Transgenic expression of human cytochrome P450 2E1 in C. elegans and rat PC-12 cells sensitizes to ethanol-induced locomotor and mitochondrial effects.

Chronic alcohol (ethanol) use is increasing in the United States and has been linked to numerous health issues in multiple organ systems including neurological dysfunction and diseases. Ethanol toxicity is mainly driven by the metabolite acetaldehyde, which is generated through three pathways: alcohol dehydrogenase (ADH2), catalase (CAT), and cytochrome P450 2E1 (CYP2E1). ADH2, while the main ethanol clearance pathway in the liver, is not expressed in the mammalian brain, resulting in CAT and CYP2E1 driving local metabolism of ethanol in the central nervous system. CYP2E1 is known to generate reactive metabolites and reactive oxygen species and localizes to the mitochondria (mtCYP2E1) and endoplasmic reticulum (erCYP2E1). We sought to understand the consequences of mtCYP2E1 and erCYP2E1 in the nervous system during acute ethanol exposure. To answer this question, we generated transgenic Caenorhabditis elegans roundworms expressing human CYP2E1 in the mitochondria, endoplasmic reticulum, or both and exposed them to ethanol. We found that at lower concentrations, wild-type and mtCYP2E1-expressing worms had a small but significant inhibition of locomotion, whereas the erCYP2E1-expressing worms showed protection from this inhibition. At higher doses, all strains had reduced locomotion, but the erCYP2E1-expressing worms recovered faster than wild-type controls. CYP2E1 expression, regardless of organellar targeting, reduced mitochondrial respiration in response to ethanol. Similarly, transgenic expression of CYP2E1 in either organelle in PC-12 rat neuronal cell lines sensitized them to ethanol-induced cell death. Together, these findings suggest that subcellular localization of CYP2E1 impacts behavioral effects of ethanol and should be further studied in the mammalian central nervous system.

Genetic polymorphisms in FABP2, CYP2E1, and TP53 genes are potentially associated with colorectal cancer susceptibility.

Colorectal cancer (CRC) is among the most prevalent cancers with a high mortality rate. Both genetic and environmental factors contribute to CRC development. This study aimed to assess the association of single nucleotide polymorphisms (SNPs) in the fatty acid binding protein-2 (rs1799883), Cytochrome P450 2E1 (rs3813865), TP53 (rs1042522), and Murine double minute 2 (rs1042522) genes with CRC. A cross-sectional case-control study was conducted at the Institute of Molecular Biology and Biotechnology from May 2020 to March 2021, involving CRC patients (N = 100) and controls (N = 100) recruited from the Multan district in Pakistan. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) were employed to investigate the studied SNPs. The association of SNPs in all genes with CRC was examined either individually or in various combinations. Genotypes at three SNPs, rs1799883 in FABP2, rs3813865 in CYP2E1, and rs1042522 in TP53, were found to be associated with the development of CRC, while rs1042522 in MDM2 was not. Patients who were married, smoked, lacked exercise habits or had a family history of CRC were at a greater risk of acquiring the disease. FABP2 gene rs1799883, CYP2E1 gene rs3813865, and TP53 gene rs1042522 polymorphisms are significant in the development of CRC in Pakistani participants.

Different ethanol exposure durations affect cytochrome P450 2E1-mediated sevoflurane metabolism in rat liver.

BACKGROUND: Chronic alcohol users often exhibit an increased minimum alveolar concentration (MAC) of sevoflurane, yet the specific mechanism remains unclear. It has been reported that ethanol exposure can upregulate the protein expression and enzyme activity of cytochrome P450 2E1 (CYP2E1). CYP2E1 is a key enzyme that converts 2-5% of sevoflurane into equimolar amounts of hexafluoroisopropanol (HFIP) and F-. This study aims to explore whether ethanol exposure could alter sevoflurane metabolism through CYP2E1 modulation, potentially explaining the increased MAC observed in alcohol users. METHODS: Eighty adult male Sprague-Dawley (SD) rats were randomly divided into two groups and received either 50% ethanol (dose: 3 g/kg) or 0.9% saline twice daily by gavage. After 1, 2, 3, and 4 weeks of gavage, ten rats were randomly selected from each group to undergo 1-hour anesthesia with 2.3% sevoflurane. Blood samples were collected after anesthesia to measure the concentration of free HFIP using gas chromatography. Additionally, the left lobe tissue of the liver was collected for the analysis of CYP2E1 protein expression by Western blot and CYP2E1 enzyme activity by colorimetric assay. Correlations between these parameters were analyzed using Pearson's correlation. RESULTS: In the ethanol group, CYP2E1 expression, activity, and the concentration of free HFIP were significantly higher at all time points compared to the control group (P < 0.05), except for protein expression in the first week (P > 0.05). Within-group comparisons indicated no significant changes in any of the parameters for the control group (P > 0.05). In the ethanol group, there was no difference in free HFIP concentration between the first and second weeks (P > 0.05), but a significant increase was observed in the third and fourth weeks (P < 0.01); protein expression and enzyme activity significantly varied over time, especially showing a notable increase from the first to the third and fourth weeks (P < 0.05). Correlation analysis revealed strong positive correlations between free HFIP concentration and CYP2E1 activity (r = 0.7898), free HFIP concentration and CYP2E1 expression (r = 0.8418), and CYP2E1 activity and expression (r = 0.8740), all with P < 0.001. CONCLUSIONS: Ethanol exposure increased both the expression and enzymatic activity of CYP2E1, consequently enhancing the metabolism of sevoflurane.

CYP2D6, CYP2E1 Gene Polymorphisms and Gastrointestinal Cancer Risk in Rural Maharashtra: A Hospital Based Case-Control Study.

BACKGROUND: Cytochrome P450 (CYP) is a family phase I metabolizing enzymes important in xenobiotics metabolism. Genetic polymorphisms of CYPs have been comprehensively studied for their association with a range of diseases including cancer risk. In this study we assessed single nucleotide polymorphism (SNP) CYP2D6 and CYP2E1 genes and their role in gastrointestinal (GI) cancer susceptibility in the rural population of Maharashtra. METHODS: Genotyping of CYP2D6*4, CYP2E1*5B, CYP2E1*6, CYP2E1*7B genes among 200 GI cancer cases and equal number of controls was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The Odds ratio (OR) with 95% confidence interval and p-value were evaluated to get the level of association of polymorphisms with risk of GI cancer, where p ≤0.005 was considered as statistically significant. RESULTS: After the analysis of CYP2D6 and CYP2E1 gene polymorphisms, we noticed that CYP2D6*4 (rs3892097) with heterozygous genotype (G/C) showed negative association with GI cancer risk (OR=0.43, 95% CI: 0.25-0.74; p=0.002) and CYP2E1*6 (rs6413432) variant genotype showed positive association (OR=2.85, 95% CI: 1.40-5.81; p=0.003) showed positive association with GI cancer risk in studied population. CONCLUSION: The findings obtained from this study concluded that the polymorphic CYP2D6 was negatively associated; however CYP2E1*6 polymorphism was significantly associated with GI cancer risk in studied population.

Polygala fallax Hemsl polysaccharides alleviated alcoholic fatty liver disease by modifying lipid metabolism via AMPK.

Alcoholic fatty liver disease (AFLD) is characterized by excessive lipid accumulation in the liver. This study aimed to investigate the protective effects and mechanisms of Polygala fallax Hemsl polysaccharides (PFPs) on AFLD. PFPs were purified and structurally characterized. An AFLD model was established in mice using alcohol and a high-fat diet. A significant reduction in hepatic steatosis was observed following PFPs treatment, evidenced by decreased fat deposition in liver tissues. Additionally, PFPs reduced various liver injury markers, increased levels of antioxidant enzymes, and improved significantly liver function. RNA sequencing revealed that PFPs improved lipid and CYP450 metabolic pathway abnormalities in AFLD mice. Furthermore, PFPs activated the AMPK pathway, reducing lipid accumulation and enhancing lipid metabolism. A HepG2 cell model treated with ethanol and oleic acid showed significant biochemical improvements with PFPs pretreatment, including reduced lipid accumulation and lower reactive oxygen species (ROS) levels. To further elucidate the AMPK and PFPs correlation in AFLD, an AMPK inhibitor (compound C) was used. In vitro and in vivo qRT-PCR and Western blot results confirmed that PFPs protected against AFLD by activating AMPK phosphorylation, regulating lipid synthesis, and inhibiting lipid accumulation. PFPs also modulated CYP2E1 and oxidative stress-related gene expression, affecting liver metabolism.

A comprehensive study of CYP2E1 and its role in carcass characteristics and chemical lamb meat quality in different Indonesian sheep breeds.

The role of CYP2E1 in oxidation is essential for its effects on meat quality. This study used 200 Indonesian sheep (Ovis aries) to determine the SNP g allele frequencies. g. 50658168 T>C of CYP2E1 gene located in 3´-UTR region and their genetic association with lamb quality traits, including carcass characteristics, retail cut carcass, physicochemical lamb, fatty acid, cholesterol, flavor and odor, and mineral content. Further, the level of CYP2E1 mRNA and CYP2E1 protein expression in muscle were determined and correlated with lamb quality traits. CYP2E1 gene polymorphisms were identified using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis. The CYP2E1 mRNA expression levels in phenotypically divergent sheep populations were analyzed using Quantitative Real Time-PCR (qRT-PCR). Immunohistochemistry (IHC) and hematoxylin-eosin (HE) staining analysis used three samples each in the high and low lamb quality groups based on pH value and tenderness. An association study of CYP2E1 gene polymorphisms was performed using General Linear Model (GLM) analysis. The genetic association between the CC, CT, and TT genotypes at the SNP g. 50658168 T>C CYP2E1 gene and lamb quality traits were significant (P<0.05), including carcass characteristics, retail cut carcass, fatty acid, cholesterol, flavor, and odor. Lambs with the CT genotype had a higher mRNA and protein expression in high lamb quality traits. The highest CYP2E1 protein expression was localized in the longissimus dorsi. The group sample with high lamb quality had a higher area and perimeter of muscle cells. CYP2E1 can be used as a genetic marker for selecting sheep with high meat quality.

Computational simulations uncover enantioselective metabolism of chiral triazole fungicides by human CYP450 enzymes: A case study of tebuconazole.

Tebuconazole (TEB), a prominent chiral triazole fungicide, has been extensively utilized for plant pathogen control globally. Despite experimental evidence of TEB metabolism in mammals, the enantioselectivity in the biotransformation of R- and S-TEB enantiomers by specific CYP450s remains elusive. In this work, integrated in silico simulations were employed to unveil the binding interactions and enantioselective metabolic fate of TEB enantiomers within human CYP1A2, 2B6, 2E1, and 3A4. Molecular dynamics (MD) simulations clearly delineated the binding specificity of R- and S-TEB to the four CYP450s, crucially determining their differences in metabolic activity and enantioselectivity. The primary driving force for robust ligand binding was identified as van der Waals interactions with CYP450s, particularly involving the hydrophobic residues. Mechanistic insights derived from quantum mechanics/molecular mechanics (QM/MM) calculations established C2-methyl hydroxylation as the predominant route of R-/S-TEB metabolism, while C6-hydroxylation and triazol epoxidation were deemed kinetically infeasible pathways. Specifically, the resulting hydroxy-R-TEB metabolite primarily originates from R-TEB biotransformation by 1A2, 2E1 and 3A4, whereas hydroxy-S-TEB is preferentially produced by 2B6. These findings significantly contribute to our comprehension of the binding specificity and enantioselective metabolic fate of chiral TEB by CYP450s, potentially informing further research on human health risk assessment associated with TEB exposure.

CYP2E1 mediated deoxynivalenol-induced hepatocyte toxicity by regulating ferroptosis.

Deoxynivalenol (DON), one of the most common mycotoxins in food and feed, can cause acute and chronic liver injury, posing a serious health risk to humans and animals. One of the important manifestations of DON-induced hepatotoxicity is ferroptosis. It has been reported that CYP2E1 can mediated ferroptosis, but the role of DON-induced CYP2E1 in DON-induced ferroptosis in hepatocytes is unknown. In the present study, we observed that DON significantly increased the expression of CYP2E1 and decreased the expression of the ferroptosis inhibitory proteins GPX4 and SLC7A11, as well as GCLC and NQO1. This resulted in an increase in the levels of cell lipid ROS and FeII, 4-HNE, which ultimately led to cell ferroptosis. Notably, knockdown of CYP2E1 resulted in an increase in DON-induced low levels of GPX4 and SLC7A11, a decrease in DON-induced high levels of lipid ROS, FeII and cell secreted 4-HNE, thus ameliorating cell ferroptosis. Moreover, the ferroptosis inhibitor ferrostatin-1 was observed to antagonise the cell growth inhibitory toxicity induced by DON exposure. This was achieved by blocking the increase in lipid ROS and FeII overload, which in turn reduced the extent of ferroptosis and increased IGF-1 protein expression. In conclusion, the present study demonstrated that CYP2E1 played a regulatory role in DON-induced ferroptosis in hepatocytes. Targeting ferroptosis may prove an effective strategy for alleviating DON-induced cell growth retardation toxicity. These findings provided a potential target and strategies to mitigate DON hepatotoxicity in the future.

Discovery of CYP2E1 as a novel target in rheumatoid arthritis and validation by a new specific CYP2E1 inhibitor.

Considerable evidence indicates that CYP2E1 is associated with a variety of inflammatory diseases. Here we evaluated CYP2E1 as a potential therapeutic target for rheumatoid arthritis (RA) and established the protective effect of a new CYP2E1 inhibitor. Gene-expression datasets were used to analyze the change in expression of CYP2E1 in RA patients; CYP2E1 activity in collagen-induced arthritis (CIA) rats was determined by HPLC. We further evaluated the protective effects of Cyp2e1 knockout and a CYP2E1-specific inhibitor, Q11, synthesized by our group, in CIA and adjuvant-induced arthritis (AIA) rats. The expression of CYP2E1 in synovial tissue was elevated in RA patients and in CIA rats and the activity of CYP2E1 in vivo and in vitro in CIA rats was greater than that of controls. Cyp2e1 knockout significantly reduced the incidence of CIA and alleviated the severity of symptoms. Treatment with different doses of Q11 decreased paw thickness, volume and arthritis scores and reduced the serum levels of IL-6, TNF-α, IL-1ß and MDA, and increased the level of GSH in CIA rats. A similar inhibitory effect was exhibited for Q11 in the AIA rats. Moreover, Q11 significantly impeded proliferation, migration, and invasion of human rheumatoid arthritis synovial fibroblasts cells. Q11 decreased the release of ROS and enhanced Nrf2 nuclear translocation and HO-1 expression in the cell nucleus. Overall, our results indicated that CYP2E1 may be a new target for RA and Q11 has potential protective effects against RA by reducing oxidative stress and opposing the inflammatory response via the ROS/Nrf2/HO-1 signaling pathway.

CYP2E1 in 1,4-dioxane metabolism and liver toxicity: insights from CYP2E1 knockout mice study.

1,4-Dioxane (DX), an emerging water contaminant, is classified as a Group 2B liver carcinogen based on animal studies. Understanding of the mechanisms of action of DX liver carcinogenicity is important for the risk assessment and control of this environmental pollution. Previous studies demonstrate that high-dose DX exposure in mice through drinking water for up to 3 months caused liver mild cytotoxicity and oxidative DNA damage, a process correlating with hepatic CYP2E1 induction and elevated oxidative stress. To access the role of CYP2E1 in DX metabolism and liver toxicity, in the current study, male and female Cyp2e1-null mice were exposed to DX in drinking water (5000 ppm) for 1 week or 3 months. DX metabolism, redox and molecular investigations were subsequently performed on male Cyp2e1-null mice for cross-study comparisons to similarly treated male wildtype (WT) and glutathione (GSH)-deficient Gclm-null mice. Our results show that Cyp2e1-null mice of both genders were resistant to DX-induced hepatocellular cytotoxicity. In male Cyp2e1-null mice exposed to DX for 3 months, firstly, DX metabolism to ß-hydroxyethoxyacetic acid was reduced to ~ 36% of WT levels; secondly, DX-induced hepatic redox dysregulation (lipid peroxidation, GSH oxidation, and activation of NRF2 antioxidant response) was substantially attenuated; thirdly, liver oxidative DNA damage was at a comparable level to DX-exposed WT mice, accompanied by suppression of DNA damage repair response; lastly, no aberrant proliferative or preneoplastic lesions were noted in DX-exposed livers. Overall, this study reveals, for the first time, that CYP2E1 is the main enzyme for DX metabolism at high dose and a primary contributor to DX-induced liver oxidative stress and associated cytotoxicity. High dose DX-induced genotoxicity may occur via CYP2E1-independent pathway(s), potentially involving impaired DNA damage repair.

Metabolism-dependent inhibition of CYP2E1 by isoniazid, a mediator of idiosyncratic liver injury.

Liver injury is a well-known adverse effect of the anti-tuberculosis drug isoniazid (INH); however, animal models that accurately replicate this effect as seen in humans have not been constructed, and the mechanism of its pathogenesis remains unclear. Recently, an immune-mediated mechanism have been proposed based on clinical studies, suggesting the involvement of cytochrome P450-mediated formation of reactive metabolites and covalent adducts in severe cases. In the present study, we investigated the role of CYP2E1 in this mechanism. Liver microsomes from humans, rats, and mice were preincubated with INH and NADPH; thereafter, residual CYP2E1 activity was measured. The inhibition of CYP2E1 by INH was potentiated by preincubation, indicating time-dependent inhibition. There were no major species-based differences in inhibition among humans, rats, and mice. Further to our findings on the inhibition kinetics, resistance of the inhibition to glutathione and catalase indicated that the reactive metabolites of INH covalently bonded to CYP2E1 in a suicidal manner. A similar time-dependent inhibition was also observed for the known metabolites acetylhydrazine and hydrazine; however, the conditions that inhibited the hydrolysis or activated the acetylation of INH did not affect inhibition by INH, suggesting that the reactive metabolites contributing to the inhibition were generated via alternative pathways. This indicates that CYP2E1 alone generates reactive INH metabolites and that haptenized CYP2E1 may be involved in immune-mediated liver injury.

Jujuboside A through YY1/CYP2E1 signaling alleviated type 2 diabetes-associated fatty liver disease by ameliorating hepatic lipid accumulation, inflammation, and oxidative stress.

Non-alcoholic fatty liver disease (NAFLD) was a chronic complication of type 2 diabetes mellitus (T2DM), and this comorbid disease lacked therapeutic drugs. Semen Ziziphi Spinosae (SZS) was the seed of Ziziphus jujuba var. Spinosa (Bunge) Hu ex H.F. Chow, and it could alleviate the symptoms of T2DM patients. As a triterpene saponin, Jujuboside A (Ju A) was the main active substance isolated from SZS and could improve hyperglycemia of diabetic mice. However, it was still unknown whether Ju A has protective effects on T2DM-associated NAFLD. Our study showed that Ju A attenuated T2DM-associated liver damage by alleviating hepatic lipid accumulation, inflammatory response, and oxidative stress in the liver of db/db mice, and high glucose (HG) and free fatty acid (FFA) co-stimulated human hepatocellular carcinomas (HepG2) cells. Along with the improved hyperglycemia and liver injury, Ju A restrained Yin Yang 1 (YY1)/cytochrome P450 2E1 (CYP2E1) signaling in vivo and in vitro. YY1 overexpression intercepted the protective effects of Ju A on T2DM-induced liver injury via promoting hepatic lipid accumulation, inflammatory response, and oxidative stress. While, the blocking effect of YY1 overexpression on Ju A's hepatoprotective effect was counteracted by further treatment of CYP2E1 specific inhibitor diethyldithiocarbamate (DDC) in vitro. In-depth mechanism research showed that Ju A through YY1/CYP2E1 signaling promoted hepatic fatty acid ß-oxidation, and inhibited inflammatory response and oxidative stress by activating peroxisome proliferator-activated receptor alpha (PPARα), leading to the improvement of T2DM-associated NAFLD. Ju A might be a potential agent in the treatment and health care of T2DM-associated liver disease, especially NAFLD.

Species-specific metabolism of triphenyl phosphate and its mono-hydroxylated product by human and rat CYP2E1 and the carp ortholog.

Organophosphorus flame retardants (PFRs) are a class of flame retardants and environmental pollutants with various biological effects. Recentstudies have evidenced activation of some PFRs by human CYP enzymes (including CYP2E1) for genotoxic effects. However, the activity of CYPs in fish species toward PFR metabolism remains unclear. This study was aimed on comparing the metabolism of triphenyl phosphate (TPHP) and 4-OH-TPHP in human, rat, and common carp, and the involvement of human CYP2E1 and its orthologs in the metabolism, by using fomepizole (4-MP, CYP2E1 inhibitor) as a modulator, in silico molecular docking and dynamics analyses. The rate of TPHP metabolism was apparently faster with human and rat, microsomes than with fish microsomes, the major metabolites were phosphodiester and hydroxylated phosphate, with 30-80 % of TPHP forming unidentified metabolites in the system of each species. 4-OH-TPHP was readily metabolized by both human and rat microsomes, whereas it was hardly metabolized in carp assays. Meanwhile, with 4-MP the transformation of TPHP to 4-OH-TPHP was enhanced in the human/rat systems while suppressed in the carp system. Moreover, the formation of unidentified metabolites in human and rat systems was mostly inhibited by 4-MP. Through molecular dynamics analysis TPHP and its primary metabolites showed high affinity for human and rat CYP2E1, as well as the carp ortholog (CYP2G1-like enzyme), however, the 4-OH-TPHP bond to the latter was too far from the heme to permit a biochemical reaction. This study suggests that the metabolism/activation of TPHP might be favored in mammals rather than carp, a fish species.

Mechanistic insights into the metabolic pathways of vanillin: unraveling cytochrome P450 interaction mechanisms and implications for food safety.

Vanillin, a key flavor compound found in vanilla beans, is widely used in the food and pharmaceutical industries for its aromatic properties and potential therapeutic benefits. This study presents a comprehensive quantum chemical analysis to elucidate the interaction mechanisms of vanillin with CYP450 enzymes, with a focus on mechanism-based inactivation. Three potential inactivation pathways were evaluated: aldehyde deformylation, methoxy dealkylation, and acetal formation. Aldehyde deformylation was identified as the most energy-efficient, involving the removal of the aldehyde group from vanillin and leading to the formation of benzyne intermediates that could react with the iron porphyrin moiety of CYP450, potentially resulting in enzyme inactivation. Further investigation into the interactions of vanillin with CYP2E1 and CYP1A2 was conducted using molecular docking and molecular dynamics (MD) simulation. The docking analyses supported the findings from DFT studies, wherein vanillin revealed high binding affinities with the studied isozymes. Moreover, vanillin occupied the main binding site in both isozymes, as evidenced by the inclusion of the heme moiety in their binding mechanisms. Employing a 100 ns molecular dynamics simulation, we scrutinized the interaction dynamics between vanillin and the two isozymes of CYP450. The assessment of various MD parameters along with interaction energies revealed that vanillin exhibited stable trajectories and substantial energy stabilization during its interaction with both CYP450 isozymes. These insights can guide future research and ensure the safe application of vanillin, especially in scenarios where it may interact with CYP450 enzymes.

Genotypic variation in CYP2E1, GCKR, and PNPLA3 among nonalcoholic steatohepatitis patients of Turkish origin.

BACKGROUND: This study examines genetic variations in CYP2E1 (rs6413432, rs3813867), GCKR (rs780094, rs1260326), and PNPLA3 (rs738409) among Turkish patients to assess their influence on nonalcoholic steatohepatitis. METHODS: Allele and genotype frequencies were compared between 245 NASH patients and 120 healthy controls using SNP genotyping via polymerase chain reaction-restriction fragment length polymorphism. Additionally, the deviation of the observed genotype frequencies from Hardy-Weinberg proportion was examined. RESULTS: No significant differences were found in the allelic and genotypic distributions of rs6413432, rs3813867, and rs780094 between NASH patients and healthy controls. However, significant disparities were noted for rs1260326 and rs738409. Gender and age-specific distributions showed no notable differences. The only observed deviation from Hardy-Weinberg proportion was in the genotype frequency of rs738409. CONCLUSIONS: Variants in GCKR (rs1260326) and PNPLA3 (rs738409) are significantly associated with increased NASH risk in the Turkish population, with the rs738409 variant potentially playing a more prominent role in NASH development.

CYP2E1 deficit mediates cholic acid-induced malignant growth in hepatocellular carcinoma cells.

BACKGROUND: Increased level of serum cholic acid (CA) is often accompanied with decreased CYP2E1 expression in hepatocellular carcinoma (HCC) patients. However, the roles of CA and CYP2E1 in hepatocarcinogenesis have not been elucidated. This study aimed to investigate the roles and the underlying mechanisms of CYP2E1 and CA in HCC cell growth. METHODS: The proteomic analysis of liver tumors from DEN-induced male SD rats with CA administration was used to reveal the changes of protein expression in the CA treated group. The growth of CA-treated HCC cells was examined by colony formation assays. Autophagic flux was assessed with immunofluorescence and confocal microscopy. Western blot analysis was used to examine the expression of CYP2E1, mTOR, AKT, p62, and LC3II/I. A xenograft tumor model in nude mice was used to examine the role of CYP2E1 in CA-induced hepatocellular carcinogenesis. The samples from HCC patients were used to evaluate the clinical value of CYP2E1 expression. RESULTS: CA treatment significantly increased the growth of HCC cells and promoted xenograft tumors accompanied by a decrease of CYP2E1 expression. Further studies revealed that both in vitro and in vivo, upregulated CYP2E1 expression inhibited the growth of HCC cells, blocked autophagic flux, decreased AKT phosphorylation, and increased mTOR phosphorylation. CYP2E1 was involved in CA-activated autophagy through the AKT/mTOR signaling. Finally, decreased CYP2E1 expression was observed in the tumor tissues of HCC patients and its expression level in tumors was negatively correlated with the serum level of total bile acids (TBA) and gamma-glutamyltransferase (GGT). CONCLUSIONS: CYP2E1 downregulation contributes to CA-induced HCC development presumably through autophagy regulation. Thus, CYP2E1 may serve as a potential target for HCC drug development.

CYP2E1 mediated advanced oxidation protein products exacerbate acetaminophen induced drug-derived liver injury in vitro and in vivo.

Drug-induced liver injury (DILI) is prevalent in the treatment of chronic kidney disease (CKD). Advanced oxidation protein products (AOPPs) are markers of CKD progression and participate in the occurrence and development of liver diseases. However, the mechanisms underlying the regulation of DILI in CKD have not been established. Herein, we demonstrate the involvement of Cytochrome p450 2E1 (CYP2E1) in DILI induced by AOPPs is exacerbated by exposure to acetaminophen (APAP). We used a adenine-induced CKD model, a model of DILI induced by APAP, and the AOPPs model was generated by intraperitoneal injection. The decline in renal function was associated with a significantly increased concentration of Scr, BUN and AOPPs, and renal tissue fibrosis. The ALT, AST, and AOPPs levels and liver tissue necrosis increased significantly in CKD model group compared with the sodium carboxymethyl cellulose (CMCNa) group. In the AOPPs model, compared to the PBS controls, ALT, AST, and AOPP levels, and liver tissue necrosis increased significantly. In HepG2 or L0-2 cell lines, cell survival was significantly reduced in the AOPP + APAP treatment and CYP2E1 protein expression was increased. FPS-ZM1 or NAC attenuated the hepatocyte toxicity induced by AOPP + APAP and suppression of CYP2E1 expression. AOPPs exacerbated APAP-induced DILI through CYP2E1 signaling pathways. Protein uremic toxins, such as AOPPs, can modify drug toxicity in patients with CKD. This study provides new a rationale to reduce the generation of DILIs in clinical treatment in patients with CKD. AOPPs targeting may present a novel approach to reduce the occurrence of DILI.

Biphasic effects of ethanol consumption on N,N-dimethylformamide-induced liver injury in mice.

N,N-Dimethylformamide (DMF) is a well-documented occupational hazardous material, which can induce occupational liver injury. The current study was designed to investigate whether ethanol consumption can affect DMF-induced hepatotoxicity and the potential underlying mechanisms involved. We found that a single dose of ethanol (1.25, 2.5, or 5 g/kg bw by gavage) significantly repressed the increase in serum alanine transaminase (ALT) and aspartate transaminase (AST) activities and alleviated the liver histopathological changes in mice challenged with 3 g/kg DMF. In contrast, long-term moderate drinking (2.5 g/kg bw) significantly aggravated the repeated DMF (0.7 g/kg bw) exposure-induced increase in the serum ALT and AST activities. Mechanistically, acute ethanol consumption suppressed DMF-induced activation of the NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome, while long-term moderate ethanol consumption promoted hepatocyte apoptosis in the mouse liver. Notably, cytochrome P4502E1 (CYP2E1) protein level and activity in mouse livers were not significantly affected by ethanol per se in the two models. These results confirm that regular drinking can increase the risk of DMF-induced hepatotoxicity, and suggest that DMF-handling workers should avoid consuming ethanol to reduce the risk of DMF-indued liver injury.

Ethylene Oxide Hemoglobin Adducts in Cord Blood and Offspring's Size at Birth: The NewGeneris European Cohort Study.

BACKGROUND: Prenatal ethylene oxide exposure may have adverse effects on fetal development. We examined the relationships between ethylene oxide hemoglobin (Hb) adduct levels and offspring's size at birth in a prospective European mother-child study. METHODS: This study included 1106 singletons from the NewGeneris project (2006-2010) with ethylene oxide Hb adducts measured in cord blood. We examined the relationships between adduct levels and offspring's size at birth among all infants and separately among infants of nonsmokers, using linear regression models for birth weight and birth head circumference and logarithmic binomial regression models for small for gestational age. We examined potential interactions between CYP2E1 single nucleotide polymorphisms in cord blood and the effects of ethylene oxide Hb adduct levels on offspring birth size. RESULTS: Higher quartiles of adduct levels as a measure of exposure were associated with decreasing birth weight and head circumference in the overall population. Compared to infants in the lowest quartile, those in the highest quartile exhibited lower birth weight (-70.73 g, 95% confidence interval = -141.16, -0.30) and reduced head circumference (-0.30 cm, 95% confidence interval = -0.58, -0.02). We observed similar, albeit less pronounced, patterns among infants of nonsmokers. There was no evidence of an association between ethylene oxide Hb adducts and risk of small for gestational age, nor consistent evidence of an interaction with CYP2E1 polymorphisms on the association between EO Hb adduct levels and offspring's size at birth. CONCLUSION: Results suggest that higher ethylene oxide Hb adduct levels in cord blood are associated with a reduction in offspring birth size.

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.