Contributing Roles of CYP2E1 and Other Cytochrome P450 Isoforms in Alcohol-Related Tissue Injury and Carcinogenesis.

The purpose of this review is to briefly summarize the roles of alcohol (ethanol) and related compounds in promoting cancer and inflammatory injury in many tissues. Long-term chronic heavy alcohol exposure is known to increase the chances of inflammation, oxidative DNA damage, and cancer development in many organs. The rates of alcohol-mediated organ damage and cancer risks are significantly elevated in the presence of co-morbidity factors such as poor nutrition, unhealthy diets, smoking, infection with bacteria or viruses, and exposure to pro-carcinogens. Chronic ingestion of alcohol and its metabolite acetaldehyde may initiate and/or promote the development of cancer in the liver, oral cavity, esophagus, stomach, gastrointestinal tract, pancreas, prostate, and female breast. In this chapter, we summarize the important roles of ethanol/acetaldehyde in promoting inflammatory injury and carcinogenesis in several tissues. We also review the updated roles of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) and other cytochrome P450 isozymes in the metabolism of various potentially toxic substrates, and consequent toxicities, including carcinogenesis in different tissues. We also briefly describe the potential implications of endogenous ethanol produced by gut bacteria, as frequently observed in the experimental models and patients of nonalcoholic fatty liver disease, in promoting DNA mutation and cancer development in the liver and other tissues, including the gastrointestinal tract.

Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury.

BACKGROUND & AIMS: Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness. METHODS: The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses. RESULTS: Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (ß-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels. CONCLUSIONS: These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease. LAY SUMMARY: Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.

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.

Indole-3-carbinol (I3C), found in Brassica family vegetables, exhibits antioxidant, anti-inflammatory, and anti-cancerous properties. Here, we aimed to evaluate the preventive effects of I3C against ethanol (EtOH)-induced liver injury and study the protective mechanism(s) by using the well-established chronic-plus-binge alcohol exposure model. The preventive effects of I3C were evaluated by conducting various histological, biochemical, and real-time PCR analyses in mouse liver, adipose tissue, and colon, since functional alterations of adipose tissue and intestine can also participate in promoting EtOH-induced liver damage. Daily treatment with I3C alleviated EtOH-induced liver injury and hepatocyte apoptosis, but not steatosis, by attenuating elevated oxidative stress, as evidenced by the decreased levels of hepatic lipid peroxidation, hydrogen peroxide, CYP2E1, NADPH-oxidase, and protein acetylation with maintenance of mitochondrial complex I, II, and III protein levels and activities. I3C also restored the hepatic antioxidant capacity by preventing EtOH-induced suppression of glutathione contents and mitochondrial aldehyde dehydrogenase-2 activity. I3C preventive effects were also achieved by attenuating the increased levels of hepatic proinflammatory cytokines, including IL1ß, and neutrophil infiltration. I3C also attenuated EtOH-induced gut leakiness with decreased serum endotoxin levels through preventing EtOH-induced oxidative stress, apoptosis of enterocytes, and alteration of tight junction protein claudin-1. Furthermore, I3C alleviated adipose tissue inflammation and decreased free fatty acid release. Collectively, I3C prevented EtOH-induced liver injury via attenuating the damaging effect of ethanol on the gut-liver-adipose tissue axis. Therefore, I3C may also have a high potential for translational research in treating or preventing other types of hepatic injury associated with oxidative stress and inflammation.

Cytochrome P450-2E1 is involved in aging-related kidney damage in mice through increased nitroxidative stress.

The aim of this study was to investigate the role of cytochrome P450-2E1 (CYP2E1) in aging-dependent kidney damage since it is poorly understood. Young (7 weeks) and aged female (16-17 months old) wild-type (WT) and Cyp2e1-null mice were used. Kidney histology showed that aged WT mice exhibited typical signs of kidney aging such as cell vacuolation, inflammatory cell infiltration, cellular apoptosis, glomerulonephropathy, and fibrosis, along with significantly elevated levels of renal TNF-α and serum creatinine than all other groups. Furthermore, the highest levels of renal hydrogen peroxide, protein carbonylation and nitration were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of iNOS and mitochondrial nitroxidative stress through altered amounts and activities of the mitochondrial complex proteins and significantly reduced levels of the antioxidant glutathione (GSH). In contrast, the aged Cyp2e1-null mice exhibited significantly higher antioxidant capacity with elevated heme oxygenase-1 and catalase activities compared to all other groups, while maintaining normal GSH levels with significantly less mitochondrial nitroxidative stress compared to the aged WT mice. Thus, CYP2E1 is important in causing aging-related kidney damage most likely through increasing nitroxidative stress and that CYP2E1 could be a potential target in preventing aging-related kidney diseases.

Diet high in fructose promotes liver steatosis and hepatocyte apoptosis in C57BL/6J female mice: Role of disturbed lipid homeostasis and increased oxidative stress.

The effects of high (H)-fructose (FR) diet (D) (HFRD) on hepatic lipid homeostasis, oxidative stress, inflammation and hepatocyte apoptosis were investigated in 6-week old female C57BL/6J mice fed a regular chow (ContD) or HFRD (35% fructose-derived calories) for 3 weeks. HFRD-fed mice exhibited increased levels of hepatic steatosis with a significant elevation of serum levels of triglyceride, cholesterol and TNFα compared to ContD-fed mice (P<0.05). HFRD-fed mice exhibited ∼2.7- fold higher levels FAS along with significantly decreased protein levels of adiponection-R2 (∼30%), P-AMPK (∼60%), P-ACC (∼70%) and RXR-α (∼55%), suggesting decreased hepatic fat oxidation compared to controls. Interestingly, hepatic fatty acid uptake into hepatocytes and lipolysis were significantly increased in HFRD-fed mice, as shown by decreased CD36 and fatty acid transporter protein-2, and increased adipose triglyceride lipase, respectively (P<0.05). Increased hepatic levels of iNOS and GSSG/GSH suggest elevated oxidative stress with a higher number of macrophages in the adipose tissue in HFRD-fed mice (P<0.05). Significantly elevated rates of hepatocyte apoptosis (∼2.4-fold), as determined by TUNEL analysis with increased Bax/Bcl2 ratio and PARP-1 levels (∼2- and 1.5-fold, respectively), were observed in HFRD-fed mice. Thus, HFRD exposure increased hepatic steatosis accompanied by oxidative stress and inflammation, leading to hepatocyte apoptosis.

Cytochrome P450-2E1 promotes fast food-mediated hepatic fibrosis.

Cytochrome P450-2E1 (CYP2E1) increases oxidative stress. High hepatic cholesterol causes non-alcoholic steatohepatitis (NASH) and fibrosis. Thus, we aimed to study the role of CYP2E1 in promoting liver fibrosis by high cholesterol-containing fast-food (FF). Male wild-type (WT) and Cyp2e1-null mice were fed standard chow or FF for 2, 12, and 24 weeks. Various parameters of liver fibrosis and potential mechanisms such as oxidative and endoplasmic reticulum (ER) stress, inflammation, and insulin resistance (IR) were studied. Indirect calorimetry was also used to determine metabolic parameters. Liver histology showed that only WT fed FF (WT-FF) developed NASH and fibrosis. Hepatic levels of fibrosis protein markers were significantly increased in WT-FF. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in FF-fed WT. Serum endotoxin, TLR-4 levels, and inflammatory markers were highest in WT-FF. FAS, PPAR-α, PPAR-γ, and CB1-R were markedly altered in WT-FF. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in FF-fed WT. Indirect calorimetry showed that Cyp2e1-null-mice fed FF exhibited consistently higher total energy expenditure (TEE) than their corresponding WT. These results demonstrate that CYP2E1 is important in fast food-mediated liver fibrosis by promoting nitroxidative and ER stress, endotoxemia, inflammation, IR, and low TEE.

Role of CYP2E1 in Mitochondrial Dysfunction and Hepatic Injury by Alcohol and Non-Alcoholic Substances.

Alcoholic fatty liver disease (AFLD) and non-alcoholic fatty liver disease (NAFLD) are two pathological conditions that are spreading worldwide. Both conditions are remarkably similar with regard to the pathophysiological mechanism and progression despite different causes. Oxidative stressinduced mitochondrial dysfunction through post-translational protein modifications and/or mitochondrial DNA damage has been a major risk factor in both AFLD and NAFLD development and progression. Cytochrome P450-2E1 (CYP2E1), a known important inducer of oxidative radicals in the cells, has been reported to remarkably increase in both AFLD and NAFLD. Interestingly, CYP2E1 isoforms expressed in both endoplasmic reticulum (ER) and mitochondria, likely lead to the deleterious consequences in response to alcohol or in conditions of NAFLD after exposure to high fat diet (HFD) and in obesity and diabetes. Whether CYP2E1 in both ER and mitochondria work simultaneously or sequentially in various conditions and whether mitochondrial CYP2E1 may exert more pronounced effects on mitochondrial dysfunction in AFLD and NAFLD are unclear. The aims of this review are to briefly describe the role of CYP2E1 and resultant oxidative stress in promoting mitochondrial dysfunction and the development or progression of AFLD and NAFLD, to shed a light on the function of the mitochondrial CYP2E1 as compared with the ER-associated CYP2E1. We finally discuss translational research opportunities related to this field.

Proteomic analysis of acetaminophen-induced hepatotoxicity and identification of heme oxygenase 1 as a potential plasma biomarker of liver injury.

PURPOSE: Overdose of acetaminophen (APAP) is a major cause of acute liver failure. This study was aimed to identify pathways related to hepatotoxicity and potential biomarkers of liver injury. EXPERIMENTAL DESIGN: Rats were treated with low (100 mg/kg) and high (1250 mg/kg) doses of APAP, and liver tissues at 6 and 24 h post-treatment were analyzed using a proteomic approach of 16O/18O labeling and 2D-LC-MS/MS. RESULTS: Molecular pathways evolved progressively from scattered and less significant perturbations to more focused and significant alterations in a dose- and time-dependent manner upon APAP treatment. Imbalanced expression of hemeoxygenase 1 (HMOX1) and biliverdin reductase A (BLVRA) was associated with hepatotoxicity. Protein abundance changes of a total of 31 proteins were uniquely correlated to liver damage, among which a dramatic increase of HMOX1 levels in plasma was observed. Liver injury-associated significant elevation of plasma HMOX1 was further validated in mice treated with APAP. CONCLUSIONS AND CLINICAL RELEVANCE: This study unveiled molecular changes associated with APAP-induced liver toxicity at the pathway levels and identified HMOX1 as a potential plasma biomarker of liver injury.

Preventive effects of dietary walnuts on high-fat-induced hepatic fat accumulation, oxidative stress and apoptosis in mice.

We hypothesized that dietary walnut would prevent high-fat-diet (HFD)-induced hepatic apoptosis based on its antioxidant properties. Male C57BL/6J mice were fed a rodent chow or HFD (45% energy-derived)±walnuts (21.5% energy-derived) for 6 weeks. Liver histological and biochemical analyses revealed significantly elevated fat accumulation in mice fed HFD compared to mice fed the chow or HFD±walnuts. Walnut supplementation prevented HFD-mediated alteration of the levels of key proteins in lipid homeostasis such as Sirt1, AMPK and FAS, leading to decreased fat accumulation. In addition, walnut supplementation to HFD significantly decreased the hepatic levels of cytochrome P450-2E1, nitrated proteins and lipid peroxidation. Furthermore, walnut supplementation decreased the activated cell-death-associated p-JNK and p-p38K accompanied with increased hepatocyte apoptosis in HFD group. The beneficial effects of dietary walnut likely result, at least partially, from its antioxidant ingredients and attenuating HFD-induced hepatic steatosis, nitroxidative stress and apoptosis.

Chronic Glutathione Depletion Confers Protection against Alcohol-induced Steatosis: Implication for Redox Activation of AMP-activated Protein Kinase Pathway.

The pathogenesis of alcoholic liver disease (ALD) is not well established. However, oxidative stress and associated decreases in levels of glutathione (GSH) are known to play a central role in ALD. The present study examines the effect of GSH deficiency on alcohol-induced liver steatosis in Gclm knockout (KO) mice that constitutively have ≈15% normal hepatic levels of GSH. Following chronic (6 week) feeding with an ethanol-containing liquid diet, the Gclm KO mice were unexpectedly found to be protected against steatosis despite showing increased oxidative stress (as reflected in elevated levels of CYP2E1 and protein carbonyls). Gclm KO mice also exhibit constitutive activation of liver AMP-activated protein kinase (AMPK) pathway and nuclear factor-erythroid 2-related factor 2 target genes, and show enhanced ethanol clearance, altered hepatic lipid profiles in favor of increased levels of polyunsaturated fatty acids and concordant changes in expression of genes associated with lipogenesis and fatty acid oxidation. In summary, our data implicate a novel mechanism protecting against liver steatosis via an oxidative stress adaptive response that activates the AMPK pathway. We propose redox activation of the AMPK may represent a new therapeutic strategy for preventing ALD.

Dietary walnut reduces hepatic triglyceride content in high-fat-fed mice via modulation of hepatic fatty acid metabolism and adipose tissue inflammation.

In this study, we evaluated the protective effects of dietary walnuts on high-fat diet (HFD)-induced fatty liver and studied the underlying mechanisms. Male C57BL/6J mice were fed either a regular rodent chow or HFD (45% energy-derived) with or without walnuts (21.5% energy-derived) for 20weeks. Walnut supplementation did not change HFD-induced increase in body weight or visceral fat mass. However, dietary walnuts significantly decreased the amounts of hepatic triglyceride (TG) observed in HFD-fed mice. The addition of walnuts significantly altered the levels of proteins, involved in the hepatic lipid homeostasis, including AMP-activated protein kinase, fatty acid synthase and peroxisome proliferator-activated receptor-α. Since adipocyte inflammation and apoptosis are reportedly important in regulating hepatic fat accumulation, we also evaluated the protective effects of walnuts on adipose tissue injury. Real-time polymerase chain reaction results revealed that adipose tissues isolated from mice fed the HFD+walnut diets showed significantly decreased levels of macrophage infiltration with suppressed expression of proinflammatory genes compared to those significantly elevated in mice fed HFD alone. These improvements also coincided with reduction of HFD-induced apoptosis of adipocytes by dietary walnuts. However, the supplemented walnuts did not significantly alter HFD-induced peripheral glucose intolerance or insulin resistance despite a trend of improvement. Collectively, these results demonstrate that the protective effects of walnuts against HFD-induced hepatic TG accumulation in mice are mediated, at least partially, by modulating the key proteins in hepatic lipid homeostasis and suppression of the genes related to adipose tissue inflammation and macrophage infiltration as well as prevention of adipocyte apoptosis.

Mitochondrial dysfunction and cell death in neurodegenerative diseases through nitroxidative stress.

Mitochondria are important for providing cellular energy ATP through the oxidative phosphorylation pathway. They are also critical in regulating many cellular functions including the fatty acid oxidation, the metabolism of glutamate and urea, the anti-oxidant defense, and the apoptosis pathway. Mitochondria are an important source of reactive oxygen species leaked from the electron transport chain while they are susceptible to oxidative damage, leading to mitochondrial dysfunction and tissue injury. In fact, impaired mitochondrial function is commonly observed in many types of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, alcoholic dementia, brain ischemia-reperfusion related injury, and others, although many of these neurological disorders have unique etiological factors. Mitochondrial dysfunction under many pathological conditions is likely to be promoted by increased nitroxidative stress, which can stimulate post-translational modifications (PTMs) of mitochondrial proteins and/or oxidative damage to mitochondrial DNA and lipids. Furthermore, recent studies have demonstrated that various antioxidants, including naturally occurring flavonoids and polyphenols as well as synthetic compounds, can block the formation of reactive oxygen and/or nitrogen species, and thus ultimately prevent the PTMs of many proteins with improved disease conditions. Therefore, the present review is aimed to describe the recent research developments in the molecular mechanisms for mitochondrial dysfunction and tissue injury in neurodegenerative diseases and discuss translational research opportunities.

Cytochrome P450-2E1 promotes aging-related hepatic steatosis, apoptosis and fibrosis through increased nitroxidative stress.

The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease.

Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury.

The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1h while liver damage was maximal at 24h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins.

Increased Sensitivity to Binge Alcohol-Induced Gut Leakiness and Inflammatory Liver Disease in HIV Transgenic Rats.

The mechanisms of alcohol-mediated advanced liver injury in HIV-infected individuals are poorly understood. Thus, this study was aimed to investigate the effect of binge alcohol on the inflammatory liver disease in HIV transgenic rats as a model for simulating human conditions. Female wild-type (WT) or HIV transgenic rats were treated with three consecutive doses of binge ethanol (EtOH) (3.5 g/kg/dose oral gavages at 12-h intervals) or dextrose (Control). Blood and liver tissues were collected at 1 or 6-h following the last dose of ethanol or dextrose for the measurements of serum endotoxin and liver pathology, respectively. Compared to the WT, the HIV rats showed increased sensitivity to alcohol-mediated gut leakiness, hepatic steatosis and inflammation, as evidenced with the significantly elevated levels of serum endotoxin, hepatic triglycerides, histological fat accumulation and F4/80 staining. Real-time PCR analysis revealed that hepatic levels of toll-like receptor-4 (TLR4), leptin and the downstream target monocyte chemoattractant protein-1 (MCP-1) were significantly up-regulated in the HIV-EtOH rats, compared to all other groups. Subsequent experiments with primary cultured cells showed that both hepatocytes and hepatic Kupffer cells were the sources of the elevated MCP-1 in HIV-EtOH rats. Further, TLR4 and MCP-1 were found to be upregulated by leptin. Collectively, these results show that HIV rats, similar to HIV-infected people being treated with the highly active anti-retroviral therapy (HAART), are more susceptible to binge alcohol-induced gut leakiness and inflammatory liver disease than the corresponding WT, possibly due to additive or synergistic interaction between binge alcohol exposure and HIV infection. Based on these results, HIV transgenic rats can be used as a surrogate model to study the molecular mechanisms of many disease states caused by heavy alcohol intake in HIV-infected people on HAART.

Translational Implications of the Alcohol-Metabolizing Enzymes, Including Cytochrome P450-2E1, in Alcoholic and Nonalcoholic Liver Disease.

Fat accumulation (hepatic steatosis) in alcoholic and nonalcoholic fatty liver disease is a potentially pathologic condition which can progress to steatohepatitis (inflammation), fibrosis, cirrhosis, and carcinogenesis. Many clinically used drugs or some alternative medicine compounds are also known to cause drug-induced liver injury, which can further lead to fulminant liver failure and acute deaths in extreme cases. During liver disease process, certain cytochromes P450 such as the ethanol-inducible cytochrome P450-2E1 (CYP2E1) and CYP4A isozymes can be induced and/or activated by alcohol and/or high-fat diets and pathophysiological conditions such as fasting, obesity, and diabetes. Activation of these P450 isozymes, involved in the metabolism of ethanol, fatty acids, and various drugs, can produce reactive oxygen/nitrogen species directly and/or indirectly, contributing to oxidative modifications of DNA/RNA, proteins and lipids. In addition, aldehyde dehydrogenases including the mitochondrial low Km aldehyde dehydrogenase-2 (ALDH2), responsible for the metabolism of acetaldehyde and lipid aldehydes, can be inactivated by various hepatotoxic agents. These highly reactive acetaldehyde and lipid peroxides, accumulated due to ALDH2 suppression, can interact with cellular macromolecules DNA/RNA, lipids, and proteins, leading to suppression of their normal function, contributing to DNA mutations, endoplasmic reticulum stress, mitochondrial dysfunction, steatosis, and cell death. In this chapter, we specifically review the roles of the alcohol-metabolizing enzymes including the alcohol dehydrogenase, ALDH2, CYP2E1, and other enzymes in promoting liver disease. We also discuss translational research opportunities with natural and/or synthetic antioxidants, which can prevent or delay the onset of inflammation and liver disease.

Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications.

Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury.

Binge alcohol promotes hypoxic liver injury through a CYP2E1-HIF-1α-dependent apoptosis pathway in mice and humans.

Binge drinking, a common pattern of alcohol ingestion, is known to potentiate liver injury caused by chronic alcohol abuse. This study was aimed at investigating the effects of acute binge alcohol on hypoxia-inducible factor-1α (HIF-1α)-mediated liver injury and the roles of alcohol-metabolizing enzymes in alcohol-induced hypoxia and hepatotoxicity. Mice and human specimens assigned to binge or nonbinge groups were analyzed for blood alcohol concentration (BAC), alcohol-metabolizing enzymes, HIF-1α-related protein nitration, and apoptosis. Binge alcohol promoted acute liver injury in mice with elevated levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and hypoxia, both of which were colocalized in the centrilobular areas. We observed positive correlations among elevated BAC, CYP2E1, and HIF-1α in mice and humans exposed to binge alcohol. The CYP2E1 protein levels (r = 0.629, p = 0.001) and activity (r = 0.641, p = 0.001) showed a significantly positive correlation with BAC in human livers. HIF-1α levels were also positively correlated with BAC (r = 0.745, p < 0.001) or CYP2E1 activity (r = 0.792, p < 0.001) in humans. Binge alcohol promoted protein nitration and apoptosis with significant correlations observed between inducible nitric oxide synthase and BAC, CYP2E1, or HIF-1α in human specimens. Binge-alcohol-induced HIF-1α activation and subsequent protein nitration or apoptosis seen in wild type were significantly alleviated in the corresponding Cyp2e1-null mice, whereas pretreatment with an HIF-1α inhibitor, PX-478, prevented HIF-1α elevation with a trend of decreased levels of 3-nitrotyrosine and apoptosis, supporting the roles of CYP2E1 and HIF-1α in binge-alcohol-mediated protein nitration and hepatotoxicity. Thus binge alcohol promotes acute liver injury in mice and humans at least partly through a CYP2E1-HIF-1α-dependent apoptosis pathway.