Cytochrome P450 2E1 potentiates ethanol induction of hypoxia and HIF-1α in vivo

Ethanol induces hypoxia and elevates HIF-1α in the liver. CYP2E1 plays a role in the mechanisms by which ethanol generates oxidative stress, fatty liver, and liver injury. This study evaluated whether CYP2E1 contributes to ethanol-induced hypoxia and activation of HIF-1α in vivo and whether HIF-1α protects against or promotes CYP2E1-dependent toxicity in vitro. Wild-type (WT), CYP2E1-knock-in (KI), and CYP2E1 knockout (KO) mice were fed ethanol chronically; pair-fed controls received isocaloric dextrose. Ethanol produced liver injury in the KI mice to a much greater extent than in the WT and KO mice. Protein levels of HIF-1α and downstream targets of HIF-1α activation were elevated in the ethanol-fed KI mice compared to the WT and KO mice. Levels of HIF prolyl hydroxylase 2, which promotes HIF-1α degradation, were decreased in the ethanol-fed KI mice in association with the increases in HIF-1α. Hypoxia occurred in the ethanol-fed CYP2E1 KI mice as shown by an increased area of staining using the hypoxia-specific marker pimonidazole. Hypoxia was lower in the ethanol-fed WT mice and lowest in the ethanol-fed KO mice and all the dextrose-fed mice. In situ double staining showed that pimonidazole and CYP2E1 were colocalized to the same area of injury in the hepatic centrilobule. Increased protein levels of HIF-1α were also found after acute ethanol treatment of KI mice. Treatment of HepG2 E47 cells, which express CYP2E1, with ethanol plus arachidonic acid (AA) or ethanol plus buthionine sulfoximine (BSO), which depletes glutathione, caused loss of cell viability to a greater extent than in HepG2 C34 cells, which do not express CYP2E1. These treatments elevated protein levels of HIF-1α to a greater extent in E47 cells than in C34 cells. 2-Methoxyestradiol, an inhibitor of HIF-1α, blunted the toxic effects of ethanol plus AA and ethanol plus BSO in the E47 cells in association with inhibition of HIF-1α. The HIF-1α inhibitor also blocked the elevated oxidative stress produced by ethanol/AA or ethanol/BSO in the E47 cells. These results suggest that CYP2E1 plays a role in ethanol-induced hypoxia, oxidative stress, and activation of HIF-1α and that HIF-1α contributes to CYP2E1-dependent ethanol-induced toxicity. Blocking HIF-1α activation and actions may have therapeutic implications for protection against ethanol/CYP2E1-induced oxidative stress, steatosis, and liver injury.     

Cytochrome P4502E1, oxidative stress, JNK, and autophagy in acute alcohol-induced fatty liver

Binge alcohol drinking induces hepatic steatosis. Recent studies showed that chronic ethanol-induced fatty liver was, at least in part, CYP2E1 dependent. The mechanism of acute alcohol-induced steatosis and whether CYP2E1 plays any role are still unclear. Increasing oxidative stress by alcohol can activate the JNK MAP kinase signaling pathway, suggesting that JNK might be a target for prevention of alcohol-induced steatosis. We used CYP2E1 knockout (KO) mice, a JNK inhibitor, and JNK1 or JNK2 knockout mice to test the role of CYP2E1, JNK, and the individual role of JNK1 and JNK2 in acute alcohol-induced steatosis. In wild-type (WT) mice, acute alcohol activates CYP2E1 and increases oxidative stress, which reciprocally increases activation of the JNK signaling pathway. Acute alcohol-induced fatty liver and oxidative stress were blunted in CYP2E1 KO mice and by the JNK inhibitor in WT mice. The antioxidant N-acetylcysteine decreased the acute alcohol-induced oxidative stress, the activation of JNK, and the steatosis but not the activation of CYP2E1. Acute alcohol decreased autophagy and increased expression of SREBP, effects blocked by the JNK inhibitor. Acute alcohol-induced fatty liver was the same in JNK1 and JNK2 KO mice as in WT mice; thus either JNK1 or JNK2 per se is sufficient for induction of steatosis by acute alcohol. The results show that acute alcohol elevation of CYP2E1, oxidative stress, and activation of JNK interact to lower autophagy and increase lipogenic SREBP resulting in fatty liver.     

CYP2E1 enhances ethanol-induced lipid accumulation but impairs autophagy in HepG2 E47 cells

The regulation and function of autophagy and lipid metabolism have recently been reported to be reciprocally related. Macroautophagy mediates the breakdown of lipids stored in lipid droplets. An inhibition of autophagy leads to the development of a fatty liver. We evaluated the ability of CYP2E1 to modulate the effects of ethanol on lipid accumulation and autophagy in vitro. The E47 HepG2 cell which expresses CYP2E1 was treated with ethanol at 50, 100 and 150 mM for 4 or 5 days. Ethanol-induced lipid accumulation and an increase of triglycerides (TG) in E47 cells to a greater extent than in control C34 cells which do not express CYP2E1. In contrast, autophagy (LC3 II/LC3 I ratio) was significantly induced by ethanol in C34 cells to a greater extent than in E47 cells. P62 was significantly increased in E47 cells after ethanol treatment. Thus, there is a reciprocal relationship between the effects of ethanol on lipid accumulation and autophagy in the CYP2E1-expressing cells. Inhibition of autophagy by 3-methyladenine (3MA), increased lipid accumulation and TG levels in C34 cells which display elevated autophagy, but enhanced lipid accumulation and TG level to a lesser extent in E47 cells which displayed lower autophagy. Ethanol induced CYP2E1 activity and oxidative stress in E47 cells compared with C34 cells. These experiments suggest that the expression of CYP2E1 may impair autophagy formation which contributes to lipid accumulation in the liver. We hypothesize that CYP2E1-induced oxidative stress promotes the accumulation of lipid droplets by ethanol and this may be responsible for the suppression of autophagy in the liver.     

Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in autophagy

Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver from progressive damage caused by alcohol. Increased oxidative stress has been reported as one mechanism underlying alcohol-induced steatosis. We evaluated whether cannabidiol, which has been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative stress-induced steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice, possibly by preventing the increase in oxidative stress and the activation of the JNK MAPK pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and in mouse liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by alcohol. In conclusion, these results show that cannabidiol protects mouse liver from acute alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-mediated oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.     

Green tea polyphenol epigallocatechin-3-gallate protects HepG2 cells against CYP2E1-dependent toxicity

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates.     

Effects of binge ethanol on lipid homeostasis and oxidative stress in a rat model of nonalcoholic fatty liver disease

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.     

Acute ethanol pretreatment increases FAS-mediated liver injury in mice: role of oxidative stress and CYP2E1-dependent and -independent pathways

This study evaluated whether acute ethanol pretreatment potentiates Fas-mediated liver injury and if oxidative stress and CYP2E1 play a role in any enhanced hepatotoxicity. There were 3-fold increases of transaminases and more extensive apoptotic necrosis of hepatocytes and focal hemorrhages of the hepatic lobule in mice treated with Jo2 Fas agonistic antibody plus ethanol compared to saline control or to mice treated with Jo2 or ethanol alone. CYP2E1 catalytic activity and protein were increased 2-fold by the acute ethanol pretreatment. There were 2- and 2.5-fold increases of caspase-8 and caspase-3 activity and 1.6-fold increases of apoptotic-positive cells in the Jo2 plus acute ethanol group compared to the Jo2 alone group. Levels of TNF-alpha, malondialdehyde, 4-hydroxynonenal, protein carbonyl formation, 3-nitrotyrosine protein adducts, and inducible nitric oxide synthase were increased in the Jo2 plus ethanol group. The enhanced hepatotoxicity of Jo2 plus ethanol and the elevated oxidative stress and TNF levels were lower in CYP2E1 knockout mice compared to wild-type mice expressing CYP2E1 but higher than saline controls. Toxicity also declined in mice treated with gadolinium chloride, an inhibitor of the inducible nitric oxide synthase or the antioxidant, N-acetyl-L-cysteine. These data indicate that acute ethanol pretreatment is capable of elevating hepatic apoptosis and liver injury induced by Jo2 Fas agonistic antibody. The enhanced hepatotoxicity involves increased oxidative and nitrosative stress, and appears to be mediated by CYP2E1-dependent and also CYP2E1-independent mechanisms.     

PAR2 promotes high-fat diet-induced hepatic steatosis by inhibiting AMPK-mediated autophagy

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptors. There are two types of PAR2 signaling pathways: Canonical G-protein signaling and β-arrestin signaling. Although PAR2 signaling has been reported to aggravate hepatic steatosis, the exact mechanism is still unclear, and the role of PAR2 in autophagy remains unknown. In this study, we investigated the regulatory role of PAR2 in autophagy during high-fat diet (HFD)-induced hepatic steatosis in mice. Increased protein levels of PAR2 and β-arrestin-2 and their interactions were detected after four months of HFD. To further investigate the role of PAR2, male and female wild-type (WT) and PAR2-knockout (PAR2 KO) mice were fed HFD. PAR2 deficiency protected HFD-induced hepatic steatosis in male mice, but not in female mice. Interestingly, PAR2-deficient liver showed increased AMP-activated protein kinase (AMPK) activation with decreased interaction between Ca²⁺/calmodulin-dependent protein kinase kinase β (CAMKKβ) and β-arrestin-2. In addition, PAR2 deficiency up-regulated autophagy in the liver. To elucidate whether PAR2 plays a role in the regulation of autophagy and lipid accumulation in vitro, PAR2 was overexpressed in HepG2 cells. Overexpression of PAR2 decreased AMPK activation with increased interaction of CAMKKβ with β-arrestin-2 and significantly inhibited autophagic responses in HepG2 cells. Inhibition of autophagy by PAR2 overexpression further exacerbated palmitate-induced lipid accumulation in HepG2 cells. Collectively, these findings suggest that the increase in the PAR2-β-arrestin-2-CAMKKβ complex by HFD inhibits AMPK-mediated autophagy, leading to the alleviation of hepatic steatosis.     

S-adenosyl-L-methionine decreases the elevated hepatotoxicity induced by Fas agonistic antibody plus acute ethanol pretreatment in mice

The current study was designed to investigate the effect and potential mechanism of exogenous administration of S-adenosyl-l-methionine (SAM) on the enhanced hepatotoxicity induced by the Fas agonistic Jo2 antibody plus acute ethanol pretreatment in C57BL/6 mice. Acute ethanol plus Jo2 treatment produces liver toxicity under conditions in which ethanol alone or Jo2 alone do not. SAM significantly attenuated this elevated hepatotoxicity in mice as manifested by a decrease of serum aminotransferases and morphological amelioration. Levels of SAM and activity of methionine adenosyltransferase were lowered by the ethanol plus Jo2 treatment but restored by administration of SAM. The ethanol plus Jo2 treatment increased activity and content of CYP2E1, iNOS content and TNF-α levels; these increases were blunted by SAM. SAM also protected against the elevated oxidative and nitrosative stress found after ethanol plus Jo2, likely due to the decreases in CYP2E1, iNOS and TNF-α. Calcium-induced swelling of mitochondria was enhanced by the ethanol plus Jo2 treatment and this was prevented by SAM. JNK and P38 MAPK were activated by the ethanol plus Jo2 treatment; JNK activation was partially prevented by SAM. It is suggested that SAM protects against the ethanol plus Jo2 toxicity by restoring hepatic SAM levels, preventing the increase in iNOS, CYP2E1 and TNF-α and there by lowering the elevated oxidative/nitrosative stress and activation of the JNK signal pathway, ultimately preventing mitochondrial damage.     

Ethanol and arachidonic acid produce toxicity in hepatocytes from pyrazole-treated rats with high levels of CYP2E1

Ethanol and polyunsaturated fatty acids such as arachidonic acid were shown to be toxic and cause apoptosis in HepG2 cells which express CYP2E1 but not in control HepG2 cell lines. The goal of the current study was to extend the observations made with the HepG2 cells to non-transformed, intact hepatocytes. Rats were treated with pyrazole to increase CYP2E1 levels, hepatocytes were isolated and placed into culture and treated for varying time points with ethanol or arachidonic acid. Comparisons were made to hepatocytes from saline-treated rats, with low CYP2E1 content. Incubation with ethanol (100 mM) or especially arachidonic acid (60 µM) resulted in loss of viability of hepatocytes from the pyrazole-treated rats, without any effect on the hepatocytes from the saline-treated rats. The toxicity appeared to be apoptotic in nature and was prevented by diallyldisulfide, an inhibitor of CYP2E1. Toxicity was reduced by trolox, an antioxidant. The treatment with ethanol or arachidonic acid resulted in release of cytochrome c into the cytosol fraction, and activation of caspase 3 (but not caspase 1) in hepatocytes from the pyrazole-treated rats but not hepatocytes from the saline-treated rats. The activation of caspase 3 was prevented by diallyldisulfide, by trolox, and by DEVD-fmk. The latter also prevented the toxicity produced by ethanol or arachidonic acid. These results extend previous observations found with HepG2 cells expressing CYP2E1 to intact hepatocytes and suggest that release of cytochrome c and activation of caspase 3 play a role in the overall pathway by which CYP2E1 contributes towards the hepatotoxic actions of ethanol and polyunsaturated fatty acids     

SCD1 deficiency protects mice against ethanol-induced liver injury

Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.     

Ethanol and tobacco smoke increase hepatic steatosis and hypoxia in the hypercholesterolemic apoE−/− mouse: Implications for a “multihit” hypothesis of fatty liver disease

Although epidemiologic studies indicate that combined exposure to cigarette smoke and alcohol increase the risk and severity of liver diseases, the molecular mechanisms responsible for hepatotoxicity are unknown. Similarly, emerging evidence indicates a linkage among hepatic steatosis and cardiovascular disease. Herein, we hypothesize that combined exposure to alcohol and environmental tobacco smoke (ETS) on a hypercholesterolemic background increases liver injury through oxidative/nitrative stress, hypoxia, and mitochondrial damage. To test this, male apoE^?/? mice were exposed to an ethanol-containing diet, ETS alone, or a combination of the two, and histology and functional endpoints were compared to filtered-air-exposed, ethanol-naïve controls. Whereas ethanol consumption induced a mild steatosis, combined exposure to ethanol + ETS resulted in increased hepatic steatosis, inflammation, α-smooth muscle actin, and collagen. Exposure to ethanol + ETS induced the largest increase in CYP2E1 and iNOS protein, as well as increased 3-nitrotyrosine, mtDNA damage, and decreased cytochrome c oxidase protein, compared to all other groups. Similarly, the largest increase in HIF1α expression was observed in the ethanol + ETS group, indicating enhanced hypoxia. These studies demonstrate that ETS increases alcohol-dependent steatosis and hypoxic stress. Therefore, ETS may be a key environmental “hit” that accelerates and exacerbates alcoholic liver disease in hypercholesterolemic apoE^?/? mice.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.     

Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione

Induction of CYP2E1 by ethanol is one mechanism by which ethanol causes oxidative stress and alcohol liver disease. Although CYP2E1 is predominantly found in the endoplasmic reticulum, it is also located in rat hepatic mitochondria. In the current study, chronic alcohol consumption induced rat hepatic mitochondrial CYP2E1. To study the role of mitochondrial targeted CYP2E1 in generating oxidative stress and causing damage to mitochondria, HepG2 lines overexpressing CYP2E1 in mitochondria (mE10 and mE27 cells) were established by transfecting a plasmid containing human CYP2E1 cDNA lacking the hydrophobic endoplasmic reticulum targeting signal sequence into HepG2 cells followed by G418 selection. A 40-kDa catalytically active NH2-terminally truncated form of CYP2E1 (mtCYP2E1) was detected in the mitochondrial compartment in these cells by Western blot analysis. Cell death caused by depletion of GSH by buthionine sulfoximine (BSO) was increased in mE10 and mE27 cells as compared with cells transfected with empty vector (pCI-neo). Antioxidants were able to abolish the loss of cell viability. Increased levels of reactive oxygen species and mitochondrial 3-nitrotyrosine and 4-hydroxynonenal protein adducts and decreased mitochondrial aconitase activity and mitochondrial membrane potential were observed in mE10 and mE27 cells treated with BSO. The mitochondrial membrane stabilizer, cyclosporine A, was also able to protect these cells from BSO toxicity. These results revealed that CYP2E1 in the mitochondrial compartment could induce oxidative stress in the mitochondria, damage mitochondria membrane potential, and cause a loss of cell viability. The accumulation of CYP2E1 in hepatic mitochondria induced by ethanol consumption might play an important role in alcohol liver disease.     

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.     

Binge Alcohol Consumption Aggravates Oxidative Stress and Promotes Pathogenesis of NASH from Obesity-Induced Simple Steatosis

The pathogenesis of nonalcoholic steatohepatitis (NASH) is a two-stage process in which steatosis is the “first hit” and an unknown “second hit.” We hypothesized that “a binge” could be a “second hit” to develop NASH from obesity-induced simple steatosis. Thirty-week-old male Otsuka Long-Evans Tokushima fatty (OLETF) rats were administered 10 mL of 10% ethanol orally for 5, 3, 2, and 1 d/wk for 3 consecutive weeks. As control, male Otsuka Long-Evans Tokushima (OLET) rats were administered the same amount of alcohol. Various biochemical parameters of obesity, steatosis and NASH were monitored in serum and liver specimens in untreated and ethanol-treated rats. The liver sections were evaluated for histopathological alterations of NASH and stained for cytochrome P-4502E1 (CYP2E1) and 4-hydroxy-nonenal (4-HNE). Simple steatosis, hyperinsulinemia, hyperglycemia, insulin resistance, hypertriglycemia and marked increases in hepatic CYP2E1 and 4-HNE were present in 30-wk-old untreated OLETF rats. Massive steatohepatitis with hepatocyte ballooning was observed in the livers of all OLETF rats treated with ethanol. Serum and hepatic triglyceride levels as well as tumor necrosis factor (TNF)-α mRNA were markedly increased in all ethanol-treated OLETF rats. Staining for CYP2E1 and 4-NHE demonstrated marked increases in the hepatic tissue of all the groups of OLETF rats treated with ethanol compared with OLET rats. Our data demonstrated that “a binge” serves as a “second hit” for development of NASH from obesity-induced simple steatosis through aggravation of oxidative stress. The enhanced levels of CYP2E1 and increased oxidative stress in obesity play a significant role in this process.