Cytochrome P450 2E1 Enzyme Activity Research Articles

Synergistic Protective Effect of Fermented Schizandrae Fructus Pomace and Hoveniae Semen cum Fructus Extracts Mixture in the Ethanol-Induced Hepatotoxicity.

Schizandrae Fructus (SF), fruits of Schisandra chinensis (Turcz.) Baill. and Hoveniae Semen cum Fructus (HSCF), the dried peduncle of Hovenia dulcis Thunb., have long been used for alcohol detoxification in the traditional medicine of Korea and China. In the current study, we aimed to evaluate the potential synergistic hepatoprotective effect of a combination mixture (MSH) comprising fermented SF pomace (fSFP) and HSCF hot water extracts at a 1:1 (w:w) ratio against ethanol-induced liver toxicity. Subacute ethanol-mediated hepatotoxicity was induced by the oral administration of ethanol (5 g/kg) in C57BL/6J mice once daily for 14 consecutive days. One hour after each ethanol administration, MSH (50, 100, and 200 mg/kg) was also orally administered daily. MSH administration significantly reduced the serum activities of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase. Histological observation indicated that MSH administration synergistically and significantly decreased the fatty changed region of hepatic parenchyma and the formation of lipid droplet in hepatocytes. Moreover, MSH significantly attenuated the hepatic triglyceride accumulation through reducing lipogenesis genes expression and increasing fatty acid oxidation genes expression. In addition, MSH significantly inhibited protein nitrosylation and lipid peroxidation by lowering cytochrome P450 2E1 enzyme activity and restoring the glutathione level, superoxide dismutase and catalase activity in liver. Furthermore, MSH synergistically decreased the mRNA level of tumor necrosis factor-α in the hepatic tissue. These findings indicate that MSH has potential for preventing alcoholic liver disease through inhibiting hepatic steatosis, oxidative stress, and inflammation.

Indole-3-carbinol ameliorated the thioacetamide-induced hepatic encephalopathy in rats

Indole-3-carbinol (I3C) is reported to have hepatic and neuroprotective properties. However, the I3C role in the protection of the liver and brain in the pathological condition of hepatic encephalopathy has not been investigated. Therefore, in the present study, we have assessed the hepatic and neuroprotective roles of I3C against thioacetamide (TAA)- induced hepatic encephalopathy in Wistar rats. TAA (300 mg/kg) was intraperitoneally administered to Wistar rats to induce hepatic encephalopathy. The elevated levels of ammonia in the blood, liver, and brain were substantially lowered by I3C treatment (25, 50, and 100 mg/kg, oral, 7 days). I3C significantly ameliorated the TAA-induced liver dysfunction by decreasing the alanine transaminase, aspartate transaminase, and alkaline phosphatase enzymes and reduced the elevated cytochrome P4502E1 (CYP2E1) activity in the liver and brain. Further, I3C alleviated mitochondrial dysfunction and oxidative stress in the brain. I3C treatment improved the anti-inflammatory cytokine interleukin (IL)− 10 while reducing inflammatory cytokines such as tumor necrosis factor-1 and IL-6 in hepatic encephalopathy rats. I3C reduced the levels of apoptotic indicators mediated by the mitochondria, including cytochrome c, caspase 9, and caspase 3. Concurrently, I3C mitigated the liver and brain histological abnormalities in hepatic encephalopathy rats. Therefore, the present study concluded that the I3C protected the liver and brain from TAA-induced hepatic encephalopathy injury by inhibiting CYP2E1 enzyme activity and decreasing ammonia, oxidative stress, inflammation, and apoptosis. The present study provides preclinical validation of I3C use as hepatic and neuroprotective for hepatic encephalopathy management.

Bortezomib alleviates drug-induced liver injury by regulating CYP2E1 gene transcription.

Acute liver failure, i.e., the fatal deterioration of liver function, is the most common indication that emergency liver transplantation is necessary. Moreover, in the USA, drug-induced liver injury (DILI), including acetaminophen (APAP)-induced hepatotoxicity, is the main cause of acute liver failure. Matching a donor for liver transplantation is extremely difficult, and thus the development of a novel therapy for DILI is urgently needed. Following recent approval by the FDA of the proteasomal inhibitor bortezomib, its therapeutic effects on various human diseases, including solid and hematologic malignancies, have been validated. However, the specific action of proteasomal inhibition in cases of DILI had not been elucidated prior to this study. To examine the effects of proteasomal inhibition in DILI experimentally, male C56Bl/6 mice were injected with 1 mg bortezomib/kg before APAP treatment. Bortezomib not only alleviated APAP-induced hepatotoxicity in a time- and dose-dependent manner, it also alleviated CCl4- and thioacetamide-induced hepatotoxicity. We also noted that bortezomib significantly reduced cytochrome P450 2E1 (CYP2E1) expression and activity in the liver, which was accompanied by the induction of endoplasmic reticulum (ER) stress. In addition, bortezomib decreased hepatocyte nuclear factor-1α-induced promoter activation of CYP2E1 in Hep3B cells. By contrast, another proteasome inhibitor, MG132, did not cause ER stress and did not markedly affect CYP2E1 enzyme activity. Liver injury induced by APAP was aggravated by MG132, possibly via elevation of connexin 32 expression. This study suggests that proteasome inhibition has different effects in cases of DILI depending on the specific inhibitor being used. Furthermore, results from the mouse model indicated that bortezomib, but not MG132, was effective in alleviating DILI. ER stress induced by proteasome inhibition has previously been shown to exert various effects on DILI patients, and thus each available proteasomal inhibitor should be evaluated individually in order to determine its potential for clinical application.

Hepatic Lipid Peroxidation and Cytochrome P-450 2E1 in Pediatric Nonalcoholic Fatty Liver Disease and Its Subtypes

To compare hepatic lipid peroxidation and cytochrome P-450 2E1 (CYP2E1) protein content in liver biopsies from children with nonalcoholic fatty liver disease (NAFLD) and 2 control groups. Elevated hepatic lipid peroxidation resulting from increased hepatic CYP2E1 enzyme activity is involved in the pathogenesis of NAFLD and nonalcoholic steatohepatitis (NASH) in adults, but studies in children are lacking. Liver biopsies from 59 children with NAFLD (49 with NASH), 10 children with normal liver histology, and 9 children with mild chronic hepatitis C (HCV) infection were examined. Hepatic malondialdehyde (a measure of lipid peroxidation) levels and CYP2E1 protein content were quantitated, as a percentage of the total area, by immunohistochemical staining of liver biopsy material followed by digital image quantitation. Lipid peroxidation was significantly greater in NAFLD liver biopsies (46.7 ± 20.8%) compared with biopsies from children with normal liver histology (7.6 ± 9.4%; P<0.001) or HCV infection (7.7 ± 7.6%; P<0.001). However, hepatic CYP2E1 expression was not different across the NAFLD, normal liver histology, and HCV groups (60.7 ± 8.7%, 53.5 ± 10.7%, and 60.0 ± 11.9%, respectively; P=0.116). Among children with NAFLD, lipid peroxidation and CYP2E1 protein content did not differ between biopsies with and without NASH. Body mass index was independently associated with hepatic lipid peroxidation levels (r=0.549; P<0.001). Hepatic lipid peroxidation is increased in children with NAFLD but this is not related to hepatic CYP2E1 expression. No difference in lipid peroxidation in pediatric NAFLD versus NASH argues against a role in disease progression.

Localization of Cytochrome P4502E1 Enzyme in Normal and Cancerous Gastric Mucosa and Association with Its Genetic Polymorphism in Unoperated and Remnant Stomach

Exposure to nitroso compounds and the activity of cytochrome P450 2E1 (CYP2E1), an activation enzyme for these carcinogens, are important factors in gastric carcinogenesis. Here, we investigated the potential correlation between genetic variation in CYP2E1 and its enzyme expression as detected with immunohistochemical (IHC) staining and cancer susceptibility in unoperated and remnant stomach. Expression of CYP2E1 in the stomach (n=117) was detected with IHC staining using a polyclonal anti-CYP2E1 antibody. Interindividual variation in CYP2E1 enzyme activity was then compared with genetic polymorphisms in the transcriptional flanking region of the CYP2E1 gene by restriction fragment length polymorphism (RFLP) detection using the Rsa I restriction enzyme. Genetic polymorphisms of Rsa I RFLP in CYP2E1 were investigated in 499 patients with gastric cancer (466 unoperated stomachs and 33 remnant stomachs) and 553 control patients with benign gastroduodenal diseases. Mucosal IHC staining for CYP2E1 was stronger in areas of intestinal metaplasia, particularly in endocrine cells, which stained consistently and strongly. Expression of CYP2E1 enzyme in areas of IHC staining were confirmed with Western blot analysis and showed a significant association between the degree of staining and the CYP2E1 genotype (p<0.01) in cancer tissues and in the foveolar epithelium of normal gastric mucosa. No association between specific CYP2E1 genotype and gastric cancer risk in the unoperated stomach was found in either the large study or the age- and gender-matched case-control study. However, the frequency of rare alleles (C1/C2 or C2/C2) was significantly higher in patients with cancer in the remnant stomach following gastrectomy than in controls subjects without cancer (odds ratio=2.8, 95% confidence interval=1.3-5.8) or those with primary gastric cancer (odds ratio=2.6, 95% confidence interval=1.3-5.5). CYP2E1 genetic polymorphisms might correlate with CYP2E1 enzyme expression levels in normal and cancerous gastric tissues. These polymorphisms do not influence the development of primary stomach cancer but may do so in specific conditions, such as the remnant stomach after gastrectomy.

Genetic Polymorphism in CYP2E1: Population Distribution of CYP2E1 Activity

Cytochrome P-450 2E1 (CYP2E1) is a key enzyme in the metabolic activation of a variety of toxicants including nitrosamines, benzene, vinyl chloride, and halogenated solvents such as trichloroethylene. CYP2E1 is also one of the enzymes that metabolizes ethanol to acetaldehyde, and is induced by recent ethanol ingestion. There is evidence that interindividual variability in the expression and functional activity of this cytochrome (CYP) may be considerable. Genetic polymorphisms in CYP2E1 were identified and linked to altered susceptibility to hepatic cirrhosis induced by ethanol and esophageal and other cancers in some epidemiological studies. Therefore, it is important to evaluate how such polymorphisms affect CYP2E1 function and whether it is possible to construct a population distribution of CYP2E1 activity based upon the known effects of these polymorphisms and their frequency in the population. This analysis is part of the genetic polymorphism database project described in the lead article in this series and followed the approach described in that article (Ginsberg et al., 2009, this issue). Review of the literature found that there are a variety of CYP2E1 variant alleles but the functional significance of these variants is still unclear. Some, but not all, studies suggest that several upstream 5′ flanking mutations affect gene expression and response to inducers such as ethanol or obesity. None of the coding-region variants consistently affects enzyme function. Part of the reason for conflicting evidence regarding genotype effect on phenotype may be due to the wide variety of exposures such as ethanol or dietary factors and physiological factors including body weight or diabetes that modulate CYP2E1 expression. In conclusion, evidence is too limited to support the development of a population distribution of CYP2E1 enzyme activity based upon genotypes. Health risk assessments may best rely upon data reporting interindividual variability in CYP2E1 function for input into physiologically based pharmacokinetic (PBPK) models involving CYP2E1 substrates.

Human hepatocytes are protected from ethanol-induced cytotoxicity by DADS via CYP2E1 inhibition

We investigated the protective effects of diallyl disulfide (DADS), a potent inhibitor of cytochrome P450 2E1 (CYP2E1), on ethanol-induced toxicity in human hepatocytes. We found a clear dose-dependent response between ethanol and CYP2E1 activity. The ethanol-dependent CYP2E1 enzyme activity and protein expression, lactate dehydrogenase and aspartate transaminase release, malondialdehyde formation and caspase-3 activity decreased dramatically in the presence of DADS. Furthermore, DADS increased the hepatocellular glutathione (GSH) content and prevented the ethanol-dependent cellular GSH depletion. Our data show that DADS reduces ethanol-induced toxicity in human hepatocytes by reducing CYP2E1 activity and/or stabilizing the cellular GSH content, which might be of therapeutic interest.

Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage

Hepatic metabolism of ethanol by cytochrome P450 2E1 (CYP2E1) is believed to contribute to alcoholic liver damage. To further evaluate CYP2E1 in alcoholic liver disease, we created a transgenic mouse containing human CYP2E1 complementary DNA (cDNA) under the control of mouse albumin enhancer-promoter. Two experiments were performed. In the first experiment, transgenic and nontransgenic mice were fed normal chow. In the second experiment, transgenic and nontransgenic mice were pair fed a nutritionally complete liquid diet for 16 weeks. The liquid diet contained 30% of calories as ethanol (or dextrose) and 25% of calories as corn oil. Liver damage was assessed by measuring serum alanine aminotransferase (ALT) levels and examining liver histology. Transgenic animals reproduced and were phenotypically normal. Hepatic levels of CYP2E1 messenger RNA (mRNA), protein, and enzyme activity did not differ between chow-fed transgenic and nontransgenic mice. Livers from transgenic mice fed the alcohol diet contained significantly more CYP2E1 protein and enzyme activity than livers from nontransgenic mice fed the same diet. Transgenic mice receiving the alcohol diet had significantly higher serum ALT levels than nontransgenic mice. Histologic examination of the livers showed higher histologic scores in transgenic mice fed ethanol compared with nontransgenic mice fed ethanol. Ballooning hepatocytes were seen in livers from transgenic mice fed ethanol. Apoptosis, as determined by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, did not differ between groups. In conclusion, we have produced a transgenic mouse that expresses human CYP2E1 in the liver. When fed a nutritionally complete alcohol diet, transgenic mice develop more liver damage than nontransgenic mice. (H EPATOLOGY 2002;36:122-134.)

Establishment of a human hepatoma cell line, HLE/2E1, suitable for detection of p450 2E1-related cytotoxicity.

By transfection of an expression vector of human cytochrome P450 2E1 (CYP2E1) into a human hepatoma cell line (HLE), a new cell line (HLE/2E1) that stably expresses activity of CYP2E1 has been established. The HLE/2E1 cell line expressed a higher level of CYP2E1 messenger ribonucleic acid than did the mother HLE cell line. CYP2E1 enzyme activity determined by a p-nitrophenol oxidation assay was also higher in HLE/2E1 cells than in HLE cells. In addition, the enzyme activity of the HLE/2E1 cells was increased by ethanol treatment. Exposure to acetaminophen (APAP) or buthionine sulfoximine (BSO) caused a greater decrease in viability of the HLE/2E1 cells than that of the HLE cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The cytotoxicity of APAP or BSO to HLE/2EI cells was inhibited by the addition of ethanol or vitamin E. However, the cytotoxicity of both APAP and BSO was enhanced by 24-h preincubation of HLE/2E1 cells with ethanol. These results show that this cell line provides a useful model for studying catalytic properties of CYP2E1 and cytotoxic mechanisms of chemicals metabolized by CYP2E1.

Zonated expression of cytokines in rat liver: effect of chronic ethanol and the cytochrome P450 2E1 inhibitor, chlormethiazole.

The release of proinflammatory cytokines by endotoxins and during oxidative stress is considered to be an early key step in the pathogenesis of alcoholic liver disease (ALD). Ethanol-inducible cytochrome P450 2E1 (CYP2E1) has potentially pro-oxidative and toxicological properties, and its expression is restricted to the perivenous region of liver. We investigated zonal differences of cytokine expression in rat liver and how these are affected by alcohol exposure and by chlormethiazole (CMZ), a transcriptional and posttranslational inhibitor of hepatic CYP2E1. Periportal and perivenous cell lysates were obtained by the digitonin pulse technique from livers of rats treated with ethanol and CMZ for 38 days. Cytokine expression on the mRNA and protein levels was quantified using competitive polymerase chain reaction (PCR) and Western blot, respectively. Chronic ethanol treatment significantly increased the expression of CYP2E1, microsomal p-nitrophenol hydroxylase activity (indicative for CYP2E1 enzyme activity), and the expression of transforming growth factor beta1 (TGF-beta1), tumor necrosis factor alpha (TNF-alpha), and interleukin (IL)-1beta (1.4- to 4.6-fold). In contrast, ethanol caused a decrease in IL-4 expression and had no influence on IL-6 expression. CMZ treatment caused a reduction in hepatic CYP2E1 expression and in the ethanol-induced cytokine expression by 40% to 60%. Expression of IL-6, IL-2, and IL-4 mRNA occurred preferentially in the periportal region, whereas ethanol caused a pronounced increase in the perivenous expression of TGF-beta1, which was inhibited by CMZ as monitored both on the mRNA and protein levels. These results show the zonated expression of several cytokines and the counteraction of CMZ on all effects of ethanol on cytokine expression. The data further strengthen a link between increased CYP2E1 expression and enhanced cytokine expression as important events in the development of ALD.

The acetaminophen regioisomer 3′-hydroxyacetanilide inhibits and covalently binds to cytochrome P450 2E1

3′-Hydroxyacetanilide has been previously studied as a nontoxic regioisomer of the analgesic acetaminophen (4′-hydroxyacetanilide). The radiolabeled derivative has been shown to covalently bind to liver proteins at levels similar to that observed with hepatotoxic doses of radiolabeled acetaminophen with no evidence of hepatic damage. Using an anti-arylacetamide antiserum the primary protein adduct detected following administration of 3′-hydroxyacetanilide (300 and 600 mg/kg) to mice was a 50 kDa microsomal protein that co-migrated with cytochrome P450 2E1. Cytochrome P450 2E1 enzyme activity ( p-nitrophenol hydroxylase) was decreased by 79% in the mice treated with 3'-hydroxyacetanilide (600 mg/kg). Incubation of 3′-hydroxyacetanilide with hepatic microsomes resulted in a time dependent 47% decrease in cytochrome P450 2E1 activity. Pre-incubation of acetaminophen with microsomes did not result in covalent binding to the cytochrome P450 nor was there a decrease in p-nitrophenol hydroxylase activity. These data suggest that 3′-hydroxyacetanilide covalently binds to cytochrome P450 2E1 with preferential loss of activity.

Covalent binding and inhibition of cytochrome P4502E1 by trichloroethylene

1. To investigate the effects of trichloroethylene on cytochrome P4502E1 (CYP2E1), an isozyme responsible for its metabolic activation, mice were treated with trichloroethylene and Western blot staining with both anti-dichloroacetyl and anti-CYP2E1 antisera detected acomigrating 50 kDaprotein band. There was adose-dependent increase in the intensity of the 50 kDa protein adduct stained immunochemically with antidichloroacetyl. 2. CYP2E1 enzyme activity was decreased from control levels in a dose-dependent manner in mice treated with 250-500 mg kg TRI. 3. Microsomal incubations with trichloroethylene resulted in covalent binding to several proteins including a 50 kDa adduct, which is in contrast with the selective binding to the 50 kDa protein observed in vivo. 4. CYP2E1 enzyme activity levels were significantly decreased following microsomal incubation with NADPH and trichloroethylene, and additionally there was a time- and NADPH- dependent decrease in enzyme activity indicating that trichloroethylene is a mechanism- based inhibitor of CYP2E1.

Transcriptional Inhibition of Cytochrome P4502E1 by a Synthetic Compound, YH439

The molecular mechanism of cytochrome P4502E1 (CYP2E1) inhibition by a synthetic compound, YH439, was studied. In rats treated with YH439,N-nitrosodimethylamine demethylase activity and the amount of immunoreactive CYP2E1 were rapidly decreased in time- and dose-dependent manners. Within 2 h after a single dose of YH439 (150 mg/kg), the CYP2E1-catalyzed activity in uninduced rats was decreased by about 30% and by 43% at 24 h after YH439 injection. YH439 treatment also reduced the elevation of CYP2E1 enzyme activity in starved (induced) animals by 34%. More profound inhibition of CYP2E1 protein levels was observed by immunoblot analysis. The level of CYP2E1 catalytic activity and immunoreactive protein remained suppressed for at least 48 h and returned to normal level at 72 h after YH439 treatment. The levels of immunoreactive CYP2B1/2 protein and catalytic activity were moderately increased while little change was observed in the levels of NADPH-dependent P450 oxidoreductase activity and its protein after treatment with YH439. Unlike competitive inhibitors of CYP2E1, YH439 rapidly (within 2 h) decreased the level of CYP2E1 mRNA, while malotilate, a structural analog of YH439, slightly suppressed its level. Nuclear run-on transcription analyses at 2, 4, and 8 h post-YH439 administration revealed that the inhibition of CYP2E1 by YH439 is at the level of transcription, indicating that YH439 is a new class of CYP2E1 inhibitor. Our data demonstrate that YH439 is a powerful inhibitor of CYP2E1 expression and is thus potentially useful as a pharmacological tool to study CYP2E1 function as well as a potential therapeutic agent.

Ethnic variation in the CYP2E1 gene: polymorphism analysis of 695 African-Americans, European-Americans and Taiwanese.

Human cytochrome P4502E1 (CYP2E1) is inducible by ethanol and is involved in metabolism of many known carcinogens including N-nitrosodimethylamine, butadiene, benzene, and carbon tetrachloride. A 50-fold variability in CYP2E1 enzyme activity in humans has been observed but it is unknown whether the basis for this variation is genetic or environmental. Recently, two restriction fragment length polymorphisms (RFLPs) within the CYP2E1 gene have been suggested as genetic markers of risk for cancer. The first was a Rsa I polymorphism in the 5' regulatory region that appeared to alter transcriptional activation of the gene and the second was a Dra I polymorphism located approximately 7000 bp downstream in an intron. Rare alleles at each of these loci have been associated with a reduced risk for lung cancer in Japanese and Swedish populations. We have used a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to determine the genotype frequency for each of these CYP2E1 RFLPs in 695 individuals of Taiwanese, African-American or European-American background. Genotype and allele frequencies for Taiwanese were significantly different from those of African-Americans and European-Americans at either Rsa I or Dra I sites (p < 0.0001). Allele frequencies for African-Americans and European-Americans were significantly different at the Rsa I site (p = 0.03). The rare alleles (c2 and C) occurred at frequencies of 0.28 and 0.24 in Taiwanese, 0.01 and 0.08 in African-Americans, and 0.04 and 0.11 in European-Americans. In addition, we describe three haplotypes common to all three population samples and a fourth haplotype that was only detected in the Taiwanese population sample. This fourth haplotype may have been caused by a recombination event between these markers.(ABSTRACT TRUNCATED AT 250 WORDS)