Effects of acetone administration on cytochrome P-450-dependent monooxygenases in hamster liver,kidney, and lung

The effects of acetone on liver, kidney, and lung monooxygenases were studied using hamsters administered 8% acetone in drinking water. Binding of aniline to liver microsomes induced a type II difference spectrum, and the spectral binding was enhanced in hamsters pretreated with acetone. Administration of acetone caused significant increases of cytochrome P-450 and cytochromeb ₅ contents in liver microsomes. The increases of the hemeproteins were associated with induction of monooxygenase activities toward test substrates, aniline, N-nitrosodimethylamine, benzphetamine, benzo(a)pyrene, and 7-ethoxycoumarin. In the kidneys, acetone administration increased microsomal contents of the hemeprotein and monooxygenase activities toward aniline, N-nitrosodimethylamine, and 7-ethoxycoumarin, but not benzphetamine or benzo(a)pyrene. In the lungs, acetone pretreatment increased aniline hydroxylase activity without affecting the levels of N-nitrosodimethylamine demethylase, cytochromes P-450 andb ₅. In marked contrast to the inductive effects in the liver, acetone administration markedly decreased lung microsomal benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities. Gel electrophoresis of liver and kidney microsomes from control and acetone-treated hamsters revealed that acetone treatment enhanced the intensity of a protein band(s) in the cytochrome P-450 molecular weight region. Immunoblotting of the microsomal proteins showed that the protein band induced by acetone in hamster liver, kidney and lung was cross-reactive with antibody raised against ethanol-inducible human liver cytochrome P-450. These results demonstrate that acetone has the ability to uniformly induce a specific form of cytochrome P-450, designated as IIE1, and to cause differential changes of monooxygenase activities in the hamster tissues. The complex effects of acetone on hepatic and extrahepatic monooxygenase systems may be important determinants of tissue-specific chemical toxicity.The nomenclature of P-450 used in this report follows the system recommended by Nebert et al. (1987, 1989). P-450 IIE1 has also been referred to as P-450ac by Patten et al. (1986), P-450j by Ryan et al. (1986), and as isozyme 3a and P-450_ALC by Coon and Koop (1987) in various species.     

Effects of pregnancy and ethanol treatment on the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by hamster liver and lung microsomes.

The tobacco-specific N-nitrosamino 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent carcinogen in adult Syrian golden hamsters and causes a high incidence of tumors in the offspring of hamsters after in utero exposure. We have investigated how pregnancy and/or ethanol treatment modulates the microsomal metabolism of NNK. Pregnancy decreased the alpha-carbon hydroxylation (activation) of NNK, whereas it increased both the pyridine N-oxidation and carbonyl reduction of NNK in liver microsomes, but not in the lung. Ethanol treatment of nonpregnant hamsters induced both the hepatic microsomal alpha-carbon hydroxylation and pyridine N-oxidation of NNK, but it increased only the formation of NNAL, the N-nitroso alcohol NNAL, in the lung. Ethanol-consuming pregnant hamsters showed no changes in the hepatic or pulmonary metabolism of NNK. In contrast, fetal hamsters exposed in utero to ethanol showed a general increase in the rate of metabolism of NNK. Immunoblot analyses demonstrated a reduction in the P-450IIE1 and total P450IIB1/IIB2 protein levels in the liver of pregnant hamsters, whereas a moderate increase of P-450IIB1 was observed in the lung. Moreover, ethanol treatment increased the amount of immunodetectable P-450IIE1 and total P-450IIB1/IIB2 in the liver of nonpregnant hamsters, but only the hepatic P-450IIE1 was induced by ethanol in pregnant hamsters. The P-450IIB1 protein levels were not affected by ethanol treatment in the lung of nonpregnant, pregnant, or fetal hamsters. In contrast, the fetal hepatic P-450IIE1 and P-450IIB1/IIB2 protein levels were increased by transplacental ethanol exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

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.     

Immunochemical detection of cytochrome P450 isozymes induced in rat liver byn-hexane, 2-hexanone and acetonyl acetone

Cytochrome P450 isozymes induced in rat liver by treatment withn-hexane, 2-hexanone and acetonyl acetone (given intraperitoneally 5 mmol/kg for 4 days) were investigated using enzyme assays (benzene, toluene, 7-ethoxyresorufin and 7-pentoxyresorufin metabolism) and monoclonal antibodies (anti-P450IA1/2, anti-P450IIB1/2, anti-P450IIC11/6, anti-P450IIE1(91) and anti-P450IIE1(98)).n-Hexane treatment enhanced the activities of low-K _m benzene aromatic hydroxylase and toluene side-chain oxidase, but not 7-ethoxyresorufin O-deethylase or 7-pentoxyresorufin O-depentylase. 2-Hexanone or acetonyl acetone treatment enhanced the activities of low-and high-K _m benzene aromatic hydroxylases, toluene side-chain oxidase and 7-pentoxyresorufin O-depentylase, but not of 7-ethoxyresorufin O-deethylase. Immunoblot analysis showed that anti-P450IA1/2 did not bind liver microsomal protein from either control and treated rats in the region of cytochrome P450s, whereas with anti-P450IIE1(98) a clear-cut band was seen in liver microsomes from control and treated rats, with intensities in the following order: 2-hexanone=acetonyl acetone n-hexane > control > phenobarbital. With anti-P450IIB1/2, a band was detected in microsomes from phenobarbital-treated rats, and to a lesser extent, in microsomes from 2-hexanone-and acetonyl acetone-treated rats. Like the immunoblot analysis, anti-P450IIE1(91) inhibited toluene side-chain hydroxylase activity in all microsomes, except in preparations from phenobarbital-treated rats and anti-P450IIB1 in microsomes from phenobarbital-, 2-hexanone- and acetonyl acetone-treated rats. Anti-P450IIC11/6 also inhibited toluene side-chain hydroxylase activity: the inhibited activity in the five different microsome preparations was as follows:n-hexane=control > acetonyl acetone=2-hexanone=phenobarbital. These results indicate thatn-hexane induces only quantitative alterations in the constitutive cytochrome P450 isozyme (P450IIE1), whereas its metabolites 2-hexanone and acetonyl acetone induce not only quantitative changes in constitutive cytochrome P450 (P450IIE1 and P450IIC11/6) but also a different type of isozyme (P450IIB1/2).     

Differences in the duration of the enhancement of liver mixed-function oxidase activities in ethanol-fed rats after withdrawal

Liver microsomal mixed-function oxidase activities were determined in female Sprague-Dawley rats after 3 weeks of ethanol feeding and for up to 10 days after withdrawal. Ethanol (36% of total calories) was administered in a high fat liquid diet and was replaced isocalorically by carbohydrates in controls. Chronic ethanol feeding similarly enhanced both microsomal cytochrome P-450 content and benzphetamine N-demethylase activity, per mg of protein, and resulted in a disproportionate increase in both aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities. A 6- to 7-day withdrawal period was apparently necessary for the overall disappearance of these effects of ethanol. Marked differences, however, were seen in the time courses of return of these variables to control levels, as also indicated by changes, during this period and specially during the first 24 hr after withdrawal, in the apparent molar activity of the microsomal fraction with the three substrates tested. The results were interpreted as indicating that the distinct ethanol-inducible cytochrome P-450 isozyme, with a high specific activity toward aniline, undergoes a very rapid turnover in liver microsomes. Induction of another form of cytochrome P-450, differing from the former by its slower turnover rate, would explain the induction by ethanol of 7-ethoxycoumarin O-deethylase activity. The withdrawal of ethanol was followed by a rapid but transient increase in benzphetamine N-demethylase activity above the ethanol-induced level, at a time when other activities were rapidly declining. This could suggest that the microsomal content of other cytochrome P-450 isozyme(s), with high specific activity toward this substrate, would also be temporarily altered during ethanol withdrawal. Important alterations in microsomal cytochrome P-450-dependent mixed-function oxidase activities occurred during the initial 24-hr period of withdrawal, even in the absence of a change in microsomal cytochrome P-450 content, indicating that the effects of chronic ethanol ingestion on hepatic drug-metabolizing enzyme activities may also be highly dependent on the proximity of ethanol intake.     

Cytochrome P-450-dependent formation of reactive oxygen radicals: isozyme-specific inhibition of P-450-mediated reduction of oxygen and carbon tetrachloride

1. Ethanol-inducible P450 IIE1 exhibits a high rate of oxygen consumption and oxidase activity. The enzyme is selectively distributed in the liver centrilobular area, the acinar region specifically destroyed after treatment with P450 IIE1 substrates/inducers such as ethanol, carbon tetrachloride, chloroform, N-nitrosodimethylamine and paracetamol. 2. Twenty substrates and ligands for cytochrome P450 IIB4 and P450 IIE1 were evaluated for their ability to inhibit microsomal and reconstituted NADPH-dependent oxidase activity, and the P450 IIE1-catalysed reduction of carbon tetrachloride to chloroform. Type I ligands and substrates did not inhibit the processes whereas nitrogen-containing compounds such as octylamine, cimetidine, imidazole and tryptamine inhibited NADPH oxidation and H2O2 formation in microsomes from starved and acetone-treated rats by around 50%. 3. Tryptamine, octylamine, isoniazid and p-chloroamphetamine inhibited reconstituted P450 IIE1-dependent oxidase activity with half maximal effects at 14-170 microM. 4. Isoniazid, cimetidine and tryptamine inhibited the P450 IIE1-dependent reduction of carbon tetrachloride, whereas acetone was without effect. 5. The oxygen dependency of microsomal oxidase activity exhibited high-affinity and low-affinity phases, with partial saturation at 20 microM of O2. 6. It is concluded that microsomal oxidase activity takes place at physiological concentrations of O2 and that isozyme-specific type II ligands compete with oxygen or carbon tetrachloride for reduction by P-450 haem.     

Liver microsomal drug metabolism in ethanol-treated hamsters

Administration of ethanol in drinking water to Syrian golden hamsters for 1-3 weeks caused alterations of microsomal cytochrome P-450-dependent monooxygenase activities in the liver accompanied by a slight elevation in cytochrome P-450 content. Ethanol treatment resulted in an increase in the activities for ethanol oxidation, aniline p-hydroxylation and dimethylnitrosamine N-demethylation. In particular, when dimethylnitrosamine was used as a substrate, the rate of formaldehyde formation was enhanced by 2- to 2.7-fold, while ethanol oxidation and aniline p-hydroxylation were increased by 1.5- to 2- and 1.2- to 1.3-fold, respectively. On the other hand, the activities of 7-ethoxycoumarin O-deethylase, benzphetamine N-demethylase and benzo[a]pyrene 3-hydroxylase were apparently decreased after ethanol treatment. These results for hamsters were significantly different from those reported for rats.     

Qualitative and quantitative differences in the induction and inhibition of hepatic benzo[a]pyrene metabolism in the rat and hamster

The present study compared the induction and inhibition of the metabolism of the prototype polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in rat and hamster liver microsomes. The production of total polar metabolites was quantitated by separating 3H-metabolites from [3H]-BaP using reverse-phase thin-layer chromatography. The rate of hepatic microsomal BaP metabolism was similar in the rat and hamster (0.81 vs 0.72 nmol/min/nmol cytochrome P-450 respectively). In the rat, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 5 micrograms/kg, i.p.) and 3-methylcholanthrene (3-MC; 50 mg/kg, i.p., X 3 days) pretreatments doubled the rate of BaP metabolism, whereas phenobarbital pretreatment (PB; 80 mg/kg, i.p., X 3 days) had no effect. In contrast, hamster hepatic microsomal BaP metabolism was elevated 2.3-fold by PB pretreatment, whereas TCDD and 3-MC pretreatments had no effect. Isosafrole pretreatment (ISO; 150 mg/kg, i.p., X 3 days) elevated the rate by almost 2-fold in each species. Another cytochrome P-448-mediated activity, 7-ethoxyresorufin O-deethylase (EROD), was induced by the same compounds that induced BaP metabolism in the rat. In hamster liver microsomes, in contrast to BaP metabolism, EROD was induced by TCDD and 3-MC but not PB or ISO pretreatments. The results suggest differences in the substrate specificity of the cytochromes P-448-450 induced by TCDD, 3-MC and PB in these species. This was supported by the different selectivity of the in vitro inhibitors, metyrapone and 7,8-benzoflavone, towards BaP metabolism and EROD in hepatic microsomes from TCDD- or PB-pretreated rats and hamsters. Reverse-phase HPLC analysis indicated that, while 3-hydroxy-BaP was the major metabolite formed by the untreated rat, untreated hamster liver microsomes formed predominantly BaP-4,5-diol. Microsomes from TCDD-treated rats generated elevated levels of all BaP-diols, diones and 3-hydroxy-BaP, with the major metabolites being BaP-9,10- and BaP-7,8-diols. In contrast, the metabolite profile from TCDD-pretreated hamsters was unchanged from the control. PB-treated hamster microsomes produced elevated levels of BaP-diones and 3-hydroxy-BaP. However, the major hepatic metabolite formed by PB-pretreated hamsters was BaP-4,5-diol, while BaP-9,10- and BaP-7,8-diols were not detected. The results of the study indicate differences in the induced cytochrome P-450s and the generation of toxic BaP metabolites in the liver of the rat and hamster.     

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.     

Oxidation of 1,1,1,2-tetrafluoroethane in rat liver microsomes is catalyzed primarily by cytochrome P-450IIE1.

1,1,1,2-Tetrafluoroethane (R-134a), a nonozone-depleting alternative air-conditioning refrigerant and propellant for pharmaceutical preparations, is oxidatively defluorinated by rat hepatic microsomes. In this report we show that induction of cytochrome P-450IIE1 in rats, by pyridine administration, resulted in an 8-fold increase in the rate of R-134a metabolism by hepatic microsomes (Vmax 47 vs. 6 nmol F-/mg microsomal protein/15 min). Furthermore, when data were normalized for P-450 content, a 4-fold increase in R-134a metabolism was noted for IIE1-enriched microsome preparations. In contrast, phenobarbital and Aroclor 1254 decreased the specific activity of hepatic microsomes for this function. The microsomal content of P-450IIE1, as evaluated by Western blot, was elevated significantly only in microsomes from pyridine-treated rats. p-Nitrophenol and aniline, which are metabolized at high rates by rat P-450IIE1, decreased the rate of R-134a defluorination by hepatic microsomes; Dixon plot analysis indicated competitive inhibition with a Ki of 36 microM p-nitrophenol or 115 microM aniline. Pyridine also potently induced defluorination of R-134a catalyzed by rabbit liver microsomes. Studies with individual P-450 isozymes purified from rabbit liver showed that the phenobarbital- and polycyclic hydrocarbon-induced isozymes (IIB1 and IA2) defluorinated R-134a at negligible rates (1.9 and 0.4 nmol F-/nmol P-450/60 min, respectively). In contrast, P-450IIE1 catalyzed defluorination of R-134a at a relatively high rate (16.2 nmol F-/nmol P-450/60 min); isozyme IA1, which also is induced by nitrogen-containing heterocycles such as pyridine, was somewhat active (5.3 nmol F-/nmol P-450/60 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acetone administration on drug-metabolizing enzymes in mice: presence of a high-affinity diethylnitrosamine de-ethylase

Treatment of CD1 mice with acetone raised activities of hepatic microsomal p-nitrophenol hydroxylase, ethoxycoumarin de-ethylase, acetone hydroxylase and diethylnitrosamine de-ethylase (DENd) several-fold. P-450IIE1-linked acetone hydroxylase showed the highest inducibility. In microsomes from acetone-pretreated mice the cytochrome b5 and P-450 content was nearly doubled and their electrophoretic profile showed induction of a protein of Mr 53,000, probably P-450IIE1. Liver phase II enzymes were not affected by acetone treatment. Kinetic analyses of DENd were performed in control or acetone-induced microsomes and Km and Vmax were determined. Two distinctly apparent Km values (0.56 and 20.3 mM) were observed for DENd of control microsomes and at least 3 apparent Km values (0.05, 0.51, 8.4 mM) were observed in acetone-induced microsomes. Thus, acetone administration to mice induces a high-affinity form of DENd which can be important in vivo at low diethylnitrosamine (DEN) exposure as this enzyme functions when DEN concentration is below 0.1 mM.     

Isozyme-specific monoclonal antibody-directed assessment of induction of hepatic cytochrome p-450 by clotrimazole

Clotrimazole, an N-substituted imidazole widely used as an antifungal agent, has been shown to both inhibit and induce hepatic cytochrome P-450 and related monooxygenase activities. In this study the profile of hepatic cytochrome P-450 isozyme(s) induced by clotrimazole treatment of male Sprague-Dawley rats was investigated. Clotrimazole administration (100 mg/kg, daily for 4 days, ig) resulted in 86% induction of spectrally detectable cytochrome P-450 in hepatic microsomes. In these microsomes 7-ethoxycoumarin O-deethylase (126%), aminopyrine N-demethylase (176%), benzphetamine N-demethylase (117%), p-nitrophenol hydroxylase (89%), and 7-ethoxyresorufin O-deethylase (62%) activities were significantly induced, whereas aryl hydrocarbon hydroxylase activity remained unchanged. Characterization of cytochrome P-450 isozyme(s) in hepatic microsomes prepared from clotrimazole-treated animals was based on the immunoreactivity of these microsomes with highly specific monoclonal antibodies (MAbs) raised against 3-methylcholanthrene-specific P-450 (MAb 1-7-1), phenobarbital-specific P-450 (MAb 2-66-3), pregnenolone-16 alpha-carbonitrile-specific P-450 (MAb C2), and ethanol-inducible P-450 (MAb 1-98-1). Western blot analysis of hepatic microsomes prepared from clotrimazole-treated animals with MAb 2-66-3, MAb 1-98-1, and MAb C2 revealed strong immunoreactive bands, whereas moderate reactivity was observed with MAb 1-7-1. MAb 2-66-3 significantly inhibited 7-ethoxycoumarin O-deethylase activity 45%), whereas MAb 1-7-1 moderately inhibited 7-ethoxyresorufin O-deethylase activity (-30%) in clotrimazole-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Influence of chronic ethanol consumption on hamster liver microsomal O-dealkylase activities and cytochrome b5 content

The effects of two different methods of administering ethanol to hamsters on liver microsomal cytochrome levels and the activities of ethoxyresorufin O-deethylase and p-nitroanisole O-demethylase have been examined. Administration of ethanol in liquid diets resulted in enhanced levels of cytochrome P-450, NADPH-supported aniline hydroxylase (Form I), and both NADPH- and NADH-supported p-nitroanisole O-demethylase. NADH-ferricyanide reductase was also increased. No change in NADPH-cytochrome c reductase or in the NADPH-supported rate of ethoxyresorufin O-deethylase was observed. In contrast, both NADH-supported ethoxyresorufin O-deethylase and cytochrome b5 levels were decreased. Administration of ethanol in the drinking water to chow-fed animals had no effect on total cytochrome P-450 levels; however, the rates of NADPH-supported aniline hydroxylase (Form I) and p-nitroanisole O-demethylase activity were increased. No changes in NADPH-cytochrome c reductase, NADH-ferricyanide reductase, or NADH-supported p-nitroanisole O-demethylase activity were noted. Cytochrome b5 levels were decreased as were both the NADPH- and NADH-supported rates of ethoxyresorufin O-deethylase. These data suggest that chronic consumption of ethanol by hamsters either in liquid diet form or as ethanol-water solutions to chow-fed animals lowers cytochrome b5 levels. When cytochrome b5 levels are lowered and total chromosome P-450 levels remain unchanged, the NADPH-supported rate of microsomal O-dealkylation of ethoxyresorufin is decreased. These data suggest that cytochrome b5 participates in the NADPH-supported microsomal O-dealkylation of ethoxyresorufin.     

New heterocyclic modifiers of oxidative drug metabolism--II. Steric factors in the interaction of isomeric 2-(naphthyl)methylbenzimidazoles with rat hepatic microsomal cytochrome P-450 and monooxygenase activities

The inhibitory potency of the two isomeric 2-(naphthyl)methylbenzimidazoles towards three monooxygenase activities (aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and aniline p-hydroxylase) was assessed in hepatic microsomal fractions from untreated, phenobarbitone-induced and beta-naphthoflavone-induced rats. The isomers were essentially equipotent with each other as inhibitors of the phenobarbitone-induced monooxygenases (the ratio of the I50s of the isomers was about 1.0 in each case) but differences between the isomers were noted in the inhibition potencies against three monooxygenase activities from beta-naphthoflavone-induced liver. The isomer 2-(1'-naphthyl)methylbenzimidazole was approximately twice as potent as the 2'-naphthyl isomer against 7-ethoxyresorufin O-deethylase activity, whereas the opposite was observed with respect to 7-ethoxycoumarin O-deethylase inhibition; aniline p-hydroxylase was poorly inhibited by both isomers. The binding affinity and extent of binding, assessed from double-reciprocal plots of spectral binding studies, of the 1'-isomer was much greater than that of the 2'-isomer in beta-naphthoflavone-induced microsomes. Inhibition data in untreated hepatic microsomes were more complex and the finding of principal interest was that the 1'-isomer was poorly inhibitory towards aniline p-hydroxylase activity whereas the 2'-isomer enhanced this activity. These studies suggest that the steric conformations of the isomeric naphthylmethylbenzimidazoles at the cytochrome P-450 active centre determines the extent to which the inhibitors modulate a specific monooxygenase activity, and that multiple binding sites with the capacity to interact to different extents with benzimidazole derivatives are present in P-450 in beta-naphthoflavone-induced hepatic microsomes. The apparent importance of steric conformation as a determinant of inhibition and enhancement of aniline p-hydroxylase in untreated microsomal fractions may well reflect specific interactions with multiple binding sites.     

Comparison of the form(s) of cytochrome P-450 induced by ethanol and glutethimide in cultured chick hepatocytes

In this study, using a combination of immunological and enzymatic characterizations, we compared the forms of cytochrome P-450 induced by ethanol and glutethimide in primary cultures of chicken embryo hepatocytes. Recently we purified a cytochrome P-450 of 50K molecular weight from chicken embryo liver using glutethimide as a prototypic inducer. Antibodies to both this chicken cytochrome P-450 and to rabbit cytochrome P-450 form 3a from the IIE subfamily detected microsomal proteins of 50K induced by either ethanol or glutethimide in cultured chick embryo hepatocytes, indicating the antigenic homology of these subfamilies of cytochromes P-450 among different animal species. However, the antibody to glutethimide-induced chick cytochrome P-450 of 50K inhibited p-nitrophenol hydroxylase and benzphetamine demethylase activities 85-90% in microsomes from both ethanol- and glutethimide-treated cells, indicating similar epitopes whose integrity is required for catalytic activity. In contrast, antibodies to rabbit cytochrome P-450 form 3a had little to no effect on these same microsomal activities. Both ethanol and glutethimide induced microsomal p-nitrophenol and aniline hydroxylase activities in cultured chick embryo hepatocytes. In microsomes from ethanol-treated cells, the turnover of p-nitrophenol per cytochrome P-450 was 2-fold greater than that induced by glutethimide treatment, suggesting that ethanol is inducing a form of cytochrome P-450 that has greater catalytic activity with this substrate than glutethimide-induced forms. Thus, in cultured chick embryo hepatocytes, ethanol may induce cytochromes P-450 from both the IIB and IIE subfamilies.     

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)     

Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes. Effects of prior treatment with ethanol and other xenobiotics

The cytochrome P-450 monooxygenase system of hamster liver microsomes and its response to prior treatment with ethanol and other xenobiotics have been examined. Male Syrian golden hamsters were administered ethanol (ETOH), phenobarbital (PB), 5,6-benzoflavone (BF) or isoniazid (INH). Each treatment resulted in a moderate increase (20-60%) in the specific content of liver microsomal cytochrome P-450 along with a unique hemeprotein ferrous carbonyl Soret maximum. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of liver microsomes revealed distinctive changes in protein banding patterns in the cytochrome P-450 (45-60 kDa) region with each treatment. NADPH: cytochrome c reductase activity was increased by both PB and INH, whereas cytochrome b5 content was increased by INH only. Microsomal oxidation of ETOH and aniline p-hydroxylation (expressed per nmol cytochrome P-450) were enhanced dramatically by ETOH and INH, whereas PB and BF had no effect on these enzymatic activities. Both ETOH and INH also increased zoxazolamine 6-hydroxylation but, in contrast to other rodent species, this drug-metabolizing activity was decreased in hamster liver microsomes after treatment with either PB or BF. Microsomal benzphetamine N-demethylation was decreased by ETOH, INH and BF administration and was only modestly enhanced after treatment with PB. ETOH and INH had no effect on the O-deethylation of 7-ethoxycoumarin, and enzymatic activity increased by BF but decreased by PB. These results demonstrate that the cytochrome P-450-dependent monooxygenase system of hamster liver microsomes responds to treatment with ETOH and other xenobiotics in a manner that is quantitatively and, in certain respects, qualitatively different from that reported for the rat, rabbit, and mouse.