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.     

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.     

A labile sulfur in trisulfide affects cytochrome P-450 dependent lipid peroxidation in rat liver microsomes

The effects of trisulfide derivatives were studied on cytochrome P-450-dependent lipid peroxidation using rat liver microsomal systems. Cytochrome P-450-dependent lipid peroxidation was induced by carbon tetrachloride or tert-butylhydroperoxide and was evident by an increase in thiobarbituric acid-reactive substances (TBA-RS) and oxygen consumption. In these cytochrome P-450-dependent lipid peroxidation systems, pretreatment of microsome with trisulfide derivatives (cystine trisulfide and thiocyclam) significantly inhibited TBA-RS formation and oxygen consumption compared with disulfide or thiol analogs (cystine, nereistoxin, or cysteine). The labile sulfur contained in trisulfide disappeared during incubation with liver microsomes. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level and NAD(P)H-cytochrome P-450 reductase activity were significantly decreased by the addition of trisulfide derivatives. Therefore, in cytochrome P-450-dependent lipid peroxidation system, the potential effects of trisulfide appear to be mediated via enzyme inhibition. These results suggest that pretreatment of the trisulfide derivatives may affect the toxic function of exogenous xenobiotics or drugs, which are reduced by the liver enzyme cytochrome P-450 to radical species.     

Interspecies homology of liver microsomal cytochrome P-450. A form of dog cytochrome P-450 (P-450-D1) crossreactive with antibodies to rat P-450-male

P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male.     

Inhibition of ethanol-inducible cytochrome P450IIE1 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.     

Inhibition of microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions by beta-lapachone and related naphthoquinones

The lipophilic o-naphthoquinones beta-lapachone, 3,4-dihydro-2-methyl-2-ethyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 8-935), 3,4-dihydro-2-methyl-2-phenyl-2H-naphtho[1,2b]pyran-5,6-dione (CG 9-442), and 3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione (CG 10-248) (a) inhibited NADPH-dependent, iron-catalyzed microsomal lipid peroxidation; (b) prevented NADPH-dependent cytochrome P-450 destruction; (c) inhibited microsomal aniline 4-hydroxylase, aminopyrine N-demethylase and 7-ethoxycoumarin deethylase; (d) did not inhibit the ascorbate- and tert-butyl hydroperoxide-dependent lipid peroxidation and the cumenyl hydroperoxide-linked aniline 4-hydroxylase reaction; and (e) stimulated NADPH oxidation, superoxide anion radical generation and Fe(III)ADP reduction by NADPH-supplemented microsomes. In the presence of ascorbate, the same o-naphthoquinones stimulated oxygen uptake and semiquinone formation, as detected by ESR measurements. The p-naphthoquinones alpha-lapachone and menadione were relatively less effective than the o-naphthoquinones. These observations support the hypothesis that, in the micromolar concentration range, o-naphthoquinones inhibit microsomal lipid peroxidation and cytochrome P-450-catalyzed reactions, by diverting reducing equivalents from NADPH to dioxygen.     

Sensitivity of the rat liver microsomal oxidation of pyrazole to antibody raised against the ethanol-inducible rabbit liver cytochrome P-450 isozyme

Pyrazole is oxidized to 4-hydroxypyrazole by rat liver microsomes in a cytochrome P-450-dependent reaction and this oxidation can be increased by prior treatment of rats with pyrazole, 4-methylpyrazole, or chronic ethanol feeding. The induction pattern suggests that pyrazole may be an effective substrate for oxidation by P-450 IIE.1. This P-450 isozyme is recognized by antibody (anti-3a IgG) raised against the ethanol-inducible P-450 in rabbits. Experiments were carried out to evaluate the ability of anti-3a IgG to inhibit pyrazole oxidation by microsomes from controls and from rats treated with inducers of P-450 IIE.1. Immunoblots with anti-3a IgG or with the anti-pyrazole P-450 IgG were identical and indicated increased staining of the pyrazole P-450 with microsomes from rats treated with pyrazole, 4-methylpyrazole, or ethanol, relative to saline controls; very little staining occurred with microsomes from pair-fed controls or phenobarbital-treated rats. Rates of pyrazole oxidation were highest with microsomes from rats treated with the inducers of P-450 IIE.1 and lowest with pair-fed controls or rats treated with phenobarbital. Anti-3a IgG produced about a 60% decrease of pyrazole oxidation in microsomes from rats treated with inducers of P-450 IIE.1 and about a 25% decrease with the saline controls; no inhibition was found with microsomes from the phenobarbital-treated rats. The anti-3a IgG-resistant rate of pyrazole oxidation was similar with all the microsomal preparations, and was not due to interaction of pyrazole with hydroxyl radicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing the free radical activation of aliphatic alcohols

Incubation of rat liver microsomes with 1-propanol and 1-butanol in the presence of NADPH and of the spin trapping agent 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) allowed the detection of free radical intermediates tentatively identified as 1-hydroxypropyl and 1-hydroxybutyl radical, respectively. Microsomes isolated from rats treated chronically with ethanol (EtOH) or with the combination of starvation and acetone treatment (SA), exhibited a two-fold increase in the ESR signal intensity as compared to untreated controls, whereas no increase was observed in phenobarbital-induced (PB) microsomes. Consistently, in reconstituted membrane vesicles, ethanol-inducible cytochrome P450IIE1 was twice as active as phenobarbital-inducible P450IIB1 in producing 1-butanol free radicals. In the microsomal preparations from EtOH and SA pretreated rats the addition of antibodies against cytochrome P450IIE1, but not of preimmune IgGs, lowered the ESR signal of 1-butanol radicals by more than 50%. The same antibodies decreased the free radical production by untreated microsomes by 35-40%, but were ineffective on microsomes from PB-treated animals. This indicated that cytochrome P450IIE1 is the major enzyme responsible for the free radical activation of alcohols in control and ethanol-fed rats. The generation of 1-hydroxybutyl radicals by EtOH microsomes was inhibited by 40, 48 and 68%, respectively, by the addition of isoniazid, tryptamine and octylamine, compounds known to specifically affect the NADPH oxidase activity of this isoenzyme. This effect was not due to the scavenging of the alcohol radical since none of these compounds affected the ESR signals originated from 1-butanol in a xanthine-xanthine oxidase system. When added to reconstituted membrane vesicles isoniazid, tryptamine and octylamine also decreased 1-butanol radical formation by P450IIE1 by 54, 38 and 66%, respectively. Such an inhibition corresponded to the effect exerted by the same compounds on O2- release from P450IIE1 containing vesicles. These results indicate that the capacity of cytochrome P450IIE1 to reduce oxygen is related to its ability to generate alcohol free radicals and suggest that ferric cytochrome P450-oxygen complex might act as oxidizing species toward alcohols.     

Rat lung and liver microsomal cytochrome P-450 isozymes involved in the hydroxylation of n-hexane

The primary metabolism of n-hexane in rat lung and liver microsomes was investigated. In liver microsomes from untreated animals the formation of each of the metabolites, 1-, 2- and 3-hexanol, was best described kinetically by a two-enzyme system, whereas for lung microsomes a one-enzyme system was indicated for each metabolite. Cytochrome P-450-PB-B, the major cytochrome P-450 isozyme induced in rat liver by phenobarbital, appeared to be responsible for the formation of 2- and 3-hexanol in lung microsomes from untreated rats as judged by antibody inhibition studies. The presence of this isozyme was confirmed by immunoblotting. In contrast, formation of 1-hexanol in rat lung was catalyzed by a cytochrome P-450 isozyme different from the major isozymes induced by either phenobarbital or beta-naphthoflavone. Similarly, formation of 2,5-hexanediol from 2-hexanol was catalyzed by a P-450 isozyme different from cytochrome P-450-PB-B and present in liver but not in lung microsomes. Furthermore, alcohol dehydrogenase activity with hexanols or hexanediol as the substrate was found exclusively in liver cytosol. These results suggest that inhaled n-hexane must be transported to the liver either intact or in the form of 2-hexanol before the neurotoxic metabolite 2,5-hexanedione can be formed.     

Diethyl ether as a substrate for acetone/ethanol-inducible cytochrome P-450 and as an inducer for cytochrome(s) P-450

The ability of diethyl ether to serve as a substrate for microsomal and purified cytochrome P-450 (P-450) and as an inducer for rat hepatic microsomal monooxygenase activities was examined. Microsomal oxidation of ether to acetaldehyde, as monitored by high pressure liquid chromatography, was elevated 3- to 5-fold by treatment of rats with acetone or ethanol, 1.5- to 2-fold by treatment with ether, and only slightly by phenobarbital treatment. Ether also induced N-nitrosodimethylamine demethylase by up to 2-fold and 7-pentoxyresorufin dealkylation by up to 10-fold. These trends agreed with immunoblot experiments in which ether was a weak inducer of the P-450 isozyme IIE1 (encoded by the rat gene P450IIE1), but a stronger inducer of IIB1. A monoclonal antibody against IIE1 inhibited the deethylation by 78% in microsomes from acetone-treated rats and by 45% in controls. N-Nitrosodimethylamine, as well as common inhibitors of IIE1 such as hexane, benzene, pyrazole, and phenylethylamine, strongly inhibited ether deethylation. Using microsomes from acetone-induced rats, the apparent Km for deethylation was 13.4 +/- 2.4 microM and the Vmax was 8.2 +/- 0.2 (nmol of acetaldehyde/min/nmol of P-450). The Km for the controls was 71.3 +/- 9.5 microM. The rates of deethylation at 1 mM ether by purified, reconstituted IIE1 and IIB1 were 4.2 and 0.42 (nmol of acetaldehyde/min/nmol of P-450), respectively. Cytochrome b5 stimulated the rate due to IIE1 apparently by a decrease in the Km. These findings, along with previous work showing marked inhibition by ether of IIE1-dependent reactions, strongly support a major role for this isozyme in ether metabolism.     

Incorporation of cytochrome b5 into rat liver microsomal membranes. Impairment of cytochrome P-450-dependent mixed function oxidase activity

Cytochrome b5 was purified to electrophoretic homogeneity from the liver microsomes of untreated rats and reincorporated into liver microsomes from phenobarbital-treated rats, resulting in an approximate three-fold enrichment of the cytochrome b5 specific content (1.5 nmol haemoprotein X mg-1 protein). Our results have shown that the N-demethylation of benzphetamine was progressively inhibited in cytochrome b5-fortified microsomal preparations. Using stopped flow, visible difference spectrophotometry, the NADPH-driven reduction kinetics of cytochrome P-450 were examined in the modified microsomes over the first few seconds of reaction. Increasing the amount of incorporated cytochrome b5 resulted in a progressive inhibition of the initial, fast phase reduction rate constant of microsomal cytochrome P-450, both in the absence and presence of the type I substrate benzphetamine. Although the initial rate of NADPH-driven cytochrome b5 reduction was the same for both native and cytochrome b5-fortified microsomes, the extent of cytochrome b5 reduction was greater in the fortified microsomes. If cytochrome b5 has a positive role to play in cytochrome P-450-dependent mixed function oxidase activity either as an effector or in electron transfer or both, the former haemoprotein must be already present in sufficient concentrations in the native microsomes.     

Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rates ofp-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent M_r of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate M_r of 50000 and 51400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.A preliminary account of this work has been presented at the Nato Advanced Study Institute on Molecular Aspects of Monooxygenases and Bioactivation of Toxic Compounds, August 27–September 7, 1989, Izmir, Turkey.     

Induction of cytochrome P-450 isozyme 3a (P-450IIE1) in rabbit olfactory mucosa by ethanol and acetone

Cytochrome P-450 isozyme 3a, the alcohol-inducible form of cytochrome P-450 (P-450IIE1), was previously identified in rabbit nasal microsomes with the use of immunochemical techniques; the occurrence of this cytochrome in the nasal mucosa was subsequently confirmed through RNA hybridization experiments. However, in contrast to the well established inducibility of isozyme 3a in liver and kidney by alcohol treatment of the animals, no induction was observed in the nasal tissue with the use of a polyclonal anti-3a antibody for immunochemical quantitation. Recently, two new P-450 isozymes, designated NMa and NMb, were identified in rabbit nasal microsomes, and were found to have overlapping substrate specificity with isozyme 3a. Moreover, the two new cytochromes cross-react with the polyclonal anti-3a antibody that was used in the earlier study for quantitation of nasal isozyme 3a. These recent findings invalidate our previous conclusion that isozyme 3a is not induced by ethanol treatment of rabbits. In the present study, immunoblot quantitation of isozyme 3a was performed with a monoclonal anti-3a antibody that does not recognize either NMa or NMb, and the nasal microsomal metabolism of butanol was examined at various substrate concentrations. We have found that the level of isozyme 3a protein in nasal mucosa is elevated about 2-fold after treatment of the animals with ethanol and about 6-fold after treatment with acetone. Furthermore, corresponding increases in the rate of microsomal butanol oxidation were observed at low substrate concentrations. Thus, we conclude that P-450 isozyme 3a is, in fact, inducible in the nasal tissues by ethanol or acetone treatment of rabbits.     

Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1

Human cytochrome P-450IIE1 has been implicated in the oxidation of a number of substrates, including protoxins and -carcinogens. To date, no drugs have been identified that are exclusive substrates for the protein and are applicable for use as noninvasive probes of the in vivo function of the enzyme in humans. Chlorzoxazone was found to be oxidized only to 6-hydroxychlorzoxazone in human liver microsomes. Results of steady-state kinetics are consistent with the view that only a single enzyme catalyzes the reaction. The microsomal reaction was strongly inhibited by rabbit anti-P-450IIE1 and, in a competitive manner, by known P-450IIE1 substrates. Rates of chlorzoxazone 6-hydroxylation in different human liver microsomal preparations were well correlated with levels of immunochemically measured P-450IIE1 and rates of (CH3)2NNO oxidation. Chlorzoxazone 6-hydroxylation was also found to be catalyzed by purified human liver P-450IIE1. These results provide strong evidence that P-450IIE1 is the primary catalyst of chlorzoxazone 6-hydroxylation in human liver. Rates of chlorzoxazone 6-hydroxylation vary considerably among human liver samples, and chlorzoxazone 6-hydroxylation may have potential use as a noninvasive probe in estimating the in vivo expression of human P-450IIE1 and its significance as a risk factor in the toxicity and carcinogenicity of a number of solvents, nitrosamines, and drugs.