Cytochrome P450 2E1 mRNA Expression Research Articles

Effect of SNPs in the Promoter Region on the Expression of Cytochrome P450 2E1 (CYP2E1) in Pig Liver.

Boar taint, an unfavorable odor in the meat of intact male pigs, is caused primarily by the accumulation of two compounds: androstenone and skatole. This multifactorial trait is regulated by numerous dietary, management and genetic factors. At the mechanistic level, there are many genes known to be involved in boar taint metabolism. Cytochrome P450 2E1 (CYP2E1) impacts boar taint through the phase I metabolism of skatole. The aim of this study was to identify single-nucleotide polymorphisms (SNPs) within the CYP2E1 gene promoter and explore their relationship with the expression of CYP2E1 mRNA and protein. Sequencing of the promoter region using pools of genomic DNA identified seven promoter region SNPs at -159, -586, -1693, -1806, -2322, -2369 and -2514 bp upstream of the ATG start site. Genomic DNA was obtained from 65 boars from the three major swine breeds: Duroc, Landrace and Yorkshire, and individual animals were genotyped for the identified SNPs. RNA was isolated from liver tissue and quantitative PCR was performed to measure CYP2E1 gene expression, while levels of CYP2E1 protein in liver were measured by Western blotting. Significant within-breed variation in CYP2E1 protein and mRNA expression was observed, indicating significant differences in gene expression among individuals. However, levels of CYP2E1 mRNA and protein were not significantly correlated. Two SNPs within the promoter were significantly associated with CYP2E1 mRNA expression, but not with protein expression. This study provides evidence of additional mutations affecting the gene expression of CYP2E1 and suggests that factors that affect the differences in translation of CYP2E1 mRNA may also be important in affecting skatole metabolism.

Combination of Cysteine and Glutathione Prevents Ethanol-Induced Hangover and Liver Damage by Modulation of Nrf2 Signaling in HepG2 Cells and Mice.

Excessive alcohol consumption increases oxidative stress, leading to alcoholic liver disease. In this study, the protective effects of a mixture of cysteine and glutathione against ethanol-induced hangover and liver damage were evaluated in mice and HepG2 cells. Ethanol (2 mL/kg) was orally administered to the mice 30 min before receiving the test compounds (200 mg/kg), and the behavioral and oxidative stress-related biochemical parameters altered by ethanol were analyzed. Acute ethanol administration increased anxiety behavior and decreased balance coordination in mice (p < 0.001); however, a mixture of cysteine and glutathione (MIX) in a 3:1 ratio improved alcohol-induced behavior more effectively than the individual compounds (p < 0.001). The MIX group showed higher ethanol-metabolizing enzyme activity than the control group (p < 0.001) and significantly suppressed the elevation of serum alcohol (p < 0.01) and acetaldehyde (p < 0.001) levels after 1 h of ethanol administration. In HepG2 cells, 2.5 mM MIX accelerated ethanol metabolism and reduced cytochrome P450 2E1 mRNA expression (p < 0.001). MIX also increased the expression of antioxidant enzymes through the upregulation of nuclear erythroid 2-related factor 2 (Nrf2) signaling and consequently suppressed the overproduction of reactive oxygen species and malondialdehyde (p < 0.001). Collectively, MIX alleviates the hangover symptoms and attenuates the alcohol-induced oxidative stress by regulating the Nrf2 pathway.

RETRACTED ARTICLE: Chemoprotective Effect of Daphnetin Against Benzene-induced Leukemia via Alteration of CYP2E1.

Leukemia is the overproduction of a large number of immature blood cells entering into the peripheral blood due to the malignancy of blood-forming tissues. The modern treatment mechanisms that are associated with leukemia comprise chemotherapy, allogeneic cell transplantation, and radiation therapy that comes with adverse side effects. Thus, medicinal herbs and their bioactive compounds are gaining more attention nowadays toward the treatment of leukemia, owing to their minimal side effects. The current study was aimed to scrutinize the antileukemic effect of daphnetin against benzene-induced leukemia in rats and explore the underlying mechanism of its action. Benzene was used for the induction of leukemia in experimental rats. The rats were divided into different groups and body weight; hematological parameters, DNA fragmentation, and cell cycle regulatory parameter were also estimated. RT-PCR was used for the estimation of mRNA expression of cytochrome P450 2E1 (CYP2E1), a membrane protein widely found in liver cells. Daphnetin-treated rats showed upregulation of body weight as compared to other groups. Moreover, daphnetin reduced the blasts (67.8%) in leukemic rats. It also altered the hematological parameters such as RBC (69.8%), WBC (54.5%), lymphocytes (47.6%), neutrophils (48.9%), monocytes (44.7%), eosinophils (48.7%), and basophils (43.5%), respectively. Daphnetin-treated rats showed an increased level of proteins p21 and p53 and reduced level of cyclins D1 and E. RT-PCR showed an upregulated mRNA expression of CYP2E1 (48.4%) of the daphnetin-treated group as compared to other groups. The current study shows the antileukemic effect of daphnetin and highlights the possibility of its use in leukemia to minimize the side effect of the usual therapy.

Bergenin Attenuates Sodium Selenite-Induced Hepatotoxicity via Improvement of Hepatic Oxidant-Antioxidant Balance in HepG2 Cells and ICR Mice

Bergenin is a C-glucoside derivative of gallic acid claimed to exhibit antioxidant and hepatoprotective activities. However, its effect on sodium selenite-induced oxidative damage has never been elucidated. We investigated the impact of bergenin on the oxidant-antioxidant balance in sodium selenite-induced oxidative stress in HepG2 cells and mouse livers. HepG2 cells were co-incubated with sodium selenite (10 μM) and either bergenin (75, 150, and 300 μM) or gallic acid (60 μM) for 24 h. Adult male ICR mice were orally administered sodium selenite (4 mg/kg/day) in combination with either bergenin (10, 50, and 250 mg/kg/day) or gallic acid (100 mg/kg/day) for 7 days. Sodium selenite injured HepG2 cells and mouse livers by disturbing the oxidant-antioxidant balance. Bergenin exerted protective effects against sodium selenite-induced oxidative stress in HepG2 cells. Moreover, bergenin attenuated sodium selenite-induced liver damage (nuclear pyknosis and necrotic areas) through a decrease in plasma aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels, and decreased production of reactive oxygen species, resulting in reduced lipid peroxidation in plasma and livers. Bergenin also restored hepatic antioxidant enzyme expression levels and activities (superoxide dismutase, catalase, and glutathione peroxidase) and reinstated hepatic glutathione homeostasis. Finally, bergenin returned cytochrome P450 2E1 mRNA expression to normal levels. In conclusion, bergenin reduced-sodium selenite induced liver damage by restoring the oxidant-antioxidant balance and reducing lipid peroxidation through multiple pathways.

Ethanol-induced CYP2E1 Expression is Reduced by Lauric Acid via PI3K Pathway in HepG2 Cells.

The metabolism of alcohol involves cytochrome P450 2E1 (CYP2E1)-induced oxidative stress, with the association of phosphatidylinositol-3-kinases (PI3K) and nuclear factor kappa B (NFκB) signalling pathways. CYP2E1 is primarily involved in the microsomal ethanol oxidising system, which generates massive reactive oxygen species (ROS) and ultimately leads to oxidative stress and tissue damage. Lauric acid, a major fatty acid in palm kernel oil, has been shown as a potential antioxidant. Here, we aimed to evaluate the use of lauric acid as a potential antioxidant against ethanol-mediated oxidative stress by investigating its effect on CYP2E1 mRNA expression and the signalling pathway in ethanol-induced HepG2 cells. HepG2 cells were firstly treated with different concentrations of ethanol, and subsequently co-treated with different concentrations of lauric acid for 24 h. Total cellular RNA and total protein were extracted, and qPCR and Western blot was carried out. Ethanol induced the mRNA expression of CYP2E1 significantly, but lauric acid was able to downregulate the induced CYP2E1 expression in a dose-dependent manner. Similarly, Western blot analysis and densitometry analysis showed that the phosphorylated PI3K p85 (Tyr458) protein was significantly elevated in ethanol-treated HepG2 cells, but co-treatment with lauric acid repressed the activation of PI3K. However, there was no significant difference in NFκB pathway, in which the normalised NFκB p105 (Ser933) phosphorylation remained constant in any treatment conditions in this study. This suggests that ethanol induced CYP2E1 expression by activating PI3K p85 (Tyr458) pathway, but not the NFκB p105 (Ser933) pathway in HepG2 cells.

Synonymous mutation rs2515641 affects CYP2E1 mRNA and protein expression and susceptibility to drug-induced liver injury.

Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 (CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results:CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.

Expression of CYP2E1 gene in paint workers occupationally exposed to organic solvents

Benzene (B), toluene (T), ethylbenzene (E) and xylene (X) are used commonly in paint industry, so workers who are a high risk of exposure to organic solvent (VOCs). CYP2E1 gene encodes CYP2E1 which plays an important role in the metabolism and bio-activation of volatile organic compounds. When workers expose to VOCs, their body will have a mechanism to metabolize the toxic. The enhancement of mRNA expression of CYP2E1 is a very sensitive and accurate biological marker, which is the basis for the next study to propose the level of mRNA expression of CYP2E1 gene as a human biological indicator to monitor to workers occupationally exposed to VOCs. In this study, we studied the level of mRNA expression of CYP2E1 gene from 118 participants that including 73 workers of exposed group from the paint factories and 45 workers of non-exposed group from garment factories by using Realtime-PCR with SYBR Green – an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The initial results, showed that the exposed group had a higher mRNA expression level of CYP2E1 than the non-exposed group approximately 10.47 times, and this difference was statistically significant (P value &lt; 0.05). And the age and the duration of exposure to organic solvents do not affect the expression level of CYP2E1.&#x0D; Keywords&#x0D; CYP2E1, the metabolisms of volatile organic compounds, mRNA expression of CYP2E1.&#x0D; References&#x0D; [1] A. Mendoza-Cantu, F. Castorena-Torres, M. Bermudez De Leon et al., Occupational toluene exposure induces cytochrome P450 2E1 mRNA expression in peripheral lymphocytes, Environmental Health Perspectives, 114 (2006) 494 - 499. https://doi.org/10.1289/ehp.8192.[2] J.H. Hartman, G. Boysen and G.P. Miller, CYP2E1 metabolism of styrene involves allostery, Drug Metabolism and Disposition, 40 (2012) 1976-1983. https://doi.org/10.1124/dmd. 112.046698. [3] S.M. Zhu, X.F. Ren, J.X. Wan et al., Evaluation in vinyl chloride monomer (VCM) - exposed workers and the relationship between liver lesions and gene polymorphisms of metabolic enzymes, World Journal of Gastroenterology,11 (2005) 5821 -5827. 10.3748/wjg.v11.i37.5821.[4] J. Wan, J. Shi, L. Hui et al., Association of genetic polymorphisms in CYP2E1, MPO, NQO1, GSTM1, and GSTT1 genes with benzene poisoning, Environmental Health Perspectives, 110 (2002), 1213-1218. https://doi.org/10.1289/ ehp.021101213.[5] U. Bernauer, B. Vieth, R. Ellrich et al., CYP2E1 - dependent benzene toxicity: the role of extrahepatic benzene metabolism, Archives of Toxicology, 73 (1999) 189 -196. https://doi.org/ 10.1007/s002040050605. [6] P.L. Sheets, G.S. Yost and G.P. Carlson. Benzene metabolism in human lung cell lines BEAS‐2B and A549 and cells overexpressing CYP2F1, Journal of Biochemical and Molecular Toxicology, 18 (2004) 92-99. https://doi.org/10. 1002/jbt.20010.[7] V. Nedelcheva, I. Gut, P. Souček et al., Metabolism of benzene in human liver microsomes: individual variations in relation to CYP2E1 expression, Archives of Toxicology, 73 (1999) 33-40. https://doi.org/10.1007/s0020400 50583.[8] W. Tassaneeyakul, D.J. Birkett, J.W. Edwards et al., Human cytochrome P450 isoform specificity in the regioselective metabolism of toluene and o-, m-and p-xylene, Journal of Pharmacology and Experimental Therapeutics, 276 (1996) 101 - 108. 10.1163/2211730x96x00063[9] A.-H. Wang, S.-M. Zhu, Y.-L. Qiu et al., CYP2E1 mRNA expression, genetic polymorphisms in peripheral blood lymphocytes and liver abnormalities in Chinese VCM-exposed workers, International Journal of Occupational Medicine and Environmental Health, 21 (2008), 141 - 146. 10.2478/v10001-008-0016-x.[10] J. Zhang, Y. Lihong, G. Liang et al., Detection of CYP2E1, a genetic biomarker of susceptibility to benzene metabolism toxicity in immortal human lymphocytes derived from the Han Chinese population, Biomedical and Environmental Sciences, 24 (2011) 300-309. https://doi.org/10. 3967/0895-3988.2011.03.014[11] American Conference of Industrial Hygienists, Guide to Occupational Exposeure Values, ACGIH, Cincinnati, 2018.[12] Bộ Y Tế, Quyết định của Bộ trưởng Bộ Y tế về việc ban hành 21 tiêu chuẩn vệ sinh lao động, 05 nguyên tắc và 07 thông số vệ sinh lao động, 2002.[13] American Conference of Industrial Hygienists, Threshold Limit Value for Chemical Substances and Physical Agents and Biological Exposure Indices, ACGIH, Cincinnati, 2018.[14] Bộ Y Tế, Thông tư 28/2006/TT- BYT hướng dẫn quản lý bệnh bệnh nghề nghiệp, 2016.[15] M. Al Zallouha, Y. Landkocz, J. Brunet et al., Usefulness of toxicological validation of VOCs catalytic degradation by air-liquid interface exposure system", Environmental Research, 152 (2017) 328-335. https://doi.org/10.1016/j.envres. 2016.10.027&#x0D;

A systematic evaluation of microRNAs in regulating human hepatic CYP2E1

Cytochrome P450 2E1 (CYP2E1) is an important drug metabolizing enzyme for processing numerous xenobiotics in the liver, including acetaminophen and ethanol. Previous studies have shown that microRNAs (miRNAs) can suppress CYP2E1 expression by binding to the 3′-untranslated region (3′-UTR) of its transcript. However, a systematic analysis of CYP2E1 regulation by miRNAs has not been described. Here, we applied in silico, in vivo, and in vitro approaches to investigate miRNAs involved in the regulation of CYP2E1. Initially, potential miRNA binding sites in the CYP2E1 mRNA transcript were identified and screened using in silico methods. Next, inverse correlations were found in human liver samples between the expression of CYP2E1 mRNA and the levels of two miRNA species, hsa-miR-214-3p and hsa-miR-942-5p. In a HepG2-derived CYP2E1 over-expression cell model, hsa-miR-214-3p exhibited strong suppression of CYP2E1 expression by targeting the coding region of its mRNA transcript, but hsa-miR-942-5p did not inhibit CYP2E1 levels. Electrophoretic mobility shift assays confirmed that hsa-miR-214-3p recruited other cellular protein factors to form stable complexes with specific sequences present in the CYP2E1 mRNA open reading frame. Transfection of HepaRG cells with hsa-miR-214-3p mimics inhibited expression of the endogenous CYP2E1 gene. Further, hsa-miR-214-3p mimics partially blocked ethanol-dependent increases in CYP2E1 mRNA and protein levels in HepG2 cells and they reduced the release of alanine aminotransferase from CYP2E1-overexpressing HepG2 cells exposed to acetaminophen. These results substantiate the suppressing effect of hsa-miR-214-3p on CYP2E1 expression.

The induction of cytochrome P450 2E1 by ethanol leads to the loss of synaptic proteins via PPARα down-regulation.

Ethanol, one of the most commonly abused substances throughout history, is a substrate and potent inducer of cytochrome P450 2E1 (CYP2E1). Our previous study showed that brain CYP2E1 was induced by chronic ethanol treatment and was associated with ethanol-induced neurotoxicity in rats. We therefore investigated the possible mechanism of brain CYP2E1 involvement in ethanol-induced neurodegeneration. Compared with the controls, chronic ethanol treatment (3.0g/kg, i.g., 160days) significantly increased CYP2E1 mRNA levels in the rat cortex, but the mRNA levels of peroxisome proliferator-activated receptor α (PPARα) and the pre- and post-synaptic proteins (synaptophysin, SYP, and drebrin1, DBN1) were decreased. Ethanol treatment dose-dependently induced CYP2E1 mRNA expression, and CYP2E1 overexpression exacerbated the ethanol-induced neurotoxicity. Pretreatment with p38 inhibitor (SB202190) and ERK1/2 inhibitor (U0126) attenuated the induction of CYP2E1 mRNA and protein levels by ethanol; however, no change was observed with JNK inhibitor pretreatment. Ethanol exposure or CYP2E1 overexpression significantly decreased PPARα, SYP, and DBN1 expression as indicated by the data from real-time RT-PCR, Western blotting and immunocytochemistry. The activation of PPARα by WY14643 increased the activity of the SYP and DBN1 promoters and attenuated the inhibition of these genes by ethanol. The specific siRNA for CYP2E1 significantly attenuated the ethanol-induced inhibition of PPARα, SYP and DBN1 mRNA levels. These results suggest that the induction of CYP2E1 by ethanol may be mediated via the p38 and ERK1/2 signaling pathways in neurons but not via the JNK pathway. The CYP2E1-PPARα axis may play a role in ethanol-induced neurotoxicity via the alteration of the genes related with synaptic function.

Activation of the Nrf2 Signaling Pathway Involving KLF9 Plays a Critical Role in Allicin Resisting Against Arsenic Trioxide-Induced Hepatotoxicity in Rats.

Arsenic trioxide (As2O3) is both the most prevalent, naturally occurring inorganic arsenical threatening human health and an efficient therapeutic for acute promyelocytic leukemia. Regretfully, As2O3-treated cancer patients often suffer from hepatotoxicity. While effective antioxidant and anticarcinogenic actions of allicin have previously been demonstrated, studies indicating how allicin affects As2O3-induced hepatotoxicity and arsenic accumulation are lacking. Our study, for the first time, elaborates potential details of the hepatoprotective mechanisms of allicin against As2O3-induced liver injury. Wistar rats were administrated allicin (30mg/kg) 1h before As2O3 (3mg/kg) by daily gavage for 2weeks. Our results indicate that allicin ameliorated As2O3-induced liver dysfunction, oxidative stress, and arsenic accumulation in the liver. Meanwhile, allicin decreased NF-κB level and upregulated expression of proteins reduced by As2O3 including nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1, nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1, and Krüppel-like factor 9 (KLF9). In addition, allicin promoted B cell lymphoma-extra large expression and suppressed B cell lymphoma-2-associated X protein levels regulated by As2O3. However, neither allicin nor As2O3 affected cytochrome P450 2E1 mRNA expression. In conclusion, allicin attenuated As2O3-induced hepatotoxicity by activating the Nrf2 signaling pathway involving KLF9 to inhibit oxidative stress and apoptosis. Our findings elucidate a detailed mechanism by which allicin provides protection against As2O3-induced liver injury and support its potential role as an adjunctive therapy for patients suffering from chronic arsenic exposure.

Association between CYP2E1 polymorphisms and risk of differentiated thyroid carcinoma.

Differentiated thyroid carcinoma (DTC) results from complex interactions between genetic and environmental factors. Known etiological factors include exposure to ionizing radiations, previous thyroid diseases, and hormone factors. It has been speculated that dietary acrylamide (AA) formed in diverse foods following the Maillard's reaction could be a contributing factor for DTC in humans. Upon absorption, AA is biotransformed mainly by cytochrome P450 2E1 (CYP2E1) to glycidamide (GA). Considering that polymorphisms within CYP2E1 were found associated with endogenous levels of AA-Valine and GA-Valine hemoglobin adducts in humans, we raised the hypothesis that specific CYP2E1 genotypes could be associated with the risk of DTC. Analysis of four haplotype tagging SNPs (ht-SNPs) within the locus in a discovery case-control study (N=350/350) indicated an association between rs2480258 and DTC risk. This ht-SNP resides within a linkage disequilibrium block spanning intron VIII and the 3'-untranslated region. Extended analysis in a large replication set (2429 controls and 767 cases) confirmed the association, with odds ratios for GA and AA genotypes of 1.24 (95% confidence interval (CI) 1.03-1.48) and 1.56 (95% CI, 1.06-2.30), respectively. Functionally, the minor allele was associated with low levels of CYP2E1 mRNA and protein expression as well as lower enzymatic activity in a series of 149 human liver samples. Our data support the hypothesis that inter-individual differences in CYP2E1 activity could modulate the risk of developing DTC suggesting that the exposure to specific xenobiotics, such as AA, could play a role in this process.

CYP2E1 in the Human Retinal Pigment Epithelium: Expression, Activity, and Induction by Ethanol.

Cytochrome p450 2E1 (CYP2E1) is a detoxifying enzyme with particular affinity for ethanol (EtOH) expressed in several tissues. Although CYP2E1 has been identified in human RPE, nothing is known about its metabolic activity. Expression of CYP2E1 and activity after EtOH exposure have been studied in human RPE and ARPE-19 cells. Ethanol-induced CYP2E1 mRNA expression was analyzed by RT-PCR and quantitative PCR (qPCR) from human donor RPE as well as from ARPE-19 cells. Expression of CYP2E1 protein was determined by Western blot. Cytoplasmic CYP2E1 location also was demonstrated by immunocytochemistry. Cell viability was studied by the colorimetric assay XTT after EtOH treatment. Diallyl sulfide (DAS) was used to inhibit CYP2E1 activity. The microsomal CYP2E1 activity assay was determined by quantification of 4-nitrocatechol (4NC) formation through HPLC. Relevant CYP2E1 mRNA levels are present in human RPE. Ethanol augmented the formation of reactive oxygen species (ROS) in ARPE-19 cells. Expression of CYP2E1 mRNA, CYP2E1 protein activity, and ROS production were induced by ethanol in a concentration-dependent manner. Interestingly, the treatment with DAS reduced all the aforementioned increased values. The presence of CYP2E1 in both hRPE and ARPE-19 cells reinforces the protective role of the RPE and strongly suggests additional roles for CYP2E1 related to vision.

Green Tea Lowers Hepatic COX-2 and Prostaglandin E2 in Rats with Dietary Fat-Induced Nonalcoholic Steatohepatitis.

Green tea extract (GTE) protects against nonalcoholic steatohepatitis (NASH) by decreasing hepatic steatosis and nuclear factor kappa B (NFκB) activation. We hypothesized that hypolipidemic and anti-inflammatory activities of GTE would protect against NASH by reducing cyclooxygenase-2 (COX-2), an NFκB-dependent enzyme, and prostaglandin E2 (PGE2) in a dietary fat-induced obese model. Male Wistar rats were fed a low-fat diet containing no GTE or a high-fat (HF) diet containing GTE at 0%, 1%, or 2% for 8 weeks. Insulin resistance and total hepatic fatty acids increased following HF feeding (P<.05) and these were normalized by GTE at 1-2%. GTE (1-2%) normalized hepatic malondialdehyde without affecting cytochrome P450 2E1 mRNA expression, which was otherwise increased by HF feeding. HF-mediated increases in hepatic COX-2 protein and activity as well as PGE2 concentrations were normalized by GTE (1-2%). COX-2 activity and PGE2 were correlated to each other, and to serum alanine aminotransferase (ALT) and hepatic NFκB-binding activity (P<.05; r=0.28-0.49). GTE attenuated HF-mediated increases in total hepatic n-6 and n-3, without affecting the n-6/n-3 ratio. GTE did not affect HF-mediated increases in n-6 in nonesterified fatty acid (NEFA) and phospholipid pools, whereas n-3 and n-6/n-3 in both pools were unaffected by GTE and HF feeding. GTE decreased total hepatic arachidonic acid without affecting HF-mediated increases in arachidonic acid in NEFA or phospholipid pools. Thus, GTE attenuates lipid peroxidation and PGE2 accumulation by decreasing COX-2 activity independent of arachidonic acid availability and supports an additional mechanism by which GTE protects against liver injury during NASH in an HF-feeding model.

Induction of brain CYP2E1 by chronic ethanol treatment and related oxidative stress in hippocampus, cerebellum, and brainstem

Ethanol is one of the most commonly abused substances, and oxidative stress is an important causative factor in ethanol-induced neurotoxicity. Cytochrome P450 2E1 (CYP2E1) is involved in ethanol metabolism in the brain. This study investigates the role of brain CYP2E1 in the susceptibility of certain brain regions to ethanol neurotoxicity. Male Wistar rats were intragastrically treated with ethanol (3.0g/kg, 30 days). CYP2E1 protein, mRNA expression, and catalytic activity in various brain regions were respectively assessed by immunoblotting, quantitative quantum dot immunohistochemistry, real-time RT-PCR, and LC–MS. The generation of reactive oxygen species (ROS) was analyzed using a laser confocal scanning microscope. The hippocampus, cerebellum, and brainstem were selectively damaged after ethanol treatment, indicated by both lactate dehydrogenase (LDH) activity and histopathological analysis. Ethanol markedly increased the levels of CYP2E1 protein, mRNA expression, and activity in the hippocampus and cerebellum. CYP2E1 protein and activity were significantly increased by ethanol in the brainstem, with no change in mRNA expression. ROS levels induced by ethanol paralleled the enhanced CYP2E1 proteins in the hippocampus, granular layer and white matter of cerebellum as well as brainstem. Brain CYP2E1 activity was positively correlated with the damage to the hippocampus, cerebellum, and brainstem. These results suggest that the selective sensitivity of brain regions to ethanol neurodegeneration may be attributed to the regional and cellular-specific induction of CYP2E1 by ethanol. The inhibition of CYP2E1 levels may attenuate ethanol-induced oxidative stress via ROS generation.

Possible Metabolic Pathways of Ethanol Responsible for Oxidative DNA Damage in Human Peripheral Lymphocytes

Ethanol abuse, especially binge drinking, can be toxic to human organs. However, there have been few studies on the genotoxicity induced by ethanol in human peripheral lymphocytes under binge drinking conditions. The purpose of this study was to investigate the oxidative DNA damage induced by ethanol in human peripheral lymphocytes in vitro and the possible mechanism associated with ethanol metabolism. The concentrations of ethanol investigated in this study were 50 and 100 mM, which are equal to the ethanol concentrations in blood after binge drinking. The maximum concentration we used was 150 mM although it is not the typical blood ethanol concentration seen during binge drinking, and most people may die at such a high concentration. The purpose of using this maximum concentration was to obtain more detailed evidence about the genotoxicity induced by ethanol. The DNA repair process was also studied. Peripheral lymphocytes were isolated from donors who were nonsmokers and not ethanol drinkers. Oxidative DNA damage, possible metabolic pathways of ethanol in human peripheral lymphocytes, and the repair system involved in the DNA auto-repair process were examined by comet assay, flow cytometry, time-of-flight mass spectrometry (TOF-MS), reverse transcription-polymerase chain reaction (RT-PCR), and western blotting. The results showed that ethanol at the concentrations of 50, 100, and 150 mM significantly induced the oxidative DNA damage in human peripheral lymphocytes in vitro, which was accompanied by a parallel increase in the generation of 8-OHdG, intracellular hydroxyl radical, and reactive oxygen species (iROS). The DNA damage induced by ethanol could be attenuated by alcohol dehydrogenase 1B (ADH1B) or acetaldehyde dehydrogenase 2 (ALDH2) inhibitor, and the mRNA expression levels of ADH1B and ALDH2 were increased markedly by ethanol. The inhibitor of cytochrome P450 2E1 (CYP2E1) had no effect on ethanol-induced DNA damage, and CYP2E1 mRNA expression was not affected by ethanol. Furthermore, ethanol-induced DNA damage could be auto-repaired by lymphocytes. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1) and the X-ray repair cross-complementation group 1 (XRCC1), 2 core enzymes in the base excision repair (BER) system, were increased in both of transcriptional and protein levels after ethanol treatment. This study provides direct evidence that ethanol can induce oxidative DNA damage in human peripheral lymphocytes in vitro, and its mechanism may be associated with the metabolism of ethanol by the ADH1B/ALDH2 pathway. Moreover, ethanol-induced DNA damage can be auto-repaired by human peripheral lymphocytes possibly mediated by the BER system.

Induction of blood lymphocyte cytochrome P450 2E1 in early stage alcoholic liver cirrhosis

To validate the induction of blood lymphocyte cytochrome P450 2E1 (CYP2E1) expression in alcoholic liver cirrhosis and mRNA and protein expression of CYP2E1 in freshly prepared blood lymphocytes of alcoholic liver cirrhotic (ACP), nonalcoholic cirrhotic patients (NACP), alcoholic controls (ACs), and nonalcoholic controls (NACs) were investigated. Registered ACP and NACP patients at Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, India along with NACs and ACs were included in the study. Real time polymerase chain reaction, enzyme-linked immunosorbent assay, and CYP2E1-dependent enzyme activity were determined in blood lymphocytes isolated from cases and controls. Significant increases in CYP2E1 mRNA and protein expression were observed in freshly prepared blood lymphocytes isolated from ACs and ACP patients as compared with respective NACs or NACP patients. A concomitant increase in N-nitrosodimethyamine demethylase activity was evident in the blood lymphocytes of ACs and ACP patients. Interestingly, the comparative increase observed in CYP2E1 expression was of greater magnitude in the blood lymphocytes isolated from ACP patients, although they abstained from alcohol drinking. Findings suggest that significant increase in the CYP2E1 mRNA and protein expression in the blood lymphocytes, isolated from early stage ACP patients, can be used to predict alcohol-induced toxicity.

The antagonistic action of epigallocatechin-3-gallate on microcystin LR-induced oxidative damage on hepatocytes of mice and the expression of cytochrome P450 2E1

To evaluate the effects of antagonistic action of epigallocatechin-3-gallate (EGCG) on microcystin LR (MC-LR) induced oxidative damage on mice and the expression of cytochrome P450 2E1 (CYP2E1) which was one of phase Iota detoxification enzymes. A total of 24 specific pathogen free (SPF) male BALB/c mice were randomly divided into four groups, including control group, MC-LR group, low concentration EGCG group, and high concentration EGCG group. Mice were sacrificed on the 15th day, body weight, and the relative organ weight, liver antioxidant enzyme level and lipid peroxidation product, liver histopathology and CYP2E1 gene and protein expression were detected and analyzed respectively. (1) EGCG could antagonise the liver injury which had been damaged by MC-LR. (2) The malonaldehyde (MDA) level ((2.87 +/- 0.03) nmol/mg prot) and superoxide dismutase (SOD) level ((168.18 +/- 2.86) U/mg prot) in MC-LR group were significantly different when compared with the two EGCG treatment groups (the MDA values of the low and high concentration EGCG group were (2.37 +/- 0.05) nmol/mg prot and (1.44 +/- 0.05) nmol/mg prot, F = 906.63, P < 0.01; the SOD values were (176.55 +/- 2.98) U/mg prot and (184.89 +/- 1.53) U/mg prot, F = 32.32, P < 0.01). (3) MC-LR up-regulated the mRNA and protein expression of CYP2E1 (the mRNA values of MC-LR group and control were 1.41 +/- 0.26, 0.86 +/- 0.13, t = -4.22, P = 0.003; the protein values of MC-LR group and control were 0.24 +/- 0.03, 0.12 +/- 0.02, t = -9.21, P < 0.05). EGCG down-regulated the mRNA (the values of the low and high concentration EGCG group were 1.09 +/- 0.08, 0.99 +/- 0.09, F = 9.03, P = 0.004) and protein expression (the values of the low and high concentration EGCG group were 0.21 +/- 0.03, 0.14 +/- 0.02, F = 24.76, P < 0.05) of CYP2E1 which activated by MC-LR. The up-regulation of CYP2E1 which induced by MC-LR was inhibited by EGCG intervention. EGCG might antagonize the oxidation damage of hepatocytes in a certain degree.

Does CYP2E1 play a major role in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes?

Isoniazid and rifampicin are first-line anti-tubercular drugs. In a recent paper, Shen et al. provided interesting findings that rifampicin exacerbated isoniazid toxicity in human hepatocytes but not in rat hepatocytes. The main conclusion was that the difference in cytochrome P450 2E1 (CYP2E1) induction by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatotoxicity by rifampicin. 4-Nitrophenol hydroxylase (4-NP) activity was the only probe of CYP2E1 activity used in the paper. The authors presented data showing that rifampicin enhanced 4-NP activity and CYP2E1 mRNA expression in human hepatocytes, but not in rat hepatocytes. However, CYP3A also makes a significant contribution to 4-NP activity in humans and rats, which has been confirmed by both CYP3A-specific inducer and inhibitors. Rifampicin is a strong inducer of human CYP3A; thus, the increase in 4-NP activity in human hepatocytes could be due to the induction of CYP3A. Rifampicin did not increase 4-NP activities in rat hepatocytes, which could reflect a lack of the induction of rat CYP3A by rifampicin. Additionally, more experiments are needed to support the conclusion that rifampicin increased CYP2E1 mRNA expression in human hepatocytes because of the small sample size and the limitations of semi-quantitative RT-PCR. The study by Shen et al. suggests that another drug-metabolizing enzyme rather than CYP2E1 could be involved in the aggravation of isoniazid toxicity by rifampicin in human hepatocytes.

Hepatotoxicity and gene expression down-regulation of CYP isozymes caused by renal ischemia/reperfusion in the rat

Renal ischemia/reperfusion (I/R) occurs in many clinical scenarios, including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the kidneys, culminating in local injury as well as distant organ dysfunction. The objectives of this study were to investigate the changes in the functions of the liver and the regulation of gene expression of cytochrome P450 (CYP) isozymes after renal I/R. Hepatoxocity was assessed by serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST) and liver glutathione-S-transferase (GST) activities, liver glutathione (GSH) level, and histopathological examination. Hepatic cytochrome P4503A1 (CYP3A1) and cytochrome P4502E1 (CYP2E1) activities were measured by erythromycin N-demethylase (ERD) and aniline hydroxylase (ANH) activities, respectively. CYP3A1 and CYP2E1 mRNA expression was determined by RT-PCR. Results showed that activities of sALT and sAST were significantly increased, while hepatic CYP3A1and CYP2E1 activities as well as their respective mRNA levels were significantly decreased after renal I/R. Moreover, hepatic tissue congestion, degeneration, and local necrosis were observed in rats after 1, 4, and 8 h renal reperfusion following 2 h renal ischemia. In conclusion, the present study suggests that renal I/R can cause hepatotoxicity and gene expression down-regulation of CYP isozymes in rats.

Rifampicin exacerbates isoniazid‐induced toxicity in human but not in rat hepatocytes in tissue‐like cultures

Rifampicin has been extensively reported to exacerbate the hepatotoxicity of isoniazid in patients with tuberculosis. However, this was controversially claimed by previous reports using rat models. This study evaluated the effect of rifampicin on isoniazid-induced hepatocyte toxicity by using human and rat hepatocytes in tissue-like culture. Hepatocytes in tissue-like gel entrapment were used to examine isoniazid toxicity, as shown by cell viability, intracellular glutathione content and albumin secretion. For demonstration of the differential effects of rifampicin on human and rat hepatocytes, induction by rifampicin of cytochrome P450 (CYP) 2E1, a major enzyme associated with isoniazid hepatotoxicity, was detected by 4-nitrocatechol formation and RT-PCR analysis. Rifampicin (12 microM) enhanced isoniazid-induced toxicity in human hepatocytes but not in rat hepatocytes. Enhanced CYP 2E1 enzymic activity and mRNA expression were similarly detected in human hepatocytes but not in rat hepatocytes. Both rat and human hepatocytes in gel entrapment were more sensitive to isoniazid treatment compared with the corresponding hepatocytes in a monolayer culture. The difference in induction of CYP 2E1 by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatocyte toxicity by rifampicin, with more significant toxicity in gel entrapment than in monolayer cultures. Thus, human hepatocytes in tissue-like cultures (gel entrapment) could be an effective model for hepatotoxicity research in vitro, closer to the in vivo situation.