Prophylactic Treatment of Probiotic and Metformin Mitigates Ethanol-Induced Intestinal Barrier Injury: In Vitro, In Vivo, and In Silico Approaches.

Ethanol depletes intestinal integrity and promotes gut dysbiosis. Studies have suggested the individual role of probiotics and metformin Met in protecting intestinal barrier function from injuries induced by ethanol. The objective of the current study is to investigate the potential mechanism by which coadministration of probiotic Visbiome® (V) and Met blocks the ethanol-induced intestinal barrier dysfunction/gut leakiness utilizing Caco-2 monolayers, a rat model with chronic ethanol injury, and in silico docking interaction models. In Caco-2 monolayers, exposure to ethanol significantly disrupted tight junction (TJ) localization, elevated monolayer permeability, and oxidative stress compared with controls. However, cotreatment with probiotic V and Met largely ameliorated the ethanol-induced mucosal barrier dysfunction, TJ disruption, and gut oxidative stress compared with ethanol-exposed monolayers and individual treatment of either agent. Rats fed with ethanol-containing Lieber-DeCarli liquid diet showed decreased expression of TJ proteins, and increased intestinal barrier injury resulting in pro-inflammatory response and oxidative stress in the colon. We found that co-administration of probiotic V and Met improved the expression of intestinal TJ proteins (ZO-1 and occludin) and upregulated the anti-inflammatory response, leading to reduced ER stress. Moreover, co-administration of probiotic V and Met inhibited the CYP2E1 and NOX gene expression, and increase the translocation of Nrf-2 as well as anti-oxidative genes (SOD, catalase, Gpx, and HO-1), leading to reduced colonic ROS content and malondialdehyde levels. The combined treatment of probiotic V and Met also improved their binding affinities towards HO-1, Nrf-2, SLC5A8, and GPR109A, which could be attributed to their synergistic effect. Our findings based on in-vitro, in-vivo, and in-silico analyses suggest that the combination of probiotic V and Met potentially acts in synergism, attributable to their property of inhibition of inflammation and oxidative stress against ethanol-induced intestinal barrier injury.

[The role of Cytochrom P4502E1 in Alcoholic Liver Disease and alcohol mediated carcinogenesis]. / Die Bedeutung von Cytochrom P4502E1 bei der alkoholischen Lebererkrankung und bei der alkoholmediierten Karzinogenese.

Various factors are involved in the pathogenesis of alcoholic liver disease (ALD) and ethanol-mediated carcinogenesis. In addition to genetic, epigenetic and immunologic mechanisms, acetaldehyde-associated toxicity, oxidative stress as well as cytokine-mediated inflammation are of major importance. Oxidative stress, with the generation of reactive oxygen species (ROS), develops either in inflammation (alcoholic hepatitis) or during oxidation of ethanol via cytochrome P4502E1 (CYP2E1). CYP2E1 is induced by ethanol, oxidizes ethanol to acetaldehyde, and generates ROS during this process. ROS results in protein damage, enhanced fibrogenesis and DNA lesions. Furthermore, CYP2E1 induction results in an enhanced activation of various procarcinogens and an increased degradation of retinol and retinoic acid (RA), a compound responsible for cell differentiation and proliferation. An inhibition of CYP2E1 results in an improvement of ALD and chemically induced carcinogenesis in animal experiments. In humans, CYP2E1 is induced following the consumption of 40 grams of ethanol per day after one week. However, the induction varies inter-individually. The mechanism for this is still unclear. Patients with ALD show a significant correlation between CYP2E1, the occurrence of highly carcinogenic etheno DNA adducts and the severity of fibrosis. First results on the effect of CYP2E1 inhibition by chlormethiazole, a specific CYP2E1 inhibitor on ALD, can be expected soon.

Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles.

BACKGROUND & AIMS: The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. METHODS: Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. RESULTS: Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.

Roles of CYP2e1 in 1,2-dichloroethane-induced liver damage in mice.

The aim of this study was to explore the roles of cytochrome P450 2E1 (CYP2E1) in 1,2-dichloroethane (1,2-DCE)-induced liver damage. Two parts were included in this study: first, effect of 1,2-DCE on microsomal expression of CYP2E1, and second, potential of an inhibitor of CYP2E1 to reduce 1,2-DCE-induced liver damage. In part one, mice were exposed to 0, 0.225, 0.45, or 0.9 g/m3 1,2-DCE for 10 days, 3.5 h per day through static inhalation. In part two, mice were divided into blank control, solvent control, inhibitor control, 1,2-DCE-poisoned group, and low or high intervention group. In part one, compared to the control, serum alanine aminotransferase (ALT) activities and hepatic malondialdehyde (MDA) levels in 0.9 g/m3 1,2-DCE group, and microsomal CYP2E1 protein expression and activity in both 0.45 and 0.9 g/m3 1,2-DCE groups increased significantly; conversely, hepatic nonprotein sulfhydryl (NPSH) levels in both 0.45 and 0.9 g/m3 1,2-DCE groups and hepatic SOD activities in 0.9 g/m3 1,2-DCE group decreased significantly. In part two, microsomal CYP2E1 protein expression and activity decreased significantly in both low and high intervention groups compared to 1,2-DCE-poisoned group. Along with the changes of CYP2E1, hepatic MDA levels and serum ALT activities decreased; conversely, hepatic NPSH levels and SOD activities increased significantly in high intervention group. Taken together, our results suggested that 1,2-DCE could enhance CYP2E1 protein expression and enzymatic activity, which could cause oxidative damage in liver, serving as an important mechanism underlying 1,2-DCE-induced liver damage. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1430-1438, 2016.

Sulforaphane induces Nrf2 and protects against CYP2E1-dependent binge alcohol-induced liver steatosis.

BACKGROUND: The mechanism(s) by which alcohol causes cell injury are still not clear but a major mechanism appears to be the role of lipid peroxidation and oxidative stress in alcohol toxicity. CYP2E1-generated ROS contributes to the ethanol-induced oxidant stress and inhibition of CYP2E1 activity decreases ethanol-induced fatty liver. The transcription factor Nrf2 regulates the expression of many cytoprotective enzymes which results in cellular protection against a variety of toxins. METHOD: The current study was designed to evaluate the ability of sulforaphane, an activator of Nrf2, to blunt CYP2E1-dependent, ethanol-induced steatosis in vivo and in vitro. RESULTS: The sulforaphane treatment activated Nrf2, increased levels of the Nrf2 target heme oxygenase-1 and subsequently lowered oxidant stress as shown by the decline in lipid peroxidation and 3-nitrotyrosine protein adducts and an increase in GSH levels after the acute ethanol treatment. It decreased ethanol-elevated liver levels of triglycerides and cholesterol and Oil Red O staining. Similar results were found in vitro as addition of sulforaphane to HepG2 E47 cells, which express CYP2E1, elevated Nrf2 levels and decreased the accumulation of lipid in cells cultured with ethanol. Sulforaphane treatment had no effect on levels of or activity of CYP2E1. CONCLUSIONS: Sulforaphane proved to be an effective in vivo inhibitor of acute ethanol-induced fatty liver in mice. GENERAL SIGNIFICANCE: The possible amelioration of liver injury which occurs under these conditions by chemical activators of Nrf2 is of clinical relevance and worthy of further study.

CYP2E1 epigenetic regulation in chronic, low-level toluene exposure: Relationship with oxidative stress and smoking habit.

BACKGROUND: CYP2E1 is a versatile phase I drug-metabolizing enzyme responsible for the biotransformation of most volatile organic compounds, including toluene. Human toluene exposure increases CYP2E1 mRNA and modifies its activity in leucocytes; however, epigenetic implications of this interaction have not been investigated. GOAL: To determine promoter methylation of CYP2E1 and other genes known to be affected by toluene exposure. METHODS: We obtained venous blood from 24 tannery workers exposed to toluene (mean levels: 10.86+/-7mg/m(3)) and 24 administrative workers (reference group, mean levels 0.21+/-0.02mg/m(3)) all of them from the city of León, Guanajuato, México. After DNA extraction and bisulfite treatment, we performed PCR-pyrosequencing in order to measure methylation levels at promoter region of 13 genes. RESULTS: In exposed group we found significant correlations between toluene airborne levels and CYP2E1 promoter methylation (r=-.36, p<0.05), as well as for IL6 promoter methylation levels (r=.44, p<0.05). Moreover, CYP2E1 promoter methylation levels where higher in toluene-exposed smokers compared to nonsmokers (p=0.009). We also observed significant correlations for CYP2E1 promoter methylation with GSTP1 and SOD1 promoter methylation levels (r=-.37, p<0.05 and r=-.34, p<0.05 respectively). CONCLUSION: These results highlight the importance of considering CYP2E1 epigenetic modifications, as well as its interactions with other genes, as key factors for unraveling the sub cellular mechanisms of toxicity exerted by oxidative stress, which can initiate disease process in chronic, low-level toluene exposure. People co-exposed to toluene and tobacco smoke are in higher risk due to a possible CYP2E1 repression.

Role of Kupffer cells in ischemic injury in alcoholic fatty liver.

BACKGROUND: This study was designed to evaluate the role of Kupffer cells (KCs) in hepatic drug metabolizing dysfunction after hepatic ischemia-reperfusion (IR) in alcoholic fatty liver. MATERIALS AND METHODS: Rats were fed the Lieber-DeCarli diet for 5 wk to develop alcoholic fatty liver, then were subjected to 90 min of hepatic ischemia and 5 h of reperfusion. For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) 48 and 24 h before the IR procedure. RESULTS: After the IR procedure, ethanol diet (ED)-fed rats had higher serum aminotransferase activity compared with the control diet-fed rats. These changes were attenuated by GdCl3. The ED-fed rats exhibited increased hepatic microsomal total cytochrome P450 (CYP) content and nicotinamide adenine dinucleotide phosphate-CYP reductase and CYP1A1, 1A2, 2B1, and 2E1 isozyme activity. After hepatic IR, these increases were reduced to lower levels than observed in the sham group, except CYP2E1 activity. Increases in CYP2E1 activity and its expression were augmented after hepatic IR in ED-fed animals, but were attenuated by GdCl3. Finally, toll-like receptor 4 and myeloid differentiation primary response gene 88 protein expression, nuclear translocation of nuclear factor-κB and activator protein 1, and levels of proinflammatory mediators were further increased in ED-fed animals compared with control diet-fed animals after IR. These increases were attenuated by GdCl3. CONCLUSIONS: We suggest that KCs contribute to hepatic drug metabolizing dysfunction during hepatic IR in alcoholic fatty liver via the toll-like receptors 4-mediated inflammatory response.

Synergistic toxic interactions between CYP2E1, LPS/TNFα, and JNK/p38 MAP kinase and their implications in alcohol-induced liver injury.

The mechanisms by which alcohol causes cell injury are not clear. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol-elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other; however, associations and interactions between them, especially in vivo, have not been extensively evaluated. We have shown that increased oxidative stress from induction of CYP2E1 in vivo sensitizes hepatocytes to LPS and TNFα toxicity and that oxidative stress, activation of p38 and JNK MAP kinases, and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNFα potentiated hepatotoxicity. This Review will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved including activation of the mitogen-activated kinase kinase kinase ASK-1 as a result of CYP2E1-derived reactive oxygen intermediates promoting dissociation of the inhibitory thioredoxin from ASK-1. This activation of ASK-1 is followed by activation of the mitogen-activated kinase kinases MKK3/MKK6 and MKK4/MMK7 and subsequently p38 and JNK MAP kinases. Synergistic toxicity occurs between CYP2E1 and the JNK1 but not the JNK2 isoform as JNK1 knockout mice are completely protected against CYP2E1 plus TNFα toxicity, elevated oxidative stress, and mitochondrial dysfunction. We hypothesize that similar interactions occur as a result of ethanol induction of CYP2E1 and TNFα.

Alcohol and cancer: an overview with special emphasis on the role of acetaldehyde and cytochrome P450 2E1.

The mechanisms by which chronic alcohol consumption enhances carcinogenesis include acetaldehyde (AA) generated by alcohol dehydrogenase and reactive oxygen species (ROS) generated predominantly by cytochrome P450 2E1 (CYP2E1), but also by other factors during inflammation. In addition, ethanol also alters epigenetics by changing DNA and histone methylation and acetylation. A loss of retinoic acid due to a CYP2E1-related enhanced degradation results in enhanced cellular proliferation and decreased cell differentiation. Changes in cancer genes and in signaling pathways (MAPK, RAS, Rb, TGFß, p53, PTEN, ECM, osteopontin, Wnt) may also contribute to ethanol-mediated mechanisms in carcinogenesis. Finally, immunosuppression may facilitate tumor spread. In the present review major emphasis is led on AA and ROS. While AA binds to proteins and DNA generating carcinogenic DNA adducts and inhibiting DNA repair and DNA methylation, ROS results in lipid peroxidation with the generation of lipid peroxidation products such as 4-hydoxynonenal which binds to DNA-forming highly carcinogenic exocyclic DNA adducts. ROS production correlates significantly with CYP2E1 in the liver but also in the esophagus, and its generation can be significantly reduced by the specific CYP2E1 inhibitor clomethiazole. Finally, CMZ also inhibits alcohol-mediated nitrosamine-induced hepatocarcinogenesis.

DIFFERENT RESPONSE OF ANTIOXIDANT DEFENSE SYSTEM TO ACAMPROSATE IN ETHANOL PREFERRING AND NON-PREFERRING RATS.

The aim of the study was to investigate whether acamprosate, an agent attenuating relapse in human alcoholics, might modulate antioxidant status in rats chronically administered ethanol. Male Wistar rats were presented with a free choice paradigm between tap water and ethanol solution for three month to distinguish two groups of animals, preferring (PRF) and non-preferring (NPF) ethanol. Then, rats were administered acamprosate, 500 mg/kg/day, per os, for 21 days. The hepatic level of enzymatically-driven lipid peroxidation was enhanced by ethanol in PRF and NPF rats by 67 and 82%, respectively. Unstimulated microsomal lipid peroxidation was increased solely in NPF rats by 33%. Acamprosate caused 36% increase in stimulated lipid peroxidation only in NPF animals. The activities of all hepatic antioxidant enzymes examined: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were decreased in rats treated with ethanol by 30 to 64% as compared to controls, however, this decrease was more distinct in ethanol preferring rats. Administration of acamprosate further reduced the activity of antioxidant enzymes only in NPF rats: catalase by 47%, glutathione peroxidase and glutathione S-transferase by 37% and glutathione reductase by 33%. No effect of acamprosate on 4-nitrophenol hydroxylase, a marker of CYP2E1 activity, was observed. As acamprosate enhanced oxidative stress only in the rats non-preferring ethanol, it could be expected that these adverse effects are not demonstrated in alcohol-dependent humans treated with acamprosate.

Evaluation of three-dimensional cultured HepG2 cells in a nano culture plate system: an in vitro human model of acetaminophen hepatotoxicity.

Overdoses of acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP) cause severe liver injury, yet there is no common or high throughput in vitro human APAP model. This study examined the characteristics and usefulness of HepG2 cells grown in a nano culture plate (NCP) system, a three-dimensional culture method, as an in vitro human model for APAP-induced hepatotoxicity. The NCP-cultured HepG2 cells showed higher expression of mRNA and protein levels of cytochrome P450 2E1, which metabolizes APAP to a toxic metabolite, APAP-cysteine adduct formation, and higher sensitivity against APAP-induced cell injury compared with conventionally cultured cells. We demonstrated that treatment of APAP in NCP-cultured HepG2 cells shows key mechanistic features of APAP-induced hepatotoxicity, such as decreases in intracellular glutathione and mitochondrial membrane potential, activation of JNK, and cellular injury; and pharmacological agents, such as Cyclosporine A (a mitochondrial permeability transition inhibitor) and SP600125 (a JNK inhibitor), prevented cell injury induced by APAP exposure. In addition, the antidote of APAP-induced hepatotoxicity, N-acetylcysteine, could attenuate cellular injury induced by APAP in NCP-cultured HepG2 cells. We suggest that cellular injury induced by APAP treatment using an NCP-HepG2 system is a useful human model to study mechanisms and screen drug candidates of APAP-induced hepatotoxicity.

The role of CYP2E1 in alcohol metabolism and sensitivity in the central nervous system.

Ethanol consumption has effects on the central nervous system (CNS), manifesting as motor incoordination, sleep induction (hypnosis), anxiety, amnesia, and the reinforcement or aversion of alcohol consumption. Acetaldehyde (the direct metabolite of ethanol oxidation) contributes to many aspects of the behavioral effects of ethanol. Given acetaldehyde cannot pass through the blood brain barrier, its concentration in the CNS is primarily determined by local production from ethanol. Catalase and cytochrome P450 2E1 (CYP2E1) represent the major enzymes in the CNS that catalyze ethanol oxidation. CYP2E1 is expressed abundantly within the microsomes of certain brain cells and is localized to particular brain regions. This chapter focuses on the discussion of CYP2E1 in ethanol metabolism in the CNS, covering topics including how it is regulated, where it is expressed and how it influences sensitivity to ethanol in the brain.

The role of cytochrome P450 2E1 in ethanol-mediated carcinogenesis.

We and others have shown that chronic alcohol consumption results in the induction of CYP2E1 in the liver. We have also detected for the first time such an induction in the mucosa of the small intestine and the colon. The overall induction of CYP2E1 shows interindividual variations and occurs already following a daily ingestion of 40 g of ethanol after 1 week. CYP2E1 induction is associated with an increased metabolism of ethanol resulting in the generation of reactive oxygen species (ROS) with direct and indirect carcinogenic action. ROS generated by CYP2E1 may lead to lipid peroxidation and lipid peroxidation products such as 4-hydroxynonenal bind to DNA forming highly carcinogenic exocyclic etheno DNA-adducts. The generation of these adducts has been shown in cell cultures in animal experiments as well as in human liver biopsies. CYP2E1 also metabolizes various procarcinogens present in diets and in tobacco smoke to their carcinogenic metabolites. Among these, nitrosamines seem to be the most important carcinogens. CYP2E1 also degrades retinoic acid and retinol to polar metabolites. Metabolism of retinoic acid not only results in the loss of retinoic acid promoting carcinogenesis through an increase in cell proliferation and dedifferentiation but also in generation of polar metabolites with apoptotic properties. We have shown that chlormethiazole is a specific CYP2E1 inhibitor in humans. Chlormethiazole inhibits CYP2E1 activity and thus blocks the formation of DNA adducts in cell cultures, restores retinoic acids in alcohol fed animals and inhibits chemical induced ethanol mediated hepatocarcinogenesis. Thus, there is increasing evidence that CYP2E1 induced by chronic alcohol consumption plays an important role in alcohol mediated carcinogenesis.

CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease.

Alcoholic (ALD) and non-alcoholic fatty liver diseases (NAFLD) are clinical conditions leading to hepatocellular injury and inflammation resulting from alcohol consumption, high fat diet, obesity and diabetes, among others. Oxidant stress is a major contributing factor to the pathogenesis of ALD and NAFLD. Multiple studies have shown that generation of reactive oxygen species (ROS) is key for the progression of fatty liver to steatohepatitis. Cytochrome P450 2E1 (CYP2E1) plays a critical role in ROS generation and CYP2E1 is also induced by alcohol itself. This review summarizes the role of CYP2E1 in ALD and NAFLD.

Cytochrome P-450 CYP2E1 knockout mice are protected against high-fat diet-induced obesity and insulin resistance.

Conventional (whole body) CYP2E1 knockout mice displayed protection against high-fat diet-induced weight gain, obesity, and hyperlipidemia with increased energy expenditure despite normal food intake and spontaneous locomotor activity. In addition, the CYP2E1 knockout mice displayed a marked improvement in glucose tolerance on both normal chow and high-fat diets. Euglycemic-hyperinsulinemic clamps demonstrated a marked protection against high-fat diet-induced insulin resistance in CYP2E1 knockout mice, with enhanced adipose tissue glucose uptake and insulin suppression of hepatic glucose output. In parallel, adipose tissue was protected against high-fat diet-induced proinflammatory cytokine production. Taken together, these data demonstrate that the CYP2E1 deletion protects mice against high-fat diet-induced insulin resistance with improved glucose homeostasis in vivo.

Chronic alcohol intake-induced oxidative stress and apoptosis: role of CYP2E1 and calpain-1 in alcoholic cardiomyopathy.

Cytochrome P-450 2E1 CYP2E1 induction has been linked to oxidative stress in a number of experimental models. The aim of this study was to investigate the relationship between CYP2E1 activity and markers of oxidative stress and cardiac cell apoptosis during the development of alcoholic cardiomyopathy (ACM). Changes in left ventricular morphology were evaluated in 4 groups of chronically instrumented dogs (control; alcohol-receiving; and alcohol-receiving plus treatment with either valsartan or carnitine) after 6 months of treatment. CYP2E1 and calpain-1 protein expression were determined by Western blotting, and apoptosis evaluated by TUNEL and immunohistochemistry. Malonyl dialdehyde levels were assessed as a marker of oxidative stress, while superoxide dismutase and glutathione peroxidase levels were evaluated as markers of antioxidant defense mechanisms. Expression of CYP2E1 was increased in the alcohol-receiving group compared with controls (P<0.05) and was associated with oxidative stress. Similarly, expression of Bad and calpain-1 protein was increased after chronic alcohol exposure, while Bcl-xL protein expression remained at a low level. Bad and calpain-1 protein expressions were significantly inhibited by treatment with valsartan or carnitine, while expression of Bcl-xL protein was increased (P<0.05). Collectively, our results indicate a possibly significant role for CYP2E1 in the oxidative stress associated with chronic alcoholism. The resulting increase in oxidative stress is accompanied by cellular apoptosis and may ultimately contribute to tissue remodeling and ACM. Importantly, these alcohol-induced effects may be abrogated by means such as angiotensin 1 receptor blockade or carnitine supplementation.

Ethanol-induced oxidative stress via the CYP2E1 pathway disrupts adiponectin secretion from adipocytes.

BACKGROUND: Adipose tissue is an important target for ethanol action. One important effect of ethanol is to reduce the secretion of adiponectin from adipocytes; this decrease is associated with lowered circulating adiponectin in rodent models of chronic ethanol feeding. Adiponectin is an insulin-sensitizing, anti-inflammatory adipokine; decreased adiponectin activity may contribute to tissue injury in response to chronic ethanol. Here, we investigated the role of cytochrome P450 2E1 (CYP2E1) and oxidative stress in the mechanism for impaired adiponectin secretion from adipocytes in response to ethanol. METHODS: Male Wistar rats were fed a liquid diet containing ethanol as 36% of calories or pair-fed a control diet for 4 weeks. 3T3-L1 adipocyte cultures, expressing CYP2E1 or not, were exposed to ethanol or 4-hydroxynonenal (4-HNE). RESULTS: Chronic ethanol feeding to rats suppressed the secretion of adiponectin from isolated epididymal adipocytes. Ethanol feeding induced the expression of CYP2E1 in adipocytes and increased markers of oxidative stress, including 4-HNE and protein carbonyls. Because adiponectin is posttranslationally processed in the endoplasmic reticulum and Golgi, we investigated the impact of ethanol on the redox status of high-density microsomes. Chronic ethanol decreased the ratio of reduced glutathione to oxidized glutathione (4.6:1, pair-fed; 2.9:1, ethanol-fed) in high-density microsomes isolated from rat epididymal adipose tissue. We next utilized the 3T3-L1 adipocyte-like cell model to interrogate the mechanisms for impaired adiponectin secretion. Culture of 3T3-L1 adipocytes overexpressing exogenous CYP2E1, but not those overexpressing antisense CYP2E1, with ethanol increased oxidative stress and impaired adiponectin secretion from intracellular pools. Consistent with a role of oxidative stress in impaired adiponectin secretion, challenge of 3T3-L1 adipocytes with 4-HNE also reduced adiponectin mRNA expression and secretion, without affecting intracellular adiponectin concentration. CONCLUSIONS: These data demonstrate that CYP2E1-dependent reactive oxygen species production in response to ethanol disrupts adiponectin secretion from adipocytes.

Ethanol induction of CYP2A5: permissive role for CYP2E1.

CYP2A5 metabolizes xenobiotics and activates hepatocarcinogens, and induction occurs in response to hepatic damage and cellular stress. We evaluated whether ethanol can elevate CYP2A5 and whether CYP2E1 plays a role in the ethanol induction of CYP2A5. Wild-type (WT), CYP2E1 knockout (KO), and CYP2E1 knockin (KI) mice were fed ethanol for 3 weeks. Ethanol increased CYP2E1 and CYP2A5 protein and activity in WT mice but not in the KO mice. Induction of CYP2A5 (and CYP2E1) was restored in the KI mice. Ethanol induction of CYP2A5 occurred only after CYP2E1 was first induced. Immunohistochemical staining revealed that CYP2E1 and CYP2A5 colocalize to the same zones in the liver. Ethanol also elevated CYP2A5 mRNA levels in WT and KI mice but not in KO mice. Induction of CYP2A5 by cadmium was partially decreased in KO mice compared with WT or KI mice. Ethanol elevated CYP2A4 mRNA levels in all mice although the extent of induction was lowest in the KO mice. In summary, ethanol elevated mouse hepatic CYP2A5 levels, which may be of toxicological significance because CYP2A5 metabolizes nicotine and other drugs and activates hepatocarcinogens. Induction of CYP2A5 by ethanol is potentiated by the induction of CYP2E1. We speculate that ethanol induction of CYP2E1 followed by increases in reactive oxygen species and activation of Nrf2 are important steps in the mechanism by which ethanol induces CYP2A5. The possibility that induction of CYP2E1 is permissive for the induction of CYP2A5 may reflect a new contribution by CYP2E1 to the actions of ethanol.

Sulforaphane prevents microcystin-LR-induced oxidative damage and apoptosis in BALB/c mice.

Microcystins (MCs), the products of blooming algae Microcystis, are waterborne environmental toxins that have been implicated in the development of liver cancer, necrosis, and even fatal intrahepatic bleeding. Alternative protective approaches in addition to complete removal of MCs in drinking water are urgently needed. In our previous work, we found that sulforaphane (SFN) protects against microcystin-LR (MC-LR)-induced cytotoxicity by activating the NF-E2-related factor 2 (Nrf2)-mediated defensive response in human hepatoma (HepG2) and NIH 3T3 cells. The purpose of this study was to investigate and confirm efficacy the SFN-induced multi-mechanistic defense system against MC-induced hepatotoxicity in an animal model. We report that SFN protected against MC-LR-induced liver damage and animal death at a nontoxic and physiologically relevant dose in BALB/c mice. The protection by SFN included activities of anti-cytochrome P450 induction, anti-oxidation, anti-inflammation, and anti-apoptosis. Our results suggest that SFN may protect mice against MC-induced hepatotoxicity. This raises the possibility of a similar protective effect in human populations, particularly in developing countries where freshwaters are polluted by blooming algae.

Proteasome inhibitor up regulates liver antioxidative enzymes in rat model of alcoholic liver disease.

Oxidative stress occurs in the liver of rats fed with alcohol chronically due to ethanol metabolism by CYP2E1, causing liver injury. The proteasome is considered as an antioxidant defense in the cell because of its activity in removing damaged and oxidized proteins, but a growing body of evidence shows that proteasome inhibitor treatment, at a non toxic low dose, provides protection against oxidative stress. In the present study, rats were fed with ethanol for 4 weeks and were treated with the proteasome inhibitor PS-341 (Bortezomib, Velcade®). Exposure to proteasome inhibitor elicited the elevation of antioxidative defense by enhancing the levels of mRNA and protein expression transcripts of glutathione reductase (GSR), glutathione synthetase (GSS), glutathione peroxidase 2 (GPX2), and superoxide dismutase 2 (SOD2) in the liver of rats fed with ethanol chronically, while ethanol alone did not increase these genes' mRNA. Our results also showed that glutamate cysteine ligase catalytic subunit (GCLC), a rate-limiting enzyme in glutathione biosynthesis, was also up regulated in the liver of rats fed with ethanol and injected with PS-431. Nrf2 mRNA level was significantly decreased in the liver of ethanol fed rats, as well as in the livers of animal fed with ethanol and treated with proteasome inhibitor, indicating that the mechanism by which proteasome inhibitor up regulates the antioxidant response element is not due to regulation of Nrf2. However, ATF4, a major regulator of antioxidant response elements, was significantly up regulated by proteasome inhibitor treatment. The beneficial effects of proteasome inhibitor treatment also reside in the reversibility of the drug because the proteasome activity was significantly increased 72 h post treatment. In conclusion, proteasome inhibitor treatment used at a non toxic low dose has potential protective effects against oxidative stress due to chronic ethanol feeding.