Metabolic aspects of alcoholic liver damage: 1984/1985 update. 2: Microsomal enzyme induction and hypermetabolism

In the second part of this review, the effect of ethanol on hepatic microsomal enzymes is primarily discussed. Since ethanol is metabolized via a cytochrome P-450 dependent biotransformation system (MEOS) in hepatic microsomes, the microsomal enzyme induction in the smooth endoplasmic reticulum has to be considered as an adaptive response. This enzyme induction results in an accelerated metabolism of ethanol. However, subsequently, the negative consequences of such a microsomal enzyme induction are predominant. Acetaldehyde production increases and oxygen consumption is enhanced leading to pericentral (perivenular) hypoxia. In addition, microsomal enzyme induction results in an enhanced metabolism of drugs, xenobiotics and hepatotoxins and thus to an increased production of toxic intermediates. Also procarcinogens are activated to a higher degree in microsomes following chronic ethanol consumption. Subsequently, an enhanced microsomal metabolism of vitamin A may explain the low serum concentrations of this vitamin in the alcoholic and may lead to toxic metabolites of retinol. The quantitative role of an enhanced reoxidation of NADH responsible for an increased oxidation of alcohol following chronic ethanol ingestion has still to be determined. However, according to recent investigations, a thyroid hormone induced hypermetabolism seems unlikely.     

Polyenylphosphatidylcholine opposes the increase of cytochrome P-4502E1 by ethanol and corrects its iron-induced decrease

Dietary iron overload damages membrane phospholipids and decreases microsomal cytochromes P-450. We wondered whether this might also pertain to cytochrome P-4502El (2E1) and whether polyenyiphosphatidylcholine (PPC), a 94-96% pure mixture of linoleate-rich polyunsaturated phosphatidylcholines that protects against alcohol-induced liver injury, also affects 2E1, either in the presence or absence of iron. Accordingly, rats were fed for 8 weeks our standard liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either PPC (3 g/1000 Cal) or equivalent amounts of linoleate (as safflower oil). 2E1 was assessed by Westem blots and by two of its characteristic enzyme activities: the microsomal ethanol oxidizing system (MEOS), evaluated by the conversion of ethanol to acetaldehyde (determined by head space GC), and p-nitrophenolhydroxylase (PNP) activity, measured by HPLC with W detection of 4nitrocatechol. With ethanol (36% of energy) replacing carbohydrates, 2E1 content increased 10-fold, with a corresponding increase in PNP and MEOS activities, but when carbonyl iron (5 g/1000 Cal) was added, the induction was significantly reduced. This iron-induced decrease was corrected by PPC. PPC is rich in linoleate, but when the latter was given as triglycerides (safflower oil), there was no effect, whereas hepatic nonheme iron content was the same in both these groups. It also was found that in the absence of iron, the ethanol-mediated induction of 2E1 and its corresponding enzyme activities were significantly less with PPC ( p < 0.001) than with safflower oil. In addition, in alcohol-fed animals, PPC decreased the oxidative stress (as determined by F₂-isoprostanes), which reflects yet another hepatoprotective effect of PPC.     

Microsomal Ethanol‐Oxidizing System: Success Over 50 Years and an Encouraging Future

Fifty years ago, in 1968, the pioneering scientists Charles S. Lieber and Leonore M. DeCarli discovered the capacity for liver microsomes to oxidize ethanol (EtOH) and named it the microsomal ethanol‐oxidizing system (MEOS), which revolutionized clinical and experimental alcohol research. The last 50 years of MEOS are now reviewed and highlighted. Since its discovery and as outlined in a plethora of studies, significant insight was gained regarding the fascinating nature of MEOS: (i) MEOS is distinct from alcohol dehydrogenase and catalase, representing a multienzyme complex with cytochrome P450 (CYP) and its preferred isoenzyme CYP 2E1, NADPH–cytochrome P450 reductase, and phospholipids; (ii) it plays a significant role in alcohol metabolism at high alcohol concentrations and after induction due to prolonged alcohol use; (iii) hydroxyl radicals and superoxide radicals promote microsomal EtOH oxidation, assisted by phospholipid peroxides; (iv) new aspects focus on microsomal oxidative stress through generation of reactive oxygen species (ROS), with intermediates such as hydroxyethyl radical, ethoxy radical, acetyl radical, singlet radical, hydroxyl radical, alkoxyl radical, and peroxyl radical; (v) triggered by CYP 2E1, ROS are involved in the initiation and perpetuation of alcoholic liver injury, consequently shifting the previous nutrition‐based concept to a clear molecular‐based disease; (vi) intestinal CYP 2E1 induction and ROS are involved in endotoxemia, leaky gut, and intestinal microbiome modifications, together with hepatic CYP 2E1 and liver injury; (vii) circulating blood CYP 2E1 exosomes may be of diagnostic value; (viii) circadian rhythms provide high MEOS activities associated with significant alcohol metabolism and potential toxicity risks as a largely neglected topic; and (ix) a variety of genetic animal models are useful and have been applied elucidating mechanistic aspects of MEOS. In essence, MEOS along with its CYP 2E1 component currently explains several mechanistic steps leading to alcoholic liver injury and has a promising future in alcohol research.     

Azide inhibits human cytochrome P -4502E1, 1A2, and 3A4

BACKGROUND: Recently, we showed that, in addition to cytochrome P-4502E1 (CYP2E1), CYP1A2 and CYP3A4 also contribute to the microsomal ethanol oxidizing system (MEOS). When MEOS activity is measured, sodium azide commonly is used to block the contaminating catalase. However, although CYP2E1 is considered insensitive to azide, its effect on the other P-450s is unknown. Therefore, the aim of the present study was to determine the effect of azide on human recombinant and hepatic CYP2E1, CYP1A2, and CYP3A4. METHODS AND RESULTS: Concentrations of sodium azide as low as 0.1 mM markedly inhibited the specific ethanol oxidation (mean +/- SEM) by recombinant CYP1A2 and CYP3A4 expressed in HepG2 cells (to 16 +/- 1% and 22 +/- 2% of control without azide, respectively; p < 0.01). By contrast, the specific activity of CYP2E1 was only slightly (and not significantly) inhibited at this azide concentration (to 79 +/- 12% of control). Similarly, in human liver microsomes (n = 6), 0.1 mM azide strongly inhibited CYP1A2-dependent (to 25 +/- 2%) and CYP3A4-dependent (to 15 +/- 2%) ethanol oxidation, whereas CYP2E1 was inhibited only at 10 mM azide (to 60 +/- 10%). Azide also strongly affected the apparent kinetic values of all three isoenzymes. Furthermore, azide inhibited the specific monooxygenase activities, both by recombinant and microsomal P-450s. CYP2E1-specific p-nitrophenol hydroxylation was the most sensitive to azide, whereas CYP1A2-dependent 7-methoxyresorufin O-dealkylation was only slightly inhibited. Judging from its effect on p-nitrophenol hydroxylation by human liver microsomes, the inhibition of azide was competitive (Ki 0.09 mM). CONCLUSIONS: Sodium azide at a concentration as low as 0.1 mM inhibited ethanol oxidation by CYP1A2 and CYP3A4. With CYP2E1, although oxidation of 50 mM ethanol was not inhibited by 0.1 mM azide, higher azide concentrations were inhibitory and 0.1 mM azide seemed to affect the kinetics of ethanol oxidation by CYP2E1. Therefore, azide should be avoided when measuring the MEOS activity because it may lead to underestimation, especially of CYP1A2- and CYP3A4-dependent ethanol oxidation.     

Attenuation of alcohol-induced apoptosis of hepatocytes in rat livers by polyenylphosphatidylcholine (PPC)

BACKGROUND: Alcohol consumption increases apoptosis of hepatocytes. This effect appears to be mediated by the induction of hepatic cytochrome P-4502E1(CYP2E1) and its generation of free radicals, which results in an enhanced lipid peroxidation that initiates apoptosis. Because polyenylphosphatidylcholine (PPC), a soybean extract rich in polyunsaturated phosphatidylcholines, decreases the induction of ethanol-specific CYP2E1 and opposes oxidative stress, we hypothesized that PPC supplementation may attenuate hepatocyte apoptosis caused by ethanol ingestion. METHODS: Twenty-eight male Sprague Dawley rats were pair-fed Lieber-DeCarli liquid diets containing 36% of energy as alcohol or an isocaloric amount of carbohydrate for 28 days. Half of the rats were given PPC (3 g/liter), whereas the other half received the same amount of linoleate (as safflower oil) and of choline as the bitartrate. An additional dose of alcohol (3 g/kg) was given intragastrically 90 min before the livers were removed. We assessed apoptosis in formalin-fixed, paraffin-embedded liver sections by using the TUNEL (terminal transferase dUTP nick end labeling) assay. Apoptotic hepatocytes were identified by positive TUNEL staining in conjunction with condensation of nucleoplasm or margination of chromatin. In each rat, 20,000 to 60,000 hepatocytes were counted by light microscopy by using Image-Pro Plus computer software, and the incidence of apoptosis was expressed as the percentage of total hepatocytes. RESULTS: Alcohol feeding resulted in a 4.5-fold increase in apoptosis of hepatocytes compared to pair-fed control rats; PPC supplementation decreased the alcohol-induced apoptosis to less than half. No difference in the incidence of apoptosis between the control and PPC-supplemented rats was found in the absence of alcohol. Apoptosis was distributed randomly in the liver lobules of the rats fed the control diet, whereas the alcohol-induced apoptosis was significantly increased in the perivenular area. PPC supplementation strikingly reduced this effect. CONCLUSIONS: PPC attenuates alcohol-induced apoptosis of hepatocytes; this effect may provide a mechanism for PPC's protection against liver injury, possibly in association with its antioxidative action via the down-regulation of ethanol-mediated CYP2E1 induction.     

Respective Roles of Human Cytochrome P-4502E1, 1A2, and 3A4 in the Hepatic Microsomal Ethanol Oxidizing System

The microsomal ethanol oxidizing system comprises an ethanolinducible cytochrome P-4502E1, but the involvement of other P-450s has also been suggested. In our study, human CYP2E1, CYP1A2, and CYP3A4 were heterologously expressed in HepG2 cells, and their ethanol oxidation was assessed using a corresponding selective inhibitor: all three P-450 isoenzymes metabolized ethanol. Selective inhibitors-4-methylpyrazole (CYP2E1), furafylline (CYP1A2), and troleandomycin (CYP3A4)?also decreased microsomal ethanol oxidation in the livers of 18 organ donors. The P-450-dependent ethanol oxidizing activities correlated significantly with those of the specific monooxygenases and the immunochemically determined microsomal content of the respective P-450. The mean CYP2E1-dependent ethanol oxidation in human liver microsomes [1.41 ± 0.11 nmol min^-1 (mg protein)^-1] was twice that of CYP1A2 (0.61 ± 0.07) or CYP3A4 (0.73 ± 0.11) (p < 0.05). Furthermore, CYP2E1 had the highest (p < 0.05) specific activity [28 ± 2 nmol min^-1 (nmol CYP2E1)^-1 versus 17 ± 3 nmol min^-1 (nmol CYP1A2)^-1, and 12 ± 2 nmol min^-1 (CYP3A4)^-1, respectively]. Thus, in human liver microsomes, CYP2E1 plays the major role. However, CYP1A2 and CYP3A4 contribute significantly to microsomal ethanol oxidation and may, therefore, also be involved in the pathogenesis of alcoholic liver disease.     

Mutations in the Exons and Exon-Intron Junction Regions of Human Cytochrome P-4502E1 Gene and Alcoholism

Cytochrome P-4502E1 (CYP2E1) is a major component of the microsomal ethanol-oxidizing system (MEOS) and is also involved in the metabolism of a variety of foreign compounds, including carcinogens. It has been shown that there are interindividual variations in the expression of human CYP2E1. Gene-environmental interactions have been suggested to account for the difference. In this study, we screened nine exons and exonintron junctions of the human CYP2E1 gene for detecting allelic variants in genomic DNA samples obtained from 115 Japanese controls, 96 alcoholics, and 44 patients with alcoholic liver diseases. A novel missense mutation in exon 2 (V72L) was found in Japanese controls, but the frequency was low (2.6%). In addition, two novel silent mutations (T303T and F420F), together with one mutation in intron 2, were found. However, no association of these mutations with alcoholism and alcoholic liver diseases was found. Our data indicate that nucleotide replacement in the open reading frame of CYP2E1 gene is not a major factor for interindividual differences in expression of CYP2E1 and susceptibility to alcohol-related disorders.     

Effect of omeprazole on ethanol oxidation and aniline hydroxylation in rat hepatic microsomes

Omeprazole, a substituted benzimidazole, is a potent gastric acid antisecretory drug, which inhibits the hepatic oxidative drug metabolism in vitro and in vivo. The effect of omeprazole on the microsomal ethanol oxidizing system (MEOS) and, since ethanol-induced cytochrome P-450 reveals a high activity for aniline hydroxylation, on aniline hydroxylase (AH) has been investigated in rat liver microsomes. Omeprazole inhibits microsomal AH activity significantly in a dose dependent manner, while this was not the case for MEOS activity. These data give indirect evidence that the microsomal metabolism of both ethanol and aniline is mediated by different isoenzymes of cytochrome P-450 and that omeprazole exhibits a different affinity to both compounds. Therefore, it must be emphasized that drug interactions with omeprazole have to be tested experimentally in each individual case, since it is impossible to predict such interactions solely on the knowledge of the drug's metabolic pathway.     

Liver Diseases by Alcohol and Hepatitis C: Early Detection and New Insights in Pathogenesis Lead to Improved Treatment

Much progress has been made in the understanding of the pathogenesis of alcoholic liver disease, resulting in improvement of treatment. Therapy must include correction of nutritional deficiencies, while taking into account changes of nutritional requirements. Methionine is normally activated to S‐adenosylmethionine (SAMe). However, in liver disease, the corresponding enzyme is depressed. The resulting deficiencies can be attenuated by the administration of SAMe but not by methionine. Similarly, phosphatidylethanolamine methyltransferase activity is depressed, but the lacking phosphatidylcholine (PC) can be administrated as polyenylphosphatidylcholine (PPC). Chronic ethanol consumption increases CYP2E1, resulting in increased generation of toxic acetaldehyde and free radicals, tolerance to ethanol and other drugs, and multiple ethanol‐drug interactions. Experimentally, PPC opposes CYP2E1 induction and fibrosis. Alcoholism and hepatitis C infection commonly co‐exist, with acceleration of fibrosis, cirrhosis, and hepatocellular carcinoma. PPC is being tested clinically as a corresponding antifibrotic agent. Available antiviral agents are contraindicated in the alcoholic. Anti‐inflammatory agents, such as steroids, may be selectively useful. Finally, anticraving agents, such as naltrexone or acamprosate, should be part of therapy.     

Aetiology and pathogenesis of alcoholic liver disease

Until the 1960s, liver disease of the alcoholic patient was attributed exclusively to dietary deficiencies. Since then, however, our understanding of the impact of alcoholism on nutritional status has undergone a progressive evolution. Alcohol, because of its high energy content, was at first perceived to act exclusively as ‘empty calories’ displacing other nutrients in the diet, and causing primary malnutrition through decreased intake of essential nutrients. With improvement in the overall nutrition of the population, the role of primary malnutrition waned and secondary malnutrition was emphasized as a result of a better understanding of maldigestion and malabsorption caused by chronic alcohol consumption and various diseases associated with chronic alcoholism. At the same time, the concept of the direct toxicity of alcohol came to the forefront as an explanation for the widespread cellular injury. Some of the hepatotoxicity was found to result from the metabolic disturbances associated with the oxidation of ethanol via the liver alcohol dehydrogenase (ADH) pathway and the redox changes produced by the generated NADH, which in turn affects the metabolism of lipids, carbohydrates, proteins and purines. Exaggeration of the redox change by the relative hypoxia which prevails physiologically in the perivenular zone contributes to the exacerbation of the ethanol-induced lesions in zone 3. In addition to ADH, ethanol can be oxidized by liver microsomes: studies over the last twenty years have culminated in the molecular elucidation of the ethanol-inducible cytochrome P450IIE1 (CYP2E1) which contributes not only to ethanol metabolism and tolerance, but also to the selective hepatic perivenular toxicity of various xenobiotics. Their activation by CYP2E1 now provides an understanding for the increased susceptibility of the heavy drinker to the toxicity of industrial solvents, anaesthetic agents, commonly prescribed drugs, ‘over the counter’ analgesics, chemical carcinogens and even nutritional factors such as vitamin A. Ethanol causes not only vitamin A depletion but it also enhances its hepatotoxicity. Furthermore, induction of the microsomal pathway contributes to increased acetaldehyde generation, with formation of protein adducts, resulting in antibody production, enzyme inactivation and decreased DNA repair; it is also associated with a striking impairment of the capacity of the liver to utilize oxygen. Moreover, acetaldehyde promotes glutathione depletion, free-radical mediated toxicity and lipid peroxidation. In addition, acetaldehyde affects hepatic collagen synthesis: both in vivo and in vitro (in cultured myofibroblasts and lipocytes), ethanol and its metabolite acetaldehyde were found to increase collagen accumulation and mRNA levels for collagen. This new understanding of the pathogenesis of alcoholic liver disease may eventually improve therapy with drugs and nutrients.     

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.     

Differences in Hepatic Microsomal Cytochrome P-450 Isoenzyme Induction by Pyrazole, Chronic Ethanol, 3-Methylcholanthrene, and Phenobarbital in High Alcohol Sensitivity (HAS) and Low Alcohol Sensitivity (LAS) Rats

High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Early Alcoholic Liver Injury: Formation of Protein Adducts with Acetaldehyde and Lipid Peroxidation Products, and Expression of CYP2E1 AND CYP3A

The formation of protein adducts with reactive aldehydes resulting from ethanol metabolism and lipid peroxidation has been suggested to play a role in the pathogenesis of alcoholic liver injury. To gain further insight on the contribution of such aldehydes in alcoholic liver disease, we have compared the appearance of acetaldehyde, malondialdehyde, and 4-hydroxynonenal adducts with the expression of cytochrome P-450IIE1, and cytochrome P-4503A enzymes in the liver of rats fed alcohol with a high-fat diet for 2 to 4 weeks according to the Tsukamoto-French procedure and in control rats (high-fat liquid diet or no treatment). Urine alcohol and serum aminotransferase levels were recorded, and the liver pathology was scored from 0 to 10 according to the presence of steatosis, inflammation, necrosis, and fibrosis. The ethanol treatment resulted in the accumulation of fat, mild necrosis and inflammation, and a mean liver pathology score of 3 (range: 1 to 5). Liver specimens from the ethanol-fed animals with early alcohol-induced liver injury were found to contain perivenular, hepatocellular acetaldehyde adducts. Malondialdehyde and 4-hydroxynonenal adducts were also present showing a more diffuse staining pattern with occasional sinusoidal reactions. In the control animals, a faint positive reaction for the hydroxynonenal adduct occurred in some of the animals fed the high fat diet, whereas no specific staining was observed in the livers from the animals receiving no treatment Expression of both CYP2E1 and CYP3A correlated with the amount of protein adducts in the liver of alcohol-treated rats. Distinct CVP2E1 -positive immunohistochemistry was seen in 3 of 7 of the ethanol-fed animals. In 5 of 7 of the ethanol-fed animals, the staining intensities for CYP3A markedly exceeded those obtained from the controls. The present findings indicate that acetaldehyde and lipid peroxidatjon-derived adducts are generated in the early phase of alcohol-induced liver disease. The formation of protein adducts appears to be accompanied by induction of both CVP2E1 and CVP3A.     

Microsomal Acetaldehyde Oxidation is Negligible in the Presence of Ethanol

The microsomal ethanol oxidizing system (MEOS), inducible by ethanol and acetone, oxidizes ethanol to acetaldehyde, which causes many toxic effects associated with excess ethanol. Recent studies reported that rat liver microsomes also oxidize acetaldehyde, thereby challenging the validity of the assessment of MEOS activity by measuring acetaldehyde production and suggesting that MEOS activity results in the accumulation not of acetaldehyde but, rather, of its less toxic metabolite, acetate. To address these issues, we compared both metabolic rates of ethanol and acetaldehyde and the effect of ethanol on the acetaldehyde metabolism. Liver microsomes were prepared from Sprague-Dawley rats induced either with acetone for 3 days or ethanol for 3 weeks. NADPH-dependent acetaldehyde (300 /μM) metabolism was measured in two ways: (1) by detection of acetaldehyde disappearance by headspace gas chromatography, and (2) by assessment of acetaldehyde oxidation by liquid scintillation counting of acetate formed from [1,2-¹⁴C]acetaldehyde. Ethanol (50 mM) oxidation was measured by gas chromatography. In acetone- and ethanol-induced rat liver microsomes, the acetaldehyde disappearance (p < 0.0001) and oxidation (p < 0.0001) rates were both significantly increased. The rates of acetaldehyde oxidation paralleled those of p-nitrophenol hydroxylation (r= 0.974, p < 0.0001), with a K_m of 82 ± 14 μM and a V_max of 4.8 ± 0.5 nmol/min/mg protein in acetone-induced microsomes. Acetaldehyde disappearance in acetone-induced microsomes and acetaldehyde oxidation in acetone-induced and ethanol-induced microsomes were significantly lower than the corresponding ethanol oxidation, with rates (nmol/min/mg protein) of 4.6 ± 0.6 versus 9.0 ± 0.8 (p < 0.005), 4.4 ± 0.3 versus 9.1 ± 0.5 (p < 0.0005), and 14.0 ± 0.9 versus 19.5 ± 1.8 (p < 0.05), respectively. The presence of 50 mM ethanol decreased this metabolism to 0.9 ± 0.3 (p < 0.005), 0.5 ± 0.1 (p < 0.001), and 1.8 ± 0.3 (p < 0.001), resulting in rates of acetaldehyde metabolism of only 9.8 ± 3.2%, 6.0 ± 0.5%, and 9.5 ± 1.2% (respectively) of those of ethanol oxidation. In conclusion, rat liver microsomes oxidize acetaldehyde at much lower rates than ethanol, and this acetaldehyde metabolism is strikingly inhibited by ethanol. Accordingly, acetaldehyde formation provides an accurate assessment of MEOS activity. Furthermore, because acetaldehyde production vastly exceeds its oxidation, the net result of MEOS activity is the accumulation of this toxic metabolite.     

Ethanol enhances retinoic acid metabolism into polar metabolites in rat liver via induction of cytochrome P4502E1

BACKGROUND & AIMS: Long-term and excessive ethanol intake results in decreased plasma and hepatic levels of retinoic acid (RA), the most active derivative of vitamin A. The decrease of RA by ethanol treatment has been proposed to be a cytochrome P450 enzyme (CYP)-dependent process. However, the role of the major ethanol-induced CYP, CYP2E1, in the metabolism of RA has not been defined. METHODS: In vitro incubations of RA with microsomal fractions of liver tissue containing CYPs from either ethanol-exposed or non-ethanol-exposed rats were carried out using chemical inhibitors and antibodies against various CYPs. In vivo, both ethanol-exposed and non-ethanol-exposed rats were treated with or without chlormethiazole, a specific CYP2E1 inhibitor, for 1 month. RA and its catabolic metabolites were analyzed by high-performance liquid chromatography and spectral analysis. RESULTS: Incubation of RA with the liver microsomal fraction from ethanol-exposed rats resulted in greater disappearance of RA and increased appearance of 18-hydroxy-RA and 4-oxo-RA compared with control rat liver microsomal fractions. The enhancement of RA catabolism by ethanol was inhibited by both CYP2E1 antibody and specific inhibitors (allyl sulfide and chlormethiazole) in a dose-dependent fashion, whereas the metabolism of RA into polar metabolites was abolished completely by nonspecific CYP inhibitors (disulfiram and liarozole). Furthermore, treatment with chlormethiazole in ethanol-fed rats in vivo restored both hepatic and plasma RA concentrations to normal levels. CONCLUSIONS: Ethanol-induced CYP2E1 plays a major role in the degradation of RA, which may provide a possible biochemical mechanism for chronic and excessive ethanol intake as a risk for both hepatic and extrahepatic cell proliferation and carcinogenesis.     

Effect of Ethanol on Lipoprotein Secretion in Two Human Hepatoma Cell Lines, HepG2 and Hep3B

The two human hepatoma cell lines, HepG2 and Hep3B, have been demonstrated to metabolize ethanol efficiently even in the absence of alcohol dehydrogenase. By using specific metabolic inhibitors, it was found that the microsomal ethanol-oxidizing system (MEOS) plays a significant role in ethanol metabolism in these two cell lines. There is a strong positive correlation between the rates of ethanol metabolism and the total cytochrome P-450 levels in the hepatoma cells. The involvement of the cytochrome P-450 system was further supported by the induction of aniline p-hydroxylase activity after ethanol treatment. However, the 3- to 4-fold elevation in aniline p-hydroxylase activity was not accompanied by an increase in cytochrome P450IIE1 mRNA level. Exposure of HepG2 and Hep3B cells to ethanol resulted in an increase of accumulation of apoA-I (15%-45% over control) in a dose-dependent manner (from 5 to 50 mM) of ethanol over a 24-hr period. All other major apolipoproteins which included apo CII, apo CIII and apoE, with the exception of apoB, were not affected by these treatments. At a concentration of ethanol of 25 mM or greater, accumulation of apoB, VLDL and LDL triglyceride were increased by 20% to 25% over the control level. Elevation of HDL cholesterol (40%-70% over control) was observed when the cells were exposed to an ethanol concentration of > or = 10 mM. Metyrapone, which inhibited the MEOS, was capable of blocking the induction of apoAI caused by ethanol treatment.     

Modulation of experimental alcohol-induced liver disease by cytochrome P450 2E1 inhibitors

This study was done to determine if a relationship exists between CYP2E1 induction by ethanol, lipid peroxidation, and liver pathology in experimental alcohol-induced liver disease in the rat. Rats were fed ethanol with or without diallyl sulfide (DAS) or phenethyl isothiocyanate (PIC) intragastrically for 1 month. CYP2E1 induction by ethanol was correlated with lipid peroxidation, liver microsomal CYP2E1 hydroxylation of paranitrophenol, and the liver pathology score using the data from the PIC-fed rats. Some of the data from the ethanol and DAS-fed rats were not included here because they have been reported elsewhere. Microsomal CYP2E1 protein levels induction by ethanol was decreased by PIC ingestion. Similarly, PIC reduced the increase microsomal reduced form of nicotinamide-adenine dinucleotide (NADPH)-dependent lipid peroxidation and p-nitrophenol hydroxylase (PNPH) activity, induced by ethanol feeding. The lipid peroxidation was reduced to below control levels; however, the pathology score was partially but not significantly reduced by isothiocyanate feeding. CYP2E1 messenger RNA (mRNA) was decreased by both inhibitors of CYP2E1. Immunohistochemical staining of liver for CYP2E1 protein showed that the lobular distribution of the isozyme changed from the centrilobular to a diffuse pattern, with an increase in the periportal region when the CYP2E1 inhibitors were fed with ethanol, and that this change correlated with the change in the distribution of fat in the lobule. The data support the idea that there is a link between CYP2E1 induction by ethanol and the early phase of ethanol-induced liver injury in this rat model. This link may involve lipid peroxidation, but other factors related to CYP2E1 induction must also be involved.     

Hepatic, Metabolic and Toxic Effects of Ethanol: 1991 Update

Until two decades ago, dietary deficiencies were considered to be the only reason for alcoholics to develop liver disease. As the overall nutrition of the population improved, more emphasis was placed on secondary malnutrition and direct hepatotoxic effects of ethanol were established. Ethanol is hepatotoxic through redox changes produced by the NADH generated in its oxidation via the alcohol dehydrogenase pathway, which in turn affects the metabolism of lipids, carbohydrates, proteins, and purines. Ethanol is also oxidized in liver microsomes by an ethanol-inducible cytochrome P-450 (P-450IIE1) that contributes to ethanol metabolism and tolerance, and activates xenobiotics to toxic radicals thereby explaining increased vulnerability of the heavy drinker to industrial solvents, anesthetic agents, commonly prescribed drugs, over-the-counter analgesics, chemical carcinogens, and even nutritional factors such as vitamin A. In addition, ethanol depresses hepatic levels of vitamin A, even when administered with diets containing large amounts of the vitamin, reflecting, in part, accelerated microsomal degradation through newly discovered microsomal pathways of retinol metabolism, inducible by either ethanol or drug administration. The hepatic depletion of vitamin A is strikingly exacerbated when ethanol and other drugs were given together, mimicking a common clinical occurrence. Microsomal induction also results in increased production of acetaldehyde. Acetaldehyde, in turn, causes injury through the formation of protein adducts, resulting in antibody production, enzyme inactivation, decreased DNA repair, and alterations in microtubules, plasma membranes and mitochondria with a striking impairment of oxygen utilization. Acetaldehyde also causes glutathione depletion and lipid peroxidation, and stimulates hepatic collagen production by the vitamin A storing cells (lipocytes) and myofibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteasome inhibition potentiates CYP2E1-mediated toxicity in HepG2 cells

Chronic ethanol consumption causes increased oxidative damage in the liver. Induction of CYP2E1 is one pathway involved in how ethanol produces oxidative stress. Ethanol can cause protein accumulation, decreased proteolysis, and decreased proteasome activity. The objective of this study was to investigate the effect of inhibition of the proteasome activity on CYP2E1-dependent toxicity. HepG2 cells over-expressing CYP2E1 (E47 cells) were treated with arachidonic acid (AA) plus iron, agents important in development of alcoholic liver injury and which are toxic to E47 cells by a mechanism dependent on CYP2E1, oxidative stress, and lipid peroxidation. Addition of various proteasome inhibitors was associated with significant potentiation of the loss of cell viability caused by AA plus iron. Potentiation of toxicity was associated with increased oxidative damage as reflected by an increase in lipid peroxidation and accumulation of oxidized and nitrated proteins in E47 cells and an enhanced decline in mitochondrial membrane potential. Antioxidants prevented the loss of viability and the potentiation of this loss of viability by proteasome inhibition. CYP2E1 levels were elevated about 3-fold by the proteasome inhibitors. Inhibition of proteasome activity also potentiated toxicity of AA alone and toxicity after treatment to remove glutathione (GSH). Similar results were found in hepatocytes from pyrazole-treated rats with high levels of CYP2E1. In conclusion, proteasome activity plays an important role in modulating CYP2E1-mediated toxicity in HepG2 cells by regulating CYP2E1 levels and by removal of oxidized proteins. Such interactions may be important in CYP2E1-catalyzed toxicity of hepatotoxins and in alcohol-induced liver injury.