Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes

The monoclonal antibody MAb 1-7-1, which specifically binds to cytochromes P-450IA1 and P-450IA2 in 3-methylcholanthrene-induced rat liver microsomes, was used to identify a cytochrome P-450IA1 homologue in human lung microsomes. Although MAb 1-7-1 had similar affinity constants for human and rat microsomes, the amount bound to human lung microsomes was severalfold lower than that bound to microsomes from untreated rat or rabbit lung and much lower than the amount bound to 3-methylcholanthrene-induced rat lung or liver microsomes. The amount bound to untreated baboon lung microsomes was similar to that bound to human lung microsomes. Three cytochrome P-450IA1-catalyzed activities, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin, O-deethylase, and aryl hydrocarbon hydroxylase, were measurable in human lung microsomes, but the cytochrome P-450IA2-dependent activity acetanilide 4-hydroxylase was not. MAb 1-7-1 inhibited, and its binding correlated strongly with, 7-ethoxyresorufin O-deethylase activity (r = 0.92, p less than 0.01) in human lung microsomes. 7-Ethoxyresorufin O-deethylase activities in human lung were similar to those measured in untreated baboon lung but considerably lower than those present in untreated rabbit lung, untreated or 3-methylcholanthrene-induced rat lung and liver, or human liver. We conclude that MAb 1-7-1 recognizes a cytochrome P-450IA1 homologue in human lung and that no cytochrome P-450IA2 homologue is detected. Cytochrome P-450IA1 is expressed in human lung at relatively low levels, similar to those observed in untreated primate (baboon) lung. The majority of the 19 human lung samples examined do not exhibit a permanent polycyclic aromatic hydrocarbon-induced state with respect to this isozyme.     

The effect of cigarette smoking on 7-ethoxyresorufin O-deethylase and other monooxygenase activities in human liver: analyses with monoclonal antibodies.

Four cytochrome P-450 enzyme activities, 7-ethoxyresorufin O-deethylase (ERDE), coumarin 7-hydroxylase (CH), 7-ethoxycoumarin O-deethylase (ECDE) and aryl hydrocarbon hydroxylase (AHH) were measured in human liver needle biopsy samples from smokers and non-smokers. Cigarette smoking was verified and quantitated by measuring plasma cotinine levels. Enzyme inhibitory monoclonal antibodies (MAb) to a 3-methylcholanthrene-induced (MAb 1-7-1) and phenobarbitone-induced (MAb 2-66-3) rat hepatic cytochrome P-450 were used to measure the contribution of MAb-defined, epitope-specific cytochromes P-450 to the total reaction measured for each of the above activities. ERDE activity was significantly elevated in the livers of cigarette smokers, whereas AHH, CH or ECDE activities were not affected by cigarette smoking. No correlation was observed between plasma cotinine concentration and ERDE activity. MAb 1-7-1 inhibited hepatic ERDE activity to a variable extent (from 0 to 65%), but had very little or no effect on AHH, CH or ECDE activities. The inhibitory effect of MAb 1-7-1 on ERDE activity was greater than 50% in the non-smokers. MAb 2-66-3 had no inhibitory effect on any of the enzyme activities studied. In contrast to liver both ERDE and AHH on human placental microsomes from cigarette smokers were inhibited by MAb 1-7-1. The MAb 2-66-3 was without effect. Cigarette smoking induces a form of P-450 in human liver, responsible for ERDE activity, that contains an epitope recognized by MAb 1-7-1. This form of cytochrome P-450 is insensitive to MAb 2-66-3 and is not contributing to AHH, CH or ECDE activities of human liver.     

Comparison of the effects of inducers of cytochrome P450 on Mongolian gerbil and rat hepatic microsomal monooxygenase activities.

1. Basal cytochrome P450 content (nmol/mg protein) was higher in gerbil (1.10 +/- 0.01) than in rat (0.81 +/- 0.05) hepatic microsomes. Pretreatment of gerbils with phenobarbitone and beta-naphthoflavone increased P450 contents by 200% and 60% respectively. 2. 7-Ethoxycoumarin O-deethylase, coumarin 7-hydroxylase and 4-nitrophenol hydroxylase activities were generally higher in gerbil liver microsomes, whereas erythromycin N-demethylase, and 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were higher in rat microsomes. Microsomal benzphetamine N-demethylase activities were similar in both species. 3. Induction of specific cytochrome P450 isozymes increased similar monooxygenase activities of rat and gerbil microsomes. Phenobarbitone, beta-naphthoflavone, isoniazid and pregnenolone 16 alpha-carbonitrile principally increased benzphetamine N-demethylase, 7-ethoxyresorufin O-deethylase, 4-nitrophenol hydroxylase and erythromycin N-demethylase activities respectively. 4. Constitutive 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were markedly lower in gerbil microsomes compared with rat microsomes, and pretreatment of gerbils with cytochrome P450 inducers did not significantly increase these activities. 5. Hepatic microsomal coumarin 7-hydroxylase activities were approximately 30-200 times greater (depending on the inducer) in the gerbil than in rat. The gerbil, due to is high coumarin 7-hydroxylase activity, would appear to be a more appropriate species than rat for investigations of coumarin metabolism and toxicity relevant to humans.     

Coumarin-induced changes in delta-aminolaevulinic acid synthase and cytochrome P-450 in chick embryo liver

Coumarin occurs naturally in the diet and inhibits several cytochrome P-450 enzymes in laboratory animals. The effect of coumarin was examined on haem biosynthesis and cytochrome P-450 activities in the 18-day-old chick embryo liver in ovo. At 40 and 50 mumol/embryo coumarin increased delta-aminolaevulinic acid synthase, porphyrins, cytochrome P-450, benzphetamine N-demethylase and benzo[a]pyrene hydroxylase. At 10 mumol/embryo coumarin decreased aniline 4-hydroxylase, and at both 10 and 50 mumol/embryo it decreased 7-ethoxyresorufin O-deethylase, coumarin 7-hydroxylase and nitrosodimethylamine N-demethylase. 7-Hydroxycoumarin and 5, 7-methoxycoumarin at 40 mumol/embryo had none of these effects. Coumarin (5-500 microM) added to liver microsomes inhibited aniline hydroxylase by 45%, but not nitrosodimethylamine N-demethylase, and inhibited 7-ethoxyresorufin O-deethylase in microsomes from 3-methylcholanthrene-treated embryos by 15 and 100% at coumarin concentrations of 250 and 500 microM, respectively. Coumarin 7-hydroxylase activity in chick embryo liver was comparable with that reported for human liver and greater than in the rat. The data indicate that coumarin can both increase and decrease cytochrome P-450 activities in chick embryo liver and can induce haem biosynthesis. Because the chick embryo liver hydroxylates coumarin at position 7 in a manner similar to humans, it may be a more suitable model than the rat for studying some of the metabolic effects of coumarin.     

Comparison of the effects of inducers of cytochrome P450 on Mongolian gerbil and rat hepatic microsomal monooxygenase activities

1. Basal cytochrome P450 content (nmol/mg protein) was higher in gerbil (1.10 ± 0.01) than in rat (0.81 ± 0.05) hepatic microsomes. Pretreatment of gerbils with phenobarbitone and β-naphthoflavone increased P450 contents by 200% and 60% respectively.

2. 7-Ethoxycoumarin O-deethylase, coumarin 7-hydrozylase and 4-nitrophenol hydroxylase activities were generally higher in gerbil liver microsomes, whereas erythromycin N-demethylase, and 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were higher in rat microsomes. Microsomal benzphetamine N-demethylase activities were similar in both species.

3. Induction of specific cytochrome P450 isozymes increased similar monooxygenase activities of rat and gerbil microsomes. Phenobarbitone, β-naphthoflavone, isoniazid and pregnenolone 16α-carbonitrile principally increased benzphetamine N-demethylase, 7-ethoxyresorufin O-deethylase, 4-nitrophenol hydroxylase and erythromycin N-demethylase activities respectively.

4. Constitutive 7-ethoxyresorufin and 7-pentoxyresorufin O-dealkylase activities were markedly lower in gerbil microsomes compared with rat microsomes, and pretreatment of gerbils with cytochrome P450 inducers did not significantly increase these activities.

5. Hepatic microsomal coumarin 7-hydroxylase activities were approximately 30–200 times greater (depending on the inducer) in the gerbil than in rat. The gerbil, due to is high coumarin 7-hydroxylase activity, would appear to be a more appropriate species than rat for investigations of coumarin metabolism and toxicity relevant to humans.     

Differential induction of cytochrome P-450 catalyzed activities by polychlorinated biphenyls and benzo [a]pyrene in B6C3F1 mouse liver and lung

The properties of some constitutive and inducible enzyme activities of liver and lung microsomes were determined in B6C3F1 mice pretreated by either intratracheal (i.t.) administration of benzo[a]pyrene (BaP) or polychlorinated biphenyl (PCBs) mixture (Aroclor 1254), or intraperitoneal (i.p.) administration with Aroclor 1254. After i.p. administration of Aroclor 1254, liver cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH), benzphetamine N-demethylase and nitroreductase activities were increased 2.8-, 2.0-, 2.2-, and 2.0-fold, respectively. Lung cytochrome P-450 content was also increased (1.9-fold) after i.p. administration of Aroclor 1254; AHH and nitroreductase activities, however, were not affected and benzphetamine N-demethylase activity was decreased. Aroclor 1254 administered i.t. did not affect liver cytochrome P-450 content. However, AHH and benzphetamine N-demethylase activities were decreased 1.4- and 1.2-fold, respectively, and nitroreductase activity was increased 1.6-fold. After i.t. administration of Aroclor 1254, lung cytochrome P-450 content and AHH activity were increased 1.4- and 2.2-fold, respectively. Benzphetamine N-demethylase activity was decreased 2.1-fold and nitroreductase activity was not affected. After i.t. administration of BaP, liver 7-ethoxyresorufin O-deethylase and nitroreductase activities were increased 2.2- and 1.5-fold, respectively, and benzphetamine N-demethylase activity was decreased 1.3-fold. Lung AHH and 7-ethoxyresorufin O-deethylase activities were increased 4.3- and 3.1-fold, respectively, and cytochrome P-450 content, benzphetamine N-demethylase and nitroreductase activities were decreased 1.4-, 1.2- and 1.3-fold, respectively, after BaP administration. These data indicate that different cytochrome P-450 isozymes induced in B6C3F1 mice are responsible for monooxygenase and nitroreductase activities, and that the route of administration of chemicals is important in the expression of cytochrome P-450 catalyzed activities.     

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.     

Pulmonary cytochromes P-450 from rabbits treated with phenobarbital

Pretreatment of rabbits with phenobarbital caused significant increases in total pulmonary cytochrome P-450 content, benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities and to a lesser extent in benzphetamine N-demethylase activity in lung microsomes. However, 7-ethoxyresorufin O-deethylase activity was not significantly altered. In addition, the pulmonary concentration of the cytochrome P-450-metyrapone complex was increased significantly. Column chromatography of the pulmonary monooxygenases demonstrated that in untreated and phenobarbital-treated rabbits, cytochromes P-450I and P-450II constituted the major forms of cytochrome P-450 isozymes. In addition, the chromatographic studies showed that pretreatment with phenobarbital caused an increase in the content of cytochrome P-450I, but not of cytochrome P-450II. These observations were confirmed by subjecting the pulmonary cytochromes to gel electrophoresis, and staining of the gels for protein and heme.     

Effects of a series of 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine on the major inducible cytochrome P-450 isozymes of rat liver

Various 4-alkyl analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) cause mechanism-based inactivation of cytochrome P-450 (P-450) by destroying the heme prosthetic group. We have examined the isozyme selectivity of representative DDC analogues with respect to the major inducible P-450 isozymes of rat liver. Hepatic microsomes from untreated, phenobarbital (PB)-treated, beta-naphthoflavone (beta NF)-treated, and dexamethasone (DEX)-treated rats were incubated with a DDC analogue and NADPH and were subsequently analyzed for P-450 and heme content, P-450 isozyme immunoreactivity, and enzyme activity. Compared with the uninduced state, 4-isopropyl-DDC caused slightly less P-450 destruction following beta NF induction and much greater destruction following DEX pretreatment. Also, 4-hexyl-DDC was found to cause less P-450 destruction following PB or DEX pretreatment, compared with results obtained with untreated rats. These results suggest that DDC analogues possess different isozyme selectivity profiles. Monoclonal antibodies (MAbs) directed against the major inducible isozymes of P-450 were used to probe Western blots of microsomal protein following DDC analogue treatment. The formation of lower molecular mass (45-55 kDa) immunoreactive proteins in microsomes from beta NF-treated rats following DDC analogue treatment was revealed by two MAbs (1-31-2 and 1-36-1), suggesting that the apoprotein of the major beta NF-inducible isozyme, P-450c, is subject to alteration by DDC analogues. In microsomes from DEX-treated rats, DDC analogues caused the formation of higher molecular mass (80, 94, and 115 kDa) proteins showing immunoreactivity with MAb 2-13-1, directed against a major DEX-inducible isozyme belonging to the P-450p family. These immunochemical findings are supported by the demonstration that DDC analogues also caused mechanism-based inhibition of the catalytic activity of P-450c (7-ethoxyresorufin O-deethylase) and P-450p (erythromycin N-demethylase) but not that of the major PB-inducible isozyme, P-450b (7-pentoxyresorufin O-dealkylase). The combined immunochemical and enzymic studies indicate that rat liver P-450 c and p are targets for mechanism-based inactivation by DDC analogues.     

Action of carbon tetrachloride in the reconstituted monooxygenase system using partially purified cytochrome P-450 and P-448

In the cytochrome P-450-reconstituted system, CCl4 stimulated NADPH-dependent lipid peroxidation of the system containing the P-450 form to a much greater extent than that of the system containing the P-448 form. When the P-450-reconstituted system was preincubated in the presence of both NADPH and CCl4, 7-ethoxycoumarin O-deethylase, aminopyrine N-demethylase and aniline hydroxylase activities were decreased by 40-60%, whereas, with P-448 form reconstituted system, no suppression was observed in these enzyme activities or in 7-ethoxyresorufin O-deethylase activity. These observations suggest that the P-450 form, but not the P-448 form, is active in metabolizing CCl4 to a reactive species that subsequently impairs the hemoprotein.     

Lack of effect of streptogramins on hepatic drug metabolism enzymes in the rat.

Male Sprague-Dawley rats were treated with streptogramin derivatives (RP 7293, RP 54476, RP 57669, and RP 59500) or with the macrolide troleandomycin. Liver cytosol and microsomes were prepared, and the in vitro transformation of several model substrates studied. Furthermore, total and complexed microsomal cytochrome P-450 levels were compared. Hepatic cytochrome P-450 metabolite complexes were detected 4 days after troleandomycin treatment (500 mg/kg/day po), whereas such effects were not observed with po RP 7293 (500 mg/kg/day, 4 days) or with iv RP 54476 (12 mg/kg/day, 7 days), RP 57669 (6 mg/kg/day, 7 days), or RP 59500 (6 and 18 mg/kg/day, 7 days). The administration of troleandomycin resulted in statistically significant increases in liver weight (+20%), microsomal protein (+70%), total cytochrome P-450 (+187%), and cytosolic glutathione S-transferase activity (+32%). The activities of aniline hydroxylase, aminopyrine N-demethylase, and the high and low phases of 7-ethoxyresorufin O-deethylase were markedly decreased by 36% to 56%. In contrast, none of these hepatic parameters was changed significantly after administration of each streptogramin. These results suggest that streptogramins have not, in contrast to many commonly used macrolide antibiotics, had potent or specific effects on hepatic drug metabolizing enzymes in rats.     

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.     

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.     

Ketoconazole: a potent inhibitor of cytochrome P-450-dependent drug metabolism in rat liver

Ketoconazole, an orally active imidazole antimycotic agent, is shown to be a potent inhibitor of drug N-demethylase activities of liver microsomes from rats pretreated with phenobarbital or pregnenolone-16 alpha-carbonitrile, and an inhibitor of 7-ethoxyresorufin O-deethylase activity of liver microsomes from rats pretreated with 5,6-benzoflavone. Spectrophotometric studies reveal that the imidazole compound binds to the cytochrome P-450 component of the monooxygenase complex, and has little effect on NADPH-cytochrome c (P-450) reductase activity. These results are strongly suggestive that cytochrome P-450 is the site of action of this potent inhibitor of drug metabolism in liver microsomes.     

Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities.

The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase activities (ERDE) in placental tissues from smoking mothers, whereas the anti-PB-B2 (to phenobarbital-induced rat liver P-450) was without significant inhibitory effect. Inhibition of 7-ethoxycoumarin O-deethylase (ECDE) by the anti-BNF-B2 was dependent on maternal smoking: the enzyme from non-smokers was not significantly inhibited, whereas the enzyme from smokers was variably inhibited by 15-60 per cent. The monoclonal antibodies towards the major 3-methylcholanthrene-inducible and phenobarbital-inducible rat liver P-450s (Mab 1-7-1 and 2-66-3, respectively) behaved similarly, except the inhibition was somewhat stronger if present. Antibody raised against rat liver NADPH-cytochrome P-450 oxido-reductase did not inhibit any activity studied. In immunoblotting experiments, the anti-reductase recognized the protein in human placental microsomes. However, neither anti-BNF-B2, anti-PB-B2 or Mab 1-7-1 or Mab 2-66-3 detected any proteins in human placental microsomes, regardless of smoking status. Northern blot hybridization analysis of placental RNA samples showed that only P-450IA1 mRNA existed in the placentas of smoking mothers with detectable ERDE activity. Despite the discrepancy between protein blotting and immunoinhibition data all other findings support the conclusion that maternal cigarette smoking induces the expression of the CYPIA1 gene (and not CYPIA2), resulting in an increased synthesis of P-450IA1 protein and increased AHH, ERDE and ECDE activities in human placenta.     

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.     

Monoclonal antibody-directed characterization of benzene, ethoxyresorufin and pentoxyresorufin metabolism in rat liver microsomes

The contribution of cytochrome P450IA, P450IIB, P450IICII and P450IIEI to the oxidative metabolism of benzene, 7-ethoxyresorufin and 7-pentoxyresorufin was investigated using monoclonal antibodies (MAb) in liver microsomes from fed, one-day fasted, phenobarbital (PB)-, 3-methylcholanthrene (MC)- and ethanol-treated rats. Overall catalytic activity varied with different pretreatments and thereby the contribution of different P450s. MAb 1-91-3 against P450IIE1 did not influence alkoxyresorufin dealkylation but inhibited benzene aromatic hydroxylase (BAH) in relation to its increasing inducibility as follows: MC, PB (less than or equal to 48%) less than fed (less than or equal to 59%) less than fasted (less than or equal to 70%) less than ethanol (less than or equal to 91%). MAbs 2-66-3, 4-7-1 and 4-29-5, all against P450IIB, had no effect on 7-ethoxyresorufin O-deethylase (EROD) but inhibited the activities of high-Km BAH (greater than or equal to 58%) and 7-pentoxyresorufin O-depentylase (PROD) (greater than or equal to 96%) in PB-treated microsomes. MAb 1-7-1 against P450IA inhibited EROD (79%), PROD (50%) and high-Km BAH (42%) activities in MC-microsomes. MAb 1-68-11 against P450IIC11 inhibited EROD, PROD and high-Km BAH activities. Thus, P450IIE1 contributed to benzene metabolism as a low-Km BAH but not to alkoxyresorufin metabolism. P450IIB was responsible besides for the major part of 7-pentoxyresorufin metabolism also, selectively, for benzene hydroxylation at high benzene concentrations. P450IA contributed primarily to 7-ethoxyresorufin metabolism and only slightly to PROD and high-Km BAH activities. P450IIC11 contributed slightly to high-Km BAH and to alkoxyresorufin metabolism.     

Selective inactivation of mouse liver cytochrome P-450IIIA by cannabidiol

Cannabidiol (CBD) inhibits hepatic drug metabolism in mice, particularly those activities known to be catalyzed by the cytochrome P-450IIIA (P-450IIIA) subfamily. CBD treatment (120 mg/kg) inhibited more than 75% of hepatic 6 beta-testosterone hydroxylase and erythromycin N-demethylase activities (functional markers of P-450IIIA) after 2 hr. An isozyme of the P-450IIIA subfamily (Mr 49,960) was purified to apparent homogeneity from hepatic microsomes of untreated mice and was found to catalyze testosterone hydroxylation at the 2 beta-, 6 beta-, and 15 beta-positions exclusively. Incubation of this isozyme with CBD in a reconstituted system resulted in a time- and concentration-dependent inactivation, with almost complete loss of P-450 chromophore and corresponding increase in P-420 content. NH2-terminal sequence analysis of the isozyme revealed an 86% similarity to the corresponding sequence of rat P-450IIIA2, a constitutive P-450 isozyme in the male rat liver. Pretreatment of mice with dexamethasone markedly (6-fold) increased the steroid-inducible P-450IIIA-dependent activities 6 beta-testosterone hydroxylation and erythromycin N-demethylation. CBD treatment of dexamethasone-pretreated animals failed to inhibit these activities, indicating that the steroid-inducible P-450IIIA was refractory to CBD-mediated inactivation. 3-Methylcholanthrene-inducible P-450IA and phenobarbital-inducible P-450IIB also appear to be refractory to CBD-mediated inactivation. On the other hand, erythromycin N-demethylase activity increased 4-fold after phenobarbital pretreatment and, as in untreated animals, was comparably inhibited by CBD, demonstrating its susceptibility to this drug. Thus, CBD appears to inactivate the P-450IIIA isozymes that are constitutively present in hepatic microsomes of untreated mice and/or inducible by phenobarbital pretreatment but not those that are steroid inducible.     

Purification and characterization of a mouse liver cytochrome P-450 induced by cannabidiol

A cytochrome P-450 isozyme (Mr = 51,600) was purified to apparent homogeneity from hepatic microsomes of mice pretreated with cannabidiol (CBD), a major constituent of marijuana. The isozyme exhibited high pentoxyresorufin O-dealkylase, hexobarbital hydroxylase, and 16 alpha- and 16 beta-testosterone hydroxylase activities and formed a Fe+2-metyrapone complex, properties characteristic of the major hepatic cytochrome P-450s previously purified from phenobarbital (PB)-pretreated animals. In addition, the CBD-induced cytochrome P-450 was immunoreactive with an antibody raised against the major rat hepatic PB-inducible cytochrome P-450 and exhibited an NH2-terminal amino acid sequence greater than 90% homologous with that of the PB-inducible rat liver isozyme. Because of the many similarities between the CBD-induced isozyme and certain other isozymes previously purified from PB-pretreated animals, a cytochrome P-450 isozyme was purified from PB-pretreated mice by a chromatographic procedure similar to that employed for purification of the CBD-induced isozyme. The PB-inducible isozyme was indistinguishable from the CBD-inducible cytochrome P-450 on the bases of apparent molecular weight, absorption spectra, NH2-terminal amino acid sequence, peptide mapping, immunoreactivity, and catalytic activity. Although the CBD- and PB-inducible P-450 isozymes appear to be qualitatively very similar, PB appears to be a quantitatively better inducer of the isozyme. Thus, CBD exposure results in the induction of an isozyme that is refractory to CBD-mediated inactivation, thereby apparently altering the cytochrome P-450 isozymal composition of mouse hepatic microsomes.     

Ontogenic development of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylase in the rat and effect of monoclonal antibodies

The O-deethylase activities with 7-ethoxycoumarin (ECDE) and 7-ethoxyresorufin (ERDE) as substrates were investigated in the liver microsomal fraction of newborn, 1 week, 2 weeks, 1 month and 2 months old rats. The rates of O-deethylation of both substrates were lowest in newborn rats at which stage the specific activities (pmol x min.-1 x mg-1 protein; mean +/- S.D.) of ECDE and ERDE were 19.0 +/- 0.2 and 6.0 +/- 0.5, respectively. In 1 week old animals, the rate of ECDE had reached the highest values (270 +/- 81 pmol x min.-1 x mg-1 protein). Thereafter, it decreased to a minimum (70.9 +/- 14.8 pmolmin.-1 x mg-1 protein) at 2 months of age. The rate of ERDE reached the highest activity (129 +/- 7.1 pmol x min.-1 x mg-1 protein) in 1 month old animals. The effects monoclonal of antibodies (MAb), on ECDE and ERDE were studied. The MAb were raised against 3-methylcholanthrene (MAb 1-7-1) or phenobarbital (MAB 2-66-3) induced rat liver microsomal cytochrome P-450. Only the former antibodies were inhibitory to the O-deethylation of 7-ethoxyresorufin. This reaction was inhibited to a similar extent (by 31-33%) in rat liver microsomes from 1 week and 2 months old animals. None of the antibodies exerted any effect on the O-deethylation of 7-ethoxycoumarin.