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)     

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.     

Contribution of NADH Increases to Ethanol's Inhibition of Retinol Oxidation by Human ADH Isoforms

Background:  A decrease in retinoic acid levels due to alcohol consumption has been proposed as a contributor to such conditions as fetal alcohol spectrum diseases and ethanol-induced cancers. One molecular mechanism, competitive inhibition by ethanol of the catalytic activity of human alcohol dehydrogenase (EC 1.1.1.1) (ADH) on all-trans-retinol oxidation has been shown for the ADH7 isoform. Ethanol metabolism also causes an increase in the free reduced nicotinamide adenine dinucleotide (NADH) in cells, which might reasonably be expected to decrease the retinol oxidation rate by product inhibition of ADH isoforms.
Methods:  To understand the relative importance of these two mechanisms by which ethanol decreases the retinol oxidation in vivo we need to assess them quantitatively. We have built a model system of 4 reactions: (1) ADH oxidation of ethanol and NAD⁺, (2) ADH oxidation of retinol and NAD⁺, (3) oxidation of ethanol by a generalized Ethanol_oxidase that uses NAD⁺, (4) NADH_oxidase which carries out NADH turnover.
Results:  Using the metabolic modeling package S crum P y , we have shown that the ethanol-induced increase in NADH contributes from 0% to 90% of the inhibition by ethanol, depending on (ethanol) and ADH isoform. Furthermore, while the majority of flux control of retinaldehyde production is exerted by ADH, Ethanol_oxidase and the NADH_oxidase contribute as well.
Conclusions:  Our results show that the ethanol-induced increase in NADH makes a contribution of comparable importance to the ethanol competitive inhibition throughout the range of conditions likely to occur in vivo, and must be considered in the assessment of the in vivo mechanism of ethanol interference with fetal development and other diseases.     

Metabolic aspects of alcoholic liver damage: 1984/5 update. 1. Epidemiology and alcohol metabolism

In western industrialized countries ethanol is an important etiologic factor in the development of cirrhosis of the liver. Metabolic, immunologic and physico-chemical alterations of the hepatocyte due to ethanol are involved in the pathogenesis of alcoholic liver disease. However, the mechanisms by which ethanol damages the liver are far from clear. During the last two decades, the effect of ethanol on multiple biochemical pathways of the hepatocyte has been investigated intensively. The present paper is focusing on the metabolic aspects of alcoholic liver disease. In the first part of the review, special emphasis has been led on the metabolites of ethanol oxidation, while in the second part microsomal enzyme induction due to alcohol has been discussed. More than 90% of ethanol metabolism takes place in the liver via cytoplasmic alcoholdehydrogenase (ADH) and via a microsomal ethanol oxidizing system (MEOS). The products of these reactions are reduced nicotinadenine dinucleotide phosphate (NADH), acetaldehyde and acetate. NADH alters the redox state of the liver cell favouring all reductive processes. This shift in metabolic pathways results in hyperlactacidaemia, lactacidosis, ketosis and hyperuricaemia. Disturbances of the carbohydrate metabolism may lead either to hypo- or hyperglycaemia. The altered redox state also influences the metabolic pathways of lipid metabolism leading to lipid accumulation within the hepatocyte which can be morphologically observed as alcoholic fatty liver. In addition, porphyrin and collagen metabolism is also affected by the increased NADH/NAD+ ratio. On the other hand, acetaldehyde damages the microtubular system and the mitochondria. Acetaldehyde may also be responsible for the increased lipidperoxidation after chronic ethanol ingestion.     

Inhibition of cytochrome P-450-dependent mixed function oxidation by ethanol

The mechanism of inhibition of cytochrome P-450-dependent mixed function oxidation by ethanol was studied. Ethanol competitively inhibited the binding of hexobarbital to liver microsomes, and increased the low spin signal of cytochrome P-450 in the electron spin resonance spectra. Therefore, ethanol decreased the substrates bound to ferric cytochrome P-450 in the first step of mixed function oxidation. The second step of mixed function oxidation is the reduction of ferric cytochrome P-450-substrate complex by NADPH-cytochrome P-450 reductase. The NADPH-dependent reduction of liver microsomal cytochrome P-450 was biphasic and composed of two first-order reactions. Ethanol decreased the rate constants of the fast and slow phases of microsomal cytochrome P-450 reduction. Thus, it is concluded that the inhibition of drug oxidation by ethanol may be due to the displacement of substrates from cytochrome P-450 and to the inhibition of reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase.     

Human Hepatic Alcohol Dehydrogenase and Human Erythrocyte Catalase Do Not Metabolize the Cytochrome P-4502E1 Substrate, Chlorzoxazone

Studies of cytochrome P-4502E1 (CYP2E1)-mediated oxidation of ethanol have been hampered by the lack of a suitable probe for in vivo human studies. Chlorzoxazone, a prescribed skeletal muscle relaxant, is metabolized to 6-hydroxychlorzoxazone by CYP2E1 and has been advocated as a specific probe of this enzyme on the basis of microsomal studies. The applications of this probe may include delineating the contribution of CYP2E1 to in vivo human ethanol metabolism. However, the activity of nonmicrosomal enzymes in metabolizing chlorzoxazone is unknown. Alcohol dehydrogenase (ADH), predominantly a hepatic cytosolic enzyme, may be more important than CYP2E1 in the oxidation of ethanol to acetaldehyde. The contribution of catalase in the in vivo oxidation of ethanol to acetaldehyde is controversial. To determine if either of these enzymes metabolizes chlorzoxazone and whether ethanol oxidation by either enzyme is inhibited by chlorzoxazone or its metabolite, multiple in vitro studies were performed. ADH enzyme kinetics were performed with human recombinant β₁β₁ and β₃β₃ ADH with ethanol and chlorzoxazone (0.5 to 2.5 mM). Neither ADH isoenzyme exhibited NAD⁺-dependent oxidation of chlorzoxazone, but displayed Michaelis-Menten kinetics for ethanol with K_m values of 89 μM and 34 mM, for β₁β₁, and β₃β₃, respectively. Typical in vivo concentrations of chlorzoxazone and its metabolite, 6-hydroxychlorzoxazone, did not alter β₁β₁, or β₃β₃ ADH-mediated oxidation of ethanol to acetaldehyde. Studies of human hepatic nonmicrosomal enzyme activity were expanded to include all nonmicrosomal NAD⁺-dependent hepatic enzymes by starch gel electrophoresis assessment. Human hepatic enzymatic activity in the presence of chlorzoxazone was similar to that observed in the control sample (no added substrate), suggesting a lack of metabolism by NAD⁺-dependent enzymes. Similarly, human erythrocyte catalase, in the presence of a hydrogen peroxide generating system, did not metabolize chlorzoxazone. Furthermore, neither chlorzoxazone nor 6-hydroxychlorzoxazone altered the catalase-induced formation of acetaldehyde from ethanol. These data are consistent with chlorzoxazone as a specific probe of CYP2E1 that may be useful to alcohol researchers.     

Ethanol Oxidation by Hydroxyl Radicals: Role of Iron Chelates, Superoxide, and Hydrogen Peroxide

Oxygen-derived free radicals such as the hydroxyl radical (.OH) have been shown to mediate the oxidation of ethanol by a variety of oxy radical-generating systems. Among these are microsomal electron transport systems (both intact and purified, reconstituted systems), the coupled oxidation of hypoxanthine or xanthine by xanthine oxidase, and the model iron-ascorbate system. The sequence of reactions leading to the oxy radical-dependent oxidation of ethanol as well as other hydroxyl radical-scavenging agents by these various systems is believed to proceed through the formation of a common intermediate, namely, hydrogen peroxide (H2O2), after dismutation of the superoxide anion radical (O2-.). The presence of iron, especially chelated iron, greatly enhances the production of .OH by serving as an oxidant for O2-. or a reductant for H2O2. Experiments were carried out to evaluate the role of iron, the chelating agent, O2-., and H2O2 in the oxidation of ethanol by a variety of in vitro systems (chemical, enzymatic, and intact membrane bound) that can produce oxy radicals via different mechanisms. The generation of .OH by all the systems studied was sensitive to catalase, which indicates that H2O2 is the precursor of .OH. Superoxide radical appears to be the reducing agent in the hypoxanthine-xanthine oxidase system, indicating an iron-catalyzed Haber-Weiss reaction. In the ascorbate, reductase, and microsomal systems, superoxide radical does not appear to be the reducing agent. However, superoxide radical probably is the precursor of H2O2. While iron plays an important role in the production of .OH by the various systems, the effect of iron depends on the nature of the iron chelate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

Ethanol Metabolism in Deermice: Role of Extrahepatic Alcohol Dehydrogenase

The relative contributions to ethanol metabolism of extrahepatic alcohol dehydrogenase (ADH) and of liver microsomes were assessed in deermice, which lack hepatic low K _m ADH (ADH^-). In vitro kinetic studies showed the existence of high K _m (>1 M) ADH activity in the liver and kidney, and an enzyme with intermediate K _m in the gastric mucosa ( K _m = 133 mM), whereas the low K _m ADH was missing. With deuterated ethanol, ADH^- deermice showed a significant exchange of reducing equivalents that had been equated with ethanol metabolism by others, whereas we found a poor correlation between the rate of exchange and the rate of metabolism. In vitro studies with subcellular fractions, isolated hepatocytes, and tissue slices revealed that neither liver, nor kidney, nor stomach from ADH^- deermice contributed to exchange of reducing equivalents. These findings clearly indicated that the ADHs with high or intermediate K _m of the tissues studied are not responsible for the exchange. Furthermore, gastrectomized ADH^- deermice still showed an exchange of reducing equivalents, thereby dissociating exchange from gastric ADH activity. Moreover, pretreatment with cimetidine (50 mg/kg body weight), an inhibitor of gastric ADH, did not alter the rate of total ethanol elimination when ethanol was given intraperitoneally. In conclusion, when ethanol is given parenterally, the microsomal ethanol-oxidizing system rather than gastric ADH is a major pathway of ethanol oxidation in ADH^- deermice, whereas both pathways contribute significantly to the metabolism of orally administered ethanol.     

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.     

Immunochemical evidence for induction of the alcohol-oxidizing cytochrome P-450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, trichloroethylene, acetone, pyrazole, and isoniazid.

Isozyme 3a of rabbit liver microsomal cytochrome P-450, also termed P-450ALC, was previously isolated in this laboratory from animals administered ethanol or imidazole, and the purified cytochrome was shown to function in the reconstituted system as an oxygenase in catalyzing the oxidation of ethanol and other alcohols. Although liver microsomes from animals treated in various ways exhibit increased alcohol-oxidizing activity, evidence was not available as to whether this was due to enzyme induction or to other factors influencing the activity. Immunochemical quantitation of P-450 isozyme 3a has now been achieved by use of purified antibody to this cytochrome in NaDodSO4/PAGE/blotting and dot-blotting techniques. The specific content of isozyme 3a in liver microsomes was found to be increased from 2- to greater than 4-fold by administration of the following agents, in increasing order of effectiveness as inducers: isoniazid, trichloroethylene, pyrazole, ethanol, imidazole, and acetone. Isozyme 3a represents about 5% of the total P-450 in control animals and is increased to as high as 27% by acetone treatment. Isozyme 3a-dependent butanol-oxidation activity, determined by the inhibitory effect of antibody on the various microsomal preparations, was found to increase proportionally with increased content of this cytochrome.     

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.     

Alterations in Brain Glucose Utilization Accompanying Elevations in Blood Ethanol and Acetate Concentrations in the Rat

Background: Previous studies in humans have shown that alcohol consumption decreased the rate of brain glucose utilization. We investigated whether the major metabolite of ethanol, acetate, could account for this observation by providing an alternate to glucose as an energy substrate for brain and the metabolic consequences of that shift. Methods: Rats were infused with solutions of sodium acetate, ethanol, or saline containing ¹³C‐2‐glucose as a tracer elevating the blood ethanol (BEC) and blood acetate (BAcC) concentrations. After an hour, blood was sampled and the brains of animals were removed by freeze blowing. Tissue samples were analyzed for the intermediates of glucose metabolism, Krebs’ cycle, acyl‐coenzyme A (CoA) compounds, and amino acids. Results: Mean peak BEC and BAcC were approximately 25 and 0.8 mM, respectively, in ethanol‐infused animals. Peak blood BAcC increased to 12 mM in acetate‐infused animals. Both ethanol and acetate infused animals had a lower uptake of ¹³C‐glucose into the brain compared to controls and the concentration of brain ¹³C‐glucose‐6‐phosphate varied inversely with the BAcC. There were higher concentrations of brain malonyl‐CoA and somewhat lower levels of free Mg²⁺ in ethanol‐treated animals compared to saline controls. In acetate‐infused animals the concentrations of brain lactate, α‐ketoglutarate, and fumarate were higher. Moreover, the free cytosolic [NAD⁺]/[NADH] was lower, the free mitochondrial [NAD⁺]/[NADH] and [CoQ]/[CoQH₂] were oxidized and the ΔG′ of ATP lowered by acetate infusion from ?61.4 kJ to ?59.9 kJ/mol. Conclusions: Animals with elevated levels of blood ethanol or acetate had decreased ¹³C‐glucose uptake into the brain. In acetate‐infused animals elevated BAcC were associated with a decrease in ¹³C‐glucose phosphorylation. The co‐ordinate decrease in free cytosolic NAD, oxidation of mitochondrial NAD and Q couples and the decrease in ΔG′ of ATP was similar to administration of uncoupling agents indicating that the metabolism of acetate in brain caused the mitochondrial voltage dependent pore to form.     

Metabolism of alcohol

Most tissues of the body contain enzymes capable of ethanol oxidation or nonoxidative metabolism, but significant activity occurs only in the liver and, to a lesser extent, in the stomach. Hence, medical consequences are predominant in these organs. In the liver, ethanol oxidation generates an excess of reducing equivalents, primarily as NADH, causing hepatotoxicity. An additional system, containing cytochromes P-450 inducible by chronic alcohol feeding, was demonstrated in liver microsomes and found to be a major cause of hepatotoxicity.     

High-level expression of rat class I alcohol dehydrogenase is sufficient for ethanol-induced fat accumulation in transduced HeLa cells

The mechanisms by which ethanol causes fatty liver are complex. Reducing equivalents generated during ethanol oxidation inhibit tricarboxylic acid cycle activity and fatty acid oxidation. In addition, ethanol inhibits lipoprotein export and increases fatty acid uptake and lipid peroxidation. To test the role that alcohol metabolism by alcohol dehydrogenase (ADH) has on cellular lipid metabolism, a cell line expressing rat ADH was generated by transducing HeLa cells with an ADH-expressing retrovirus. The cells expressed high levels of ADH protein and had ADH activity similar to that of liver. Exposure of the cells to 20 mmol/L ethanol for 24 hours led to substantial accumulation of free fatty acids and triacylglycerol in the transduced, but not wild-type, HeLa cells. The rate of synthesis of saponifiable lipid was increased significantly by ethanol under these conditions. Ethanol exposure also promoted triacylglycerol accumulation when the cells were incubated with linoleic acid. This was associated with a decrease in the rate at which the cells oxidized 1-[14-C]-linoleic acid. Fat accumulation was not prevented by including alpha-tocopherol in the medium, arguing against a role for lipid peroxidation. However, the presence of methylene blue completely prevented the fat accumulation. This was associated with a return of the elevated lactate/pyruvate ratio toward normal. These data suggest that generation of reducing equivalents by ADH was sufficient to cause fat accumulation in this cell model.     

Studies on the metabolic activation of diethanolnitrosamine in animal-mediated and in vitro assays using Escherichia coli K-12 343/113 as an indicator

Summary The mutagenic activity of diethanolnitrosamine (NDELA), a carcinogenic compound which leads to inconsistent results in standard in vitro procedures was tested in vitro and in animal-mediated assays with the indicator strain Escherichia coli (E. coli) K-12 343/113. This strain allows the simultaneous detection of forward and back mutations arising in several genes of the E. coli chromosome. In animal-mediated assays in which mice were used as hosts for i.v. injected E. coli indicator cells, s.c. application of NDELA induced a dose dependent increase of galactose fermenting mutants in cells recovered from galactose fermenting mutants in cells recovered from the livers of animals exposed for 3 h to the mutagen.Comparison with results obtained with diethylnitrosamine (DENA) in the same test system revealed that the two compounds apparently cause different types of mutagenic lesions. Induction of arg⁺ mutations by DENA and several other aliphatic nitrosamines is mainly due to base pair substitutions, whereas NDELA is rather mutagenic in the galR^s system. This latter system is, in addition, sensitive to frameshifts and deletions. These differences in mutagenic specifity suggest that NDELA and DENA, although structurally closely related, are activated via different molecular mechanisms.In fact, evidence is accumulating that alcohol dehydrogenase (ADH) could be involved in the activation of NDELA. On the other hand, the effective mutagenesis of NDELA obtained in vitro with E. coli upon addition of rat liver microsomal fraction would not be expected if ADH is involved in the activation since the S-9 Mix used in the present experiments was devoid of cofactors (NAD, NADP), necessary to accomplish oxidation by ADH.Therefore, further in vivo studies were performed, in which pyrazole, a potent blocker of ADH, was administered prior (1 and 24 h) to the injection of the mutagen. The observation that a dose dependent increase of mutants in the liver (and to a lower extent in the spleens) of treated animals takes place under conditions in which ADH activity is blocked, whereas several microsomal enzymes are stimulated, indicated that besides oxidation of NDELA by ADH other metabolic activation pathways are involved.Apparently enzymes contained in the liver homogenate, possibly NADPH dependent enzymes of the microsomal ethanol oxidizing system, play an important role in the formation of mutagenic metabolites of NDELA.     

Concentration Dependence of Ethanol Metabolism In Vivo in Rats and Man

The metabolism of ethanol to acetaldehyde has been widely considered to be almost independent of concentration (i.e., "pseudolinear") except when blood ethanol was near the K_m (0.5–1.0 m/ M ) of alcohol dehydrogenase (ADH). On the contrary, we report the concentration dependency of ethanol metabolism, in rats and man, at blood ethanol levels several fold the K_m of ADH. After intravenous loading, blood ethanol disappearance in 10 rats at blood levels between 38 and 17 m/ M ethanol exceeded that between 17 and 4 m/ M (14.09 ± 1.38 versus 8.80 ± 0.86 mmoles/liter blood water/hr ± SEM; p < 0.001). Similarly, in 12 men, blood ethanol disappeared faster at 30–17 m/ M ethanol compared to 17–4 m/ M (5.96 ± 0.33 versus 4.96 7plusmn; 0.28 mmole/liter blood water/hr ± SEM; p < 0.001). When ethanol was maintained at either the 30–60 m/ M or 3–19 m/ M ethanol range in the blood of 14 fasted rats by constant intravenous infusion, ethanol oxidation was 25% greater at the higher concentration (9.08 ± 0.50 versus 7.23 ± 0.41 mmole/liter blood water/hr ± SEM; p < 0.02). Faster ethanol oxidation at high ethanol concentration could be due to the presence of the microsomal ethanol-oxidizing system (MEOS), which would only be fully engaged, because of its K_m (8.5 m/ M ), at ethanol levels exceeding those necessary to saturate ADH. The concentration dependency of ethanol metabolism casts doubt on the validity of the common medicolegal practice of calculating prior blood ethanol levels by linear extrapolation of subsequent ones, on the false assumption that metabolism is unaffected by concentration.     

Converting NADH to NAD+ by nicotinamide nucleotide transhydrogenase as a novel strategy against mitochondrial pathologies during aging

Mitochondrial DNA defects are involved supposedly via free radicals in many pathologies including aging and cancer. But, interestingly, free radical production was not found increased in prematurely aging mice having higher mutation rate in mtDNA. Therefore, some other mechanisms like the increase of mitochondrial NADH/NAD⁺ and ubiquinol/ubiquinone ratios, can be in action in respiratory chain defects. NADH/NAD⁺ ratio can be normalized by the activation or overexpression of nicotinamide nucleotide transhydrogenase (NNT), a mitochondrial enzyme catalyzing the following very important reaction: NADH + NADP⁺ ↔ NADPH + NAD⁺. The products NAD⁺ and NADPH are required in many critical biological processes, e.g., NAD⁺ is used by histone deacetylase Sir2 which regulates longevity in different species. NADPH is used in a number of biosynthesis reactions (e.g., reduced glutathione synthesis), and processes like apoptosis. Increased ubiquinol/ubiquinone ratio interferes the function of dihydroorotate dehydrogenase, the only mitochondrial enzyme involved in ubiquinone mediated de novo pyrimidine synthesis. Uridine and its prodrug triacetyluridine are used to compensate pyrimidine deficiency but their bioavailability is limited. Therefore, the normalization of the ubiquinol/ubiquinone ratio can be accomplished by allotopic expression of alternative oxidase, a mitochondrial ubiquinol oxidase which converts ubiquinol to ubiquinone.     

NMR Investigation of the Futile Cycling of Ethanol in Chronic Alcoholic Rats

Weight gain efficiency differences previously reported between alcohol-fed rats and their controls were investigated. Additionally, the futile cycling of ethanol proposed to explain such differences was studied by NMR spectroscopy. Male Sprague-Dawley rats were fed a nutritionally adequate diet containing 36% of the calories as alcohol, and their paired controls were fed an isocaloric diet for 1 f weeks to establish conditions of chronic alcohol feeding. Normalized metabolic efficiencies varied significantly during the initial 2-week period (6.86 ± 0.51 vs. 2.83 ± 0.18 g/kcal × 10⁻²) for control and alcohol-fed groups, respectively, and to a lesser extent over the entire feeding period (6.41 ± 0.78 vs. 4.60 ± 0.27 g/kcal × 10⁻²) for control and alcohol-fed groups, respectively. Alcohol-induced weight gain inefficiency in metabolism has previously been studied and explained by a variety of different biochemical and physiological mechanisms. One possible pathway of energy wastage may occur due to ethanol futile cycling from ethanol to acetaldehyde through the microsomal ethanol oxidation system pathway, and simultaneously from acetaldehyde to ethanol via the ADH pathway. This futile cycle represents a net loss of 6 ATP/cycle, corresponding to the loss of two reducing equivalents (NADH and NADPH). 1H NMR spectroscopy was used to test for this cycling in blood extracts after administration of 1,1-²H₂ ethanol. No futile cycling was detected either during the initial 2 weeks of feeding or after the entire feeding period.