Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. XII. Enantioselectivity and temperature dependence in microsomes and reconstituted cytochrome P-450 systems from rat liver.

Formation of metabolic intermediate (MI) complexes was studied with the enantiomers of amphetamine, 1-phenyl-2-pentanamine, N-hydroxyamphetamine, and 2-nitroso-1-phenylpropane (the C-nitroso analogue of amphetamine). Three different enzyme systems were used; liver microsomes from phenobarbital pretreated rats and two reconstituted systems containing the P450 2B1 and P450 2C11 forms of cytochrome P-450. Enantioselective complex formation in microsomes was shown for the amines and the nitroso compound, but not for the hydroxylamine. The highly purified P450 2B1 system formed the MI complex with all substrates tested, and the enantioselectivity observed with the microsomal system was reproduced. In the P450 2C11 system the nitroso compounds were completely inactive, whereas the enantiomers of N-hydroxyamphetamine still produced the complex at a high rate. Changes in temperature were shown to affect (R)-2-nitroso-1-phenylpropane more than its enantiomer. Both enantiomers showed biphasic Arrhenius plots for MI complex formation in microsomes (breaks around 22 degrees C), but the activation energies of the (R)-isomer were about five times higher than those of the (S)-isomer. A theory is presented which suggests different modes of interaction with the active site of P-450 to account for the different behaviour of the various substrates.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

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 H2O2 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.     

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.     

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol-inducible form of liver microsomal cytochrome P-450 (P-450 LMeb), caused, compared to controls, 3-25-fold enhanced rates of CCl4-dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4-dependent lipid peroxidation was expressed relative to the amount of P-450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P-450 was a 100-fold more effective catalyst of CCl4 metabolism than either of the cytochromes P-450 LM2 or P-450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4-dependent hepatotoxicity.     

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.     

Pyrazole is different from acetone and ethanol as an inducer of the polysubstrate monooxygenase system in mice: evidence that pyrazole-inducible P450Coh is distinct from acetone-inducible P450ac

The induction of liver microsomal monooxygenase activities elicited by pyrazole, ethanol, and acetone, all shown to be inducers of rat P450j and rabbit P450LM3a, has been compared in inbred strains of DBA/2N, AKR/J, and Balb/c mouse. Pyrazole strongly increases coumarin 7-hydroxylase (COH) activity in DBA/2N but much less in other strains. The effect of pyrazole on aniline p-hydroxylase and ethanol oxidase activities is also strain dependent: an increase was seen only in the DBA/2N strain. Ethanol and acetone were unable to induce COH, whereas aniline p-hydroxylase and ethanol oxidase were elevated about 1.4- to 3.3-fold in all strains. No strain difference could be detected in aniline p-hydroxylase or ethanol oxidase inducibility. There was a strong correlation between aniline p-hydroxylase and ethanol oxidase activities in every strain, whereas no positive correlation could be found between COH and aniline p-hydroxylase activities. Immunoinhibition experiments showed that a polyclonal antibody against purified pyrazole-inducible COH (P450Coh) blocked about 90% of COH activity, but only about 10% of aniline p-hydroxylase or ethanol oxidase in mouse liver microsomes. Monoclonal antibody 1-91-3 (raised against rat acetone-inducible P450ac) did not inhibit COH, whereas aniline p-hydroxylase was blocked 46-76% and ethanol oxidase 25-70%, depending on the source of microsomes. In immunoblots, anti-P450Coh recognized only its own antigen but not the P450ac, whereas monoclonal antibody 1-98-1 against P450ac detected P450ac and a corresponding form in the D2 mouse liver, but not the P450Coh. The purified P450ac and P450Coh had molecular masses of 52 and 50 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These antigens were expressed differentially in response to pyrazole, ethanol, and acetone: P450Coh was increased only after pyrazole treatment, but 1-98-1-detectable protein was elevated in D2 mouse liver microsomes by ethanol and acetone, but not by pyrazole. We conclude that mouse P450Coh and rat P450ac are not corresponding forms of the same isozyme, and that a P450ac-like protein, responsible for most of aniline p-hydroxylation and ethanol oxidation, is present in the D2 mouse liver. These two P450 isozymes are also dissimilarly expressed in the mouse liver in response to inducer administration.     

Pyrazole and 4-methylpyrazole inhibit oxidation of ethanol and dimethyl sulfoxide by hydroxyl radicals generated from ascorbate, xanthine oxidase, and rat liver microsomes

Pyrazole and 4-methylpyrazole, which are potent inhibitors of alcohol dehydrogenase, inhibited the oxidation of ethanol and of dimethyl sulfoxide by two model hydroxyl radical-generating systems. The systems used were the iron-catalyzed oxidation of ascorbic acid and the coupled oxidation of xanthine by xanthine oxidase. Pyrazole and 4-methylpyrazole were more effective inhibitors at lower substrate concentrations than at higher substrate concentrations; the oxidation of ethanol was inhibited to a greater extent than the oxidation of dimethyl sulfoxide. These results are consistent with competition between pyrazole or 4-methylpyrazole with the substrates for the generated hydroxyl radicals. Pyrazole and 4-methylpyrazole appear to be equally effective in reacting with hydroxyl radicals. An approximate rate constant of about 8 × 10⁹m^?1 s^?1 was calculated from the inhibition curves, indicating that pyrazole and 4-methylpyrazole are potent scavengers of the hydroxyl radical. Previous studies have implicated a role for hydroxyl radicals in the microsomal pathway of ethanol oxidation. In the presence of azide (to inhibit catalase), pyrazole and 4-methylpyrazole inhibited the NADPH-dependent microsomal oxidation of ethanol, as well as several other hydroxyl radical-scavenging agents. This inhibition by pyrazole and by 4-methylpyrazole may reflect a mechanism involving competition for hydroxyl radicals generated by the microsomes. However, the kinetics of inhibition by pyrazole were mixed, not competitive, and pyrazole and 4-methylpyrazole also inhibited aminopyrine demethylase activity. Pyrazole has been shown by others to interact with cytochrome P-450. It is suggested that pyrazole and 4-methylpyrazole affect microsomal oxidation of ethanol via effects on the mixed-function oxidase system and via competition for the generated hydroxyl radicals. In view of these results, low concentrations of pyrazole and 4-methylpyrazole should be used in studies on pathways of alcohol metabolism, and caution should be made in interpreting the actions of these compounds when used at high concentrations.     

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.     

Modulation of the activity of 5′-nucleotidase by the transfer of non-esterified cholesterol to rat-liver microsomal fraction and evidence for regulation of the concentration of non-esterified cholesterol in plasma membranes in vivo

The transfer of non-esterified cholesterol to rat-liver microsomal fraction resulted in a considerable decrease in the activity of 5′-nucleotidase and in changes in the characteristics of the Arrhenius plots of the enzyme. The decrease in the activity of 5′-nucleotidase and the increase in the concentration of non-esterified cholesterol in the serum-treated preparations were serum-concentration-dependent and incubation-time-dependent. The enzyme in serum-treated preparations with high non-esterified cholesterol content showed Arrhenius plots with a constant activation energy between 37 and 19°C, whereas the enzyme in the non-treated microsomal fraction or the lipoprotein-deficient serum-treated preparations showed a break at about 28°C, with activation energies higher below and lower above the break. These changes in the temperature-induced kinetics are consistent with an increase in the concentration of non-esterified cholesterol in the plasma membrane vesicles of the serum-treated preparations. The Arrhenius plots of 5′-nucleotidase in liver microsomal fraction from rats fed cholesterol-supplemented diet showed constant activation energy between 37 and 19°C and had similar characteristics with the plots for 5′-nucleotidase in serum-treated preparations. Since the changes in the characteristics of Arrhenius plots of the enzyme in microsomal fraction from rats that had been denied food for 36 h were in the opposite direction to those produced by feeding cholesterol, these results are consistent with a lower concentration of non-esterified cholesterol in hepatic plasma membranes from fasted rats relative to that in plasma membranes from fed rats. The isolation of a plasma membrane preparation with negligible contamination of endoplasmic reticular membranes from rats fed the standard or cholesterol-supplemented diet and from fasted rats showed that the ratio of cholesterol to phospholipid has increased in the preparation from rats fed cholesterol and decreased in that from rats that had been denied food relative to the ratio in the preparation from rats fed the standard diet. The Arrhenius plots of 5′-nucleotidase in these preparations showed characteristics similar to the corresponding plots of the enzyme in the microsomal fraction from the rats in the three experimental conditions.     

Increased catalytic activity of cytochrome P-450IIE1 in pericentral hepatocytes compared to periportal hepatocytes isolated from pyrazole-treated rats.

Cytochrome P-450IIE1 is induced by a variety of agents, including acetone, ethanol and pyrazole. Recent studies employing immunohistochemical methods have shown that P-450IIE1 was expressed primarily in the pericentral zone of the liver. In order to evaluate whether catalytic activity of P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, the oxidation of aniline and p-nitrophenol, two effective substrates for P-450IIE1, by periportal and pericentral hepatocytes isolated from pyrazole-treated rats was determined. Periportal and pericentral hepatocytes were prepared by a digitonin-collagenase procedure; the marker enzymes glutamine synthetase and gamma-glutamyl transpeptidase indicated reasonable separation of the two cell populations. Viability, yield and total cytochrome P-450 content were similar for the periportal and pericentral hepatocytes. Pericentral hepatocytes oxidized aniline and p-nitrophenol at rates that were 2-4-fold greater than periportal hepatocytes under a variety of conditions. Carbon monoxide inhibited the oxidation of the substrates with both preparations and abolished the increased oxidation found with the pericentral hepatocytes. Pyrazole or 4-methylpyrazole, added in vitro, effectively inhibited the oxidation of aniline and p-nitrophenol and prevented the augmented rate of oxidation by the pericentral hepatocytes. Western blots carried out using isolated microsomes revealed a more than 2-fold increase in immunochemical staining with microsomes isolated from the pericentral hepatocytes, which correlated to the 2-4-fold increase in the rate of oxidation of aniline or p-nitrophenol by the pericentral hepatocytes. These results suggest that functional catalytic activity of cytochrome P-450IIE1 is preferentially localized in the pericentral zone of the liver acinus, and that most of the induction by pyrazole of P-450IIE1 appears to occur within the pericentral zone.     

Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation

Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a.     

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)     

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased NADH-dependent production of reactive oxygen intermediates by microsomes after chronic ethanol consumption: comparisons with NADPH.

Microsomes from chronic ethanol-fed rats were previously shown to catalyze the NADPH-dependent production of reactive oxygen intermediates at elevated rates compared to controls. Recent studies have shown that NADH can also serve as a reductant and promote the production of oxygen radicals by microsomes. The current study evaluated the influence of chronic ethanol consumption on NADH-dependent microsomal production of reactive oxygen intermediates, and compared the results with NADH to those of NADPH. Microsomal oxidation of chemical scavengers, taken as a reflection of the production of hydroxyl radical (.OH)-like species was increased about 50% with NADH as cofactor and about 100% with NADPH after chronic ethanol consumption. The potent inhibition of the production of .OH-like species by catalase suggests a precursor role for H2O2 in .OH production. Rates of NADH- and NADPH-dependent H2O2 production were increased by about 50 and 70%, respectively, after chronic ethanol consumption. A close correlation between rates of H2O2 production and generation of .OH-like species was observed for both NADH and NADPH, and increased rates of H2O2 production appear to play an important role in the elevated generation of .OH-like species after chronic ethanol treatment. Microsomal lipid peroxidation was elevated about 60% with NADH, and 120% with NADPH, after ethanol feeding. With both types of microsomal preparations, the characteristics of the NADH-dependent reactions were similar to the NADPH-dependent reactions, e.g., sensitivity to antioxidants and free radical scavengers and catalytic effectiveness of ferric complexes. However, rates with NADPH exceeded the NADH-dependent rates by 50 to 100%, and the increased production of reactive oxygen intermediates by microsomes after ethanol treatment was greater with NADPH (about twofold) than with NADH (about 50%). Oxidation of ethanol results in an increase in hepatic NADH levels and interaction of NADH, iron, and microsomes can produce potent oxidants capable of initiating lipid peroxidation and oxidizing .OH scavengers. These acute metabolic interactions produced by ethanol-derived NADH are increased, not attenuated, in microsomes from chronic ethanol-fed rats, and it is possible that such increases in NADH (and NADPH)-dependent production of reactive oxygen species play a role in the development of oxidative stress in the liver as a consequence of ethanol treatment.     

Modulation of rat liver cytochrome P450 activity by prolonged red wine consumption

Cytochrome P450-dependent oxidation of lauric acid, p-nitrophenol and ethanol by liver microsomal fractions were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total cytochrome P450 and the isoenzyme 2E1 content, as well as the p-nitrophenol hydroxylation and ethanol oxidation. These effects of ethanol treatment were attenuated by red wine administration. Red wine increased the total antioxidant capacity of plasma, whereas the alcohol-free red wine decreased the cytochrome P450 content and decreased the oxidation of lauric acid, p-nitrophenol and ethanol to values lower than control. It is concluded that red wine administration attenuates the ethanol-induced enhancement in liver microsomal parameters dependent on cytochrome P450 2E1 activity, an affect that seems to be accomplished by the non-alcoholic constituents of red wine known to have antioxidant properties.     

Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical

Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were the iron-catalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates H2O2 as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress.     

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimirystoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18–28°C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots ofK _m also show breaks in the same temperature interval, with the exception of the dioleoylenzyme vesicles, whereK _m is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24°C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3–18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.