Alcohol (ethanol)-mediated cell and tissue damage is partly caused by increased oxidative and nitrative stress. The majority of reactive oxygen and nitrogen species in alcohol-exposed cells/tissues is known to be produced through direct inhibition of the mitochondrial respiratory chain and induction/activation of ethanol-inducible cytochrome P450-2E1 (CYP2E1), NADPH-oxidase, xanthine oxidase, inducible nitric oxide synthase (iNOS), etc. Combination of activated CYP2E1, a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), responsible for the removal of toxic acetaldehyde and lipid peroxides, leads to increased nitroxidative stress. Alcohol-induced nitroxidative stress not only elevates the amounts of acetaldehyde and lipid peroxides but also promotes many different post-translational modifications (PTMs) of cellular proteins, contributing to mitochondrial dysfunction and tissue damage. In collaboration with Dr. Ali Keshavarzian at Rush Medical Center in Chicago, we investigated the role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and advanced inflammatory liver disease. Binge alcohol administration (6 g/kg oral gavage for 3 times at 12-h intervals) increased the levels of serum endotoxin, hepatic enterobacterial contents, and hepatic fat accumulation with inflammatory foci in WT but not in the corresponding Cyp2e1-null mice. The increased fat accumulation, serum endotoxin, and hepatic contents of enterobacteria were significantly reduced when ethanol-exposed WT mice were co-treated with a specific CYP2E1 inhibitor chlormethiazole (CMZ) or an antioxidant N-acetylcysteine (NAC). Binge alcohol markedly elevated the levels of nitroxidative marker proteins CYP2E1 and iNOS in intestinal epithelial cells of WT mice. In contrast, these changes were not observed in the ethanol-exposed Cyp2e1-null mice or ethanol-exposed WT mice pretreated with CMZ and NAC, suggesting a critical role of CYP2E1 in gut leakiness and endotoxemia following binge alcohol exposure. The role of different PTMs of tight junction proteins in binge alcohol-mediated gut leakiness and endotoxemia is being investigated. Based on our results of alcohol-induced tissues injury, we also studied the role of CYP2E1 in alcohol-induced hypoxia and hepatotoxicity in mice and human specimens. Our results showed that binge alcohol promoted acute liver injury in WT mice with elevated levels of CYP2E1 and hypoxia, both of which were co-localized in centrilobular areas. Positive correlations among elevated blood alcohol concentration (BAC), CYP2E1, and hypoxia-inducible factor-1α(HIF-1α) in mice and autopsied human livers exposed to binge alcohol. Binge alcohol promoted protein nitration and apoptosis with significant correlations observed between the levels of iNOS and BACs, CYP2E1, or HIF-1αin human specimens. Binge alcohol-induced HIF-1αactivation and subsequent protein nitration or apoptosis seen in WT mice were significantly alleviated in the corresponding Cyp2e1-null mice while pretreatment with an HIF-1αinhibitor PX-478 prevented HIF-1αelevation with a trend of decreased levels of 3-nitrotyrosine and apoptosis. These results suggest that binge alcohol promotes acute liver injury in mice and humans partly through a CYP2E1-HIF-1α-dependent protein nitration and apoptosis pathway. Having studied the roles of CYP2E1 and different PTMs in alcohol-induced gut leakiness and inflammatory NASH, we also aimed to study the role of another PTM in alcohol-induced brain damage. In continued collaboration with Dr. Bong-Hee Lee at Gachon University, Korea, we investigated the mechanism of alcohol-induced neurodegeneration by studying the role of activated microglia cells in producing advanced glycation end adduct with albumin (AGE-albumin). WT rats were exposed to binge alcohol administrations (5 g/kg/day oral gavage for 10 consecutive days). Our results revealed that microglial activation and neuronal damage were found in the hippocampus and entorhinal cortex in alcohol-exposed rats. Increased AGE-albumin synthesis and secretion were also observed in activated microglial cells after alcohol exposure. The expressed levels of the receptor for AGE (RAGE)-positive neurons and RAGE-dependent neuronal death were markedly elevated by AGE-albumin through the stress-activated protein kinases p-JNK and p-p38kinase. Treatment with soluble RAGE or AGE inhibitors significantly reduced neuronal damage in the alcohol-exposed rats. Consistently, the elevated levels of activated microglial cells, AGE-albumin and neuronal loss were observed in autopsied human brain specimens from alcoholic individuals compared to normal controls. These results indicated that increased AGE-albumin from activated microglial cells induces neuronal death, and that efficient regulation of its synthesis and secretion is a therapeutic target for preventing alcohol-induced neurodegeneration. We recently demonstrated an important role of CYP2E1 in high-fat diet (HFD)-induced mitochondrial dysfunction, insulin resistance and the development of nonalcoholic steatohepatitis (NASH) progressed from nonalcoholic fatty liver disease (NAFLD). In continuation, we further studied the role of CYP2E1 in high fat-western diet (HFWD)-induced liver fibrosis in mice. Aged-matched male wild-type (WT) and Cyp2e1-null mice were fed a control diet or HFWD without or with 15% (v/v) ethanol in drinking water for 9 and 19 weeks for determination of various parameters. Liver histology and insulin resistance test showed that HFWD-fed WT (WT-HFWD) developed insulin resistance and NASH earlier than the HFWD-fed Cyp2e1-null mice (null-HFWD). Electron microscopy data also suggests greater alterations in mitochondria and ER structures in WT-HFWD compared to their controls. CYP2E1 was not elevated in WT-HFWD while iNOS and lipid peroxidation were differentially regulated in WT-HFWD and null-HFWD, as they were down-regulated in the WT-HFWD but not in null-HFWD. The development of hepatic fibrosis in WT-HFWD was confirmed by sirius red staining and the elevated levels of TGF-β, α-SMA and collagen-1. Further, serum endotoxin, TLR4, p-NFKβ, IL-1β, P-JNK, P-SMAD-3, all pro-fibrogenic factors, were either increased earlier or persistently elevated in the WT-HFWD than the null-HFWD. In contrast, PPAR-α, P-STAT1, P-STAT3, β-catenin, IRS-1 and IRS-2, all reportedly anti-fibrogenic factors, were persistently higher in the null-HFWD than the WT-HFWD. Interestingly, addition of ethanol (EtOH) in drinking water to HFWD led to the development of hepatic fibrosis in both strains, as evidenced by sirius red staining and collagen-1 levels. Hepatic fibrosis occurred despite the fact that hepatic steatosis and liver injury, determined by ALT levels and inflammatory foci, were markedly higher in WT-HFWD with EtOH than those without EtOH. These results suggest that CYP2E1 is important in the early development of HFWD-induced hepatic fibrosis where multiple factors are involved in this process. In fact, the absence of CYP2E1 seems to serve as a protective threshold to delay the development of hepatic fibrosis in HFWD-exposed mice without EtOH. However, CYP2E1 absence is no longer protective when the body system is over-whelmed by the additive or synergistic effects between HFWD and EtOH, as in the fibrotic case observed in null-HFWD with EtOH in drinking water. Based on our mechanistic studies on alcoholic fatty liver disease (AFLD) and NAFLD/NASH in our mouse strains, we plan to perform translational research by evaluating the beneficial effects of a few anti-oxidants from nuts and fruits containing n-3 fatty acids against AFLD and NAFLD/NASH in WT and Cyp2e1-null mice.
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