Induction of cytochrome P450 enzymes and generation of protein-aldehyde adducts are associated with sex-dependent sensitivity to alcohol-induced liver disease in micropigs.

To assess possible links between ethanol-induced oxidant stress, expression of hepatic cytochrome P450 (CYP) enzymes, and sex steroid status, we used immunohistochemical methods to compare the generation of protein adducts of acetaldehyde (AA), malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE) with the amounts of CYP2E1, CYP2A, and CYP3A in the livers of castrated and noncastrated male micropigs fed ethanol for 12 months. In castrated micropigs, ethanol feeding resulted in accumulation of fat, hepatocellular necrosis, inflammation, and centrilobular fibrosis, whereas only minimal histopathology was observed in their noncastrated counterparts. CYP2A and CYP3A were more prominent in the castrated animals than in the noncastrated micropigs. Ethanol feeding increased the hepatic content of all CYP forms. The most significant increases occurred in CYP2E1 and CYP3A in the noncastrated animals and in CYP2E1 and CYP2A in the castrated animals. Ethanol-fed castrated animals also showed the greatest abundance of perivenular adducts of AA, MDA, and HNE. In the noncastrated ethanol-fed micropigs a low expression of each CYP form was associated with scant evidence of aldehyde-protein adducts. Significant correlations emerged between the levels of different CYP forms, protein adducts, and plasma levels of sex steroids. The present findings indicate that the generation of protein-aldehyde adducts is associated with the induction of several cytochrome enzymes in a sex steroid-dependent manner. It appears that the premature, juvenile, metabolic phenotype, as induced by castration, favors liver damage. The present findings should be implicated in studies on the gender differences on the adverse effects of ethanol in the liver.     

Immunohistochemical characterization of hepatic malondialdehyde and 4-hydroxynonenal modified proteins during early stages of ethanol-induced liver injury

BACKGROUND: Chronic ethanol consumption is associated with hepatic lipid peroxidation and the deposition or retention of aldehyde-adducted proteins postulated to be involved in alcohol-induced liver injury. The purpose of this study was to characterize hepatocellular formation of aldehyde-protein adducts during early stages of alcohol-induced liver injury. METHODS: Female Sprague Dawley(R) rats were subjected to the intragastric administration of a low-carbohydrate/high-fat total enteral nutrition diet or a total enteral nutrition diet containing ethanol for a period of 36 days. Indexes of hepatic responses to ethanol were evaluated in terms of changes in plasma alanine aminotransferase activity, hepatic histopathologic analysis, and induction of cytochrome P-4502E1 (CYP2E1). Immunohistochemical methods were used to detect hepatic proteins modified with malondialdehyde (MDA) or 4-hydroxynonenal (4-HNE) for subsequent quantitative image analysis. RESULTS: After 36 days of treatment, rats receiving the alcohol-containing diet displayed hepatic histopathologies characterized by marked micro- and macrosteatosis associated with only minor inflammation and necrosis. Alcohol administration resulted in a 3-fold elevation of plasma alanine aminotransferase activity and 3-fold increases (p < 0.01) in hepatic CYP2E1 apoprotein and activity. Quantitative immunohistochemical analysis revealed significant (p < 0.01) 5-fold increases in MDA- and 4-HNE modified proteins in liver sections prepared from rats treated with alcohol. The MDA- or 4-HNE modified proteins were contained in hepatocytes displaying intact morphology and were colocalized primarily with microvesicular deposits of lipid. Aldehyde-modified proteins were not prevalent in parenchymal or nonparenchymal cells associated with foci of necrosis or inflammation. CONCLUSIONS: These results suggest that alcohol-induced lipid peroxidation is an early event during alcohol-mediated liver injury and may be a sensitizing event resulting in the production of bioactive aldehydes that have the potential to initiate or propagate ensuing proinflammatory or profibrogenic cellular events.     

Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the liver of patients with alcoholic and non-alcoholic liver diseases

BACKGROUND/AIMS: Interaction between CYP2E1, ethanol metabolites, and enhanced lipid peroxidation is linked to the pathogenesis of alcoholic liver disease. This study was conducted to compare the expression of various cytochrome enzymes and the appearance of aldehyde adducts in humans. METHODS: Acetaldehyde- and lipid peroxidation-derived protein adducts and CYP2A6, 2E1, and 3A4/5 were examined immunohistochemically from liver specimens of 12 alcohol abusers with either mild (n=7) or severe (n=5) liver disease, and from nine non-drinking patients with non-alcoholic steatosis (n=4), or hepatitis (n=5). RESULTS: Ethanol-inducible CYP2E1 was present in all alcoholic livers. While CYP2A6 in zone 3 hepatocytes was also abundant in the alcoholic patients with various degrees of liver disease, CYP3A415 was most prominent in alcoholic cirrhosis. The sites of CYP2E1 and CYP2A6 immunoreactivity co-localized with fatty deposits, and with the sites of acetaldehyde and lipid peroxidation-derived protein adducts. The CYP enzymes were also abundant in the centrilobular hepatocytes of patients with fatty liver due to obesity or diabetes. CONCLUSIONS: Alcohol-induced liver damage is associated with a generalized induction of CYP2A6, CYP2E1 and CYP3A4 and generation of acetaldehyde and lipid peroxidation-derived protein-aldehyde adducts. However, CYP induction also occurred in patients with non-alcoholic steatosis.     

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased 4-hydroxynonenal levels in experimental alcoholic liver disease: association of lipid peroxidation with liver fibrogenesis.

The precise role of lipid peroxidation in the pathogenesis of alcoholic liver disease is still being debated. To explore the issue, this study was undertaken to investigate the status of lipid peroxidation, antioxidants and prooxidants at two discrete stages of experimental alcoholic liver disease. Male Wistar rats were intragastrically fed a high-fat diet plus ethanol for 5 or 16 wk (the duration that resulted in initiation of centrilobular liver necrosis or liver fibrosis, respectively). Lipid peroxidation was assessed in isolated microsomes and mitochondria with three parameters: malondialdehyde equivalents as determined by thiobarbituric acid assay, conjugated diene formation and 4-hydroxynonenal as a 2,4-dinitrophenylhydrazone derivative. To assess antioxidant systems, hepatic concentrations of glutathione, methionine and alpha-tocopherol were determined. The concentration of nonheme iron, a known prooxidant, was also measured. At wk 5, centrilobular liver necrosis was already evident in the ethanol-fed animals, with two- or threefold increases in plasma AST and ALT levels. At this stage, neither malondialdehyde equivalents nor conjugated diene values were elevated, and the 4-hydroxynonemal level was below 0.2 nmol/mg protein. Hepatic concentrations of methionine and alpha-tocopherol in these animals were increased two- and threefold, respectively, whereas the reduced glutathione level remained unchanged. When alcoholic liver disease had progressed to perivenular or bridging fibrosis at wk 16, all three parameters of lipid peroxidation showed consistent increases that were accompanied by significant reductions in the hepatic glutathione and methionine levels. Interestingly, the control animals pair-fed with the high-fat diet also had significantly elevated 4-hydroxynonenal levels at wk 16 compared to the wk 5 level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Testosterone-Dependent Enzyme, Carbonic Anhydrase III, and Oxidative Stress in Experimental Alcoholic Liver Disease

We studied the sequential immunohistochemicalappearance of androgen-dependent carbonic anhydrase (CAIII) during the development of ethanol-induced liverinjury using liver samples from castrated andnoncastrated male micropigs. In castrated micropigs, thebaseline expression of CA III was either low or absent,while distinct positive immunoreactions were found inzone 3 hepatocytes at 5 and 12 months after the initiation of the ethanol diet. The CA IIIenzyme and protein adducts of lipid peroxidation-derivedaldehydic products, malondialdehyde and4-hydroxynonenal, appeared together in the perivenousregion, suggesting that the enzyme functions in anoxidative environment. The positive staining became moreabundant and widespread during the progression ofalcoholic liver disease. After 12 months, CA III was significantly more abundant in both theethanol-fed noncastrated and castrated micropigs than inthe control animals (P < 0.001, P < 0.05,respectively). CA III content was strikingly high in the ethanol-fed noncastrated animals, consistentwith a potential role of androgens in the regulation ofethanol-induced CA III expression. The strongly positiveCA III immunoreactions in the ethanol-fed noncastrated micropigs were associated with scant evidenceof aldehydic protein adducts and minimal histopathology.Thus, enhanced expression of CA III during ethanolconsumption may also account in part for gender differences in the susceptibility foralcohol-induced liver injury.     

Hepatic mitochondrial glutathione depletion and progression of experimental alcoholic liver disease in rats.

Long-term ethanol feeding has been shown to selectively reduce hepatic mitochondrial glutathione content by impairing mitochondrial uptake of this thiol. In this study, we assessed the role of this defect in evolution of alcoholic liver disease by examining the mitochondrial glutathione pool and lipid peroxidation during progression of experimental alcoholic liver disease to centrilobular liver necrosis and fibrosis. Male Wistar rats were intragastrically infused with a high-fat diet plus ethanol for 3, 6 or 16 wk (the duration that resulted in induction of liver steatosis, necrosis and fibrosis, respectively). During this feeding period, the cytosolic pool of glutathione remained unchanged in the ethanol-fed animals compared with that in pair-fed controls. In contrast, the mitochondrial pool of glutathione selectively and progressively decreased in rats infused with ethanol for 3, 6 or 16 wk, by 39%, 61% and 85%, respectively. Renal mitochondrial glutathione level remained unaffected throughout the experiment. Serum ALT levels increased significantly in the ethanol-fed rats at 6 wk and remained elevated at 16 wk. In the mitochondria with severely depleted glutathione levels at 16 wk, enhanced lipid peroxidation was evidenced by increased malondialdehyde levels. Thus a progressive and selective depletion of mitochondrial glutathione is demonstrated in the liver in this experimental model of alcoholic liver disease and associated with mitochondrial lipid peroxidation and progression of liver damage.     

Mitochondria-targeted ubiquinone (MitoQ) decreases ethanol-dependent micro and macro hepatosteatosis

Chronic alcohol-induced liver disease results in inflammation, steatosis, and increased oxidative and nitrosative damage to the mitochondrion. We hypothesized that targeting an antioxidant to the mitochondria would prevent oxidative damage and attenuate the steatosis associated with alcoholic liver disease. To test this we investigated the effects of mitochondria-targeted ubiquinone (MitoQ) (5 and 25 mg/kg/day for 4 weeks) in male Sprague-Dawley rats consuming ethanol using the Lieber-DeCarli diet with pair-fed controls. Hepatic steatosis, 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE), hypoxia inducible factor α (HIF1α), and the activity of the mitochondrial respiratory chain complexes were assessed. As reported previously, ethanol consumption resulted in hepatocyte ballooning, increased lipid accumulation in the form of micro and macrovesicular steatosis, and induction of cytochrome P450 2E1 (CYP2E1). MitoQ had a minor effect on the ethanol-dependent decrease in mitochondrial respiratory chain proteins and their activities; however, it did decrease hepatic steatosis in ethanol-consuming animals and prevented the ethanol-induced formation of 3-NT and 4-HNE. Interestingly, MitoQ completely blocked the increase in HIF1α in all ethanol-fed groups, which has previously been demonstrated in cell culture models and shown to be essential in ethanol-dependent hepatosteatosis. CONCLUSION: These results demonstrate the antioxidant capacity of MitoQ in alleviating alcohol-associated mitochondrial reactive oxygen species (ROS) and several downstream effects of ROS/RNS (reactive nitrogen species) production such as inhibiting protein nitration and protein aldehyde formation and specifically ROS-dependent HIF1α stabilization.     

Effects of light and dark beer on hepatic cytochrome P-450 expression in male rats receiving alcoholic beverages as part of total enteral nutrition

BACKGROUND: Alcoholic beverages contain many congeners in addition to ethanol. Therefore, consumption of alcoholic beverages may have considerably different effects on expression of hepatic microsomal monooxygenases than the relatively selective induction of cytochrome P-450 (CYP) 2E1 observed after consumption of pure ethanol. METHODS:: In the current study, we compared the effects of two beers: lager (a light roasted beer) and stout (a dark roasted beer) with those of an equivalent amount of pure ethanol on hepatic CYP expression. Beer or pure ethanol was part of a complete total enteral nutrition diet that was infused intragastrically into male Sprague Dawley rats for 21 days. At the end of the infusion period, rats were euthanized, and liver and intestinal microsomes were prepared. Expression and activity of CYP1A1/2, CYP2B1, CYP2E1, CYP3A, and CYP4A were assessed by Western immunoblotting and by using CYP enzyme-specific substrates, respectively. RESULTS: mRNA and protein levels of CYP4A1 were elevated only in stout-treated animals. However, lauric acid 12-hydroxylase activity (a CYP4A-specific activity) was reduced (p < or = 0.05) in microsomes from lager- and stout-fed rats. After oxidation with potassium ferricyanide, this activity was significantly increased in microsomes from stout-fed animals. The relative expression of CYP2E1 and CYP2B1 and the activities toward p-nitrophenol, pentoxyresorufin, or benzyloxyresorufin did not differ between beers or compared with pure ethanol or controls. However, the mean expression of CYP1A2, CYP3A, and CYP4A apoproteins was greater in liver microsomes from stout-infused rats than in those from lager-infused rats, ethanol-infused rats, and diet controls (p < or = 0.05). In addition, although no significant differences were observed in ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), midazolam, or testosterone hydroxylase activities between groups, stout-infused rats had greater hepatic microsomal erythromycin N-demethylase activity than other groups (p < or = 0.05). CONCLUSIONS: Stout contains components other than ethanol that interact in a complex fashion with the monooxygenase system.     

Microsomal Ethanol-Oxidizing System (MEOS): The First 30 Years (1968-1998)–A Review

Oxidation of ethanol via alcohol dehydrogenase (ADH) explains various metabolic effects of ethanol but does not account for the tolerance and a number of associated disorders that develop in the alcoholic. These were elucidated by the discovery of the microsomal metabolism of ethanol. The physiologic role of this system comprises gluconeogenesis from ketones, fatty acid metabolism, and detoxification of xenobiotics, including ethanol. After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases, with an associated rise in cytochromes P-450, especially CYP2E1. This induction is associated with proliferation of the endoplasmic reticulum, both in experimental animals and in humans. The role of MEOS in vivo and its increase after chronic ethanol consumption was shown most conclusively in alcohol dehydrogenase-negative deer mice. Enhanced ethanol oxidation is associated with cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, there is increased conversion of known hepatotoxic agents (such as CCl₄) to toxic metabolites, which may explain the enhanced susceptibility of alcoholics to the adverse effects of industrial solvents. CYP2E1 also has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis (e.g., from dimethylnitrosamine). Moreover, catabolism of retinol is accelerated and there also is induction of microsomal enzymes involved in lipoprotein production, resulting in hyperlipemia. Contrasting with the chronic effects of ethanol consumption, acute ethanol intake inhibits the metabolism of other drugs through competition for the at least partially shared microsomal pathway. In addition, metabolism by CYP2E1 results in a significant free radical release and acetaldehyde production which, in turn, diminish reduced glutathione (GSH) and other defense systems against oxidative stress. Acetaldehyde also forms adducts with proteins, thereby altering the functions of mitochondria and of repair enzymes. Increases of CYP2E1 and its mRNA prevail in the perivenular zone, the area of maximal liver damage. CYP1A2 and CYP3A4, two other perivenular P-450s, can also sustain the metabolism of ethanol, thereby contributing to MEOS activity and possibly liver injury. By contrast, CYP2E1 inhibitors oppose alcohol-induced liver damage, but heretofore available compounds were too toxic for clinical use. Recently, however, polyenylphosphatidylcholine (PPC), an innocuous mixture of polyunsaturated lecithins extracted from soybeans, was discovered to decrease CYP2E1 activity. PPC (and its active component dilino-leoylphosphatidylcholine) also oppose hepatic oxidative stress and fibrosis. PPC is now being tested clinically for the prevention and treatment of liver disease in the alcoholic.     

Induction of cytochrome P450 2E1 [corrected] promotes liver injury in ob/ob mice

Cytochrome P450 2E1 (CYP2E1) activates several hepatotoxins and contributes to alcoholic liver damage. Obesity is a growing health problem in the United States. The aim of the present study was to evaluate whether acetone- or pyrazole-mediated induction of CYP2E1 can potentiate liver injury in obesity. CYP2E1 protein and activity were elevated in acetone- or pyrazole-treated obese and lean mice. Acetone or pyrazole induced distinct histological changes in liver and significantly higher aminotransferase enzymes in obese mice compared to obese controls or acetone- or pyrazole-treated lean mice. Higher caspase-3 activity and numerous apoptotic hepatocytes were observed in the acetone- or pyrazole-treated obese mice. Increased protein carbonyls, malondialdehyde, 4-hydroxynonenal-protein adducts, elevated levels of inducible nitric oxide synthase, and higher 3-nitrotyrosine protein adducts were found in livers of acetone- or pyrazole-treated obese animals, suggesting elevated oxidative and nitrosative stress. Liver tumor necrosis factor alpha levels were higher in pyrazole-treated animals. The CYP2E1 inhibitor chlormethiazole and iNOS inhibitor N-(3-(aminomethyl)-benzyl) acetamidine abrogated the toxicity and the oxidative/nitrosative stress elicited by the induction of CYP2E1. CONCLUSION: These results show that obesity contributes to oxidative stress and liver injury and that induction of CYP2E1 enhances these effects.     

Induction of Cytochrome P-4502E1 by Ethanol in Rat Kupffer Cells

Ethanol has been shown to affect several Kupffer cell functions, but the mechanisms underlying these changes are unknown. One possible mediator is cytochrome P-4502E1 (CYP2E1), an ethanol-inducible enzyme that has been associated with toxic effects in the liver, as well as in many extrahepatic organs. To assess whether CYP2E1 can be induced by ethanol in Kupffer cells, male rats pair-fed ethanol-containing or control Lieber-DeCarli diets for 3 weeks were studied. Immunoblotting experiments showed that ethanol-treatment caused a 7-fold increase in CYP2E1 content both in Kupffer cells and hepatocytes. When expressed per milligram of S9 protein, the content of CYP2E1 in Kupffer cells was, however, 10 times lower than in hepatocytes. Immunohistochemical studies revealed that CYP2E1 is located in the endoplasmic reticulum of Kupffer cells in vivo and that it is also present in isolated Kupffer cells. In both Kupffer cells and hepatocytes, ethanol feeding increased the hydroxylation of p -nitrophenol, a relatively specific substrate for CYP2E1, demonstrating that the induced CYP2E1 was catalytically active. This reaction was significantly inhibited by anti-CYP2E1 IgG in both types of cells. Although CYP2E1 may not be the predominant pathway for ethanol metabolism in hepatocytes, it is possibly the major one in Kupffer cells. Thus, the induction of CYP2E1 by ethanol in these cells could cause significant changes in intracellular acetaldehyde concentrations which, together with increased lipid peroxidation, may contribute to the development of alcoholic liver injury.     

Fatty Acid ω- and (ω-1)-Hydroxylation in Experimental Alcoholic Liver Disease: Relationship to Different Dietary Fatty Acids

Arachidonic acid concentrations in liver are decreased in response to ethanol administration. In addition, the oxygenated products of arachidonic acid metabolites could affect the severity of alcoholic liver injury. Selective utilization of arachidonic acid by the cyto-chrome P-450 system could, in part, account for the decrease in arachidonic acid. To evaluate this pathway further, male Wistar rats were fed different dietary fats: medium chain triglycerides, palm oil, and corn oil or fish oil with either ethanol or isocaloric amounts of dextrose. Histopathology, cytochrome P-45ME1 (CYP2E1) and cy-tochrome P-4504A (CYP4A), and ω- and (ω-1)-hydroxylation products of lauric and arachidonic acids were evaluated. Ethanol induction of CYP2El was related to the concentration of polyunsaturated fatty acids in the diet; induction of CYP4A by ethanol was seen in all groups. The highest levels of 11-hydroxy-lauric acid and 19-hydroxyarachidonic acid (ω-1) were seen in rats fed ethanol with palm oil and corn oil. Highly significant correlations were seen between the (ω-1)-hydroxylation products and CYP2E1 activity. No correlation was seen between the ω-hydroxylation products and CYP2E1 activity. In contrast, the levels of ω-hydroxylation products correlated with CYP4A. The overall results showed a significant increase in (ω-1)-hydroxylation products in rats fed diets containing significant amounts of linoleic acid (i.e., palm oil and corn oil).     

Markedly Enhanced Cytochrome P450 2E1 Induction and Lipid Peroxidation Is Associated with Severe Liver Injury in Fish Oil—Ethanol-Fed Rats

We evaluated the role of changes in cytochrome P-450 2E1 (CYP 2E1) and lipid peroxidation in relation to development of severe liver injury in fish oil–ethanol-fed rats. The experimental animals (male Wistar rats) were divided into 5 rats/group and were fed the following diets for 1 month: corn oil and ethanol (CO+E) or corn oil and dextrose (CO+D), and fish oil and ethanol (FO+E) or fish oil and dextrose (FO+D). For each animal, microsomal analysis of CYP 2E1 protein, aniline hydroxylase activity, fatty acid composition, and conjugated dienes was conducted. Also, evaluation of severity of pathology was done for each rat. The mean ± SD of the pathology score was significantly higher ( p < 0.01) in the FO+E (6.0 ± 1.3) group than in the CO+E group (3.0 ± 0.5). No pathological changes were evident in the dextrose-fed controls. The CYP 2E1 protein levels (mean ± SD) were significantly higher ( p < 0.01) in the FO+E group (13.1 ± 2.0) compared with the CO+E (4.7 ± 1.2) and FO+D (1.8 ± 0.5) groups. Higher levels of eicosapentaenoic and docosa-hexaenoic acids and lower levels of arachidonic acid were detected in liver microsomes from rats fed fish oil compared with corn oil. A significant correlation was obtained between CYP 2E1 protein and conjugated diene levels ( r = 0.78, p < 0.01). Our results showing markedly increased CYP 2E1 induction and lipid peroxidation in the FO+E group provides one possible explanation for the greater severity of liver injury in this group.     

Metabolism of Acetaldehyde to Acetate by Rat Hepatic P-450s: Presence of Different Metabolic Pathway from Acetaldehyde Dehydrogenase System

NADPH-dependent activity of acetaldehyde oxidation was investigated in microsomes by assaying [¹⁴C]acetic acid produced from [¹⁴C]acetaldehyde with ion-exchange column. Rat hepatic microsomes exhibited acetaldehyde oxidation activity in the presence of NADPH. This activity was induced 2-fold by the treatment of rats with ethanol. We designated this NADPH-dependent oxidation system as microsomal acetaldehyde-oxidizing system (MAOS), to distinguish from the NAD-dependent acetaldehyde oxidation system by acetaldehyde in mitochondria and cytsol. We further investigated essential enzymes contributing to MAOS activity. Acetaldehyde oxidation activity was investigated in eight forms of purified P-450 in a reconstituted system. Cytochrome P-450 (CYP) 2E1 had the highest oxidation activity and CYP1A2 and CYP4A2 had the next highest activity. Other forms had low activity. To assess the contribution of these forms to MAOS activity, immunoblot was done. CYP2E1 was induced 2-fold by ethanol treatment, but CYP1A2 and CYP4A2 were not, reflecting the MAOS activity increased by ethanol treatment. These results suggest that CYP2E1 is the essential enzyme in the MAOS of rats.     

Increased hepatocyte CYP2E1 expression in a rat nutritional model of hepatic steatosis with inflammation

BACKGROUND & AIMS: Nonalcoholic steatohepatitis is morphologically identical to alcoholic hepatitis and has multiple etiologic associations and an unknown pathogenesis. The present study used a rat nutritional model of hepatic steatosis with inflammation to test the hypothesis that induction of the alcohol-inducible hepatic cytochrome P450 (CYP) 2E1 is associated with production of steatohepatitis. METHODS: Rats received a diet devoid of methionine-choline. CYP2E1 protein was detected in liver sections by immunohistochemistry and in hepatic microsomal fractions by immunoblotting; CYP2E1 activity was detected by N-demethylation of N,N-dimethylnltrosamine (NDMA). CYP2E1 messenger RNA was analyzed by Northern blotting and slot blot hybridization. RESULTS: After 4 weeks of methionine-choline devoid diet, macrovesicular steatosis and an inflammatory infiltrate were prominent in hepatic acinar zone 3. CYP2E1 immunostaining was increased and had a more extensive acinar distribution corresponding to that of the steatosis. Microsomal CYP2E1 protein, NDMA activity, and hepatic CYP2E1 messenger RNA levels were all correspondingly increased. CONCLUSIONS: CYP2E1 is induced, partly at a pretranslational level, in this experimental form of steatohepatitis. The finding of biochemical and histological similarities between this nutritional model of hepatic steatosis with inflammation and alcoholic hepatitis indicates possible clues to common pathogenetic mechanisms. The relevance of this finding to human nonalcoholic steatohepatitis remains uncertain and requires further investigation of human liver specimens. (Gastroenterology 1996 Dec;111(6):1645-53)     

Inhibition of rat hepatic mitochondrial aldehyde dehydrogenase-mediated acetaldehyde oxidation by trans-4-hydroxy-2-nonenal

The hepatic oxidation of ethanol has been demonstrated to cause peroxidation of cellular membranes, resulting in the production of aldehydes that are substrates for hepatic aldehyde dehydrogenases. It was the purpose of this study to evaluate the cooxidation of the lipid peroxidation product, trans-4-hydroxy-2-nonenal, and acetaldehyde by high-affinity mitochondrial aldehyde dehydrogenase, which is of prominent importance in the oxidation of ethanol-derived acetaldehyde. Experiments were performed for determination of kinetic parameters for uninhibited acetaldehyde and 4-hydroxynonenal oxidation by semi-purified mitochondrial aldehyde dehydrogenase prepared from male Sprague-Dawley rat liver. The affinity of the enzyme for the substrate at low substrate concentrations and the Michaelis-Menten constant of mitochondrial aldehyde dehydrogenase for acetaldehyde were 25 and 10 times greater, respectively, than those determined for 4-hydroxynonenal. Coincubation of acetaldehyde with physiologically relevant concentrations of 4-hydroxynonenal (0.25 to 5.0 mumol/L) with mitochondrial aldehyde dehydrogenase demonstrated that 4-hydroxynonenal is a potent competitive or mixed-type inhibitor of acetaldehyde oxidation, with concentration of 4-hydroxynonenal required for a twofold increase in the slope of the Lineweaver-Burk plot for acetaldehyde oxidation by ALDH of 0.48 mumol/L. The results of this study suggest that the aldehydic lipid peroxidation product, trans-4-hydroxy-2-nonenal, is a potent inhibitor of hepatic acetaldehyde oxidation and may potentiate the hepatocellular toxicity of acetaldehyde proposed to be an etiological factor of alcoholic liver disease.     

CYP2E1 activity in patients with alcoholic liver disease

Background/Aims: In addition to the possible toxicological impact of cytochrome P4502E1 (CYP2E1) in alcohol-induced liver damage, its activity can be regarded as a variable for dug action in patients with alcoholic liver disease as CYP2E1 is involved in the metabolism of several drugs, for example, paracetamol and halogenated anesthetics. The purpose of our study was to acquire detailed knowledge of CYP2E1 activity in patients with progressingly severe manifestations of alcoholic liver disease.Methods: The concentration ratio of 6-hydroxy-chlorzoxazone/chlorzoxazone in plasma 2 h after ingestion of 500 mg chlorzoxazone (so-called metabolic ratio) has been shown to reflect CYP2E1 activity in vivo. We examined CYP2E1 activity in 56 Caucasian inpatients with minor (n=20), more pronounced (n=14) and severe alcoholic liver disease (n=22). Alcohol abusers were compared to healthy teetotallers (n=14).Results: Metabolic ratios were increased 3-fold in actively drinking (ethanol-induced) compared to abstaining (non-induced) patients with alcoholic liver disease (1.19±0.84 vs. 0.44±0.45, mean±SD, (p<0.0001). CYP2E1 activity was significantly lower in non-induced patients with severe alcoholic liver disease (0.19±0.10) than in healthy controls (0.50±0.28, p<0.01), abstaning alcohol abusers with minor (0.67±0.60, p<0.01) and more pronounced alcoholic liver disease (0.53±0.31, p<0.01). When non-induced patients with alcoholic liver disease were arranged in progressing order of liver damage (minor, more pronounced, severe alcoholic liver disease), there was a significant decline in CYP2E1 activity (p=0.0008).Conclusions: In non-induced patients, CYP2E1 activity decreases in line with severity of alcoholic liver disease. CYP2E1-mediated drug metabolism is significantly impaired in severe alcoholic liver disease.     

Genetic Repeat Polymorphism in the Regulating Region of CYP2E1: Frequency and Relationship With Enzymatic Activity in Alcoholics

BACKGROUND: Differences in the regulatory region of the CYP2E1 gene could be responsible for the interindividual variation in the cytochrome P-450 2E1 (CYP2E1) involved in ethanol oxidation. Recently, a polymorphic repeat sequence in the human gene was described between -2178 and -1945 base pairs. Its frequency seemed to vary among different ethnic populations, and it was suspected to be related to an increased inducibility to further ethanol intake. In the study reported here, the frequency of this polymorphism was investigated in a white French population. Its relationship with the previously described PstI/RsaI or DraI CYP2E1 polymorphisms, alcoholism, alcoholic liver disease, and inducibility of CYP2E1 by ethanol was examined. METHODS: The polymorphic region was characterized by polymerase chain reaction in 103 controls, 148 alcoholic subjects without liver diseases, and 98 others with liver cirrhosis. By using in vivo chlorzoxazone (CHZ) metabolism, CYP2E1 phenotype was assessed in 36 non-ethanol-induced subjects (17 controls and 19 withdrawn alcoholics) and in 14 ethanol-induced subjects (10 controls after ingestion of 0.8 g/kg ethanol and four alcoholics with 100 g of daily intake). This phenotype was expressed as the 6-hydroxy CHZ/CHZ ratio. RESULTS: The rare allele frequency was found to be 1.58% in whites (n = 349). Neither significant association with alcoholism or alcoholic liver diseases, nor relationship with the PstI/RsaI polymorphism, was observed. But the DraI polymorphism was more frequent among the heterozygous subjects when compared with wild-type homozygous ones (p < 0.05). The CYP2E1 phenotype was similar in wild-type homozygotes and in heterozygotes at the constitutive level, as well as after induction with ethanol. CONCLUSIONS: Our data suggest that CYP2E1 repeat polymorphism does not seem to constitute a major factor for interindividual differences in CYP2E1 expression and susceptibility to alcohol-related disorders in whites.