Targeting of CYP2E1 by miRNAs in alcohol-induced intestine injury.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.

The Benzene-induced Hepatic Cytochrome P450 2E1 Expression and Activity are Reduced by Quercetin Administration in Mice.

BACKGROUND: Benzene as an environmental and industrial agent induces adverse effects that are mainly metabolism-dependent. OBJECTIVES: Effects of Quercetin (QCN) on Benzene (BNZ)-induced changes in the hepatic Cytochrome P450 2E1 expression and activity were investigated. METHODS: Thirty-six adult male mice were divided into 6 groups (n = 6) and nominated as control, BNZ (exposed to BNZ: 30 ppm), QCN (received QCN: 50 mg/kg, orally), and the fourth, fifth and sixth groups were exposed to 30 ppm BNZ and received 10, 50 and 100 mg/kg QCN respectively, for 28 days. The microsomal subcellular fraction was isolated from the liver samples and the activity of CYP 2E1 was measured based on the hydroxylation rate of 4-nitrophenol. The hepatic activity of myeloperoxidase also was assessed. Total antioxidant capacity and nitric oxide contents of the liver were determined. Expression changes of CYP 2E1 at the mRNA level were examined by qPCR technique. RESULTS: QCN lowered significantly (p < 0.05) the BNZ-increased hepatic nitric oxide levels and restored the BNZ-reduced antioxidant capacity. The BNZ-elevated activity of myeloperoxidase was declined in QCN-received mice. QCN downregulated the expression and activity of hepatic CYP 2E1 in BNZ-exposed animals. CONCLUSION: Our results suggest that QCN could be a novel hepatoprotective compound for BNZ-induced hepatotoxicities, which is attributed to its capability in the down-regulation of CYP 2E1 expression and activity.

New insights into the downregulation of cytochrome P450 2E1 via nuclear factor κB-dependent pathways in immune-mediated liver injury.

The extent of immune-mediated hepatic damage (such as in viral hepatitis) is characterised by the downregulation of cytochrome P450s (CYPs), a class of drug-metabolising enzymes. However, whether this downregulation aids liver cells in maintaining their homeostasis or whether the damage is aggravated remains largely unexplored. Herein, we evaluated the effects of phosphorylation mediated by the protein kinase C (PKC)/cAMP-response element binding protein (CREB) and nitration mediated by inducible nitric oxide synthase (iNOS) on the downregulation of CYP2E1 during immune-mediated liver injury. Additionally, we investigated the regulatory mechanism mediated by the nuclear factor κB (NF-κB). The rat model of immune-mediated liver injury was replicated by administering a single i.v. injection of Bacillus Calmette-Guerin (BCG, 125 mg/kg) vaccine and three i.p. injections of ammonium pyrrolidine dithiocarbamate (25, 50, 100 mg/kg/d, days 11, 12, and 13); blood was then collected on day 14. Subsequently, the livers were extracted to identify the different pharmacokinetic and biochemical indicators involved in the process. Our study reports new findings on the dependence between PKC-mediated CREB phosphorylation in the anti-inflammatory pathway and nitration emergency induced by iNOS in pro-inflammatory pathways in the NF-κB pathway. The interaction of these two pathways leads to the downregulation and recovery of CYP2E1, thus alleviating inflammation and nitration stress. Our results confirm that BCG-mediated downregulation of CYP2E1 is linked to iNOS-induced nitration and PKC/NF-κB-mediated CREB phosphorylation, and that NF-κB is an important molecular target in this process. These findings suggest that the downregulation of CYP2E1 may be an autonomous process characteristic of liver cells, helping them adapt to environmental changes, alleviate further hypoxia in inflamed tissues, and minimise exposure to toxic and harmful metabolites.

Pathogenesis of Alcoholic Fatty Liver a Narrative Review.

Alcohol effect hepatic lipid metabolism through various mechanisms, leading synergistically to an accumulation of fatty acids (FA) and triglycerides. Obesity, as well as dietary fat (saturated fatty acids (FA) versus poly-unsaturated fatty acids (PUFA)) may modulate the hepatic fat. Alcohol inhibits adenosine monophosphate activated kinase (AMPK). AMPK activates peroxisome proliferator activated receptor a (PPARα) and leads to a decreased activation of sterol regulatory element binding protein 1c (SRABP1c). The inhibition of AMPK, and thus of PPARα, results in an inhibition of FA oxidation. This ß-oxidation is further reduced due to mitochondrial damage induced through cytochrome P4502E1 (CYP2E1)-driven oxidative stress. Furthermore, the synthesis of FAs is stimulated through an activation of SHREP1. In addition, alcohol consumption leads to a reduced production of adiponectin in adipocytes due to oxidative stress and to an increased mobilization of FAs from adipose tissue and from the gut as chylomicrons. On the other side, the secretion of FAs via very-low-density lipoproteins (VLDL) from the liver is inhibited by alcohol. Alcohol also affects signal pathways such as early growth response 1 (Egr-1) associated with the expression of tumour necrosis factor α (TNF α), and the mammalian target of rapamycin (mTOR) a key regulator of autophagy. Both have influence the pathogenesis of alcoholic fatty liver. Alcohol-induced gut dysbiosis contributes to the severity of ALD by increasing the metabolism of ethanol in the gut and promoting intestinal dysfunction. Moreover, pathogen-associated molecular patterns (PAMPS) via specific Toll-like receptor (TLR) bacterial overgrowth leads to the translocation of bacteria. Endotoxins and toxic ethanol metabolites enter the enterohepatic circulation, reaching the liver and inducing the activation of the nuclear factor kappa-B (NFκB) pathway. Pro-inflammatory cytokines released in the process contribute to inflammation and fibrosis. In addition, cellular apoptosis is inhibited in favour of necrosis.

The Wnt/ß-catenin signaling pathway plays a role in drug-induced liver injury by regulating cytochrome P450 2E1 expression.

Drug-induced liver injury (DILI) is a major cause of acute liver failure and drug withdrawal. Cytochrome P450 (CYP) 2E1 is involved in the metabolism of several drugs, and can induce liver injury through the production of toxic metabolites and the generation of reactive oxygen species. This study aimed to elucidate the role of Wnt/ß-catenin signaling in CYP2E1 regulation for drug-induced hepatotoxicity. To achieve this, mice were administered cisplatin or acetaminophen (APAP) 1 h after treatment with the CYP2E1 inhibitor dimethyl sulfoxide (DMSO), and histopathological and serum biochemical analyses were performed. APAP treatment induced hepatotoxicity, as evidenced by an increase in liver weight and serum ALT levels. Moreover, histological analysis indicated severe injury, including apoptosis, in the liver tissue of APAP-treated mice, which was confirmed by TUNEL assay. Additionally, APAP treatment suppressed the antioxidant capacity of the mice and increased the expression of the DNA damage markers γ-H2AX and p53. However, these effects of APAP on hepatotoxicity were significantly attenuated by DMSO treatment. Furthermore, the activation of Wnt/ß-catenin signaling using the Wnt agonist CHIR99021 (CHIR) increased CYP2E1 expression in rat liver epithelial cells (WB-F344), whereas treatment with the Wnt/ß-catenin antagonist IWP-2 inhibited nuclear ß-catenin and CYP2E1 expression. Interestingly, APAP-induced cytotoxicity in WB-F344 cells was exacerbated by CHIR treatment and suppressed by IWP-2 treatment. Overall, these results showed that the Wnt/ß-catenin signaling is involved in DILI through the upregulation of CYP2E1 expression by directly binding the transcription factor ß-cat/TCF to the Cyp2e1 promoter, thus exacerbating DILI. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00180-6.

8-methoxypsoralen protects against acetaminophen-induced liver injury by antagonising Cyp2e1 in mice.

This study aimed to investigate the effect and mechanism of 8-methoxypsoralen (8-MOP) on acetaminophen (APAP)-induced hepatotoxicity in mice. The study found that 1 h after intraperitoneal injection of 300 mg/kg APAP, treatment with 40 mg/kg, 80 mg/kg and 120 mg/kg 8-MOP could reduce serum transaminase level and histopathological liver necrosis area. Elevated mRNA expression of liver inflammatory mediators caused by excessive APAP was also reversed. 8-MOP significantly reduced APAP-induced hepatotoxicity dose-dependently, and the highest therapeutic dose of 8-MOP (120 mg/kg) had no harmful effects on the liver. Cocktail probe assay revealed that 8-MOP can inhibit Cyp2e1 enzymatic activities of mice, thereby reducing the production of acetaminophen-cysteine (APAP-CYS), a toxic metabolite of APAP. 8-MOP had no significant effect on the protein and gene expression of Cyp2e1. The three-dimensional structures of mouse Cyp2e1 were constructed by homologous modeling. Molecular docking showed that 8-MOP had a good binding effect on the enzyme activity site of Cyp2e1. In summary, 8-MOP dose-dependently attenuated APAP-induced hepatotoxicity by binding to Cyp2e1 and occupying the active center of the enzyme, thus competitively inhibiting the oxidative metabolism of APAP, and reducing the generation of toxic product APAP-CYS.

Hydroxytyrosol attenuates ethanol-induced liver injury by ameliorating steatosis, oxidative stress and hepatic inflammation by interfering STAT3/iNOS pathway.

Objective: Hydroxytyrosol (HT) is a polyphenol with a wide range of biological activities. Excessive drinking can lead to oxidative stress and inflammation in the liver, which usually develop into alcohol liver disease (ALD). At present, there is no specific drug to treat ALD. In this paper, the protection effect of HT on ALD and the underline mechanism were studied.Methods: HepG2 cells were exposed to ethanol in vitro and C57BL/6J mice were fed with a Lieber-DeCarli ethanol liquid diet in vivo.Results: triglyceride (TG) level in serum and the expression of fatty acid synthase (FASN) were reduced significantly by the treatment with HT The acetaldehyde dehydrogenase (ALDH) activity was increased, the serum level of malondialdehyde (MDA) was decreased, catalase (CAT) and glutathione (GSH) were increased, suggesting that HT may reduce its oxidative damage to the body by promoting alcohol metabolism. Furthermore, according to the mRNA levels of tnf-α, il-6 and il-1ß, HT inhibited ethanol-induced inflammation significantly. The anti-inflammatory mechanism of HT may be related to suppress the STAT3/iNOS pathway.Dissussion: Our study showed that HT could ameliorate ethanol-induced hepatic steatosis, oxidative stress and inflammation and provide a new candidate for the prevention and treatment of ALD.

Cathelicidin promotes liver repair after acetaminophen-induced liver injury in mice.

Background & Aims: Acetaminophen (APAP)-induced acute liver injury (AILI) is a leading cause of acute liver failure (ALF). N-acetylcysteine (NAC) is only effective within 24 h after APAP intoxication, raising an urgent need for alternative approaches to treat this disease. This study aimed to test whether cathelicidin (Camp), which is a protective factor in chronic liver diseases, protects mice against APAP-induced liver injury and ALF. Methods: A clinically relevant AILI model and an APAP-induced ALF model were generated in mice. Genetic and pharmacological approaches were used to interfere with the levels of cathelicidin in vivo. Results: An increase in hepatic pro-CRAMP/CRAMP (the precursor and mature forms of mouse cathelicidin) was observed in APAP-intoxicated mice. Upregulated cathelicidin was derived from liver-infiltrating neutrophils. Compared with wild-type littermates, Camp knockout had no effect on hepatic injury but dampened hepatic repair in AILI and reduced survival in APAP-induced ALF. CRAMP administration reversed impaired liver recovery observed in APAP-challenged Camp knockout mice. Delayed CRAMP, CRAMP(1-39) (the extended form of CRAMP), or LL-37 (the mature form of human cathelicidin) treatment exhibited a therapeutic benefit for AILI. Co-treatment of cathelicidin and NAC in AILI displayed a stronger hepatoprotective effect than NAC alone. A similar additive effect of CRAMP(1-39)/LL-37 and NAC was observed in APAP-induced ALF. The pro-reparative role of cathelicidin in the APAP-damaged liver was attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced neutrophil phagocytosis of necrotic cell debris in an autocrine manner. Conclusions: Cathelicidin reduces APAP-induced liver injury and ALF in mice by promoting liver recovery via facilitating inflammation resolution, suggesting a therapeutic potential for late-presenting patients with AILI with or without ALF. Impact and implications: Acetaminophen-induced acute liver injury is a leading cause of acute liver failure. The efficacy of N-acetylcysteine, the only clinically approved drug against acetaminophen-induced acute liver injury, is significantly reduced for late-presenting patients. We found that cathelicidin exhibits a great therapeutic potential in mice with acetaminophen-induced liver injury or acute liver failure, which makes up for the limitation of N-acetylcysteine therapy by specifically promoting liver repair after acetaminophen intoxication. The pro-reparative role of cathelicidin, as a key effector molecule of neutrophils, in the APAP-injured liver is attributed to an accelerated resolution of inflammation at the onset of liver repair, possibly through enhanced phagocytic function of neutrophils in an autocrine manner.

Acetaminophen-Induced Hepatotoxicity in Obesity and Nonalcoholic Fatty Liver Disease: A Critical Review.

The epidemic of obesity, type 2 diabetes and nonalcoholic liver disease (NAFLD) favors drug consumption, which augments the risk of adverse events including liver injury. For more than 30 years, a series of experimental and clinical investigations reported or suggested that the common pain reliever acetaminophen (APAP) could be more hepatotoxic in obesity and related metabolic diseases, at least after an overdose. Nonetheless, several investigations did not reproduce these data. This discrepancy might come from the extent of obesity and steatosis, accumulation of specific lipid species, mitochondrial dysfunction and diabetes-related parameters such as ketonemia and hyperglycemia. Among these factors, some of them seem pivotal for the induction of cytochrome P450 2E1 (CYP2E1), which favors the conversion of APAP to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). In contrast, other factors might explain why obesity and NAFLD are not always associated with more frequent or more severe APAP-induced acute hepatotoxicity, such as increased volume of distribution in the body, higher hepatic glucuronidation and reduced CYP3A4 activity. Accordingly, the occurrence and outcome of APAP-induced liver injury in an obese individual with NAFLD would depend on a delicate balance between metabolic factors that augment the generation of NAPQI and others that can mitigate hepatotoxicity.

Pathogenesis of Alcohol-Associated Liver Disease.

Excessive alcohol consumption is a global healthcare problem with enormous social, economic, and clinical consequences. While chronic, heavy alcohol consumption causes structural damage and/or disrupts normal organ function in virtually every tissue of the body, the liver sustains the greatest damage. This is primarily because the liver is the first to see alcohol absorbed from the gastrointestinal tract via the portal circulation and second, because the liver is the principal site of ethanol metabolism. Alcohol-induced damage remains one of the most prevalent disorders of the liver and a leading cause of death or transplantation from liver disease. Despite extensive research on the pathophysiology of this disease, there are still no targeted therapies available. Given the multifactorial mechanisms for alcohol-associated liver disease pathogenesis, it is conceivable that a multitherapeutic regimen is needed to treat different stages in the spectrum of this disease.

Phosphodiesterase 4 mRNA Level Suppression is Important for Extract of Black Carrot to Protect Against Hepatic Injury Induced by Ethanol.

Excessive alcohol use often results in alcoholic liver disease (ALD). An early change in the liver due to excessive drinking is hepatic steatosis, which may ultimately progress to hepatitis, liver fibrosis, cirrhosis, and liver cancer. Among these debilitating processes, hepatic steatosis is reversible with the appropriate treatment. Therefore, it is important to find treatments and foods that reverse hepatic steatosis. Black carrot has antioxidant and anti-inflammatory effects. In this study, we examined the effectiveness of black carrot extract (BCE) on hepatic steatosis in in vivo and in vitro ethanol-induced liver injury models. For the in vivo experiments, serum aminotransferase activities enhanced by ethanol- and carbon tetrachloride were significantly suppressed by the BCE diet. Furthermore, morphological changes in the liver hepatic steatosis and fibrosis were observed in the in vivo ethanol-induced liver injury model, however, BCE feeding resulted in the recovery to an almost normal liver morphology. In the in vitro experiments, ethanol treatment induced reactive oxygen species (ROS) levels in hepatocytes at 9 h. Conversely, ROS production was suppressed to control levels and hepatic steatosis was suppressed when hepatocyte culture with ethanol were treated with BCE. Furthermore, we investigated enzyme activities, enzyme protein levels, and messenger RNA levels of alcohol dehydrogenase (ADH), cytochrome p450 2E1 (CYP2E1), and aldehyde dehydrogenase (ALDH) using enzyme assays, western blot, and quantitative reverse transcription-polymerase chain reaction analyses. We found that the activities of ADH, CYP2E1, and ALDH were regulated through the cAMP-PKA pathway at different levels, namely, translational, posttranslational, and transcriptional levels, respectively. The most interesting finding of this study is that BCE increases cAMP levels by suppressing the Pde4b mRNA and PDE4b protein levels in ethanol-treated hepatocytes, suggesting that BCE may prevent ALD.

A combined metabolomics and molecular biology approach to reveal hepatic injury and underlying mechanisms after chronic l-lactate exposure in mice.

This study aimed to explore whether chronic l-lactate exposure could affect the peripheral tissues of mice and to determine the underlying pathogenesis. Herein, male C57BL/6 mice were divided into control and l-lactate groups. After l-lactate treatment for eight weeks (1 g/kg), metabolic changes in liver, kidney, muscle, and serum samples were determined by 1H nuclear magnetic resonance (1H NMR)-based metabolomics. Additionally, organ function was evaluated by serum biochemical and histopathological examinations. Reactive oxygen species (ROS) levels were measured using dihydroethidium staining; levels of signals involved in lactate metabolism and ROS-related pathways were detected using western blotting or polymerase chain reaction. Apoptosis was detected by TUNEL-fluorescence staining. Metabolomic analysis revealed that l-lactate mice showed decreased levels of glutathione (GSH), taurine, ATP, and increased glucose content, compared to control mice. Furthermore, l-lactate mice presented significantly higher serum levels of alanine aminotransferase and aspartate aminotransferase and increased glycogen content in hepatic tissues, compared to control mice. l-lactate mice also had a greater number of apoptotic nuclei in the livers than controls. Moreover, l-lactate exposure reduced mRNA and protein levels of superoxide dismutase-2 and c-glutamylcysteine ligase, elevated levels of cytochrome P450 2E1 and NADPH oxidase-2, and increased the protein expressions of LDHB, Bax/Bcl-2, cleaved caspase-3, and sirtuin-1 in hepatic tissues. Together, these results indicate that chronic l-lactate exposure increases oxidative stress and apoptosis in hepatocytes via upregulation of Bax/Bcl-2 expression and the consequent mitochondrial cytochrome-C release and caspase-3 activation, which contributes to the pathogenesis of hepatic dysfunction.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Increased serum anti-CYP2E1 IgG autoantibody levels may be involved in the pathogenesis of occupational trichloroethylene hypersensitivity syndrome: a case-control study.

Occupational exposure to trichloroethylene (TCE) causes a systemic skin disorder with hepatitis known as TCE hypersensitivity syndrome (TCE-HS). Human Leukocyte Antigen (HLA)-B*13:01 is its susceptibility factor; however, the immunological pathogenesis of TCE-HS remains unknown. We herein examined the hypothesis that autoantibodies to CYP2E1 are primarily involved in TCE-HS. A case-control study of 80 TCE-HS patients, 186 TCE-tolerant controls (TCE-TC), and 71 TCE-nonexposed controls (TCE-nonEC) was conducted to measure their serum anti-CYP2E1 antibody (IgG) levels. The effects of TCE exposure indices, such as 8-h time-weighted-average (TWA) airborne concentrations, urinary metabolite concentrations, and TCE usage duration; sex; smoking and drinking habits; and alanine aminotransferase (ALT) levels on the antibody levels were also analyzed in the two control groups. There were significant differences in anti-CYP2E1 antibody levels among the three groups: TCE-TC > TCE-HS patients > TCE-nonEC. Antibody levels were not different between HLA-B*13:01 carriers and noncarriers in TCE-HS patients and TCE-TC. The serum CYP2E1 measurement suggested increased immunocomplex levels only in patients with TCE-HS. Multiple regression analysis for the two control groups showed that the antibody levels were significantly higher by the TCE exposure. Women had higher antibody levels than men; however, smoking, drinking, and ALT levels did not affect the anti-CYP2E1 antibody levels. Anti-CYP2E1 antibodies were elevated at concentrations lower than the TWA concentration of 2.5 ppm for TCE exposure. Since HLA-B*13:01 polymorphism was not involved in the autoantibody levels, the possible mechanism underlying the pathogenesis of TCE-HS is that TCE exposure induces anti-CYP2E1 autoantibody production, and HLA-B*13:01 is involved in the development of TCE-HS.

Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis.

BACKGROUND: Hepatic steatosis is an early pathology of alcohol-associated liver disease (ALD). Fatty acid-binding protein-4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. METHODS: Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1-inhibitor; CMZ) and/or EX-527 (a sirtuin-1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4 mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], ß-catenin, peroxisome proliferator-activated receptor-α [PPARα], and lipin-1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. RESULTS: CYP2E1-dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased ß-catenin, PPARα, and lipin-1α protein levels in CYP2E1-expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1 mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1-expressing cells. Pharmacological inhibition of ß-catenin, PPARα, or lipin-1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. CONCLUSION: CYP2E1-dependent ethanol metabolism inhibits SIRT1-FOXO1 signaling, which leads to increased FABP4 mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.

Role of Mitochondrial Cytochrome P450 2E1 in Healthy and Diseased Liver.

Cytochrome P450 2E1 (CYP2E1) is pivotal in hepatotoxicity induced by alcohol abuse and different xenobiotics. In this setting, CYP2E1 generates reactive metabolites inducing oxidative stress, mitochondrial dysfunction and cell death. In addition, this enzyme appears to play a role in the progression of obesity-related fatty liver to nonalcoholic steatohepatitis. Indeed, increased CYP2E1 activity in nonalcoholic fatty liver disease (NAFLD) is deemed to induce reactive oxygen species overproduction, which in turn triggers oxidative stress, necroinflammation and fibrosis. In 1997, Avadhani's group reported for the first time the presence of CYP2E1 in rat liver mitochondria, and subsequent investigations by other groups confirmed that mitochondrial CYP2E1 (mtCYP2E1) could be found in different experimental models. In this review, we first recall the main features of CYP2E1 including its role in the biotransformation of endogenous and exogenous molecules, the regulation of its expression and activity and its involvement in different liver diseases. Then, we present the current knowledge on the physiological role of mtCYP2E1, its contribution to xenobiotic biotransformation as well as the mechanism and regulation of CYP2E1 targeting to mitochondria. Finally, we discuss experimental investigations suggesting that mtCYP2E1 could have a role in alcohol-associated liver disease, xenobiotic-induced hepatotoxicity and NAFLD.

Diallyl disulfide prevents 1,3-dichloro-2-propanol-induced hepatotoxicity through mitogen-activated protein kinases signaling.

We investigated whether diallyl disulfide (DADS) has protective effects against 1,3-dichloro-2-propanol (1,3-DCP)-induced hepatotoxicity and oxidative damage in rats and HepG2 cells. DADS was administered to rats once daily for 7 days at doses of 30 and 60 mg/kg/day. One hour after the final DADS treatment, the rats were administered 90 mg/kg 1,3-DCP to induce acute hepatotoxicity. DADS treatment significantly suppressed the increase in serum aminotransferase levels induced by 1,3-DCP administration, and reduced histopathological alterations in the liver. DADS treatment reduced 1-3-DCP-induced apoptotic changes in the liver, as revealed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining and immunohistochemistry for caspase-3. DADS treatment competitively inhibited or reduced cytochrome p450 2E1 (CYP2E1) expression, which is involved in the metabolic activation of 1,3-DCP, and enhanced antioxidant properties. Furthermore, DADS treatment inhibited phosphorylation of mitogen-activated protein kinases (MAPKs) and apoptotic signaling. In in vitro experiments, MAPKs inhibitors reduced the expression of Bax/Bcl-2/Caspase 3 signaling, which effects were more significant in co-treated cells with DADS and MAPKs inhibitors. In conclusion, the protective effect of DADS against 1,3-DCP-induced hepatotoxicity may be related to blocking the metabolic activation of 1,3-DCP by suppressing CYP2E1 expression, inducing antioxidant enzyme activity, and reducing apoptotic activity by inhibiting phosphorylation of MAPKs.

Behavioral consequences of the downstream products of ethanol metabolism involved in alcohol use disorder.

Research concerning Alcohol Use Disorder (AUD) has previously focused primarily on either the behavioral or chemical consequences experienced following ethanol intake, but these areas of research have rarely been considered in tandem. Compared with other drugs of abuse, ethanol has been shown to have a unique metabolic pathway once it enters the body, which leads to the formation of downstream metabolites which can go on to form biologically active products. These metabolites can mediate a variety of behavioral responses that are commonly observed with AUD, such as ethanol intake, reinforcement, and vulnerability to relapse. The following review considers the preclinical and chemical research implicating these downstream products in AUD and proposes a chemobehavioral model of AUD.

Echinacoside alleviates acetaminophen-induced liver injury by attenuating oxidative stress and inflammatory cytokines in mice.

This study evaluates the protective effect of Echinacoside on acute liver toxicity induced by acetaminophen in mice and the mechanism behind it. Echinacoside and N-Acetyl Cysteine were intragastrically administrated for 7 days, and acetaminophen was intraperitoneally injected into mice 1 h after the last treatment on day 7. At the end of the experimental period, histological examination, parameters for the level of oxidative damage, hepatic malondialdehyde, serum pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6, and interleukin-1ß), UDP-glucuronosyltransferases, and sulfotransferases changes were examined using enzyme-linked immunosorbent assay and standard biochemical procedures. The expression of cytochrome P450 2E1 protein was assessed by western blot, followed by in silico molecular docking. Acetaminophen treatment obviously increased the levels of ALT and AST, changed hepatic histopathology, promoted oxidative stress, decreased antioxidant enzyme activities, and elevated the pro-inflammatory cytokines. Echinacoside significantly attenuated Acetaminophen-induced liver damage in a dose-dependent manner, with the most effective dose at 100 mg/kg. The pretreatments of Echinacoside in different concentrations altered the Acetaminophen-induced hepatotoxicity levels by decreasing the level of liver enzymes, reducing the liver necrosis with vacuolization, decreasing the hepatic malondialdehyde formation, increasing hepatic antioxidants activities, suppressing the pro-inflammatory cytokines (Tumor Necrosis Factor, Interleukin-6 and Interleukin-1beta), inhibiting Nitric Oxide production, enhancing sulfotransferases and UDP-glucuronosyltransferases activities. Notably, the expression of cytochrome P450 2E1 was inhibited by Echinacoside in a dose-dependent manner and the binding energy was -214.3 MeV. Echinacoside showed a significant protective effect against Acetaminophen-induced hepatotoxicity through the inhibition of oxidative stress, the expression of pro-inflammatory cytokines and cytochrome P450 2E1 protein expression.

Triptolide regulates oxidative stress and inflammation leading to hepatotoxicity via inducing CYP2E1.

Triptolide (TP), the main active compound extracted from medicine-tripterygium wilfordii Hook f. (TWHF). It has anti-tumor and immunomodulatory properties. Our study aimed to investigate the mechanisms of hepatotoxicity treated with TP in vivo and in vitro, as well as their relationship with the NF-κB (p65) signal pathway; and to assess TP-induced hepatotoxicity after CYP2E1 modulation by the known inhibitor, clomethiazole, and the known inducer, pyrazole. Mice were given TP to cause liver injury and IHHA-1 cells were given TP to cause hepatocyte injury. The enzyme activity and hepatotoxicity changed dramatically when the CYP2E1 inhibitor and inducer were added. In comparison to the control group, the enzyme inducer increased the activity of CYP2E1, whereas the enzyme inhibitor had the opposite effect. Our findings suggest that TP is an inducer of CYP2E1 via a time-dependent activation mechanism. In addition, TP can promote oxidative stress, inflammatory and involving the NF-κB (p65) signal pathway. Therefore, we used triptolide to stimulate C57 mice and IHHA-1 cells to determine whether TP can promote oxidative stress and inflammation by activating CYP2E1 in response to exacerbated liver damage and participate in NF-κB (p65) signaling pathway.