P-nitrophenol Hydroxylation Research Articles

The Effect of Phenobarbital and Dexamethasone Coadministration on the Activity of Rat Liver P450 System

Phenobarbital, the potent inducer of CYP2B and CYP3A, and dexamethasone, that induces CYP3A, are not able to elevate p-nitrophenol hydroxylase activity of CYP2E1. However, rats treated with phenobarbital and dexamethasone in combination showed threefold increase in p-nitrophenol hydroxylation and the activity correlates with an elevated amount of a 53.000 dalton protein. Biosynthesis of mRNA and P450 protein is required for the induction. 3-amino-1,2,4-triazole and anti CYP2E1 IgG inhibition studies show that CYP2E1 is not responsible for enhanced p-nitrophenol hydroxylation, but the residual activity indicates the participation of other isozyme(s). As a result of double induction, changes in the amount of CYP2E1 of microsomes were not detected by Western blot analysis compared to untreated rat liver microsomes.

An evaluation of cytochrome P450 isoform activities in the female dark agouti (DA) rat: Relevance to its use as a model of the CYP2d6 poor metaboliser phenotype

The female dark agouti (DA) rat lacks CYP2D1, the equivalent enzyme in the rat to human CYP2D6 (debrisoquine hydroxylase), and shows impaired metabolism of a number of CYP2D6 substrates. However, from the data available in the literature it is not entirely clear whether the enzyme deficiency in the DA rat is restricted to CYP2D1, and whether factors such as age and substrate concentration are important determinants of interstrain differences in the activity of this enzyme. Given that the female DA rat is used as a model of the human CYP2D6 poor metaboliser phenotype, there is a need for a systematic evaluation of the P450 activities in the DA rat, and of its suitability as a model of the PM phenotype. In the present study metoprolol was used as a probe substrate to investigate CYP2D1 activity since both the α-hydroxylation and O-demethylation of this drug are catalysed by CYP2D6 in man. Formation of α-hydroxymetoprolol (AHM) and O-demethylmetoprolol (ODM) was 10- and 2.5-fold lower in liver microsomes from female DA rats compared with microsomes from agematched female Wistar rats, the latter representing the extensive metaboliser strain. Kinetic analysis suggested that in both strains of rat both the α-hydroxylation and O-demethylation of metoprolol were catalysed by more than one enzyme. By using quinine as a specific inhibitor of the enzyme, CYP2D1 was identified as an intermediate affinity site in the Wistar strain and was shown to have impaired activity in the DA strain. The activities of lower and higher affinity sites were similar in the two strains. Thus, the only difference between the two strains with respect to both routes of metoprolol metabolism appeared to be in the activity of CYP2D1. Interstrain differences were found to be highly dependent on the choice of substrate concentration, being more marked at lower concentrations. We have also investigated the metabolism of a number of probe compounds for some of the other P450 isoforms commonly involved in drug metabolism to determine the selectivity of the deficiency in the DA strain. p-Nitrophenol hydroxylation and erythromycin N-demethylation were catalysed at higher rates by DA than by Wistar liver microsomes, indicating higher levels of activity of CYP2E1 and CYP3A in the former strain. Felodipine oxidation, tolbutamide hydroxylation and both the hydroxylation and N-demethylation of S-mephenytoin were catalysed at similar rates by microsomes from the two strains, indicating similar activities of enzymes in the CYP2C and CYP3A families. However, both the hydroxylation and N-demethylation of R-mephenytoin were impaired in the DA strain. This indicates that at least one other isoform of P450, thought also to be a member of the CYP2C or CYP3A families, in addition to CYP2D1 is deficient in the DA strain. Our findings indicate that whilst the female DA rat could be used as a preliminary screen to identify CYP2D6 substrates, because of interspecies differences in metabolism it could not be used to provide quantitative information regarding the contribution of CYP2D6 to an oxidation in man. In addition, a small number of false positives would be identified owing to other enzyme deficiencies; no false negatives would be expected. Comparisons between strains should be performed using female, age-matched animals and low substrate concentrations.

Species differences in the biotransformation of ethyl chloride. I. Cytochrome P450-dependent metabolism.

Groups of male and female F-344 rats and B6C3F1 mice were exposed to 15,000 ppm ethyl chloride (monochloroethane, ECL) or to air for 5 days (6 h/day). In this report, features of the P450-dependent ECL metabolism in the animals are described. A concurrent report describes the in vitro and in vivo features of the GSH-dependent ECL metabolism (Fedtke et al. 1994). ECL is oxidatively dechlorinated in an NADPH- and O2-dependent reaction, resulting in the formation of acetaldehyde (AC). The oxidative ECL metabolism rates in microsomal incubations were measured. The results indicated induction of the oxidative ECL metabolism by ECL itself in mice and female rats. The hydroxylation of p-nitrophenol, which was used as an indicator of P450IIE1 activity, was also induced in microsomal incubations from ECL-exposed mice and female rats, but, corresponding to the ECL metabolism, not in male rats. In contrast, catalytic activities related to P450IA and IIB subfamilies were not induced by ECL treatment. Additional experiments with the P450IIE1-specific inhibitor 3-amino-1,2,4-triazole and induction experiments with acetone, phenobarbital and methylcholanthrene confirmed that the isoenzyme mainly involved in the dechlorination reaction is cytochrome P450IIE1. AC was not detected in serum of ECL exposed animals and only slightly enhanced amounts were detected in urine samples from ECL exposed mice, reflecting the high capacities of the AC metabolizing pathways in vivo. The data are discussed with regard to the results of a 2-year bioassay with F-344 rats and B6C3F1 mice exposed to 15,000 ppm ECL (NTP 1989a).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of P450 inhibition and induction on the olfactory toxicity of beta,beta'-iminodipropionitrile (IDPN) in the rat.

In addition to the neurotoxic effects of beta,beta'-iminodipropionitrile (IDPN) which have been previously reported by other investigators, the olfactory toxicity of this compound has recently been uncovered in this laboratory. Due to the apparently conflicting observations that the IDPN-induced lesion in the olfactory mucosa is very focal in nature (suggesting site-specific activation) and the observation by other investigators that the behavioral effects of IDPN appear to be due to the parent compound, we initiated studies into the possible role of the cytochrome P450 enzymes in the olfactory toxicity of IDPN. Immunohistochemical studies with antibodies raised against several different P450 isoforms revealed good correlation between IDPN-induced olfactory mucosal degeneration and the localization of a protein immunoreacting with an antibody to P450 2E1. Enzymatic studies revealed that there is approximately five-fold more p-nitrophenol hydroxylation activity in the olfactory mucosa than in the liver on a per milligram microsomal protein basis. Administration of 1% acetone in the drinking water increased the levels of olfactory mucosal 2E1, and the increase in enzyme levels corresponded to increased olfactory toxicity of IDPN; inhibition of P450 activities with either metyrapone or carbon tetrachloride eliminated or significantly decreased the olfactory toxicity of IDPN, respectively. These studies suggest a role for cytochrome P450, specifically the 2E1 isoform, in the activation of IDPN within the nasal mucosa.

Determination of p-Nitrophenol Hydroxylase Activity of Rat Liver Microsomes by High-Pressure Liquid Chromatography

p-Nitrophenol hydroxylation to p-nitrocatechol is a useful metabolic marker for the presence of functional cytochrome P450 2E1 in mammalian cell microsomes, but the assay is limited by the sensitivity of the spectrophotometric method used to monitor p-nitrocatechol formation. In this paper, a reverse-phase high-pressure liquid chromatography method, which is nearly 20 times more sensitive than the spectrophotometric method and more specific for p-nitrocatechol determination, is described. The method involves monitoring the presence of p-nitrocatechol in the trifluoroacetic acid-quenched reaction mixtures at 345 am. The utility of the method was demonstrated with rat liver microsomes, where p-nitrocatechol formation was found to be NADPH dependent, was linear with incubation times (2.5 to 30.0 min) and protein concentrations (0.03-0.48 mg/incubation), and exhibited typical Michaelis-Menton kinetics ( K m = 197 μM, V max = 2.8 nmol/mg protein/min).

In Vivo Induction of Hepatic P4502E1 by Ethanol: Role of Increased Enzyme Synthesis

P4502E1 (2E1), an ethanol-inducible P450 enzyme, plays an important role in the bioactivation of certain hepatotoxins and chemical carcinogens. Different mechanisms of 2E1 induction by ethanol and other agents (e.g., acetone) have been proposed, ranging from enhanced de novo enzyme synthesis caused by an increase in 2E1 mRNA and/or the efficiency with which it is translated to decreased enzyme degradation stemming from substrate stabilization. To evaluate these mechanisms, we first examined the time course of hepatic 2E1 protein induction in rats pair-fed liquid diets containing 36% of total calories as either ethanol or dextrin-maltose (controls) for 28 days. Western blot analysis with anti-2E1 immunoglobulins revealed that 2E1 reached a new steady-state level (eightfold greater than that found with controls) after ethanol feeding for 10 days and remained elevated for the duration of treatment. Microsomal p-nitrophenol hydroxylation, a 2E1-catalyzed reaction, exhibited a similar induction time course, with the maximal increase in enzyme activity also observed on Day 10 of ethanol administration. We then determined steady-state 2E1 protein turnover in ethanol-fed and control animals that were given [35S]methionine plus [3H]aminolevulinate to radiolabel 2E1 apoprotein and the prosthetic heme group, respectively. Monophasic exponential decay curves showed that hepatic 2E1 protein and heme half-lives (27-28 h and 17 h, respectively) did not differ between the treatment groups. However, rates of 2E1 synthesis, assessed by measuring initial rates of incorporation of [35S]methionine and [3H]aminolevulinate into 2E1 apoprotein and heme, were increased in animals fed ethanol. Our results indicate that the in vivo induction of hepatic 2E1 protein by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation. This enhancement of de novo 2E1 synthesis most likely entails the ethanol-mediated increase of steady-state levels of 2E1 mRNA and/or the stimulation of its translational efficiency.

Effects of ether anaesthesia and fasting on various cytochromes P450 of rat liver and kidney

Fed and fasted, male, Wistar albino rats exposed to light ether anaesthesia and killed immediately or after 30 or 120 min recovery were compared with non-anaesthetized rats for changes in liver and kidney cytochrome P450 (CYP) activities. In fed rats, liver total CYP (nmol/mg protein) decreased by 30% immediately after ether, but was restored to normal levels after 30 min recovery; in fasted rats, liver total CYP increased by 20% by fasting alone, then decreased by 65% immediately after ether, and recovered to only 70% of control at 2 hr after ether. Rat liver cytochrome P4501A (CYP1A; 7-ethoxyresorufin O-deethylase or EROD activity) and cytochrome P4502B (CYP2B; 7-pentoxyresorufin O-dealkylase or PROD activity) were decreased after ether anaesthesia, similar to those for total CYP. In contrast, rat liver cytochrome P4502E1 (CYP2E1), determined by p-nitrophenol hydroxylation, increased by 40% by ether anaesthesia alone, 70% by fasting alone and 140% by ether plus fasting; these increases were confirmed by the CYP2E1-mediated activation of nitrosopyrrolidine and by immunoblot analysis using antibody to CYP2E1. In rat kidney, losses of total CYP, CYP1A and CYP2B, and increases of CYP2E1, induced by ether anaesthesia, were much more marked in fasted (90% loss in total CYP, 30% increase in CYP2E1) than in fed rats (slight loss in total cytochrome P450, 30% increase in CYP2E1). As maximum losses of total CYP in liver of fasted rats exposed to ether occurred at the time of maximum increase of CYP2E1 and maximum rate of generation of reactive oxygen species (ROS), it is suggested that the increase of CYP2E1, resulting from its stabilization by fasting and ether, leads to generation of ROS, increase in lipid peroxidation and consequent loss of total CYP, associated with the hepatic and renal necrosis seen in ether intoxication and surgical trauma.

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of cytochrome P450 2E1: Selective loss after labilization of the enzyme

Mechanism-based inactivation of cytochrome P450 can result in the chemical modification of the heme, the protein, or both as a result of covalent binding of modified heme to the protein. In the present study we took advantage of different modes of inactivation of P450 2E1 by CCl 4, 1-aminobenzotriazole, or 3-amino-1,2,4-triazole to investigate parameters which target P450 2E1 for proteolysis from the microsomal membrane. Treatment of mice with CCl 4 at the point of maximal induction of P450 2E1 after a single oral dose of acetone resulted in the complete loss of P450 2E1-dependent p-nitrophenol hydroxylation and a 75% loss of immunochemically detectable protein within 1 h of administration. Treatment with 1-aminobenzotriazole at the point of maximal induction caused a complete loss of P450 2E1-dependent p-nitrophenol hydroxylation but only a 12% loss of immunochemically detectable protein 1 h after administration. Treatment of mice with 3-amino-1,2,4-triazole caused a rapid loss of both catalytic activity and microsomal p-nitrophenol hydroxylase activity. However, unlike CCl 4 treatment, the activity and enzyme level rebounded 5 and 9 h after treatment. The P450 2E1 ligand, 4-methylpyrazole, administered at the point of maximal induction maintained the acetone-induced catalytic and immunochemical level of P450 2E1. These results suggest that differentially modified forms of P450 2E1 show a characteristic susceptibility to degradation. While there are many potential pathways for protein degradation, the loss of P450 2E1 was associated with increased formation of hieh molecular weight microsomal ubiquitin conjugates. The formation of ubiquitin-conjugated microsomal protein which correlates with P450 2E1 loss suggests that ubiquitination may represent a proteolytic signal for the rapid and selective proteolysis of certain labilized conformations of P450 2E1 from the endoplasmic reticulum.

Oxidative and reductive metabolism by cytochrome P450 2E1

We are constantly exposed to many potentially toxic chemicals. Most require metabolic activation to species responsible for cell injury. Although cytochrome P450 2E1 is only one of many different forms of cytochrome P450 that catalyze these reactions, it has an important role in human health as a result of being readily induced by acute and chronic alcohol ingestion. The enzyme efficiently catalyzes the low Km metabolism of compounds commonly used as solvents in industry and at home as well as components found in cigarette smoke, many of which are established carcinogens and hepatotoxins. As a result, there is the potential for increased risk to low level exposure to such chemicals while cytochrome P450 2E1 is induced. Many substrates have been identified for cytochrome P450 2E1. Of the 52 substrates for the enzyme identified in this review, the demethylation of N,N-dimethylnitrosamine and the hydroxylation of p-nitrophenol and chlorzoxazone are the most effective for monitoring the level of this enzyme. In addition to oxidative reactions, cytochrome P450 2E1 is also an efficient catalyst of reductive reactions. CCl4-induced hepatotoxicity is one of the best-documented cases for the participation of cytochrome P450 2E1 in a toxicologically important reductive reaction. The reduction of oxygen to superoxide and peroxide are also important reductive reactions of the enzyme and could be important in lipid peroxidation. However, the role of this reaction in vivo remains controversial.

Induction of rat hepatic mixed-function oxidases by acetone and other physiological ketones: their role in diabetes-induced changes in cytochrome P450 proteins.

1. To evaluate the role of ketone bodies in diabetes-induced changes in hepatic cytochrome P450 composition, rats were treated with acetone, 3-hydroxybutyrate or 1,3-butanediol. 2. Treatment with acetone enhanced the rat hepatic O-dealkylations of ethoxyresorufin and methoxyresorufin, and the hydroxylation of p-nitrophenol, but had no effect on lauric acid hydroxylation and ethylmorphine N-demethylation. Neither 3-hydroxybutyrate nor 1,3-butanediol modulated the metabolism of the above substrates. 3. Immunoblot analysis of hepatic microsomal proteins revealed that treatment with acetone increased the apoprotein levels of P4501A2, P4502B1/2 and P4502E1. 4. It is concluded that acetone is responsible, at least partly, for the diabetes-induced increase in hepatic microsomal P4501A2, P4502B1/2 and P4502E1 proteins but does not mediate the increases in the P4503A1 and P4504A1 proteins. On the basis of work from our own and other laboratories a mechanism for the diabetes-induced changes in hepatic cytochrome P450 proteins is proposed.

Ethanol‐Induced Enhancement of Cocaine Bioactivation and Irreversible Protein Binding: Evidence Against a Role of Cytochrome P‐450IIE1

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.

Influence of ethanol on microsomalp‐nitrophenol hydroxylation and ethoxyresorufin deethylation in rat liver and lung

The ability of ethanol to induce the deethylation of ethoxyresorufin and hydroxylation of p-nitrophenol in rat lung and liver was investigated. The administration of 10% ethanol in the drinking water for 7 d increased hepatic p-nitrophenol hydroxylation twofold whereas ethanol ip (3 ml/kg/d for 7 d) decreased the activity. Both ethanol treatments decreased the activity in the lung. Ethoxyresorufin deethylation was induced in the liver by both ethanol treatments, and in the lung the bolus dosing ip increased the activity four-fold. The results indicate that the lung microsomes have p-nitrophenol hydroxylase and ethoxyresorufin deethylase activities, but only the latter was increased by ethanol treatment.

Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes

In intact rats, ethanol treatment has been associated with increases in hepatic levels of both P450IIB1/2 and P450IIE. When rat hepatocytes were cultured on an extracellular tumor matrix (Matrigel), exposure to ethanol from 48 to 96 h in culture resulted in increases in cytochromes P450IIE, IIB1/2, and IIIA. Cytochrome P450IIE was detected immunologically and enzymatically, using two activities associated with cytochrome P450IIE, p-nitrophenol hydroxylation, and acetaminophen activation to a metabolite that binds to glutathione. The content of cytochrome P450IIE in freshly isolated cells decreased when the cells were placed in culture. Exposure of the cultured hepatocytes to ethanol from 48 to 96 h after inoculation resulted in an increase in cytochrome P450IIE compared to untreated cultured cells. In addition, in culture, the amount of enzymatically active protein after ethanol treatment was equal to that in hepatocytes freshly isolated from intact animals. Ethanol treatment resulted in increases in cytochrome P450IIB1/ 2 compared to untreated cells, as shown immunologically and by increased benzyloxyresorufin dealkylase activity. However, phenobarbital induced cytochrome P450IIB1/ 2 to higher levels, compared to ethanol. Ethanol and phenobarbital treatments both increased P450IIIA, as determined immunologically and by the amount of propoxycoumarin depropylase activity that is inhibited by triacetyloleandomycin. However, the amount of P450IIIA increased after ethanol treatment was less than that increased after treatment with dexamethasone in these cells. The ethanol-mediated increases in all four forms of cytochrome P450 in culture suggest that these increases in the intact animal result from direct effects of ethanol on the liver.

Ethanol‐Inducible Cytochrome P‐450: Assessment of Substrates' Specific Chemical Probes in Rat Liver Microsomes

The capacity of liver microsomes to oxidize various substrates known to be specific of alcohol-inducible cytochrome P-450 was studied in rats treated with different xenobiotics such as 3-methylcholanthrene, phenobarbital, acetone, and ethanol. Analysis of results showed a significantly marked increase following ethanol and acetone treatments of the p-nitrophenol hydroxylation (283 +/- 19% and 304 +/- 21%), N-nitrosodimethylamine (NDMA) demethylation (280 +/- 105% and 228 +/- 95%), benzene hydroxylation (258 +/- 60% and 236 +/- 61%), butanol oxidation (173 +/- 34% and 154 +/- 32%), aniline hydroxylation (147 +/- 22% and 95 +/- 8%), and ether de-ethylation (95 +/- 17% and 83 +/- 17%) and a not significant increase of N-nitrosodiethylamine (NDEA) de-ethylation (34 +/- 11% and 9 +/- 8%) in rat microsomes, respectively, versus control animals (mean +/- SD, values expressed as nmol/min/nmole P-450). All of these activities significantly decreased after 3-MC treatment, except for the p-nitrophenol hydroxylation. PB treatment markedly enhanced NDEA de-ethylation, p-nitrophenol, and benzene hydroxylations (106 +/- 38%, 109 +/- 14%, and 153 +/- 62%, respectively) versus controls. These results suggest that NDMA and especially 1-butanol are the most specific and useful probes of alcohol-inducible cytochrome P-450 in crude liver microsomes.

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.

Inhibition of ethanol-inducible cytochrome P 450IIE1 by 3-amino-1,2,4-triazole

The p-nitrophenol hydroxylase activity of hepatic microsomes from acetone-treated rabbits was inhibited by 3-amino-1,2,4-triazole in a time- and NADPH-dependent manner. The loss of p-nitrophenol hydroxylation, an activity catalyzed predominately by P450IIE1, displayed a number of characteristics consistent with suicide inhibition of the enzyme. These include irreversibility, saturability, similarity of the effect of pH on the rate constant for inactivation and catalysis by the isozyme, protection by substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. At pH 6.8, the KI for 3-amino-1,2,4-triazole for inactivation was 57 mM and the maximal rate of inactivation was 0.43 min-1. The inactivation of hepatic microsomes resulted in a loss of spectrally detectable P450 which was correlated with the concentration of P450IIE1 in various microsomal preparations. Purified P450IIE1 was rapidly autoinactivated (kinact of about 0.1 min-1) in the presence of NADPH and P450 reductase. However, the autoinactivation was completely prevented by the addition of catalase. In the presence of catalase, purified P450IIE1 was inactivated in a time- and concentration-dependent manner by 3-amino-1,2,4-triazole (KI was 10 mM and the maximal rate of inactivation was 0.44 min-1). The inactivation resulted in the loss of spectrally detectable P450 but did not cause the formation of P420 or a loss of heme as determined by the reduced pyridine hemochrome. The spectrum of the inactivated enzyme exhibited a decreased extinction in the Soret region with a broad maximum at 378 nm and a shoulder around 428 nm. Inactivated P450IIE1 did not show a characteristic low-spin spectrum in the presence of 4-methylpyrazole. When 3-amino-1,2,4-[5-14C]triazole was used in the inactivation reaction, there was no significant incorporation of radioactivity into the protein or heme; these results suggest that the inactivation may be due to covalent binding of the heme to the protein or the modification of residues near the heme, which prevent substrate interaction. The effective inhibition of P450IIE1 by 3-amino-1,2,4-triazole suggests that the compound may be useful for the identification of P450IIE1-dependent microsomal catalysis.

Relative contributions of the cytochrome P450 and flavin-containing monooxygenases to the microsomal oxidation of phorate following treatment of mice with phenobarbital, hydrocortisone, acetone, and piperonyl butoxide

The relative contributions of the cytochrome P450-dependent monooxygenase system (P450) and the flavin-containing monooxygenase (FMO) in the microsomal oxidation of phorate, O,O-diethyl S-[(ethylthio)methyl]phosphorodithioate, were investigated in mice following treatment with phenobarbital (PB), piperonyl butoxide (PBO), acetone, or hydrocortisone. PBO treatment produced a distinct biphasic effect (initial inhibition and subsequent induction) on several hepatic enzyme activities, including phorate sulfoxidation. The relative contribution by P450 to phorate sulfoxidation decreased from 76% in control to 58% at 2 hr after treatment and increased to 89% at 12 hr. Hydrocortisone treatment caused an increase in FMO activity in liver microsomes, but not in lung microsomes. Administration of acetone caused an increase in benzphetamine N-demethylation and p-nitrophenol hydroxylation in the liver, but no change in phorate sulfoxidase activity was observed, nor was the relative contributions due to P450 and FMO altered. Treatment with phenobarbital produced a large increase in both benzphetamine N-demethylase and phorate sulfoxidase activity in liver microsomes, with the percentage of phorate sulfoxidase activity due to P450 being increased from 76% in the control livers to 85% in the treated livers.

Acetone-dependent regulation of cytochrome P-450j (IIE1) and P-450b (IIB1) in rat liver.

1. Concomitant changes in the concentration of P450 IIB1 (P-450b) and the rate of O-depentylation of 7-pentoxyresorufin was observed in rat liver microsomes after a single intragastric dose of acetone. 2. In contrast the concentration of P450 IIE1 (P-450j) did not coincide with changes in the rate of P450 IIE1-dependent p-nitrophenol hydroxylation or metabolic activation of carbon tetrachloride. 3. Quantification of the proteins in lysosomes indicated that both P450 IIB1 and P450 IIE1 are degraded via an autophagosomal/autolysosomal pathway. 4. It is concluded that P450 IIE1 is catalytically inactivated in microsomes prior to the degradation of this protein.

Catalase-dependent ethanol oxidation in perfused rat liver. Requirement for fatty-acid-stimulated H2O2 production by peroxisomes.

The purpose of this study was to measure rates of catalase-dependent ethanol uptake and rates of H2O2 generation in perfused rat livers in the presence of fatty acids of varying chain length. Rates of ethanol uptake in livers from fasted rats, perfused in a recirculating system, of about 80 mumol g-1 h-1 were decreased to about 10 mumol g-1 h-1 by the addition of an inhibitor of alcohol dehydrogenase (ADH), 4-methylpyrazole. The medium-chain-length fatty acid, laurate (12:0; 1 mM), increased rates of 4-methylpyrazole-insensitive ethanol uptake maximally to 80-85 mumol g-1 h-1. Rates of ethanol uptake diminished as the chain length of fatty acid was decreased [hexanoate (6:0) = 23 mumol g-1 h-1; octanoate (8:0) = 55 mumol g-1 h-1; decanoate (10:0) = 65 mumol g-1 h-1] or increased [myristate (14:0) = 77 mumol g-1 h-1; palmitate (16:0) = 80 mumol g-1 h-1; stearate (18:0) = 29 mumol g-1 h-1; oleate (18:1) = 60 mumol g-1 h-1; erucate (22:3) = 22 mumol g-1 h-1] from 12:0. Oleate did not increase rates of hydroxylation of p-nitrophenol, a substrate for the ethanol-inducible form of cytochrome P-450, indicating that the stimulation of ethanol uptake by fatty acids was not due to increased mixed-function oxidation. The increase of ethanol uptake was also not due to displacement of 4-methylpyrazole from ADH by fatty acids, since oleate stimulated ethanol uptake by about 50% in perfused livers from deermice genetically deficient in ADH. The increase in 4-methylpyrazole-insensitive ethanol uptake by fatty acids was blocked by the catalase inhibitor, aminotriazole, indicating the involvement of catalase. Rates of H2O2 generation by livers perfused in a non-recirculating system with 1.7% albumin were increased from 6 +/- 1 to 23 +/- 5 mumol g-1 h-1 by oleate (1 mM). Because of the discrepancy between rates of ethanol metabolism and H2O2 production, methods were developed to measure H2O2 production in a recirculating perfusion system. H2O2 generation was determined from the time necessary for steady-state level of catalase-H2O2, measured spectrophotometrically (660-640 nm) through a lobe of the liver, to return to basal values after the addition of a known quantity of methanol, which is not metabolized by ADH in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)