Microsomal ethanol-oxidizing system

Advances in our knowledge of the microsomal metabolism of ethanol enable us to understand a number of complications that develop in the alcoholic. After chronic ethanol consumption, microsomal ethanol-oxidizing system (MEOS) activity increases with an associated rise in microsomal cytochrome P-450, including a form different from that induced by phenobarbital and methylcholanthrene and which has a high affinity for ethanol, as shown in reconstituted systems. The role of this MEOS in vivo and its increase after chronic ethanol consumption was most conclusively shown in alcohol dehydrogenase-negative deer mice. Microsomal induction is also associated with enhanced metabolism of other drugs, resulting in metabolic drug tolerance. Furthermore, there is increased conversion to toxic metabolites of known hepatotoxic agents (such as CCl4), which may explain the enhanced susceptibility of alcoholics to the toxicity of industrial solvents. Furthermore, the ethanol-induced form of cytochrome P-450 has a high capacity for the conversion to toxic metabolites of some commonly used drugs, such as acetaminophen, and also carcinogens, such as dimethylnitrosamine which is activated at concentrations much lower than those required for other microsomal inducers. Moreover, catabolism of retinol is accelerated through a newly discovered microsomal pathway, thereby contributing to hepatic vitamin A depletion and possibly vitamin A toxicity. There is also induction of microsomal enzymes involved in lipoprotein production, resulting in hyperlipemia. Contrasting with the chronic effects of ethanol consumption, acutely, ethanol inhibits the metabolism of other drugs through competition for an at least partially shared microsomal detoxification pathway.     

Effect of mesitylene on ethanol metabolism in rat liver microsomes.

Increased catalase activity was observed in the liver microsomal fraction of ethanol-treated rats (10% v/v aqueous ethanol solution per os for 5 weeks). In contrast, cytochrome P-450 concentration and specific activity of NADPH-cytochrome c reductase remained at the same level as in the liver of control rats (drinking water). The ratio of microsomal H2O2-generation to catalase activity was lower in the "ethanol" group than in the control one. This phenomenon seems to be related to the increased contribution of the "peroxidatic" reaction (increased rate of ethanol oxidation). Administration of mesitylene (1,3,5-trimethylbenzene) by gastric tube for 3 days (5 mmoles per kg daily) increased cytochrome P-450 concentration, specific activity of NADPH-cytochrome c reductase and ethanol metabolism.     

Tissue specificity of induction of hamster monooxygenase activity by ethanol

The effects of ethanol on liver, kidney and intestine monooxygenases were studied using hamsters chronically fed with isocaloric control and ethanol-containing liquid diets. The inductive effects of ethanol on liver and kidney aniline hydroxylase activities began to approach plateau level after the animals were fed ethanol for two weeks. Intestinal aniline hydroxylation was refractory to ethanol induction. In control and ethanol-fed hamsters, CO-difference spectra of hepatic and extrahepatic microsomes differed in absorption maxima. Chronic alcohol consumption caused significant increases of cytochrome P-450 and cytochrome b5 contents of liver and kidney microsomes. The increases of the heme proteins were associated with the induction of aniline hydroxylase, N-nitrosodimethylamine demethylase and 7-ethoxycoumarin 0-deethylase activities. In contrast to the liver and kidney, intestinal microsomal cytochromes P-450 and b5 contents in ethanol-treated animals were lower than the controls. Ethanol pretreatment was without effect on intestinal monooxygenase activities toward the metabolism of aniline, N-nitrosodimethylamine, 7-ethoxycoumarin and benzo(a)pyrene. Gel electrophoresis of tissue microsomes from control and ethanol-treated hamsters revealed that ethanol treatment enhanced the intensity of the protein band(s) in the cytochrome P-450 molecular weight region in the liver and kidney, but not in the intestine. These results demonstrate that in hamsters the response of monooxygenase to ethanol may vary from tissue to tissue and it is difficult to make a generalization regarding the inducing property of ethanol. The differential effect on cytochrome P-450 may be an important factor in determining the interaction between ethanol and xenobiotic metabolism in animal tissues.     

Identification of P-450ALC in microsomes from alcohol dehydrogenase-deficient deermice: contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH-) and -positive (ADH+) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH- than from naive ADH+ animals. Ethanol treatment induced the protein about 3-fold in the ADH+ strain and about 4-fold in the ADH- strain. The antibody to rabbit P-450ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450ALC homolog, increased about 3-fold upon ethanol treatment in the ADH+ strain and about 4-fold in the ADH- strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH+ strain and 1.9-fold and 3.3-fold, respectively, in the ADH- strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH+ and ADH- strains, respectively. After chronic ethanol treatment, P-450ALC could account maximally for 8% of the total ethanol elimination in the ADH+ strain and 22% in the ADH- strain. Further, cytochrome P-450ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

Effect of a single oral dose of methanol, ethanol and propan-2-ol on the hepatic microsomal metabolism of foreign compounds in the rat.

Methanol and ethanol administered to rats as a single oral dose increased aniline hydroxylation by the hepatic microsomal fraction by a maximum of 169 and 66% respectively, whereas aminopyrine demethylation was inhibited by 51 and 61%. The concentration of microsomal cytochrome P-450, and the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were unchanged. Propan-2-ol, administered as a single oral dose, increased microsomal aniline hydroxylation by 165% and increased aminopyrine demethylation by 83%. The concentration of cytochrome P-450 was unchanged whereas NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase were both increased by 38%. Methanol, ethanol and propan-2-ol administration resulted in a decreased type I spectral change but had no effect on the reverse type I spectral change. Methanol administration decreased the type II spectral change whereas ethanol and propan-2-ol had no effect. Cycloheximide blocked the increases in aniline hydroxylation and aminopyrine demethylation but could not completely prevent the decreases in aminopyrine demethylation. The increases in aniline hydroxylation were due to an increase in V, but Km was unchanged. The ability of acetone to enhance and compound SKF 525A to inhibit microsomal aniline hydroxylation was decreased by the administration of all three alcohols. The decrease in the metabolism of aminopyrine may result from a decrease in the binding to the type I site with a consequent failure of aminopyrine to stimulate the reduction of cytochrome P-450. Methanol administration may lead to an increase in aniline hydroxylation because of a failure of aniline to inhibit cytochrome P-450 reduction.     

Role of the microsomal ethanol-oxidizing system in regulating linoleoyl CoA desaturase activity in long-term ethanol loading

Long-term ethanol load resulted in a decrease of the rat liver linoleyl desaturase activity and the activation of MEOS accompanied by an increase in the activity of NADPH-dependent chain and the initial steps of NADH-dependent chain of microsomal electron transport, indicating electron transfer from NADH to cytochrome P-450. It is suggested that, when the main potential of NADH- and NADPH-dependent chains is transferred to microsomal ethanol oxidation, insufficient electron supply for linoleyl-CoA desaturase decreases the activity of this process.     

Mechanisms of hydroxyl radical formation and ethanol oxidation by ethanol-inducible and other forms of rabbit liver microsomal cytochromes P-450

The hydroxyl radical-mediated oxidation of 5,5-dimethyl-1-pyrroline N-oxide, benzene, ketomethiolbutyric acid, deoxyribose, and ethanol, as well as superoxide anion and hydrogen peroxide formation was quantitated in reconstituted membrane vesicle systems containing purified rabbit liver microsomal NADPH-cytochrome P-450 reductase and cytochromes P-450 LM2, P-450 LMeb , or P-450 LM4, and in vesicle systems devoid of cytochrome P-450. The presence of cytochrome P-450 in the membranes resulted in 4-8-fold higher rates of O-2, H2O2, and hydroxyl radical production, indicating that the oxycytochrome P-450 complex constitutes the major source for superoxide anions liberated in the system, giving as a consequence hydrogen peroxide and also, subsequently, hydroxyl radicals formed in an iron-catalyzed Haber-Weiss reaction. Depletion of contaminating iron in the incubation systems resulted in small or negligible rates of cytochrome P-450-dependent ethanol oxidation. However, small amounts (1 microM) of chelated iron (e.g. Fe3+-EDTA) enhanced ethanol oxidation specifically when membranes containing the ethanol and benzene-inducible form of cytochrome P-450 (cytochrome P-450 LMeb ) were used. Introduction of the Fe-EDTA complex into P-450 LMeb -containing incubation systems caused a decrease in hydrogen peroxide formation and a concomitant 6-fold increase in acetaldehyde production; consequently, the rate of NADPH consumption was not affected. In iron-depleted systems containing cytochrome P-450 LM2 or cytochrome P-450 LMeb , an appropriate stoichiometry was attained between the NADPH consumed and the sum of hydrogen peroxide and acetaldehyde produced. Horseradish peroxidase and scavengers of hydroxyl radicals inhibited the cytochrome P-450 LMeb -dependent ethanol oxidation both in the presence and in the absence of Fe-EDTA. The results are not consistent with a specific mechanism for cytochrome P-450-dependent ethanol oxidation and indicate that hydroxyl radicals, formed in an iron-catalyzed Haber-Weiss reaction and in a Fenton reaction, constitute the active oxygen species. Cytochrome P-450-dependent ethanol oxidation under in vivo conditions would, according to this concept, require the presence of non-heme iron and endogenous iron chelators.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

Evidence that covalent binding of metabolically activated phenol to microsomal proteins is caused by oxidised products of hydroquinone and catechol

The metabolic activation of [14C]phenol resulting in covalent binding to proteins has been studied in rat liver microsomes. The covalent binding was dependent on microsomal enzymes and NADPH and showed saturation kinetics for phenol with a Km-value of 0.04 mM. The metabolites hydroquinone and catechol were formed at rates which were 10 or 0.7 times that of the binding rate of metabolically activated phenol. The effects of cytochrome P-450 inhibitors and cytochrome P-450 inducers on the metabolism and binding of phenol to microsomal proteins, suggest that cytochrome P-450 isoenzyme(s) other than P-450 PB-B or P-450 beta NF-B catalyses the metabolic activation of phenol. Furthermore, reconstituted mixed-function oxidase systems containing cytochrome P-450 PB-B and P-450 beta NF-B were (on basis of cytochrome P-450 content) 6 and 11 times less active in catalysing the formation of hydroquinone than microsomes. The isolated metabolites hydroquinone and catechol bound more extensively to microsomal proteins than phenol and the binding of these was not stimulated by NADPH. The binding occurring during the metabolism of phenol could be predicted by the rates of formation of hydroquinone and catechol and the rates by which the isolated metabolites were bound to proteins.     

Significant pathways of hepatic ethanol metabolism.

Rat liver microsomes oxidized ethanol two to three times faster than propanol when incubated with either an NADPH- or an H2O2-generating system. In addition, solubilized, purified microsomal subfractions were found to contain protein with an electrophoretic mobility identical to rat liver catalase on SDS polyacrylamide gels, suggesting that the separation of catalase from cytochrome P-450 and other microsomal components may not be feasible. These data support the postulate that catalase is responsible for NADPH-dependent microsomal ethanol oxidation. Direct read-out techniques for pyridine nucleotides, the catalase-H2O2 complex, and cytochrome P-450 were utilized to evaluate the specificity of inhibitors of alcohol dehydrogenase (4-methylpyrazole; 4 mM) and catalase (aminotriazole; 1.0 g/kg) qualitatively in perfused rat livers. 4-Methylpyrazole and aminotriazole are specific inhibitors for alcohol dehydrogenase and catalase, respectively, under these conditions. Neither inhibitor nor a combination of them altered the mixed function oxygen of p-nitroanisole to p-nitrophenol as observed by oxygen uptake and product formation. When ethanol utilization was measured over the concentration range 20-80 mM in perfused liver, a concentration dependence was observed. At low concentrations of ethanol, ethanol oxidation was almost totally abolished by 4-methylpyrazole; however, the contribution of 4-methylpyrazole-insensitive ethanol uptake increased as a function of ethanol concentration. At 80 mM ethanol, ethanol utilization was nearly 50% methylpyrazole-insensitive. This portion of ethanol oxidation, however, was abolished by aminotriazole. The data indicate that alcohol dehydrogenase and catalase-H2O2 are responsible for hepatic ethanol oxidation. At low ethanol concentrations (less than 20 mM), alcohol dehydrogenase is predominant; however, at higher ethanol concentrations (up to 80 mM), the contribution of catalase-H2O2 to overall ethanol utilization is significant. No evidence that the endoplasmic reticulum is involved in ethanol metabolism in the perfused liver emerged from these studies.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

Spin trapping of free radical species produced during the microsomal metabolism of ethanol

Liver microsomes incubated with a NADPH regenerating system, ethanol and the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) produced an electron spin resonance (ESR) signal which has been assigned to the hydroxyethyl free radical adduct of 4-POBN by using 13C-labelled ethanol. The free radical formation was dependent upon the activity of the microsomal monoxygenase system and increased following chronic feeding of the rats with ethanol. The production of hydroxyethyl free radicals was stimulated by the addition of azide, while catalase and OH. scavengers decreased it. This suggested that hydroxyl radicals (OH.) produced in a Fenton-type reaction from endogenously formed hydrogen peroxide were involved in the free radical activation of ethanol. Consistently, the supplementation of iron, under various forms, also increased the intensity of the ESR signal which, on the contrary, was inhibited by the iron-chelating agent desferrioxamine. Microsomes washed with a solution containing desferrioxamine and incubated in a medium treated with Chelex X-100 in order to remove contaminating iron still produced hydroxyethyl radicals, although at a reduced rate. Under these conditions the free radical formation was apparently independent from the generation of OH. radicals, whereas addition of cytochrome P-450 inhibitors decreased the hydroxyethyl radical formation, suggesting that a cytochrome P-450-mediated process might also be involved in the activation of ethanol. Reduced glutathione (GSH) was found to effectively scavenge the hydroxyethyl radical, preventing its trapping by 4-POBN. The data presented suggest that ethanol-derived radicals could be generated during the microsomal metabolism of alcohol probably through two different pathways. The detection of ethanol free radicals might be relevant in understanding the pathogenesis of the liver lesions which are a consequence of alcohol abuse.     

Age-dependent changes in in vivo ethanol metabolism and in the activity of hepatic enzymes involved in ethanol oxidation and microsomal functions

The study of the influence of the age of the animals (13 to 53 weeks) on the rate of ethanol metabolism in vivo and the total activity of liver alcohol dehydrogenase and microsomal ethanol oxidizing system showed a progressive decline with age. These effects were observed concomitantly with a diminution in the content of cytochrome P-450 and microsomal functions related to oxidative and free-radical mediated reactions, namely, NADPH oxidase activity, NADPH-dependent oxygen uptake and NADPH-or t-butyl hydroperoxide-induced chemiluminescence. It is concluded that ageing is accompanied by a diminution in the total oxidative activity of the liver tissue, which would explain the depression in basal and ethanol-induced lipid peroxidation found in the oldest group of rats studied.     

Ethanol metabolism in the yeasts Yarrowia and Torulopsis (a review)

Results of research into ethanol metabolism in yeast organisms with highly pronounced aerobic metabolism are reviewed. The low activity of NAD-dependent alcohol dehydrogenase (EC 1.1.1.1), observed under the conditions of aerobic yeast growth on ethanol, demonstrates that alternative enzyme systems--alcohol oxidase (EC 1.1.3.13), microsomal ethanol-oxidizing system (including cytochrome P-450), and catalase (EC 1.11.1.6)--may be involved in the alcohol oxidation. The role of these systems in alcohol oxidation and conditions favoring their operation in this processes are analyzed. It is concluded that iron ions are important regulators of ethanol metabolism the microorganisms of this group.     

Effect of oxygen concentration on microsomal oxidation of ethanol and generation of oxygen radicals.

The iron-catalysed production of hydroxyl radicals, by rat liver microsomes (microsomal fractions), assessed by the oxidation of substrate scavengers and ethanol, displayed a biphasic response to the concentration of O2 (varied from 3 to 70%), reaching a maximal value with 20% O2. The decreased rates of hydroxyl-radical generation at lower O2 concentrations correlates with lower rates of production of H2O2, the precursor of hydroxyl radical, whereas the decreased rates at elevated O2 concentrations correlate with lower rates (relative to 20% O2) of activity of NADPH-cytochrome P-450 reductase, which reduces iron and is responsible for redox cycling of iron by the microsomes. The oxidation of aniline or aminopyrine and the cytochrome P-450/oxygen-radical-independent oxidation of ethanol also displayed a biphasic response to the concentration of O2, reaching a maximum at 20% O2, which correlates with the dithionite-reducible CO-binding spectra of cytochrome P-450. Microsomal lipid peroxidation increased as the concentration of O2 was raised from 3 to 7 to 20% O2, and then began to level off. This different pattern of malondialdehyde generation compared with hydroxyl-radical production probably reflects the lack of a role for hydroxyl radical in microsomal lipid peroxidation. These results point to the complex role for O2 in microsomal generation of oxygen radicals, which is due in part to the critical necessity for maintaining the redox state of autoxidizable components of the reaction system.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.