Effects of Dietary Fat Composition on Activities of the Microsomal Ethanol Oxidizing System and Ethanol-Inducible Cytochrome P450 (CYP2E1) in the Liver of Rats Chronically Fed Ethanol

Abstract: We studied the effects of dietary fat composition on the activities of the microsomal ethanol oxidizing system (MEOS), paranitrophenol hydroxylase (PH) activity and ethanol-inducible cytochrome P450 isozymes (CYP2E1 and CYP2B1) in the liver of rats to determine the role of this ethanol metabolizing pathway in the pathogenesis of alcoholic liver disease (ALD). Wistar male rats were pair-fed a liquid diet, containing either tallow (TF) or corn oil (CF) as the fat component, and ethanol or an isocaloric amount of dextrose, through an implanted intragastric cannula. Liver pathology of rats fed ethanol (CF-ALC) and CF diet showed severe fatty change whereas the rats fed TF-alcohol and the TF and CF controls did not. MEOS activity of the CF-ALC group was 8 times of that in the CF-CTL group (P < 0.01). In TF-ALC rats, MEOS activity was increased to 2.6 times compared to that of TF-CTL (P< 0.01). ApoCYP2E1 in CF-ALC and TF-ALC were 818 ± 63 and 433 ± 17 pmol/mg protein, respectively, and these values were significantly higher when compared with those of the pair-fed controls (P< 0.005). In contrast, apoCYP2B1 was increased to an equal degree in both CF-ALC and TF-ALC. When PH-activity was measured, the level of activity on TF-ALC rats did not differ from that of CF-ALC rats. Thus, ethanol-induction of apoCYP2Bl (2 x) and PH (6–8 x) were the same for CF and TF (2 x); but not for apoCYP2E1 (21 and 8 x, respectively) and MEOS activity (8 and 2.6 x, respectively). These results indicate that the dietary effect on the expression of CYP2E1 correlates with the induction of centrilobular liver damage seen in the corn oil fed rats. The centrilobular distribution of this isozyme also correlates with the site of liver cell injury further suggesting a pathogenic link to alcohol-induced cell injury.     

Brain CYP2E1 is induced by nicotine and ethanol in rat and is higher in smokers and alcoholics

1. Ethanol and nicotine are commonly coabused drugs. Cytochrome P450 2E1 (CYP2E1) metabolizes ethanol and bioactivates tobacco-derived procarcinogens. Ethanol and nicotine can induce hepatic CYP2E1 and we hypothesized that both centrally active drugs could also induce CYP2E1 within the brain. 2. Male rats were treated with saline, ethanol (3.0 g kg(-1) by gavage) or nicotine (1.0 mg kg(-1) s.c.) for 7 days. Ethanol treatment significantly increased CYP2E1 in olfactory bulbs (1.7-fold), frontal cortex (2.0-fold), hippocampus (1.9-fold) and cerebellum (1.8-fold), while nicotine induced CYP2E1 in olfactory bulbs (2.3-fold), frontal cortex (3.0-fold), olfactory tubercle (3.1-fold), cerebellum (2.5-fold) and brainstem (2.0-fold). Immunocytochemical analysis revealed that the induction was cell-type specific. 3. Consistent with the increased CYP2E1 found in rat brain following drug treatments, brains from alcoholics and alcoholic smokers showed greater staining of granular cells of the dentate gyrus and the pyramidal cells of CA2 and CA3 hippocampal regions as well as of cerebellar Purkinje cells compared to nonalcoholic nonsmokers. Moreover, greater CYP2E1 immunoreactivity was observed in the frontal cortices in the alcoholic smokers in comparison to nonalcoholic nonsmokers and alcoholic nonsmokers. 4 To investigate if nicotine could contribute to the increased CYP2E1 observed in alcoholic smokers, we treated human neuroblastoma IMR-32 cells in culture and found significantly higher CYP2E1 immunostaining in nicotine-treated cells (0.1-10 nM). 5. CYP2E1 induction in the brain, by ethanol or nicotine, may influence the central effects of ethanol and the development of nervous tissue pathologies observed in alcoholics and smokers.     

Absence of a differential induction of cytochrome P450 2E1 by different alcoholic beverages in rats: implications for the aetiology of human oesophageal cancer

Alcohol is an important risk factor for human oesophageal cancer. There is evidence from epidemiological studies that some specific alcoholic drinks, e.g. Calvados apple brandy, are associated with a greater risk than others. Alcohol induces cytochrome P450 2E1 (CYP2E1) and the hypothesis was tested that different alcoholic beverages, containing a variety of alcoholic compounds, could differentially induce expression of cytochrome P450 enzymes. Twelve groups of five rats each were treated for 3 days with different alcoholic beverages (ethanol alone, whisky, farm-produced or commercial Calvados brandy, beer, cider, wine) adjusted to 4, 10 or 20% of ethanol in drinking water. Immunoblotting using a monoclonal antibody specific for rat CYP2E1 revealed a single protein band in liver microsomes. Densitometric quantitation of microsomal proteins demonstrated a significant two-, three- and sixfold increase in band intensity after treatment with ethanol concentrations of 4, 10 and 20% respectively, compared to control rats drinking water alone. There was a dose-dependent increase in liver microsomal metabolism of CYP2E1 substrates (para-nitrophenol and dimethylnitrosamine) in ethanol-treated rats. However, there were no significant differences in the level of CYP2E1 protein or enzymatic activity between the different alcoholic beverages at the same ethanol concentration. There was a slight increase in hepatic CYP1A-related enzymatic activities in the alcohol-treated rats compared to the controls, but no difference between the treated groups either with dose of ethanol or type of beverage. These data show that induction of CYP2E1 with acute alcohol treatment is predominantly determined by the ethanol content of the beverage. Received: 10 February 1997 / Accepted: 26 May 1997     

CYP2E1--biochemical and toxicological aspects and role in alcohol-induced liver injury

Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. CYP2E1 is of interest because of its ability to metabolize and activate many toxicological substrates, including ethanol, to more reactive toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, it produces powerful oxidants such as the hydroxyl radical. This review article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines developed to constitutively express the human CYP2E1 in assessing the actions of CYP2E1. Regulation of CYP2E1 is quite complex and will be briefly reviewed. Future directions in research, which may help clarify the actions of CYP2E1 and its role in alcoholic liver injury, are suggested.     

Mechanisms of hepatotoxicity.

This review addresses recent advances in specific mechanisms of hepatotoxicity. Because of its unique metabolism and relationship to the gastrointestinal tract, the liver is an important target of the toxicity of drugs, xenobiotics, and oxidative stress. In cholestatic disease, endogenously generated bile acids produce hepatocellular apoptosis by stimulating Fas translocation from the cytoplasm to the plasma membrane where self-aggregation occurs to trigger apoptosis. Kupffer cell activation and neutrophil infiltration extend toxic injury. Kupffer cells release reactive oxygen species (ROS), cytokines, and chemokines, which induce neutrophil extravasation and activation. The liver expresses many cytochrome P450 isoforms, including ethanol-induced CYP2E1. CYP2E1 generates ROS, activates many toxicologically important substrates, and may be the central pathway by which ethanol causes oxidative stress. In acetaminophen toxicity, nitric oxide (NO) scavenges superoxide to produce peroxynitrite, which then causes protein nitration and tissue injury. In inducible nitric oxide synthase (iNOS) knockout mice, nitration is prevented, but unscavenged superoxide production then causes toxic lipid peroxidation to occur instead. Microvesicular steatosis, nonalcoholic steatohepatitis (NASH), and cytolytic hepatitis involve mitochondrial dysfunction, including impairment of mitochondrial fatty acid beta-oxidation, inhibition of mitochondrial respiration, and damage to mitochondrial DNA. Induction of the mitochondrial permeability transition (MPT) is another mechanism causing mitochondrial failure, which can lead to necrosis from ATP depletion or caspase-dependent apoptosis if ATP depletion does not occur fully. Because of such diverse mechanisms, hepatotoxicity remains a major reason for drug withdrawal from pharmaceutical development and clinical use.     

Metabolism of ethanol and associated hepatotoxicity

Over the last three decades, direct hepatotoxic effects of ethanol were established, some of which were linked to redox changes produced by NADH generated via the alcohol dehydrogenase (ADH) pathway and shown to affect the metabolism of lipids, carbohydrates, proteins, and purines. It was also determined that ethanol can be oxidized by a microsomal ethanol oxidizing system (MEOS) involving a specific cytochrome P-450; this newly discovered ethanol-inducible cytochrome P-450 (P-450 IIEi) contributes to ethanol metabolism, tolerance, energy wastage (with associated weight loss), and the selective hepatic perivenular toxicity of various xenobiotics. Their activation by P-450IIEi now provides an understanding of the increased susceptibility of the heavy drinker to the toxicity of industrial solvents, anaesthetic agents, commonly prescribed drugs, over-the-counter analgesics, and chemical carcinogens. P-450 induction also explains depletion (and toxicity) of nutritional factors such as vitamin A. As a consequence, treatment with vitamin A and other nutritional factors is beneficial, but must take into account a narrowed therapeutic window in alcoholics who have increased needs for nutrients and also display an enhanced susceptibility to some of their adverse effects. Acetaldehyde (the metabolite produced from ethanol by either ADH or MEOS) impairs hepatic oxygen utilization and forms protein adducts, resulting in antibody production, enzyme inactivation, and decreased DNA repair. It also stimulates collagen production by the vitamin A storing cells (lipocytes) and myofibroblasts, and causes glutathione depletion. Supplementation with S-adenosyl-L-methionine partly corrects the depletion and associated mitochondrial injury, whereas administration of polyunsaturated lecithin opposes the fibrosis. Thus, at the cellular level, the classic dichotomy between the nutritional and toxic effects of ethanol has now been bridged. The understanding of how the ensuing injury eventually results in irreversible scarring or cirrhosis may provide us with improved modalities for treatment and prevention.     

Paracetamol, alcohol and the liver

It is claimed that chronic alcoholics are at increased risk of paracetamol (acetaminophen) hepatotoxicity not only following overdosage but also with its therapeutic use. Increased susceptibility is supposed to be due to induction of liver microsomal enzymes by ethanol with increased formation of the toxic metabolite of paracetamol. However, the clinical evidence in support of these claims is anecdotal and the same liver damage after overdosage occurs in patients who are not chronic alcoholics. Many alcoholic patients reported to have liver damage after taking paracetamol with 'therapeutic intent' had clearly taken substantial overdoses. No proper clinical studies have been carried out to investigate the alleged paracetamol-alcohol interaction and acute liver damage has never been produced by therapeutic doses of paracetamol given as a challenge to a chronic alcoholic. The paracetamol-alcohol interaction is complex; acute and chronic ethanol have opposite effects. In animals, chronic ethanol causes induction of hepatic microsomal enzymes and increases paracetamol hepatotoxicity as expected (ethanol primarily induces CYP2E1 and this isoform is important in the oxidative metabolism of paracetamol). However, in man, chronic alcohol ingestion causes only modest (about twofold) and short-lived induction of CYP2E1, and there is no corresponding increase (as claimed) in the toxic metabolic activation of paracetamol. The paracetamol-ethanol interaction is not specific for any one isoform of cytochrome P450, and it seems that isoenzymes other than CYP2E1 are primarily responsible for the oxidative metabolism of paracetamol in man. Acute ethanol inhibits the microsomal oxidation of paracetamol both in animals and man. This protects against liver damage in animals and there is evidence that it also does so in man. The protective effect disappears when ethanol is eliminated and the relative timing of ethanol and paracetamol intake is critical. In many of the reports where it is alleged that paracetamol hepatotoxicity was enhanced in chronic alcoholics, the reverse should have been the case because alcohol was actually taken at the same time as the paracetamol. Chronic alcoholics are likely to be most vulnerable to the toxic effects of paracetamol during the first few days of withdrawal but maximum therapeutic doses given at this time have no adverse effect on liver function tests. Although the possibility remains that chronic consumption of alcohol does increase the risk of paracetamol hepatotoxicity in man (perhaps by impairing glutathione synthesis), there is insufficient evidence to support the alleged major toxic interaction. It is astonishing that clinicians and others have unquestion-ingly accepted this supposed interaction in man for so long with such scant regard for scientific objectivity.     

Cytochrome P450 and liver diseases

Cytochrome P-450 (CYPs) are involved in the metabolism of drugs, chemicals and endogenous substrates. The hepatic CYPs are also involved in the pathogenesis of several liver diseases. CYP-mediated activation of drugs to toxic metabolites induces hepatotoxicity. Well-known examples include acetaminophen and halothane. In some instances, covalent binding of the toxic metabolite to CYP leads to the formation of anti-CYP antibodies and immune-mediated hepatotoxicity (hydralazine, tienilic acid). Anti-CYP2D6 antibodies are also present in the serum of patients with type II autoimmune hepatitis, but the mechanism leading to their presence and their pathogenic significance remains unclear. Several studies support a role for CYP2E1 in the pathogenesis of alcoholic liver disease and non-alcoholic steatohepatitis. In these conditions, enhanced CYP2E1 activity is associated with lipid peroxidation and the production of reactive oxygen species with secondary damage to cellular membranes and mitochondria. Because of its ability to activate carcinogens, a role for CYP2E1 as a cofactor for hepatocellular carcinoma has also been postulated. On the other hand, drug metabolism is impaired in patients with liver disease, particularly that mediated by CYPs. The content and activity of CYP1A, 2C19 and 3A appear to be particularly vulnerable to the effect of liver disease while CYP2D6, 2C9 and 2E1 are less affected. The pattern of CYPs isoenzymes alterations also differs according to the etiology of liver disease. A strong relationship between the activity of CYPs and the severity of cirrhosis has been demonstrated, but the usefulness of measuring CYP activity to assess hepatic functional reserve remains uncertain.     

CYP2E1-dependent oxidative stress and toxicity: role in ethanol-induced liver injury

Ethanol-induced oxidative stress plays a major role in the mechanisms by which ethanol causes liver injury. Many pathways contribute to how ethanol induces a state of oxidative stress. One central pathway appears to be the induction, by ethanol, of the CYP2E1 form of cytochrome P450 enzymes. CYP2E1 is of interest because it metabolises and activates many toxicological substrates, including ethanol, to more reactive products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions. CYP2E1 is an effective generator of reactive oxygen species. This review summarises some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines in assessing the actions of CYP2E1. Future directions, which may help to better understand the actions of CYP2E1 and its role in alcoholic liver injury, are suggested.     

Hepatic and pulmonary microsomal benzene metabolism in CYP2E1 knockout mice.

Benzene is an occupational and environmental toxicant. The major health concern for humans is acute myelogenous leukemia. To exert its toxic effects, benzene must be metabolized via cytochrome P450. CYP2E1 has been identified as the most important cytochrome, P450 isozyme in hepatic benzene metabolism in mice, rats, and humans. In pulmonary microsomes CYP2E1 and members of the CYP2F subfamily are both significantly involved. In the current study CYP2E1 knockout mice and wild-type controls were used to further examine the cytochrome P450 isozymes involved in metabolism of 24 microM benzene. The results show that CYP2E1 is the most important isozyme in the liver, accounting for 96% of the total hydroxylated metabolite formation. However, in the lung CYP2E1 was responsible for only 45% of the formation of total hydroxylated metabolite. Chemical inhibitors of CYP2E1 and CYP2F2 were used to further examine the contributions of these isozymes to benzene metabolism. The results confirmed the finding that while CYP2E1 is the most important isozyme in the liver, CYP2F2 and CYP2E1 are both significantly involved in the lung.     

Metabolic oxidation and toxification of N-methylformamide catalyzed by the cytochrome P450 isoenzyme CYP2E1.

Alkylformamides, for example N-methylformamide, are hepatotoxic in rodents and humans. The mechanism by which N-methylformamide exerts its hepatotoxicity involves metabolic oxidation at the formyl moiety to yield a short-lived intermediate, perhaps methyl isocyanate, which reacts with glutathione to afford S-(N-methylcarbamoyl)glutathione. The hypothesis that the cytochrome P450 isozyme CYP2E1 catalyzes the metabolic toxification of N-methylformamide was tested. Hepatocytes obtained from mice that had received acetone, an inducer of CYP2E1, were incubated for up to 4 hr with N-methylformamide (5 and 10 mM). Whereas N-methylformamide caused cytotoxicity in these cells, as measured by release from the cells of lactate dehydrogenase, it was barely toxic, under these conditions, to cells from untreated mice. Coincubation of N-methylformamide with dimethylsulfoxide (10 mM), a CYP2E1 inhibitor, for 4 or 6 hr abolished the hepatocytotoxicity of N-methylformamide. Metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was measured in incubates with liver microsomes from rats, mice, or humans in the presence of glutathione. Pretreatment of rodents with acetone or ethanol induced the rate of metabolism of N-methylformamide and of p-nitrophenol, a known CYP2E1 substrate, but it did not increase aminopyrine N-demethylation. Metabolism of N-methylformamide and p-nitrophenol was elevated in microsomes from animals that had received acetone (1%) in their drinking water for 1 week to 230% and 200%, respectively, of control values in mouse microsomes and to 310% and 240%, respectively, of control values in rat microsomes. Pretreatment of animals with 4-methylpyrazole (200 mg/kg intraperitoneally, once daily for 3 days) increased metabolism of N-methylformamide to 410% of control values in rat liver microsomes but was without effect on murine microsomal metabolism of N-methylformamide. The metabolism of this compound was strongly inhibited by the CYP2E1 substrates or inhibitors dimethylsulfoxide (1-100 mM), p-nitrophenol (100 microM), and diethyldithiocarbamate (100 microM), which did not affect aminopyrine N-demethylation. A polyclonal antibody against rat CYP2E1 (10 mg of IgG/nmol of cytochrome P450) inhibited N-methylformamide metabolism in liver microsomes from rats and from a human by 75% and 80%, respectively. The rate of metabolism of N-methylformamide to S-(N-methylcarbamoyl) glutathione was determined in liver microsomes from six humans and correlated with extent of metabolic hydroxylation of chlorzoxazone, a CYP2E1 probe, and with amount of immunodetectable enzyme using an anti-rat CYP2E1 antibody (r = 0.81 and 0.80, respectively). The results suggest that CYP2E1 is the predominant, if not sole, cytochrome P450 isozyme responsible for the metabolic toxification of hepatotoxic N-alkylformamides.     

Cytochrome P450 isozymes involved in the metabolism of phenol, a benzene metabolite.

Benzene is an occupational and environmental toxicant. The major health concern for humans is acute myelogenous leukemia. To exert its toxic effects, benzene must be metabolized by cytochrome P450 to phenol and subsequently to catechol and hydroquinone. Previous research has implicated CYP2E1 in the metabolism of phenol. In this study the cytochrome P450 isozymes involved in the metabolism of phenol were examined in hepatic and pulmonary microsomes utilizing chemical inhibitors of CYP2E1, CYP2B, and CYP2F2 and using CYP2E1 knockout mice. CYP2E1 was found to be responsible for only approximately 50% of 20 microM phenol metabolism in the liver. This suggests another isozyme(s) is involved in hepatic phenol metabolism. In pulmonary microsomes both CYP2E1 and CYP2F2 were significantly involved.     

Effects of theanine on alcohol metabolism and hepatic toxicity

We previously showed that theanine, is a major amino acid in green tea, enhanced doxorubicin (DOX) induced antitumor activity. Besides, theanine induced the elevation of glutathione (GSH) level attributable to the increase of glutamate in the liver of mice, namely theanine would reduce the adverse reaction of DOX. Consequently, theanine was thought to be effective against the tissue changes with GSH level reduction. On the other hand, it is suggested excessive uptake of alcohol causes a production of free radicals, a decrease of GSH level, and an increase in the amount of lipid peroxide (LPO) in liver, and shifting to an alcoholic liver injury. Then, aiming at the prevention and medical treatment of a hepatic toxicity by the food components with little toxicity, we have studied the effect of theanine (i.p.) on ethanol metabolism and hepatic toxicity using ethanol (p.o.) single-administered mice. On the 1st hour after ethanol administration, the ethanol concentrations in blood of the theanine combined groups decreased compared with the ethanol-alone group. The alcohol dehydrogenase and aldehyde dehydrogenase activities in the liver increased by combined theanine. Since the elevation of cytochrome P450 (CYP) 2E1 activity was controlled in the theanine-combined groups, it was considered that these disorders attributable to CYP2E1 in ethanol long-term uptake might be avoidable by theanine. Although LPO increased in 3 h after by single-administration of ethanol, the increase was controlled by theanine-administration and was improved until the normal level. In conclusion, it was indicated that theanine was effective against alcoholic liver injury.     

An improved in vitro method for screening toxin and medicine targeting CYP2E1

The cytochrome P450 enzyme 2E1 (CYP2E1) presents in both microsome and mitochondrion, which influences the metabolism of many xenobiotics. The mice active liver homogenate was prepared for the medicinal incubation and mitochondrion was extracted for chemical screening targeting CYP2E1 enzyme. Representative CYP2E1 inducers (ethanol and pyrazole) and inhibitors (diallyldisulfide and kaempferol) were applied to evaluate the effectiveness of homogenate-mitochondrial system. In parallel, the in-vitro microsomal method targeting CYP2E1 was also operated for comparison. The results showed that in homogenate-mitochondrial method, the protein level and activity of CYP2E1 were increased by ethanol and pyrazole; reduced by diallyldisulfide and kaempferol, and this homogenate-mitochondrial method is convenient with good repeatability and reproducibility in screening chemicals targeting CYP2E1, especially for the inducers. Thus, the homogenate-mitochondrial method might be effective in screening both CYP2E1 inhibitor and inducer.     

Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-L-methionine

The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5'-Adenosyl)-l-homocysteine (SAH), 5'-Deoxy-5'-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC(50) around 1.5-5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to alpha-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the K(i) for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.     

Ethanol induced induction of cytochrome P450 2E1 and activation of mitogen activated protein kinases in peripheral blood lymphocytes

Cytochrome P450 2E1 (CYP2E1), which induces oxidative stress that leads to alcohol-mediated toxicity in liver, is expressed in peripheral blood lymphocytes. To validate blood lymphocyte CYP2E1 as a biomarker of alcohol-induced diseases, studies were initiated to investigate similarities in CYP2E1 induction and associated cell signalling pathways in freshly prepared blood lymphocytes with the liver in rats exposed to alcohol. Acute or chronic treatment of ethanol produced significant increase in enzyme activity and lipid peroxidation in blood lymphocytes. As observed in liver, this increase was associated with the enrichment of CYP2E1 protein and mRNA. Similar pattern of increase in the mRNA and protein expression of c-jun and c-fos was also observed in blood lymphocytes and liver. Acute exposure to ethanol activated ERK and JNK MAP kinases and c-jun in the blood lymphocytes and liver. The present data demonstrating similarities in the induction of CYP2E1 and lipid peroxidation and activation of MAP Kinases in blood lymphocytes with liver after acute or chronic exposure of ethanol have suggested that blood lymphocytes could be used to monitor ethanol induced CYP2E1 induction and associated oxidative stress in liver.     

CYP2 E1在酒精性肝病中的作用

酒精性肝病(ALD)是一种长期大量饮酒所致的慢性肝脏疾病。乙醇经肝脏代谢产生大量的自由基和活性氧。氧化应激在乙醇引起肝损伤的机制发挥关键作用。在肝脏,细胞色素P450 2E1(CYP2E1)可被乙醇诱导并参与乙醇代谢,CYP2E1是一种有效的活性氧产生酶,产生超氧阴离子自由基和过氧化氢,铁催化剂的存在下,产生羟基自由基。该文主要总结了CYP2E1在ALD发病过程中的作用及机制,为ALD的临床治疗提供思路。     

Metabolic basis of ethanol-induced cytotoxicity in recombinant HepG2 cells: role of nonoxidative metabolism

Chronic alcohol abuse, a major health problem, causes liver and pancreatic diseases and is known to impair hepatic alcohol dehydrogenase (ADH). Hepatic ADH-catalyzed oxidation of ethanol is a major pathway for the ethanol disposition in the body. Hepatic microsomal cytochrome P450 (CYP2E1), induced in chronic alcohol abuse, is also reported to oxidize ethanol. However, impaired hepatic ADH activity in a rat model is known to facilitate a nonoxidative metabolism resulting in formation of nonoxidative metabolites of ethanol such as fatty acid ethyl esters (FAEEs) via a nonoxidative pathway catalyzed by FAEE synthase. Therefore, the metabolic basis of ethanol-induced cytotoxicity was determined in HepG2 cells and recombinant HepG2 cells transfected with ADH (VA-13), CYP2E1 (E47) or ADH + CYP2E1 (VL-17A). Western blot analysis shows ADH deficiency in HepG2 and E47 cells, compared to ADH-overexpressed VA-13 and VL-17A cells. Attached HepG2 cells and the recombinant cells were incubated with ethanol, and nonoxidative metabolism of ethanol was determined by measuring the formation of FAEEs. Significantly higher levels of FAEEs were synthesized in HepG2 and E47 cells than in VA-13 and VL-17A cells at all concentrations of ethanol (100-800 mg%) incubated for 6 h (optimal time for the synthesis of FAEEs) in cell culture. These results suggest that ADH-catalyzed oxidative metabolism of ethanol is the major mechanism of its disposition, regardless of CYP2E1 overexpression. On the other hand, diminished ADH activity facilitates nonoxidative metabolism of ethanol to FAEEs as found in E47 cells, regardless of CYP2E1 overexpression. Therefore, CYP2E1-mediated oxidation of ethanol could be a minor mechanism of ethanol disposition. Further studies conducted only in HepG2 and VA-13 cells showed lower ethanol disposition and ATP concentration and higher accumulation of neutral lipids and cytotoxicity (apoptosis) in HepG2 cells than in VA-13 cells. The apoptosis observed in HepG2 vs. VA-13 cells incubated with ethanol appears to be mediated by release of mitochondrial cytochrome c via activation of caspase-9 and caspase-3. These results strongly support our hypothesis that diminished hepatic ADH activity facilitates nonoxidative metabolism of ethanol and the products of ethanol nonoxidative metabolism cause apoptosis in HepG2 cells via intrinsic pathway.     

CYP2E1-dependent and leptin-mediated hepatic CD57 expression on CD8 + T cells aid progression of environment-linked nonalcoholic steatohepatitis

Environmental toxins induce a novel CYP2E1/leptin signaling axis in liver. This in turn activates a poorly characterized innate immune response that contributes to nonalcoholic steatohepatitis (NASH) progression. To identify the relevant subsets of T-lymphocytes in CYP2E1-dependent, environment-linked NASH, we utilized a model of diet induced obese (DIO) mice that are chronically exposed to bromodichloromethane. Mice deficient in CYP2E1, leptin (ob/ob mice), or both T and B cells (Pfp/Rag2 double knockout (KO) mice) were used to delineate the role of each of these factors in metabolic oxidative stress-induced T cell activation. Results revealed that elevated levels of lipid peroxidation, tyrosyl radical formation, mitochondrial tyrosine nitration and hepatic leptin as a consequence of metabolic oxidative stress caused increased levels of hepatic CD57, a marker of peripheral blood lymphocytes including NKT cells. CD8 + CD57 + cytotoxic T cells but not CD4 + CD57 + cells were significantly decreased in mice lacking CYP2E1 and leptin. There was a significant increase in the levels of T cell cytokines IL-2, IL-1β, and IFN-γ in bromodichloromethane exposed DIO mice but not in mice that lacked CYP2E1, leptin or T and B cells. Apoptosis as evidenced by TUNEL assay and levels of cleaved caspase-3 was significantly lower in leptin and Pfp/Rag2 KO mice and highly correlated with protection from NASH. The results described above suggest that higher levels of oxidative stress-induced leptin mediated CD8 + CD57 + T cells play an important role in the development of NASH. It also provides a novel insight of immune dysregulation and may be a key biomarker in NASH.     

The effects of alcohol and diallyl sulphide on CYP2E1 activity in humans: a phenotyping study using chlorzoxazone.

The effects of acute administration of dietary levels of ethanol and the garlic oil extract, diallyl sulphide (DAS), on cytochrome P450 2E1 (CYP2E1) activity in volunteers were studied using the selective probe substrate, chlorzoxazone (CZX). The ratio of the CZX metabolite 6- hydroxychlorzoxazone (6-OHCZX) to CZX was taken to indicate CYP2E1 activity. The mean differences between the baseline and DAS-treated (0.2 mg/kg) CYP2E1 activities were significantly different (two-tailed p value = 0.0242, n = 8). Likewise, the mean differences between the baseline and ethanol-treated (0.8 g/kg) CYP2E1 activities were also significantly different (two-tailed p value = 0.0005, n = 7). The reduction in in vivo CYP2E1 activity by DAS is consistent with reported inhibition observed in vitro. The marked reduction in CYP2E1 activity following acute ingestion of ethanol is consistent with a competitive inhibition mechanism of CZX metabolism. The inhibitory effect of DAS maybe additive with daily consumption of Allium vegetables in particular. This may explain the lower 6-OHCZX/CZX metabolic ratios measured in various European and Mexican cohorts and is consistent with the lower incidence of stomach, liver and colon cancers observed in southern Europeans.