Microsomal Ethanol Oxidation Research Articles

Effects of ethanol on microsomal drug metabolism in aging female rats. II. Inhibition

The effect of aging on the inhibition by ethanol of drug metabolism activity was examined in liver microsomes of female Fischer 344 rats aged 4, 14 and 24 months. Inhibition of aniline hydroxylase activity in microsomes from 4-month-old females occurred at low concentrations of ethanol (0.1 mM) and was predominentaly competitive. Aging was associated with a significat increase in apparent K m for aniline in the absence of ethanol (24 ± 2, 20 ± 2 and 32 ± 1 μM in microsomes from 4-, 14- and 24-month-old rats, respectively) and a change from competitive to non-competitive inhibition by ethanol. Inhbition of benphetamine N-demethylase activity occurred only at high concentrations of ethanol (100 mM) and was non-competitive in nature. There was no significant effects of aging on the kinetics of the reaction or the type of inhibition produced by ethanol. Microsomal ethanol oxidation rates were measured in liver microsomes of 4-, 15- and 25-month-old Fischer 344 rats of both sexes. Ethanol oxidation in males was greater than in females and was decreased significantly in old age. Ethanol oxidation in female rats was unaffected by aging. The results suggest that significant changes in drug/ethanol interactions can occur as a consequence of aging.

Deuterium Isotope Effects as a Tool in the Study of Ethanol Oxidation in Rat Liver Microsomes

The apparent kinetic deuterium isotope effect (I) on the oxidation of ethanol to acetaldehyde by washed rat liver microsomes was measured with (1-R)-[1-2H2, 1-14C]-ethanol (I1) and [1-2H2, 2-14C]-ethanol (I2) as substrates by a competitive technique involving only measurements of radioactivity. The average values were for non-induced rats, I1 = 1.57 and I2 = 2.23. When these two substrates were used with stereospecific enzymes (alcohol dehydrogenase and catalase) a small secondary effect was observed, causing I2 to be about 10% higher than I1. With non-stereospecific systems I2 was much larger than I1, and the values were connected by a simple formula. This relation in combination with use of the inhibitors, sodium azide and thiourea, made it possible to calculate tentatively the contribution to microsomal ethanol oxidation of catalase, a non-identified stereospecific enzyme, and non-stereospecific catalytic systems, as well as the isotope effects of the latter two systems. Measurements were made in microsomes from normal, phenobarbital treated, and acetone treated rats. For the stereospecific component an isotope effect of 1.4-1.5 was calculated for all three groups. For the non-stereospecific enzyme in acetone treated rats a value of 4.0 was found. Both the other groups showed a value about 2.7. The activity of the non-stereospecific system was about twice the normal in barbiturate treated, and 3 times the normal in the acetone treated group, where it contributed 70% of the total activity. The isotope effects on the changes in ethanol oxidation (the 'differential isotope effect') caused by inhibitors and activators were utilized to decide whether inhibitors were specific for a single reaction. Thus azide while inhibiting catalase completely, also inhibited other reactions. The large increase (5-6 times) in rate caused by Fe-ESDTA has an I2 of 1.6, equal to that for oxidation of ethanol by hydroxyl radicals.

Dehydrogenase-dependent Ethanol Metabolism in Deer Mice (Peromyscus maniculatus) Lacking Cytosolic Alcohol Dehydrogenase: Reversibility and isotope effects in vivo and in subcellular fractions

Elimination of [2H]ethanol in vivo as studied by gas chromatography/mass spectrometry occurred at about half the rate in deer mice reported to lack alcohol dehydrogenase (ADH−) compared with ADH+ deer mice and exhibited kinetic isotope effects on Vmax and Km (D(V/K] of 2.2 ± 0.1 and 3.2 ± 0.8 in the two strains, respectively. To an equal extent in both strains, ethanol elimination was accompanied by an ethanol-acetaldehyde exchange with an intermolecular transfer of hydrogen atoms, indicating the occurrence of dehydrogenase activity. This exchange was also observed in perfused deer mouse livers. Based on calculations it was estimated that at least 50% of ethanol elimination in ADH- deer mice was caused by the action of dehydrogenase systems. NADPH-supported cytochrome P-450-dependent ethanol oxidation in liver microsomes from ADH+ and ADH− deer mice was not stereoselective and occurred with a D(V/K) of 3.6. The D(V/K) value of catalase-dependent oxidation was 1.8, whereas a kinetic isotope effect of cytosolic ADH in the ADH+ strain was 3.2. Mitochondria from both ADH+ and ADH− deer mice catalyzed NAD+-dependent ethanol oxidation and NADH-dependent acetaldehyde reduction. The kinetic isotope effects of NAD+-dependent ethanol oxidation in the mitochondrial fraction from ADH+ and ADH− deer mice were 2.0 ± 0.1 and 2.3 ± 0.3, respectively. The results indicate only a minor contribution by cytochrome P-450 to ethanol elimination, whereas the isotope effects are consistent with ethanol oxidation by the catalase-H2O2 system in ADH− deer mice in addition to the dehydrogenase systems.

Temperature dependence of the microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent reactions

The temperature dependence and activation energies for the oxidation of ethanol by microsomes from controls and from rats treated with pyrazole was evaluated to determine whether the overall mechanism for ethanol oxidation by microsomes was altered by the pyrazole treatment. Arrhenius plots of the temperature dependence of ethanol oxidation by pyrazole microsomes were linear and exhibited no transition breaks, whereas a slight break was observed at about 20 ± 2.5 °C with control microsomes. Energies of activation (about 15–17 kcal/mol) were identical for the two microsomal preparations. Although transition breaks were noted for the oxidation of substrates such as dimethylnitrosamine and benzphetamine, activation energies for these two substrates were similar for control microsomes and microsomes from the pyrazole-treated rats. The addition of ferric-EDTA to the microsomes increased the rate of ethanol oxidation by a hydroxyl radical (.OH)-dependent pathway. Arrhenius plots of the .OH-dependent oxidation of ethanol by both microsomal preparations were linear with energies of activation (about 7 kcal/mol) that were considerably lower than values found for the P450-dependent pathway. These results suggest that, at least in terms of activation energy, the increase in microsomal ethanol oxidation by pyrazole treatment is not associated with any apparent change in the overall mechanism or rate-limiting step for ethanol oxidation but likely reflects induction of a P450 isozyme with increased activity toward ethanol. The lower activation energy for the .OH-dependent oxidation of ethanol suggests that different steps are rate limiting for oxidation of ethanol by .OH and by P450, which may reflect the different enzyme components of the microsomal electron transfer system involved in these reactions.

Human liver microsomal cytochrome P-450IIE1. Imnunological evaluation of its contribution to microsomal ethanol oxidation, carboti tetrachloride reduction and NADPH oxidase activity

Human liver microsomal cytochrome P-450IIE1. Imnunological evaluation of its contribution to microsomal ethanol oxidation, carboti tetrachloride reduction and NADPH oxidase activity

Obesity as a risk factor for drug-induced organ injury. VI. Increased hepatic P450 concentration and microsomal ethanol oxidizing activity in the obese overfed rat

The obese overfed rat effectively models many of the pharmacological changes in human obesity. Recent data show that the obese rat is unusually susceptible to liver damage by several metabolically activated drugs that may be more toxic in obese humans. Results of the present study suggest a specific molecular locus for this interaction. In obese rats, P450 content of liver and the microsomal concentration of P450 were elevated 88% and 31%, respectively, over nonobese controls. Increases in microsomal ethanol oxidation were of identical magnitude. The ethanol-inducible form of P450 that is responsible for microsomal ethanol oxidation, P450IIE1, bioactivates several drugs that are shown to cause increased injury in obese rats. Collectively, these findings indicate that specific forms of P450 may become up-regulated in obesity, increasing the risk of a biochemically defined spectrum of drug-induced organ injuries.

The effect of di-isopropyl 1,3 dithiol-2-ylidenemalonate (Malotilate) on the hepatic changes induced by ethanol administration in the rat

The sulphur-containing drug, di-isopropyl-1,3-dithiol-2-ylidenemalonate (Malotilate) protects against the increase in hepatic triglyceride concentration after acute ethanol administration (either 6 g/kg p.o. or 2 g/kg i.p.)inrats. The compound had no influence on the increased hepatic NADH: NAD ratio (measured as the lactate : pyruvate and 3-hydroxybutyrate : acetoacetate ratios) after acute ethanol dosing (2 g/kg i.p.), but was found to lower hepatic acetaldehyde concentrations and prevent some of the disturbances in lipid metabolism observed in liver slices from ethanol-treated animals (e.g. decreased oxidation of [1- 14C]palmitate to 14CO 2) after this ethanol dose. The drug did not inhibit ethanol metabolism in this acute experiment. Administration of Malotilate to Wistar rats (100 mg/kg/day orally) during chronic feeding of ethanol as 36% of the total calorie intake in a liquid diet, resulted in a lower intake of the alcohol-containing diet by ethanol-fed animals and reduced body weight gain in rats which received the drug, without blood ethanol levels or the ethanol intake (expressed in g/kg body weight/day) being affected. In ethanol-fed animals, Malotilate prevented the production of fatty liver and the adaptive increase in the ethanol elimination rate (EER) normally seen in ethanol-fed animals, although the drug actually caused a slight increase in EER in glucose pair-fed controls. Malotilate did not significantly decrease the degree of induction of microsomal cytochrome P-450 by ethanol, but the increase in aniline hydroxylation was much less marked in animals receiving ethanol and Malotilate, suggesting that the activity of the inducible microsomal ethanol oxidising system (MEOS) may be reduced by the compound. Determination of hepatic acetaldehyde concentrations during ethanol feeding, and during an acute ethanol challenge test following long-term ethanol treatment showed that the compound significantly lowered the level of this ethanol metabolite in the liver under both circumstances. This reduction of hepatic acetaldehyde concentrations, probably resulting in part from the reduced EER as well as increased low- K m aldehyde dehydrogenase activities and glutathione contents seen in the livers of Malotilate-treated rats, are possible mechanisms by which the drug protects against triglyceride accumulation after ethanol administration.

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

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, 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-450 ALC 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-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, 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-450 ALC 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-450 ALC 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-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC 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-450 ALC 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. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.

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.

Molecular regulation of ethanol-inducible cytochrome P450-IIEI in hamsters

Liver polysomal poly(A) + RNA, isolated from hamsters treated with ethanol or pyrazole, was translated in vitro to determine the effect of these compounds on specific mRNA encoding P450-IIEI, an ethanol-inducible P450 isozyme. As assessed by immunoprecipitation of translation products, ethanol and pyrazole increased hepatic P450-IIEI mRNA levels by 160% and 45%, respectively, when compared to controls. In liver microsomes from the same animals, ethanol and pyrazole caused a two-fold increase in microsomal P450-IIEI protein and a two- to three-fold enhancement of microsomal ethanol oxidation and p -nitrophenol hydroxylation. Our results show that the induction of P450-IIEI protein in hamsters by ethanol and pyrazole, an “ethanol-like” inducer, is accompanied by an increase in translatable P450-IIEI mRNA.

Increased sensitivity of the microsomal oxidation of ethanol to inhibition by pyrazole and 4-methylpyrazole after chronic ethanol treatment

Pyrazole and 4-methylpyrazole, inhibitors of the oxidation of ethanol by alcohol dehydrogenase, also inhibit microsomal metabolism of ethanol. The inhibitory effectiveness of these agents was increased in microsomes isolated from rats treated chronically with ethanol as compared to microsomes from pair-fed controls or from rats treated with other cytochrome P-450 inducers such as phenobarbital or 3-methylcholanthrene. Pyrazole and 4-methylpyrazole produced type II binding spectra with all the microsomal preparations. However, there was an increased affinity (lower K s value) for these agents by the microsomes from the ethanol-fed rats. A correlation between K s values and inhibitory effectiveness against ethanol oxidation by the various microsomal preparations could be observed. This suggests that an increase in affinity, which may reflect the induction of an alcohol-preferring isozyme of cytochrome P-450, is responsible for the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole towards ethanol oxidation by microsomes after chronic ethanol treatment. One difference between pyrazole and 4-methylpyrazole was the increased affinity and inhibitory effectiveness of the latter but not the former with microsomes from rats treated with 3-methylcholanthrene. This could be due to the ability of 4-methylpyrazole, compared to pyrazole, to interact with and induce several isozymes of cytochrome P-450. Pyrazole and 4-methylpyrazole are often utilized to evaluate ethanol metabolism by alcoholdehydrogenase-dependent and -independent pathways. However, the sensitivity of microsomal ethanol oxidation to inhibition by these agents, especially after chronic ethanol treatment, would suggest that their use in this regard is complex and could tend to underestimate the contribution of the microsomal pathway towards the metabolic tolerance found after ethanol treatment.

Involvement of iron and iron-catalyzed free radical production in ethanol metabolism and toxicity.

Lipoperoxidation, a degradative process of membranous polyunsaturated fatty acids, has been suggested to represent an important mechanism in the pathogenesis of ethanol toxicity on the liver and possibly also on the brain. Catalysis by transition metals, especially iron, is involved in the biosynthesis of free radicals contributing to lipid peroxidation. Although the exact nature of the redox-active iron implicated in this catalysis is still unknown, it has been well established that lipid peroxidation can be prevented in vitro by iron chelators such as desferrioxamine. Deprivation of redox-active iron through desferrioxamine inhibits by about 50% the microsomal oxidation of ethanol in vitro and reduces very significantly in vivo the overall ethanol elimination rate in rats. Administration of desferrioxamine together with ethanol also reduces the ethanol-induced disturbances in the antioxidant defense mechanisms of the hepatocyte. It also reduces in mice both the severity of physical dependence on ethanol and lethality following the acute administration of a narcotic dose of ethanol. Chronic overloading of rats with iron results, on the opposite, in an increased rate of ethanol elimination, although alcohol dehydrogenase and catalase activities are reduced and cytochrome P-450 depleted in the liver of such iron-overloaded animals. The magnitude of the ethanol-induced increase in lipid peroxidation and decrease in the major membranous antioxidant, alpha-tocopherol, is exacerbated in iron-overloaded rats. Several disturbances of iron metabolism have been reported in human alcoholics. Their contribution to ethanol toxicity appears very likely in the case of hepatic siderosis associated with alcohol abuse. Ethanol could however disturb iron metabolism even in the absence of gross abnormalities of the total iron stores. It is suggested that ethanol intoxication could increase cellular redox-active iron, thus contributing to an enhanced steady-state concentration of reactive-free radicals. This oxidative stress would lead to lipoperoxidative damage and cellular injury.

Inhibition of microsomal oxidation of ethanol by pyrazole and 4-methylpyrazole in vitro. Increased effectiveness after induction by pyrazole and 4-methylpyrazole.

Pyrazole and 4-methylpyrazole, which are inhibitors of alcohol dehydrogenase, were also found to be effective inhibitors of the oxidation of ethanol by liver microsomes (microsomal fractions) in vitro. Ethanol oxidation by microsomes from rats previously treated for 2 or 3 days with either pyrazole or 4-methylpyrazole appeared to be especially sensitive to inhibition in vitro by pyrazole or 4-methylpyrazole. The kinetics of inhibition by pyrazole or 4-methylpyrazole in all microsomal preparations were mixed, as the Km for ethanol was elevated while Vmax was lowered. However, Ki values for pyrazole (about 0.35 mM) and especially 4-methylpyrazole (about 0.03-0.10 mM) were much lower than those found with the saline controls (about 0.7-1.1 mM). In contrast, Ki values for dimethyl sulphoxide as an inhibitor of microsomal ethanol oxidation were similar in all microsomal preparations. Pyrazole and 4-methylpyrazole reacted with microsomes to produce type II spectral changes whose magnitude increased after treatment with either pyrazole or 4-methylpyrazole. Thus the increased inhibitory effectiveness of pyrazole and 4-methylpyrazole appears to be associated with increased interactions with the cytochrome P-450 isoenzyme(s) induced by these compounds. These isoenzymes have properties similar to those of the isoenzyme induced by chronic ethanol treatment. Therefore, caution is needed in the use of pyrazole or 4-methylpyrazole to assess pathways of ethanol metabolism, especially after chronic ethanol treatment, since these agents, besides inhibiting alcohol dehydrogenase, are also effective inhibitors of microsomal ethanol oxidation.

The combined effects of protein deficiency and chronic ethanol administration on rat ethanol metabolism.

This investigation was performed to examine the combined effects of protein deficiency and chronic ethanol consumption on ethanol clearance and hepatic ethanol metabolism of the rat. Protein deficiency alone was associated with reduced ethanol clearance and decreased activity of hepatic alcohol dehydrogenase and the microsomal ethanol-oxidizing system. However, when ethanol (as 36% of energy) was administered concurrently with protein-deficient diets, accelerated ethanol clearance and increased microsomal oxidation of ethanol was observed. Furthermore, in protein-deficient animals fed ethanol, liver alcohol dehydrogenase levels were less decreased when compared with values observed in animals fed protein-deficient diets without ethanol, and this effect was associated with markedly reduced serum testosterone levels in the former group.

Effect of the variations of S-adenosyl- l-methionine liver content on fat accumulation and ethanol metabolism in ethanol-intoxicated rats

The protective effect of S-adenosyl- l-methionine against rat liver steatosis induced by chronic ethanol ingestion was investigated. S-Adenosyl- l-methionine given during ethanol treatment prevented steatosis and accelerated recovery from steatosis when given after ethanol withdrawal. It also caused a slight inhibition of blood ethanol consumption in both acutely and chronically intoxicated rats. About 30% inhibition of alcohol dehydrogenase, but not of the microsomal ethanol oxidation system, occurred in rats subjected to acute ethanol toxicity as well as in normal rats as a consequence of S-adenosyl- l-methionine treatment. A comparison between S-adenosyl- l-methionine and pyrazole, as concerns inhibition of ethanol oxidation and fat accumulation, revealed that a greater inhibition of ethanol metabolism by pyrazole was associated with incomplete prevention of steatosis, while a lower inhibition by S-adenosyl- l-methionine was coupled to a complete prevention. Ethanol induced a drastic decrease of reduced glutathione liver content as well as 630 and 133% increases of blood and liver acetaldehyde contents, respectively. S-Adenosyl- l-methionine treatment almost completely reconstituted the liver reduced glutathione pool and caused a large decrease of the liver and blood acetaldehyde contents. 1-Chloro-2,4-dinitrobenzene, which depletes the cellular reduced glutathione, and diethylethanolamine, an inhibitor of the phosphatidylethanolamine methylation, abolished the S-adenosyl- l-methionine-induced modifications of the reduced glutathione, acetaldehyde, and triacylglycerol contents in the liver of ethanol-treated rats. Neither S-adenosyl- l-methionine nor reduced glutathione inhibitors affected the liver acetaldehyde dehydrogenase activity. It is suggested that, although S-adenosyl- l-methionine induced a small inhibition of ethanol metabolism in the liver, its antisteatosic effect could largely depend on its role as a modulator of the reduced glutathione liver content.

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.

The distribution of cytochrome P-450-mediated drug oxidation and glutathione in periportal and perivenous rat hepatocytes after phenobarbital treatment

The effect of phenobarbital pretreatment on acinar distribution of microsomal drug metabolizing enzymes was investigated by analysis of periportal (pp) and perivenous (pv) enriched rat hepatocytes isolated by collagenase gradient perfusion. In untreated animals the activities of cytochrome P-450, NADPH-cytochrome c reductase and microsomal ethanol oxidation were significantly higher in pv cells. Phenobarbital produced a 45% increase of the yielded microsomes related to the hepatocytic protein but did not change their relative distribution. The content of reduced glutathione (GSH) was lower in hepatocytes from the pv area. The GSH content was more than 20% increased after phenobarbital treatment in both subclasses of cells, but the distribution pattern remained unchanged. The higher activity of drug metabolizing enzymes in the pv area of untreated animals may account for the higher cytotoxicity of numerous drugs to the perivenous hepatocytes. A 3-day treatment with phenobarbital equalized the pp-pv difference by producing more induction of the periportal cytochrome P-450-mediated drug and ethanol oxidation capacities in microsomes derived from periportally enriched hepatocytes.

Microsomal interactions between iron, paraquat, and menadione: Effect on hydroxyl radical production and alcohol oxidation

The addition of menadione or paraquat to rat liver microsomes resulted in about a threefold increase in the production of hydroxyl radical (.OH) as reflected by the increased oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene. This increase was not sensitive to Superoxide dismutase but was blocked by catalase. The increase occurred in the absence of added iron and was not affected by the potent iron chelating agent, desferrioxamine, which suggests the possibility that .OH was produced from an interaction between H 2O 2 and the paraquat or menadione radical. Menadione and paraquat were especially effective in stimulating the oxidation of KMBA in the presence of certain iron chelates such as ferric-ADP, -ATP, or -EDTA, but not ferric-desferrioxamine, -citrate, or -histidine, or unchelated iron. In fact, ferric-ADP or -ATP only stimulated .OH production in the presence of menadione or paraquat. In the presence of ferric-EDTA, the greater than additive increase of .OH production was sensitive to catalase, but not to superoxide dismutase, suggesting the possibility of reduction of ferric-EDTA by paraquat or menadione radical. The interactions with ferric adenine nucleotides may increase the catalytic effectiveness of menadione or paraquat in producing potent oxidants such as the hydroxyl radical, and thus play a role in the toxicity associated with these agents. Paraquat and menadione had little effect on the overall oxidation of ethanol by microsomes. Microsomal drug metabolism was decreased by menadione or paraquat. As a consequence, the effect of these agents on the microsomal oxidation of ethanol was complex since it appeared that paraquat and menadione stimulated the oxidation of ethanol by a .OH-dependent mechanism, but inhibited the oxidation of ethanol by a cytochrome P-450-dependent oxidation pathway. Experiments with carbon monoxide, ferric-EDTA, and 2-butanol plus catalase tended to verify that microsomal oxidation of alcohols was increased by a .OH-dependent pathway when menadione or paraquat were added to microsomes.

Role of alcohol P-450-oxygenase (APO) in microsomal ethanol oxidation

The form of liver microsomal cytochrome P-450 induced by chronic administration of ethanol to rabbits, designated as P-450 alc or P-450 isozyme 3a, has been purified to homogeneity as judged by several criteria, including NH 2- and COOH-terminal amino acid sequence determination. The reconstituted alcohol-P-450 oxygenase (APO) system containing P-450 alc and NADPH-cytochrome P-450 reductase catalyzes the oxidation of a variety of primary and secondary alcohols to aldehydes and ketones, including methanol, ethanol, n-propanol, n-butanol, 2-butanol, n-pentanol, and cyclohexanol. Other purified P-450 cytochromes, including isozymes 2, 3b, 3c, 4, and 6, are much less active than P-450 alc in the oxidation of alcohols. That P-450 alc functions in ethanol oxidation in liver microsomal membranes as well as in the reconstituted system was shown by immunochemical experiments involving inhibition by sheep anti-P-450 alc antibodies. We conclude that P-450 alc is the predominant ethanol-oxidizing cytochrome present after induction by chronic alcohol administration and that the other P-450 cytochromes have low but significant activity in both control and ethanol-induced animals.

Induction of rabbit hepatic microsomal cytochrome P-450 by imidazole: Enhanced metabolic activity and altered substrate specificity

Pretreatment of rabbits with imidazole resulted in a twofold increase in hepatic microsomal cytochrome P-450 content, with the apparent induction of two or more distinct forms of the cytochrome [ K. K. Hajek and R. F. Novak (1982) Biochem. Biophys. Res. Commun. 108, 664–672 ]. The metabolic properties of imidazole-induced microsomes have been compared to those of uninduced, phenobarbital- and β-naphthoflavone-induced preparations. Metabolic activity was enhanced as a consequence of increased P-450 content and as a result of the presence of different forms of the cytochrome. When rates were expressed per nanomole P-450 the following were observed: (a) p-nitroanisole O-demethylation was comparable in all preparations; (b) N,N-dimethylaniline N-demethylation was comparable in imidazole- and β-naphtho-flavone-induced, and uninduced microsomes; (c) polycyclic aromatic hydrocarbon hydroxylase activity was approximately twofold greater in imidazole-induced relative to phenobarbital-induced microsomes, but was only one-half that of β-naphthoflavone-induced microsomes; and (d) metabolism of N,N-dimethylnitrosamine was enhanced fivefold, alcohol oxidation increased three- to fivefold, and aniline hydroxylation was threefold greater in imidazole-induced microsomes compared to phenobarbital- or β-naphthoflavone-induced preparations. Eadie-Scatchard analysis yielded a single K m value for dimethylnitrosamine N-demethylase activity in imidazole-induced microsomes; in contrast, both high- and low- K m values were obtained for phenobarbital- or β-naphthoflavone-induced microsomal preparations. Dimethylnitrosamine N-demethylase activity was P-450 dependent; neither flavin monooxygenase nor monoamine oxidase appeared to contribute significantly to dimethylnitrosamine metabolism. Dimethyl sulfoxide was a competitive inhibitor of dimethylnitrosamine N-demethylase activity in imidazole-, phenobarbital-, and β-naphthoflavone-induced microsomes. Dimethyl sulfoxide competitively inhibited ethanol oxidation in imidazole-induced microsomes; it was a noncompetitive inhibitor of ethanol oxidation in phenobarbitalor β-naphthoflavone-induced microsomes.