the PR treatment. Together, these findings demonstrate that PR mediates hepatoprotection against alcohol-induced liver injury. The mechanisms underlying the cytoprotective effects of PR are associated with inhibiting immunotoxicity in hepatocytes and regulating the GSK-3β/NF-κB pathway, thereby maintaining metabolic homeostasis in the liver tissue.International immunopharmacology 06/2013; 17(1). DOI:10.1016/j.intimp.2013.05.023