Co-induction of cytochrome P-450 isozymes in rat liver by 2,4,5,2',4',5'-hexachlorobiphenyl or 3-methoxy-4-aminoazobenzene

A multitude of xenobiotics have been demonstrated to co-induce either cytochromes P-450c and P-450d or cytochromes P-450b and P-450e in rat hepatic microsomes. Recently, the compounds 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) and 3-methoxy-4-aminoazobenzene (3-MeO-AAB) have been suggested as selective inducers of cytochrome P-450b (Eur. J. Biochem. 151:67 (1985)) and P-450d (Biochem. Biophys. Res. Commun. 133:1072 (1985)), respectively. Since the identification of inducers with such unique characteristics would have implications with regard to the mechanism of induction of all four isozymes, we have examined the induction of cytochromes P-450b and P-450e by HCB and cytochromes P-450c and P-450d by 3-MeO-AAB in liver microsomes from adult male rats. Immunoblot analysis with monoclonal antibodies directed against cytochromes P-450b and P-450e indicate that HCB induces both isozymic species at the three dosage levels examined (10, 90, and 180 mg/kg). Similarly, 3-MeO-AAB does not appear to represent a unique inducer. Immunoblots of hepatic microsomes from animals treated with three different dosage regimens of 3-MeO-AAB demonstrate that, even at the lowest dosage level (50 mg/kg), both cytochromes P-450c and P-450d are induced. Moreover, immunoinhibition of 7-ethoxyresorufin O-deethylase (EROD) activity by monospecific antibody against either cytochrome P-450c or P-450d confirms this result. 3-MeO-AAB increases this enzyme activity 10-fold; approximately one-third of this induced activity is inhibited with monospecific anti-P-450c, while two-thirds is inhibited with monospecific anti-P-450d. This study also demonstrates that hepatic EROD activity is not an accurate estimate of cytochrome P-450c content since the majority of this enzyme activity in control and 3-MeO-AAB-treated rats is inhibited with monospecific anti-P-450d but not with monospecific anti-P-450c.     

Effect of glutathione on the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes

Rat liver microsomes catalyze both the N-demethylation and the 4'-hydroxylation of the azo dye carcinogen N,N-dimethyl-4-aminoazobenzene (DAB). It was found that addition of glutathione (GSH) to the microsomal system markedly stimulated both metabolic pathways. This occurred in the presence of either added NADPH or an NADPH-generating system. It was necessary that GSH be present when the reaction began; if added later, stimulation did not occur. This suggested a direct effect on microsomes rather than a chemical interaction with metabolic intermediates of DAB. Since stimulation occurred even in the presence of EDTA, the GSH effect cannot be satisfactorily explained in terms of suppression of lipid peroxidation which is totally inhibited by EDTA. Cysteine and cysteamine also stimulated both pathways but were less potent than was GSH; oxidized GSH was without significant effect. Dithiothreitol and beta-mercaptoethanol stimulated 4'-hydroxylation but inhibited N-demethylation, even in the presence of stimulatory concentrations of GSH. Apparently, the synthetic sulfhydryl compounds act through a mechanism different from that of GSH. Inhibition by dithiothreitol is consistent with formation of an N-oxide intermediate during N-demethylation. These observations also support previous findings that N-demethylation and 4' hydroxylation are, in the main, catalyzed by different isozymes of cytochrome P-450.     

Cytosolic factors that alter the metabolism of N,N-dimethyl-4-aminoazobenzene by rat liver microsomes

N,N-Dimethyl-4-aminoazobenzene (DAB), an azo dye carcinogen, is N-demethylated and 4'-hydroxylated by rat liver microsomes. Addition of hepatic cytosol to the microsomal system stimulated both pathways. This occurred in the presence of added NADPH or an NADPH-generating system. Cytosol was effective only when present prior to addition of substrate; no stimulation was seen when added after the reaction had begun. This suggested a direct effect on the microsomes rather than a chemical interaction with one or more metabolic intermediates of DAB. The degree of stimulation was somewhat different when using microsomes from phenobarbital- or beta-naphthoflavone-treated animals, implying a selectivity of the cytosolic effect for various isozymes of cytochrome P-450. Some loss of stimulatory activity occurred with dialysis. Activity was restored by adding back glutathione (GSH) which can stimulate DAB metabolism even in the absence of cytosol. DAB metabolism is also stimulated by EDTA. Although both EDTA and cytosol inhibit lipid peroxidation, cytosol stimulated DAB metabolism even in the presence of EDTA. Therefore, suppression of lipid peroxidation does not explain satisfactorily the cytosolic effect. Separation of cytosolic proteins by gel filtration revealed a factor which inhibits N-demethylation but not 4'-hydroxylation of DAB. Heating at 100 degrees partially inactivated the stimulatory activity. However, inhibitory activity was less susceptible to heat inactivation than was stimulatory activity. These results indicate that, in the whole cell, microsomal metabolism of xenobiotics is regulated to an appreciable extent by macromolecular cytosolic substances.     

Oxidative N-demethylation of N,N-dimethylaniline by purified isozymes of cytochrome P-450

The metabolism of N,N-dimethylaniline (DMA) by rabbit liver microsomes results in the formation of N-methylaniline (NMA) and formaldehyde. The N-oxide of DMA (DMA N-oxide) has been suggested as an intermediate in the cytochrome P-450-catalyzed demethylation reaction. The role of DMA N-oxide as an intermediate in demethylation has been investigated in a reconstituted system consisting of NADPH-cytochrome P-450 reductase, phospholipid, and several different purified isozymes of cytochrome P-450. The abilities of several cytochrome P-450 isozymes from rabbit liver (P-450 form 2 and P-450 form 4) and rat liver (P-450b and P-450c) to catalyze N-oxide formation and their abilities to catalyze demethylation of the N-oxide were determined and compared with their abilities to catalyze the demethylation of DMA. The metabolism of DMA by the purified isozymes of cytochrome P-450 in the reconstituted system did not result in the formation of measurable amounts of the N-oxide. The turnover numbers for the metabolism of DMA and DMA N-oxide to formaldehyde by the reconstituted system containing cytochrome P-450 form 2 were 25.6 and 3.4 nmol/min/nmol cytochrome P-450, respectively. The three other isozymes (P-450 form 4, P-450b, and P-450c) also exhibited significantly greater rates for the demethylation of DMA than for the N-oxide. If the N-oxide were an intermediate in the demethylation reaction, it should be metabolized at a rate greater than or at least equal to DMA. Therefore, these data, along with the inability to detect N-oxide formation during the cytochrome P-450-catalyzed demethylation of DMA, suggest that the N-oxide of DMA is not an intermediate in demethylation of DMA by these forms of cytochrome P-450 and that DMA N-oxidase activity is not associated with these isozymes.     

Drug metabolism in cirrhosis. Selective changes in cytochrome P-450 isozymes in the choline-deficient rat model

The effect of a choline-deficient diet on microsomal cytochrome P-450 and mixed-function oxidase (MFO) activity was investigated in relation to the development of nutritional cirrhosis. In rats that received the choline-deficient diet for 28 weeks cirrhosis was evident macroscopically and histologically; control rats that received an identical diet supplemented with choline had normal livers. Microsomal cytochrome P-450 and cytochrome b5 were reduced in cirrhotic liver to 50% of control levels. Three MFO activities (ethylmorphine N-demethylase, aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase) were also reduced to 40-70% of control levels. However, the turnover number for the O-deethylation of 7-ethoxycoumarin was not reduced in cirrhotic liver. This finding suggested that certain drug oxidations may be selectively depressed in nutritional cirrhosis. To examine the possibility that selective changes in MFO activity may reflect the suppression of certain cytochrome P-450 isozymes, partially purified fractions of the cytochrome were prepared after solubilisation and hydrophobic affinity chromatography (on n-octylamino-Sepharose 4B) of cirrhotic and control liver microsomes. Analysis of these fractions by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and laser densitometry indicated that a protein band of apparent minimum molecular weight 50.5 kD was primarily affected in cirrhotic rat liver microsomes. Levels of two other bands (apparent minimum molecular weight 48 and 52.5 kD) appeared essentially unaltered. Additional electrophoretic studies, conducted under non-reduced conditions, indicated the haemoprotein nature of protein bands in the 48-55 kD region. These data strongly suggest that cirrhosis produced in rats by a choline-deficient diet is associated with selective decreases in oxidative drug metabolism and individual cytochrome P-450 isozymes.     

Role of different cytochrome P-450 isozymes in the demethylation of various substrates

Phenobarbital treatment of rats enhanced 2-fold the hepatic aninopyrine (AP) and dimethylhydrazine (DMH) demethylation but not that of N-nitrosodimethylamine (NDMA). Pyrazole enhanced the demethylation rate of DMH and NDMA but not that of AP. The in vitro effects of metyrapone and SKF-525A on the demethylation rate of various substrates were dependent on the substrate and treatment of rats. The data suggest that the demethylation of various substrates might be catalyzed by different cytochrome P-450 isozymes.     

Role of isozymes of rabbit microsomal cytochrome P-450 in the metabolism of retinoic acid, retinol, and retinal.

The metabolism of retinoic acid, retinol, and retinal has been investigated with eight purified rabbit cytochrome P-450 (P-450) isozymes, including the major forms in nasal and liver microsomes. Retinoids hydroxylated at the 4-position were found to be major metabolites with each of the isozymes examined. Only two of the isozymes, polycyclic aromatic hydrocarbon-inducible P-450 1A2 and antibiotic-inducible P-450 3A6, also catalyze the oxidation of retinal to retinoic acid, a reaction not previously attributed to P-450. P-450 1A2 showed high activities in both the 4-hydroxylation and aldehyde oxidation reactions. Phenobarbital-inducible P-450 2B4 also had high activity in the 4-hydroxylation reaction of retinoids, and cytochrome b5 was found to increase the activity of P-450 2B4 with each substrate but to increase the activity of P-450 1A2 only with retinoic acid. In microsomes, retinoic acid is converted in an NADPH-dependent manner to both 4-hydroxyretinoic acid and 4-oxoretinoic acid, but none of the isozymes investigated was found to convert the 4-hydroxy derivative to the 4-oxo derivative. Microsomes from animals treated with phenobarbital were more active than those from untreated animals in the 4-hydroxylation reaction and, consequently, showed an increase in the ratio of 4-hydroxy to 4-oxo derivatives produced. These results show that the individual forms of P-450 metabolize retinoic acid, retinol, and retinal to multiple products, and they indicate that the amounts formed may be dependent on the exposure of animals to various inducers of P-450.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24?h after the last treatment.

2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene.

3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes.

4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes.

5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30?min after treatment with the drug.     

Effect of nutritional imbalances on cytochrome P-450 isozymes in rat liver

Male Sprague-Dawley rats were fed for six weeks either a control diet containing 22% casein (C) and 5% fat (F) or a low-protein diet (6% C, 5% F) or high-lipid diet (30% C, 30% F). A group of rats received a control diet containing 50 ppm of Phenoclor DP6. Three major forms of cytochrome P-450, UT 50, BP 3a and MC 2 were purified from livers of DP6-fed rats and only two forms, UT 50 and PB 3a, were purified from control and dietary groups. The amino acid composition and the catalytic activities towards all substrates tested were only significantly modified in the purified UT 50 P-450 isozyme from rats fed the low-protein diet. The N-terminal sequence analysis shows that cytochrome P-450 UT 50 (from control group) and UT 501 (from low-protein group) are two distinct proteins.     

Effect of cannabidiol on cytochrome P-450 isozymes

Cannabidiol (CBD) has been shown to inhibit mouse hepatic mixed-function oxidations of several drugs after acute treatment, whereas repetitive treatment resulted in the restoration of drug-metabolizing capabilities. We have found that acute CBD treatment modestly decreased cytochrome P-450 content but markedly decreased hexobarbital hydroxylase, erythromycin N-demethylase, and 6 beta-testosterone hydroxylase activities. Repetitive CBD treatment, on the other hand, resulted in the restoration of cytochrome P-450 content as well as hexobarbital hydroxylase and erythromycin N-demethylase activities. However, after such repeated treatments a fresh dose of CBD can once again inactivate erythromycin N-demethylase activity but not hexobarbital hydroxylase activity. The resistance of hexobarbital hydroxylase to re-inactivation by CBD was paralleled by stimulation of pentoxyresorufin O-dealkylase activity and the appearance of a 50 kD protein that was immunoreactive to an antibody raised against rat hepatic cytochrome P-450b. CBD metabolism in vitro by microsomes prepared from such CBD-"induced" animals, resulted in a pattern of metabolites different from that observed from comparable incubations with liver microsomes from either untreated or phenobarbital-treated animals. Thus, it appears that CBD initially inactivates at least one cytochrome P-450 isozyme, but after repetitive CBD treatment, an isozyme is induced that is resistant to further re-inactivation by CBD. This isozyme appears to be immunochemically similar to, but somewhat functionally distinct from, the isozyme induced by phenobarbital treatment in mice.     

Metabolism of lidocaine by purified rat liver microsomal cytochrome P-450 isozymes

The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB-5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P less than 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT-2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P-450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5.     

Uptake, metabolism, and secretion of 3'-methyl-N,N-dimethyl-4-aminoazobenzene by isolated perfused rat liver

Uptake, metabolism, and biliary secretion of 3'-methyl-N,N-dimethyl-4-aminoazobenzene (3'-Me-DAB) were studied in isolated rat liver which was perfused with protein-free fluorocarbon medium. [14C]3'-Me-DAB (5-10 nmol) was injected into the portal vein and allowed to recirculate. The recovery of radioactivity in bile was 7.5, 14, and 20% at 15, 30, and 45 min of injection, respectively. At 45 min, the liver contained an additional 17% of injected radioactivity. Azo dye metabolites in perfused liver differed from those in vivo; metabolites co-migrating with 3'-CHO-DAB and 3'-methyl-n,n-methyl aminoazobenzene (Me-MAB) (and the parent compound 3'-Me-DAB) were present, while metabolites co-migrating with 3'-Me-4'-OH-AB and 3'-CH2OH-MAB were increased and metabolites co-migrating with 3'-CH2OH-DAB were decreased. In bile from perfused liver, metabolites co-migrating with 3'-CHO-DAB and 3'-Me-MAB were undetectable. When proteins from normal rat bile were injected into portal vein 15 min after the administration of 3'-Me-DAB, the compounds co-migrating with 3'-Me-MAB, 3'-CHO-DAB, 3'-Me-4'-OH-AB, and 3'-CH2OH-MAB decreased, and compounds co-migrating with 3'-CH2OH-DAB increased in the liver; in bile, there was increase in 3'-Me-MAB, 3'-CHO-DAB, and 3'-Me-4'-OH-MAB, which there was a decrease in N-Ac-3'-Me-4'-OH-AB and 3'-COOH-DAB. Appearance of protein-metabolite adducts in bile was also observed after addition of normal bile proteins to the perfusate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselectivity of cytochrome P-450 isozymes and epoxide hydrolase in the metabolism of polycyclic aromatic hydrocarbons

Enantiomeric compositions of epoxides formed in the metabolism of planar benz[a]anthracene (BA), benzo[a]pyrene (BaP), and chrysene (CR), and nonplanar benzo[c]phenanthrene (BcPh), 12-methylbenz[a]anthracene (12-MBA) and 7,12-dimethylbenz[a]anthracene (7,12-DMBA) by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are determined either by direct chiral stationary phase HPLC analysis or by the enantiomeric compositions of metabolically formed trans-dihydrodiols. Cytochrome P-450 isozymes contained in various liver microsomal preparations have varying degrees of stereoselectivity in catalyzing the epoxidation reactions at various formal double bonds of the polycyclic aromatic hydrocarbons studied. In general, cytochrome P-450c, the major cytochrome P-450 isozyme contained in liver microsomes from 3-methylcholanthrene-treated rats, has the highest degree of stereoselectivity. Regardless of absolute configuration, non-K-region epoxides are converted to trans-dihydrodiols by epoxide hydrolase-catalyzed water attack at the allylic carbon. The S-center of K-region S,R-epoxide enantiomers derived from planar BA, BaP and CR is the major site of epoxide hydrolase-catalyzed water attack. In contrast, the R-center of K-region S,R-epoxide enantiomers derived from nonplanar BcPh, 12-MBA and 7,12-DMBA is the major site of epoxide hydrolase-catalyzed water attack. However, the K-region R,S-epoxide enantiomers of the six polycyclic aromatic hydrocarbons studied are hydrated by microsomal epoxide hydrolase with varying degrees of regioselectivity. Thus the enantiomeric composition of a metabolically formed dihydrodiol is determined by (i) the stereoselective epoxidation at a formal double bond of a parent hydrocarbon by microsomal cytochrome P-450 isozymes and (ii) the enantioselective and regioselective hydration of the metabolically formed epoxide by microsomal epoxide hydrolase.     

Stereoselective formations of K-region and non-K-region epoxides in the metabolism of chrysene by rat liver microsomal cytochrome P-450 isozymes

The K-region 5,6-epoxide and non-K-region 1,2- and 3,4-epoxides of chrysene were isolated by normal phase high performance liquid chromatography (HPLC) from a mixture of products formed in the metabolism of chrysene by liver microsomes from untreated (control), phenobarbital-treated, or 3-methylcholanthrene-treated rats in the presence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide. Epoxides were characterized by ultraviolet, mass, and circular dichroism spectral and chiral stationary phase HPLC analyses. Each of the metabolically formed epoxides was hydrated by rat liver microsomal epoxide hydrolase to a trans-dihydrodiol. The metabolically formed chrysene 5,6-epoxides were determined by chiral stationary phase HPLC and were found to contain (5S,6R):(5R,6S) enantiomer ratios of 68:32 (control), 71:29 (phenobarbital), and 5:95 (3-methylcholanthrene), respectively. The enantiomers of chrysene 1,2-epoxide and 3,4-epoxide were also resolved by chiral stationary phase HPLC. However, the enantiomeric compositions of the metabolically formed chrysene 1,2- and 3,4-epoxides, which racemized rapidly at room temperature, could not be directly determined. By using molecular oxygen-18 in the in vitro incubation of chrysene and by mass spectral analyses of the resulting oxygen-18-containing dihydrodiol metabolites and their acid-catalyzed dehydration (phenolic) products, both 1,2-epoxide and 3,4-epoxide were found to be converted by microsomal epoxide hydrolase-catalyzed water attack at predominantly (greater than or equal to 97%) the allylic carbons.     

Induction of cytochrome P-450, cytochrome b-5, NADPH-cytochrome c reductase and change of cytochrome P-450 isozymes with long-term trichloroethylene treatment

Several reports have described the effects of trichloroethylene (TCE) on the microsomal mixed function oxidase system (MFOS). These studies suggest that repeated TCE administration induces MFOS, especially cytochrome P-450 and NADPH-cytochrome c reductase. However, it is uncertain what isozymes are induced by TCE treatment, and it is not clear how microsomal enzymes or cytochrome P-450 isozymes are altered when TCE is administered for a duration longer than 28 days. We investigated the changes of MFOS by long-term TCE treatment. Male Wistar rats were injected with TCE, 1.0 g/kg body weight once a day for 5 continuous days or 2.0 g/kg body weight twice a week for 15 days. The mean body weight of the rats treated with TCE for 15 weeks was slightly, but not significantly, less than that of the control rats. Relative liver weights (liver wt/body wt) of the TCE-treated group were however significantly larger (21%) than those of the control group. The weights of the other organs were not changed by long-term TCE treatment. Trichloroethylene treatments for 5 days and 15 weeks caused significant increases in microsomal protein, cytochrome P-450, cytochrome b-5 and NADPH-cytochrome c reductase. TCE treatments produced an increase in a polypeptide band at 52,000 molecular weight range observed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This increase in similar to, but less pronounced than that induced by phenobarbital (PB) treatment. There were no remarkable changes at 56,000 molecular weight range where a band appeared after the treatment with 3-methylcholanthrene (MC). It is likely that the induction of cytochrome P-450 by TCE is relatively similar to that by PB.     

Inactivation of rat hepatic cytochrome P-450 isozymes by 3,5-dicarbethoxy- 2,6-dimethyl-4-ethyl-1,4-dihydropyridine

We have reported [Correia et al. (1987) Arch. Biochem. Biophys. 258, 436-443] that administration of 3,5-dicarbethoxy-4-ethyl-2,6-dimethyl-1,4-dihydropyridine (DDEP) to untreated, phenobarbital (PB) pretreated, or dexamethasone (DEX) pretreated rats results in relatively selective inactivation of cytochrome P-450 (P-450) isozymes h (CYP2C11), k (CYP2C6), and p (CYP3A). Such inactivation involves destruction of P-450 prosthetic heme predominantly by N-ethylation in untreated and PB-pretreated rats, whereas in DEX-pretreated rats, it also appears to be associated with prosthetic heme alkylation of the apocytochrome presumably at the active site. The cause for this differential course of DDEP-mediated P-450 heme destruction is unclear. Since this process is absolutely dependent on NADPH-mediated DDEP metabolism and can be reproduced in vitro, in search of mechanistic clues, we have examined DDEP metabolism by liver microsomes from the three rat sources as well as by isolated purified rat liver P-450h and P-450k. HPLC analyses of microsomal incubations of DDEP with NADPH, in the presence of an esterase inhibitor, revealed the presence of two major products: deethylated pyridine (DP) and 4-ethylpyridine (4-EDP) with product ratios (DP/4-EDP) of 1.4, 1.4, and 0.7 for reactions catalyzed by liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats, respectively. The corresponding mean product ratios for P-450h- and P-450k-catalyzed reactions were 4.2 and 5.5, respectively. On the other hand, partition ratios (DP formed/P-450 destroyed) ranged from 12.0, 10.5, and 4.8, respectively, for incubations of liver microsomes from untreated, PB-pretreated, and DEX-pretreated rats to 9.5 and 28.9 for purified P-450h- and P-450k-catalyzed reactions, respectively. However, DP formation in all these microsomal systems was comparable, and although 4-EDP formation was greatly stimulated by DEX pretreatment, it does not appear to be a destructive pathway. In view of this, our findings reported herein suggest that the active site environment of P-450's h, k, and p apparently determines not only the pattern of DDEP metabolism but also the differential course of prosthetic heme destruction.     

Role of human hepatic cytochrome P-450s in territrem A metabolism

The ability of human liver microsomal preparations (HLM1, 2, 3, and 5), microsomes from human lymphoblasts expressing different cytochrome P-450 (CYP450) isoforms, and CYP3A4 cDNA-transfected V79 Chinese hamster cells to metabolize territrem A (TRA) was studied. The only metabolite generated by any of these preparations was 6beta-hydroxymethyl-6beta-demethylterritrem A (MA(1)). MA(1) formation was observed with all four human liver microsomal samples. Of the eight microsomal preparations from human lymphoblasts expressing different cytochrome P-450 enzymes (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, and 3A4) examined, only those expressing CYP2C9, CYP2D6, or CYP3A4 metabolized TRA, with that expressing CYP3A4 being the most active. No TRA metabolites were formed by control V79MZ cells, but MA(1) was formed by CYP3A4 cDNA-transfected V79 Chinese hamster cells. In order to investigate which CYP450 isoforms were involved in MA(1) formation in the human liver microsomal preparations, the effects of six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketaconazole, and diethyldithiocarbamate) and anti-3A4, anti-2C9, and anti-2D6 antibodies on TRA metabolism by HLM2 and HLM5 were examined. MA(1) formation was markedly inhibited by ketaconazole, with quinidine and sulfaphenazole having less of an effect. Anti-CYP3A4 antibody markedly inhibited MA(1) formation, while antibodies against CYP2C9 or CYP2D6 had little effect. The amount of MA(1) formed using different HLM preparations was related to the 6beta-testosterone hydroxylase activity and CYP3A4 protein content of the preparations. These results suggest that CYP3A4 is the major enzyme involved in TRA metabolism by human liver microsomes, with CYP2C9 and CYP2D6 playing a minor role.