Ethanol-inducible Cytochrome P450 2E1 Research Articles

Contributing Roles of CYP2E1 and Other Cytochrome P450 Isoforms in Alcohol-Related Tissue Injury and Carcinogenesis.

The purpose of this review is to briefly summarize the roles of alcohol (ethanol) and related compounds in promoting cancer and inflammatory injuryin many tissues. Long-term chronic heavy alcohol exposure is known to increase the chances of inflammation, oxidative DNA damage,and cancer development in many organs. The rates of alcohol-mediated organ damage and cancer risks are significantly elevated in the presence of co-morbidity factors such as poor nutrition, unhealthy diets, smoking, infection with bacteria orviruses, and exposure to pro-carcinogens. Chronic ingestion of alcohol and its metabolite acetaldehyde may initiate and/or promote the development of cancer in the liver, oral cavity, esophagus, stomach, gastrointestinal tract, pancreas, prostate, and female breast. In this chapter, we summarize the important roles of ethanol/acetaldehyde in promoting inflammatory injuryand carcinogenesis in several tissues. We also review the updated roles of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) and other cytochrome P450 isozymes in the metabolism of various potentially toxic substrates, and consequent toxicities, including carcinogenesis in different tissues. We also briefly describe the potential implications of endogenous ethanol produced by gut bacteria, as frequently observed in the experimental models and patients of nonalcoholic fatty liver disease, in promoting DNA mutation and cancer development in the liver and other tissues, including the gastrointestinal tract.

Cytochrome P450-2E1 promotes aging-related hepatic steatosis, apoptosis and fibrosis through increased nitroxidative stress

The role of ethanol-inducible cytochrome P450-2E1 (CYP2E1) in promoting aging-dependent hepatic disease is unknown and thus was investigated in this study. Young (7 weeks) and aged female (16 months old) wild-type (WT) and Cyp2e1-null mice were used in this study to evaluate age-dependent changes in liver histology, steatosis, apoptosis, fibrosis and many nitroxidative stress parameters. Liver histology showed that aged WT mice exhibited markedly elevated hepatocyte vacuolation, ballooning degeneration, and inflammatory cell infiltration compared to all other groups. These changes were accompanied with significantly higher hepatic triglyceride and serum cholesterol in aged WT mice although serum ALT and insulin resistance were not significantly altered. Aged WT mice showed the highest rates of hepatocyte apoptosis and hepatic fibrosis. Further, the highest levels of hepatic hydrogen peroxide, lipid peroxidation, protein carbonylation, nitration, and oxidative DNA damage were observed in aged WT mice. These increases in the aged WT mice were accompanied by increased levels of mitochondrial nitroxidative stress and alteration of mitochondrial complex III and IV proteins in aged WT mice, although hepatic ATP levels seems to be unchanged. In contrast, the aging-related nitroxidative changes were very low in aged Cyp2e1-null mice. These results suggest that CYP2E1 is important in causing aging-dependent hepatic steatosis, apoptosis and fibrosis possibly through increasing nitroxidative stress and that CYP2E1 could be a potential target for translational research in preventing aging-related liver disease.

Translational Implications of the Alcohol-Metabolizing Enzymes, Including Cytochrome P450-2E1, in Alcoholic and Nonalcoholic Liver Disease.

Fat accumulation (hepatic steatosis) in alcoholic and nonalcoholic fatty liver disease is a potentially pathologic condition which can progress to steatohepatitis (inflammation), fibrosis, cirrhosis, and carcinogenesis. Many clinically used drugs or some alternative medicine compounds are also known to cause drug-induced liver injury, which can further lead to fulminant liver failure and acute deaths in extreme cases. During liver disease process, certain cytochromes P450 such as the ethanol-inducible cytochrome P450-2E1 (CYP2E1) and CYP4A isozymes can be induced and/or activated by alcohol and/or high-fat diets and pathophysiological conditions such as fasting, obesity, and diabetes. Activation of these P450 isozymes, involved in the metabolism of ethanol, fatty acids, and various drugs, can produce reactive oxygen/nitrogen species directly and/or indirectly, contributing to oxidative modifications of DNA/RNA, proteins and lipids. In addition, aldehyde dehydrogenases including the mitochondrial low Km aldehyde dehydrogenase-2 (ALDH2), responsible for the metabolism of acetaldehyde and lipid aldehydes, can be inactivated by various hepatotoxic agents. These highly reactive acetaldehyde and lipid peroxides, accumulated due to ALDH2 suppression, can interact with cellular macromolecules DNA/RNA, lipids, and proteins, leading to suppression of their normal function, contributing to DNA mutations, endoplasmic reticulum stress, mitochondrial dysfunction, steatosis, and cell death. In this chapter, we specifically review the roles of the alcohol-metabolizing enzymes including the alcohol dehydrogenase, ALDH2, CYP2E1, and other enzymes in promoting liver disease. We also discuss translational research opportunities with natural and/or synthetic antioxidants, which can prevent or delay the onset of inflammation and liver disease.

Binge alcohol promotes hypoxic liver injury through a CYP2E1–HIF-1α-dependent apoptosis pathway in mice and humans

Binge drinking, a common pattern of alcohol ingestion, is known to potentiate liver injury caused by chronic alcohol abuse. This study was aimed at investigating the effects of acute binge alcohol on hypoxia-inducible factor-1α (HIF-1α)-mediated liver injury and the roles of alcohol-metabolizing enzymes in alcohol-induced hypoxia and hepatotoxicity. Mice and human specimens assigned to binge or nonbinge groups were analyzed for blood alcohol concentration (BAC), alcohol-metabolizing enzymes, HIF-1α-related protein nitration, and apoptosis. Binge alcohol promoted acute liver injury in mice with elevated levels of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and hypoxia, both of which were colocalized in the centrilobular areas. We observed positive correlations among elevated BAC, CYP2E1, and HIF-1α in mice and humans exposed to binge alcohol. The CYP2E1 protein levels (r = 0.629, p = 0.001) and activity (r = 0.641, p = 0.001) showed a significantly positive correlation with BAC in human livers. HIF-1α levels were also positively correlated with BAC (r = 0.745, p < 0.001) or CYP2E1 activity (r = 0.792, p < 0.001) in humans. Binge alcohol promoted protein nitration and apoptosis with significant correlations observed between inducible nitric oxide synthase and BAC, CYP2E1, or HIF-1α in human specimens. Binge-alcohol-induced HIF-1α activation and subsequent protein nitration or apoptosis seen in wild type were significantly alleviated in the corresponding Cyp2e1-null mice, whereas pretreatment with an HIF-1α inhibitor, PX-478, prevented HIF-1α elevation with a trend of decreased levels of 3-nitrotyrosine and apoptosis, supporting the roles of CYP2E1 and HIF-1α in binge-alcohol-mediated protein nitration and hepatotoxicity. Thus binge alcohol promotes acute liver injury in mice and humans at least partly through a CYP2E1–HIF-1α-dependent apoptosis pathway.

Mitochondrial dysfunction and tissue injury by alcohol, high fat, nonalcoholic substances and pathological conditions through post-translational protein modifications

Mitochondria are critically important in providing cellular energy ATP as well as their involvement in anti-oxidant defense, fat oxidation, intermediary metabolism and cell death processes. It is well-established that mitochondrial functions are suppressed when living cells or organisms are exposed to potentially toxic agents including alcohol, high fat diets, smoking and certain drugs or in many pathophysiological states through increased levels of oxidative/nitrative stress. Under elevated nitroxidative stress, cellular macromolecules proteins, DNA, and lipids can undergo different oxidative modifications, leading to disruption of their normal, sometimes critical, physiological functions. Recent reports also indicated that many mitochondrial proteins are modified via various post-translation modifications (PTMs) and primarily inactivated. Because of the recently-emerging information, in this review, we specifically focus on the mechanisms and roles of five major PTMs (namely oxidation, nitration, phosphorylation, acetylation, and adduct formation with lipid-peroxides, reactive metabolites, or advanced glycation end products) in experimental models of alcoholic and nonalcoholic fatty liver disease as well as acute hepatic injury caused by toxic compounds. We also highlight the role of the ethanol-inducible cytochrome P450-2E1 (CYP2E1) in some of these PTM changes. Finally, we discuss translational research opportunities with natural and/or synthetic anti-oxidants, which can prevent or delay the onset of mitochondrial dysfunction, fat accumulation and tissue injury.

CYP2E1 potentiates binge alcohol-induced gut leakiness, steatohepatitis, and apoptosis

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) contributes to increased oxidative stress and steatosis in chronic alcohol-exposure models. However, its role in binge ethanol-induced gut leakiness and hepatic injury is unclear. This study was aimed at investigating the role of CYP2E1 in binge alcohol-induced gut leakiness and the mechanisms of steatohepatitis. Female wild-type (WT) and Cyp2e1-null mice were treated with three doses of binge ethanol (WT-EtOH or Cyp2e1-null–EtOH) (6g/kg oral gavage at 12-h intervals) or dextrose (negative control). Intestinal histology of only WT-EtOH exhibited epithelial alteration and blebbing of lamina propria, and liver histology obtained at 6h after the last ethanol dose showed elevated steatosis with scattered inflammatory foci. These were accompanied by increased levels of serum endotoxin, hepatic enterobacteria, and triglycerides. All these changes, including the intestinal histology and hepatic apoptosis, determined by TUNEL assay, were significantly reversed when WT-EtOH mice were treated with the specific inhibitor of CYP2E1 chlormethiazole and the antioxidant N-acetylcysteine, both of which suppressed oxidative markers including intestinal CYP2E1. WT-EtOH also exhibited elevated amounts of serum TNF-α, hepatic cytokines, CYP2E1, and lipid peroxidation, with decreased levels of mitochondrial superoxide dismutase and suppressed aldehyde dehydrogenase 2 activity. Increased hepatocyte apoptosis with elevated levels of proapoptotic proteins and decreased levels of active (phosphorylated) p-AKT, p-AMPK, and peroxisome proliferator-activated receptor-α, all of which are involved in fat metabolism and inflammation, were observed in WT-EtOH. These changes were significantly attenuated in the corresponding Cyp2e1-null–EtOH mice. These data indicate that both intestinal and hepatic CYP2E1 induced by binge alcohol seems critical in binge alcohol-mediated increased nitroxidative stress, gut leakage, and endotoxemia; altered fat metabolism; and inflammation contributing to hepatic apoptosis and steatohepatitis.

P.1.g.058 The influence of brain serotonergic system on the expression and activity of cytochrome P450 in the liver

The aldehyde dehydrogenase inhibitor disulfiram (DS) has been used to deter drinking in alcoholics, but it also precipitates pharmacokinetic interactions with coadministered drugs. From previous experiments conducted in vitro, it has been proposed that the ethanol-inducible cytochrome P450 2E1 (CYP2E1) is the major target for inhibition by DS, but the inference from reported drug interactions is that the drug inhibits multiple CYPs. The aim of the present study was to evaluate the inhibition of major constitutive CYPs in rat liver by DS. Thus, the effects of DS on activities mediated by CYPs 2A1/2, 2C11, 2E1, and 3A, which constitute ∼80% of total CYPs in male rat liver, were evaluated. It was found that CYP2E1-mediated aniline 4-hydroxylase activity was weakly inhibited by DS in vitro, but that preincubation of the drug with NADPH-supplemented microsomes to generate metabolites of DS enhanced the extent of inhibition somewhat. In contrast, constitutive testosterone hydroxylases were inhibited effectively at low concentrations of DS (20 μM decreased the activities of all hydroxylation pathways to 40–60% of control), and a preincubation step between DS and NADPH-fortified microsomes enhanced the inhibition of CYP2C11 and 3A2 activities. In vivo studies were undertaken in which a single dose of DS (100 mg/kg, i.p.) was administered to rats; 24 hr later, CYP2E1-mediated aniline 4-hydroxylase activity was decreased to about 50% of the activity in untreated control rats. CYP2E1 apoprotein and mRNA were also decreased to 38% of the respective control, and CYP3A apoprotein and CYP3A2 mRNA responded similarly. In contrast, CYP2C11 apoprotein was decreased to 66% of control after DS administration, and CYP2A1 expression was unchanged. These findings establish that multiple CYPs are targets for inhibition by DS and provide a basis for clinically significant drug interactions involving CYPs other than 2E1. In addition, the in vivo modulation of CYP function by DS administration is not restricted to enzyme inactivation and may also include down-regulatory effects mediated at a pretranslational level.

Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-induced non-alcoholic steatohepatitis

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) activity contributes to oxidative stress. However, CYP2E1 may have an important role in the pathogenesis of high-fat mediated non-alcoholic steatohepatitis (NASH). Thus, the role of CYP2E1 in high-fat mediated NASH development was evaluated. Male wild type (WT) and Cyp2e1-null mice were fed a low-fat diet (LFD, 10% energy-derived) or a high-fat diet (HFD, 60% energy-derived) for 10 weeks. Liver histology and tissue homogenates were examined for various parameters of oxidative stress and inflammation. Liver histology showed that only WT mice fed a HFD developed NASH despite the presence of increased steatosis in both WT and Cyp2e1-null mice fed HFD. Markers of oxidative stress such as elevated CYP2E1 activity and protein amounts, lipid peroxidation, protein carbonylation, nitration, and glycation with increased phospho-JNK were all markedly elevated only in the livers of HFD-fed WT mice. Furthermore, while the levels of inflammation markers osteopontin and F4/80 were higher in HFD-fed WT mice, TNFα and MCP-1 levels were lower compared to the corresponding LFD-fed WT. Finally, only HFD-fed WT mice exhibited increased insulin resistance and impaired glucose tolerance. These data suggest that CYP2E1 is critically important in NASH development by promoting oxidative/nitrosative stress, protein modifications, inflammation, and insulin resistance.

PPARα Expression Protects Male Mice from High Fat–Induced Nonalcoholic Fatty Liver1–3

Emerging evidence suggests that the lack of PPARα enhances hepatic steatosis and inflammation in Ppara-null mice when fed a high-fat diet (HFD). Thus, the aim of this study was to determine whether Ppara-null mice are more susceptible to nonalcoholic steatohepatitis (NASH) than their wild-type (WT) counterparts following short-term feeding with a HFD. Age-matched male WT and Ppara-null mice were randomly assigned to consume ad libitum a standard Lieber-DeCarli liquid diet (STD) (35% energy from fat) or a HFD (71% energy from fat) for 3 wk. Liver histology, plasma transaminase levels, and indicators of oxidative/nitrosative stress and inflammatory cytokines were evaluated in all groups. Levels of lobular inflammation and the NASH activity score were greater in HFD-exposed Ppara-null mice than in the other 3 groups. Biochemical analysis revealed elevated levels of ethanol-inducible cytochrome P450 2E1 and TNFα accompanied by increased levels of malondialdehyde as well as oxidized and nitrated proteins in Ppara-null mice. Elevated oxidative stress and inflammation were associated with activation of c-Jun-N-terminal kinase and p38 kinase, resulting in increased hepatocyte apoptosis in Ppara-null mice fed a HFD. These results, with increased steatosis, oxidative stress, and inflammation observed in Ppara-null mice fed a HFD, demonstrate that inhibition of PPARα functions may increase susceptibility to high fat-induced NASH.

Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes – Alcohol dehydrogenase and Cytochrome P450 2E1

Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes – alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and over-whelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis.

Mitochondria-targeted Cytochrome P450 2E1 Induces Oxidative Damage and Augments Alcohol-mediated Oxidative Stress

The ethanol-inducible cytochrome P450 2E1 (CYP2E1) is also induced under different pathological and physiological conditions. Studies including ours have shown that CYP2E1 is bimodally targeted to both the endoplasmic reticulum (microsomes) (mc CYP2E1) and mitochondria (mt CYP2E1). In this study we investigated the role of mtCYP2E1 in ethanol-mediated oxidative stress in stable cell lines expressing predominantly mt CYP2E1 or mc CYP2E1. The ER+ mutation (A2L, A9L), which increases the affinity of the nascent protein for binding to the signal recognition particle, preferentially targets CYP2E1 to the endoplasmic reticulum. The Mt+ (L17G) and Mt++ (I8R, L11R, L17R) mutant proteins, showing progressively lower affinity for signal recognition particle binding, were targeted to mitochondria at correspondingly higher levels. The rate of GSH depletion, used as a measure of oxidative stress, was higher in cells expressing Mt++ and Mt+ proteins as compared with cells expressing ER+ protein. In addition, the cellular level of F(2)-isoprostanes, a direct indicator of oxidative stress, was increased markedly in Mt++ cells after ethanol treatment. Notably, expression of Mt++ CYP2E1 protein in yeast cells caused more severe mitochondrial DNA damage and respiratory deficiency than the wild type or ER+ proteins as tested by the inability of cells to grow on glycerol or ethanol. Additionally, liver mitochondria from ethanol-fed rats containing high mt CYP2E1 showed higher levels of F(2)-isoprostane production. These results strongly suggest that mt CYP2E1 induces oxidative stress and augments alcohol-mediated cell/tissue injury.

Role of microsomal enzymes in development of alcoholic liver diseases

Chronic ethanol consumption results in the proliferation of the membranes of the smooth endoplasmic reticulum. Although these microsomal changes can be interpreted as adaptive alterations secondary to induction of the membranes after chronic ethanol ingestion, some injurious consequences may ensue. Accelerated ethanol metabolism results in enhanced production of acetaldehyde and exacerbation of its various toxic manifestations including enhanced lipid peroxidation. The latter may also be promoted more directly through enhanced free radical formation by the induced microsomes and cytochrome P4502E1 (CYP2E1). Ethanol-inducible CYP2E1 is of interest because of its ability to metabolize and activate many toxicologically important substrates including ethanol, CCl(4), acetaminophen, and N-nitrosodimethylamine, to more toxic products. Major interest in CYP2E1 reflects the ability of this enzyme to oxidize ethanol, to generate reactive products from ethanol oxidation (e.g. acetaldehyde and 1-hydroxyethyl radical), to activate various agents including CCl(4) and acetaminophen into reactive products, and to generate reactive oxygen species. There is considerable interest in the role of ethanol-induced oxidative stress and generation of reactive oxygen species in the mechanisms by which ethanol becomes hepatotoxic. To understand the basic effects and actions of CYP2E1, an approach has been established to utilize the cell lines that constitutively express human CYP2E1. This review article briefly describes a role of microsomal enzymes in the development of alcoholic liver injury as well as the usefulness of this cell line to further clarify the mechanisms of CYP2E1-related hepatotoxicity.

Detection of auto-antibodies against cytochrome P4502E1 (CYP2E1) in chronic hepatitis C

Chronic hepatitis C (CHC) is often associated with auto-immune reactions. In the light of the role of alcohol in promoting CHC progression, we have investigated the possible presence of auto-reactivity against the ethanol-inducible cytochrome P4502E1 (CYP2E1) in CHC patients with and without alcohol consumption. The IgG reactivity against recombinant human CYP2E1 was evaluated by solid-phase immunoassays in 102 CHC patients with different alcohol consumption and 59 HCV-free controls. Auto-antibodies against CYP2E1 were significantly (p<0.0001) increased in CHC patients as compared to controls. Anti-CYP2E1 IgG above the 97th percentile in the controls were evident in 41 (40%) CHC patients. Competition experiments revealed that CYP2E1 recognition was not due to the cross-reactivity with CYP2D6. The detection of anti-CYP2E1 IgG was unrelated to alcohol consumption and no difference in gender, age, aminotransferase levels and virus genotype was evident among the patients with or without anti-CYP2E1 auto-antibodies. However, anti-CYP2E1 auto-reactivity was significantly (p=0.025) associated with the severity of periportal/periseptal interface hepatitis. Moreover, confocal microscopy demonstrated that anti-CYP2E1 IgG associated with CHC recognized CYP2E1 exposed on the outer side of hepatocyte plasma membranes. HCV infection favours the breaking of self-tolerance against CYP2E1 that might contribute to hepatocyte injury.

Proteasome-dependent degradation of cytochromes P450 2E1 and 2B1 expressed in tetracycline-regulated HeLa cells

The degradation of ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible cytochrome P450 2B1 (CYP2B1) expressed in tetracycline (Tc)-inducible HeLa cell lines was characterized. A steady-state pulse-chase analysis was used to determine a half-life of 3.8 h for CYP2E1 while the half-life of CYP2B1 was 2.3-fold greater in the same cell line. In contrast, NADPH cytochrome P450 reductase which is constitutively expressed in Tc-HeLa cells had a half-life of about 30 h. Lactacystin and other selective proteasome inhibitors including N-benzyloxycarbonyl-leucyl-leucyl-leucinal (MG132) and N-benzyloxycarbonyl- l-leucyl- l-leucyl- l-norvalinal (MG115) significantly inhibited both CYP2E1 and CYP2B1 degradation. The turnover of CYP2E1 was slightly inhibited by calpain inhibitors while CYP2B1 turnover was not altered. Inhibitors of lysosomal proteolysis had no effect on the degradation of either protein. Treatment of cells with brefeldin A did not alter the degradation of either P450 which suggested the degradation occurred in the endoplasmic reticulum (ER). Even in the presence of proteasome inhibitors high molecular weight ubiquitin conjugates were not observed. Mutagenesis of two putative ubiquitination sites (Lys 317 and 324) did not alter the degradation of CYP2E1. The role of ubiquitination in the degradation of CYP2E1 was also examined in a Chinese hamster mutant cell line E36ts20 that contains a thermolabile ubiquitin-activating enzyme (E1). The turnover of CYP2E1 was not significantly different at the nonpermissive temperature in the ts20 when compared to the control E36 cells. Furthermore, the addition of the hsp90 inhibitors geldanamycin, herbimycin, and radicicol had no effect on the turnover of CYP2E1, differentiating the degradation of CYP2E1 from other substrates for proteasome-dependent degradation.

Ethanol upregulates pro-fibrogenic connective tissue growth factor (CTGF) gene expression in HepG2 cells via cytochrome P450 2E1-mediated ethanol oxidation

Connective tissue growth factor (CTGF) is a pro-fibrogenic molecule involved in several human fibrotic disorders. CTGF overexpression in the liver, through upregulation of CTGF mRNA in hepatic stellate cells (HSCs), is reported to correlate with the degree of fibrosis. Although alcohol is a major cause of hepatic fibrosis, the role of CTGF in alcoholic hepatic fibrogenesis, has been poorly understood. Oxidative stress, mediated by ethanol-inducible Cytochrome P-4502E1(CYP2E1), has been implicated as a crucial factor in alcoholic hepatic fibrogenesis. Therefore, we investigated the contribution of CYP2E1-mediated ethanol oxidation to CTGF mRNA expression by using a well-established HepG2 cell line constitutively expressing CYP2E1 (E9 cells). CTGF mRNA quantitation by a real-time reverse polymerase chain reaction (RT-PCR) showed a significant increase in CTGF mRNA levels in ethanol-treated E9 cells. Phorbol myristate acetate (PMA), which enhances CYP2E1 expression, increased CTGF mRNA levels. This increase was diminished after co-incubation with 4-methylpyrazole (4MP), a CYP2E1 inhibitor, or an antioxidant, N -acetylcysteine. We visualized the state of oxidative stress only in ethanol-treated E9 cells by a newly-established anti-acrolein-modified antibody. In conclusion, our study has first identified a possible role of CYP2E1-mediated ethanol oxidation in CTGF gene upregulation, suggesting a considerable role of CTGF in alcoholic hepatic fiberogenesis.

Cytochrome P450 2E1 (CYP2E1)-Dependent Production of a 37-kDa Acetaldehyde–Protein Adduct in the Rat Liver

Ethanol-inducible cytochrome P450 2E1 (CYP2E1) has been shown to be involved in the metabolism of both ethanol and acetaldehyde. Acetaldehyde, produced from ethanol metabolism, is highly reactive and can form various protein adducts. In this study, we investigated the role of CYP2E1 in the production of a 37-kDa acetaldehyde–protein adduct. Rats were pair-fed an isocaloric control or an alcohol liquid diet with and without cotreatment of YH439, an inhibitor of CYP2E1 gene transcription, for 4 weeks. The soluble proteins from rat livers of each group were separated on SDS–polyacrylamide gels followed by immunoblot analysis using specific antibodies against the 37-kDa protein acetaldehyde adduct. In addition, catalytic activities of the enzymes involved in alcohol and acetaldehyde metabolism were measured and compared with the adduct level. Immunoblot analysis revealed that the 37-kDa adduct, absent in the pair-fed control, was evident in alcohol-fed rats but markedly reduced by YH439 treatment. Immunohistochemical analysis also showed that the 37-kDa adduct is predominantly localized in the pericentral region of the liver where CYP2E1 protein is mainly expressed. This staining disappeared in the pericentral region after YH439 treatment. The levels of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase isozymes were unchanged after YH439 treatment. However, the level of the 37-kDa protein adduct positively correlated with the hepatic content of P4502E1. These data indicate that the 37-kDa adduct could be produced by CYP2E1-mediated ethanol metabolism in addition to the ADH-dependent formation.

Effect of Simultaneous Exposure to Benzene and Ethanol on Urinary Phenol Excretion in Mice

Benzene has been widely used as a solvent, but its use is declining in most developed countries because it is a well established human carcinogen. An association between occupational exposure to benzene and health effects such as aplastic anaemia and leukaemia has been reported in studies of workers in various industries. For many solvent uses benzene has been replaced by other less toxic organic solvents and is considered a toxic impurity in other industrial solvents, but it is still used as a starting material in numerous chemical syntheses. In addition to industrial sources, benzene is present in the environment as a component of cigarette smoke and automobile emissions. Benzene induces hematotoxicity as a result of chronic exposure. Acute exposure to benzene causes neurotoxic and hepatotoxic effects. Toxic effects of benzene, chronic as well as acute, are the consequence of the unknown reactive metabolite produced by cytochrome P-450. Benzene is metabolized mainly by ethanol inducible cytochrome P-4502E1 (CYP2E1) in animal and human liver microsomes. Ethanol is consumed worldwide in tremendous amounts and is an effective inducer of hepatic xenobiotic metabolism, especially involving pathways accomplished by the isoform CYP2E1 of cytochrome P-450. Therefore, whenever xenobiotics that are substrates of CYP2E1, such as many organic solvents, are taken in by an individual who is also chronically consuming ethanol, the accelerated metabolism of these agents has to be considered. In contrast to the long-term consumption of ethanol, which induces the hepatic metabolism of xenobiotics, short-term consumption inhibits their metabolism because of direct competition for CYP2E1. Ethanol, when given to a living body, exerts dual effects on the xenobiotic metabolizing enzymes, i.e., inhibition and stimulation. Which one is more predominant over the other depends on the time that has elapsed after ethanol ingestion. In an early period when ethanol exists in the body in high concentrations, it may preferentially act as an inhibitor . In this study, effect of simultaneous exposure to benzene and ethanol on benzene metabolism in mice were investigated by measuring the concentration of phenol, the main metabolite of benzene.

Molecular Basis for the Transport of Cytochrome P450 2E1 to the Plasma Membrane

Endoplasmic reticulum-resident cytochrome P450 enzymes that face the cytosol are present on the plasma membrane of hepatocytes, but the molecular origin for their transport to this compartment has until now remained unknown. The molecular basis for the transport of rat ethanol-inducible cytochrome P450 2E1 (CYP2E1) to the plasma membrane was investigated by transfection of several different mutant cDNAs into mouse H2.35 hepatoma cells. Two NH(2)-terminal CYP2E1 mutants were constructed: N(++)2E1, which carried two positive charges in the NH(2) terminus, and 2C-2E1, in which the transmembrane domain of CYP2E1 was replaced with that of CYP2C1, which was previously described to cause retention of CYP2C1 in the endoplasmic reticulum, as well as CYP2E1 COOH-terminally tagged with the vesicular stomatitis virus G protein (VSV-G) epitope (2E1-VSV-G). Immunofluorescent microscopy and cell surface biotinylation experiments revealed that all CYP2E1 variants were present on the extracellular side of the plasma membrane. The VSV-G epitope on CYP2E1 was detected on the outside of the plasma membrane using VSV-G-specific antibodies, indicating that the large COOH-terminal part of CYP2E1 is indeed exposed on the outside of the plasma membrane. The relative levels of CYP2E1, 2C-2E1, and 2E1-VSV-G on the cell surface were found to be about 2% of total cellular enzyme, whereas twice this amount of N(++)2E1 was recovered at the cell surface. Protease protection experiments performed on microsomes isolated from cDNA transfected cells revealed that a small fraction of CYP2E1 and all variant proteins was found to be located in the lumen of the endoplasmic reticulum (type II orientation), whereas the majority of the proteins were in the expected cytosolic or type I orientation. It is concluded that the NH(2)-terminal transmembrane domain of CYP2E1 plays a critical role in directing the protein to the cell surface and that topological inversion of a small fraction of CYP2E1 in the endoplasmic reticulum directs the protein to the plasma membrane.

Ethanol and Arachidonic Acid Increase α2(I) Collagen Expression in Rat Hepatic Stellate Cells Overexpressing Cytochrome P450 2E1: ROLE OF H2O2 AND CYCLOOXYGENASE-2

The ability of ethanol and arachidonic acid (AA), as inducers of oxidative stress and key factors in alcoholic liver disease, to up-regulate alpha 2 collagen type I (COL1A2) gene expression was studied in a hepatic stellate cell line overexpressing the ethanol-inducible cytochrome P450 2E1 (CYP2E1) (E5 cells). A time- and dose-dependent induction in COL1A2 mRNA by ethanol or AA was observed that was prevented by diallylsulfide, a CYP2E1 inhibitor. Nuclear run-on experiments showed transcriptional activation of the COL1A2 gene by ethanol and AA. Catalase abrogated the increase in COL1A2 mRNA suggesting an H(2)O(2)-dependent mechanism. Cyclooxygenase-2 (COX-2) levels and production of prostaglandin E(2) upon addition of AA were elevated in the E5 cells. Incubation with NS-398, a COX-2 inhibitor, blocked the effect of AA, but not of ethanol, on COL1A2 expression suggesting that CYP2E1 activates COX-2 expression, and the oxidation of AA by COX-2 is responsible for the increase in COL1A2. Activity of a reporter construct driven by -378 base pairs of the proximal promoter region of the COL1A2 gene increased in E5 but not control cells and was further increased by ethanol or AA. These experiments link CYP2E1-dependent oxidative stress to induction of COX-2 and the actions of ethanol and AA on activation of collagen gene expression in hepatic stellate cells.

Alcohol and drug interactions

Alcohol metabolism occurs mainly in the liver, where in abstainers the alcoholdehydrogenase (ADH) pathway plays the major role. After chronic alcohol consumption, the microsomal ethanol-oxidizing system (MEOS), involving the ethanol-inducible cytochrome P450 2E1, increases in importance with a four- to ten-fold increase in the contribution to alcohol metabolism. Because of the fact that this enzyme system catalyses not only the metabolism of ethanol but also activates a great number of drugs, it is a very common site of alcohol-drug interactions. Clinically relevant interactions will be discussed. Only a small amount of alcohol is metabolized outside the liver, mainly in the stomach by gastric ADH, which leads to the so-called first-pass metabolism of ethanol. Its significance in alcohol metabolism is reviewed. The only way to prevent severe alcohol-drug interactions is to make medical doctors as well as their patients more aware of these possible secondary effects.