Ascorbic acid deficiency decreases specific forms of cytochrome P-450 in liver microsomes of guinea pigs

Ascorbic acid (VC) deficiency resulted in a decrease in the activities of aminopyrine N-demethylase, aniline hydroxylase, and p-nitroanisole O-demethylase and in the content of cytochrome P-450, as spectrally determined, whereas it caused an increase in the activities of 6 beta-hydroxylases for testosterone and progesterone in liver microsomes of guinea pigs. Western blot analysis of liver microsomes with antibodies to rat P-448-H (P-4501A2), P-450j (P-450IIE), P-450 PB-1 (P-450IIIA), and P-450b (P-450IIB1) showed that VC deficiency decreased the amount of cytochrome P-450 immunochemically related to P-450IA2 and P-450IIE but did not change the amount of the form that was cross-reactive with antibodies to P-450IIB1 and tended to slightly increase (not statistically significantly) the amount of the form of the cytochrome immunochemically related to P-450IIIA. The larger decrease by VC deficiency in the amount of cytochrome P-450 that was cross-reactive to the rat P-450IA2 resulted in a lower capacity of liver microsomes to activate promutagens, such as 2-amino-3-methyl-imidazo(4,5-f)quinoline and aflatoxin B1. These results indicate that VC deficiency in guinea pigs differentially affects the content of individual forms of cytochrome P-450.     

Cytochromes P-450 of sea birds: Cross-reactivity studies with purified rat cytochromes

1. Polyclonal antibodies against rat cytochrome P-450c (IA1), P-450d (IA2), P-450b (IIB1), P-450h (IIC11) and P-450j (IIE1), were used to probe liver microsomes prepared from six sea bird species collected from the Irish Sea between 1978 and 1988.

2. Significant cross-reactivity in all the sea bird species was seen only with antibodies to P450 IA1. Expression of cross-reactive proteins was highly variable between individual birds, which show evidence of environmental induction.

3. Shared epitopes to P450 IA1 and IA2 were seen on a single protein expressed in liver microsomes from the cormorant (Phalacrocorax carbo).

4. Antibodies against members of rat P450 gene family II showed a small degree of cross-reactivity with sea bird microsomes. Antibodies against P450 IIB1 and IIC11 showed weak cross-reactivity in all species with little inter-individual variation. Antibodies to P450 IIE1 showed no cross-reactivity in any bird species.

5. P450 gene family I appears to be well represented in sea birds while P450 gene family II is not well developed in this group of lower vertebrates.     

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.     

Tolbutamide hydroxylation by human, rabbit and rat liver microsomes and by purified forms of cytochrome P-450

Tolbutamide hydroxylation has been investigated in human, rabbit and rat liver microsomes and by six purified forms of hepatic rabbit cytochromes P-450. These studies were carried out to investigate whether an appropriate animal model could be developed for the human cytochrome(s) P-450 metabolizing tolbutamide. Selective induction was used in rats and rabbits to indicate the isozymes primarily responsible for tolbutamide hydroxylation in these species. Microsomal tolbutamide hydroxylase activity was significantly induced only by phenobarbital pretreatment in the rat which induces P-450 forms b (P-450IIB1) and/or e (P-450IIB2). Only pretreatment of rabbits with rifampicin, which induces cytochrome P-450 form 3c (P-450IIIA6), significantly increased the microsomal hydroxylation of tolbutamide. However, the increase in tolbutamide hydroxylase activity in rifampicin-induced microsomes (congruent to 50%) appears low compared to known levels of induction of P-450IIIA6 following rifampicin pretreatment (5-10-fold). These data suggest that P-450IIIA6 is at least partially involved in tolbutamide hydroxylation in rabbit liver but that other form(s) may be relatively more important. Reconstitution experiments with six purified forms of rabbit cytochrome P-450 indicated that the highest activity occurred with P-450IIIA6 (form 3c). As isozymes from different gene families or subfamilies appeared to metabolize tolbutamide in the three species studied, catalytic similarities between the P-450s with respect to inhibition was further investigated in microsomes using sulfaphenazole, alpha-naphthoflavone and mephenytoin. These studies showed that the catalytic characteristics in relation to inhibition differ markedly between species. Hence, it appears that the animal model approach is not likely to be successful in the identification and characterization of the cytochrome P-450 form(s) metabolizing tolbutamide in humans.     

Cytochromes P-450 of sea birds: cross-reactivity studies with purified rat cytochromes

1. Polyclonal antibodies against rat cytochrome P-450c (IA1), P-450d (IA2), P-450b (IIB1), P-450h (IIC11) and P-450j (IIE1), were used to probe liver microsomes prepared from six sea bird species collected from the Irish Sea between 1978 and 1988. 2. Significant cross-reactivity in all the sea bird species was seen only with antibodies to P450 IA1. Expression of cross-reactivity proteins was highly variable between individual birds, which show evidence of environmental induction. 3. Shared epitopes to P450 IA1 and IA2 were seen on a single protein expressed in liver microsomes from the cormorant (Phalacrocorax carbo). 4. Antibodies against members of rat P450 gene family II showed a small degree of cross-reactivity with sea bird microsomes. Antibodies against P450 IIB1 and IIC11 showed weak cross-reactivity in all species with little inter-individual variation. Antibodies to P450 IIE1 showed no cross-reactivity in any bird species. 5. P450 gene family I appears to be well represented in sea birds while P450 gene family II is not well developed in this group of lower vertebrates.     

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.     

Induction of cytochrome P-450 isozymes upon repeated administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine administration at a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450IIA1, P-450IIB1, and P-450IIIA1, respectively. The induction of cytochrome P-450IIIB1 was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450IIB1/B2.     

Comparison of cytochrome P450 isoenzyme profiles in rat liver and hepatocyte cultures. The effects of model inducers on apoproteins and biotransformation activities

The metabolic profile of seven subfamilies of cytochrome P450 (P450IA, IIA, IIB, IIC, IIE, IIIA, IVA) was studied in rat liver (in vivo) and in primary hepatocyte cultures (in vitro) after treatment with various inducers. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD), aniline 4-hydroxylation and the regio- and stereoselective hydroxylation of testosterone were measured to characterize the isoenzyme pattern in intact hepatocytes and in liver microsomes. Occurrence of isoenzyme apoproteins was determined using Western blotting. Primary cultures of rat hepatocytes retain the capacity to respond to inducers of isoenzymes belonging to six different subfamilies (P450IA, IIA, IIB, IIC, IIIA and IVA). Treatment of cells with beta-naphthoflavone revealed a P450-activity profile similar to in vivo, namely a highly induced EROD (P450IA1), a small enhancement of testosterone 7 alpha-hydroxylation (P450IIA) and a marked reduction in 2 alpha- and 16 alpha-hydroxylation (P450IIC11). Exposure of cultured cells to phenobarbital resulted in a higher testosterone 16 beta-hydroxylation (reflecting P450IIB), though to a lesser extent than in vivo. The induction of P450IIIA due to both phenobarbital and dexamethasone, as mirrored by 6 beta- and 15 beta-hydroxylation of testosterone, was the same in cultured hepatocytes and in vivo. Treatment of cells with clofibric acid resulted in an induction profile similar to the one observed in liver microsomes from clofibrate-treated rats: the apoprotein P450IVA as well as the apoprotein P450IIB1/2 and its associated activities (PROD and testosterone 16 beta-hydroxylation) were induced. Isoniazid, a known in vivo inducer of P450IIE1 and aniline 4-hydroxylation, did not change any of the determined P450-dependent activities in vitro.     

Identification of the rabbit and human cytochromes P-450IIIA as the major enzymes involved in the N-demethylation of diltiazem

Oxidative metabolism of diltiazem (DTZ), a calcium channel blocker, was investigated in rabbit and human liver microsomes as well as in primary cultures of human hepatocytes. DTZ N-demethylation, the major metabolic pathway in man, was strongly increased by treatment of animals, patients, and hepatocyte cultures with rifampicin and other inducers of the P-450IIIA subfamily. In a reconstituted system with purified forms of P-450 and NADPH cytochrome P-450 reductase, P-450IIIA7 exhibited the highest DTZ N-demethylase activity. In both rabbit and human liver microsomes, this activity was highly correlated with erythromycin demethylase, a characteristic substrate of P-450IIIA, or with an immunoquantitated level of P-450IIIA, and was specifically inhibited by anti-P-450IIIA7 polyclonal and monoclonal antibodies. Cyclosporin A, another specific substrate of P-450IIIA in rabbit and human, competitively inhibited DTZ N-demethylase in both species. In primary cultures of human hepatocytes treated with various inducers, including rifampicin, dexamethasone, phenobarbital, phenylbutazone or beta-naphthoflavone, the rate of release of N-demethyl-DTZ in the extracellular medium was highly correlated with the intracellular level of P-450IIIA, which appeared to be strongly induced by rifampicin and phenobarbital and to a lesser extent by dexamethasone and phenylbutazone. In aggregate, these results are consistent with the view that in both rabbit and human, cytochromes P-450 from the P-450IIIA subfamily are the major enzymes involved in the N-demethylation of DTZ. Accordingly, drugs which may be specific substrates or inducers of this P-450 are likely to influence both the side effects and the efficacy of this molecule.     

Interspecies homology of cytochrome P-450: inhibition by anti-P-450-male antibodies of testosterone hydroxylases in liver microsomes from various animal species including man

P-450-male is one of the male-specific forms of cytochrome P-450 in liver microsomes of adult rats and functions as testosterone 2 alpha- and 16 alpha-hydroxylases. The purpose of this study was to examine whether forms of cytochrome P-450 cross-reactive with anti-P-450-male antibodies are present in liver microsomes of other animal species, such as male and female mice, rabbits, guinea pigs, hamsters, dogs and humans. The antibodies cross-reacted with a protein(s) in the liver microsomes of all animal species to show one or more bands on nitrocellulose paper by Western blot peroxidase-anti-peroxidase staining analysis. Qualitative as well as quantitative species differences were noted in the testosterone hydroxylases. The antibodies inhibited 2 alpha-hydroxylase in male rats, 16 alpha-hydroxylase in male rats and male and female dogs, 7 alpha-hydroxylase in female rats and male and female mice and hamsters, and 15 alpha-hydroxylase in female mice and hamsters. No clear inhibition of 6 beta-hydroxylase, which was present in all animal species, was observed. These results indicate that forms of cytochrome P-450 that are immunochemically related with P-450-male catalyze the hydroxylation of testosterone at varying positions depending on the animal species, with the exception of 6 beta-hydroxylation, which may be catalyzed by a distinct form of cytochrome P-450.     

Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA

1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied. 2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm3/m3 (2100 mg/m3). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four. 3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%. 4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylation (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced. 5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrome P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450IIE1 (NDMAD, 60%) and P450IIB (PROD, 3000%). 6. Our conclusions are that styrene metabolism in vivo in both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.     

Characterization of human liver cytochromes P-450 involved in theophylline metabolism.

Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antioxidant effects of albumin-bound sulfur in lipid peroxidation of rat liver microsomes.

The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by gamma-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically. The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disappearance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl4-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.     

Three N-aralkylated derivatives of 1-aminobenzotriazole as potent and isozyme-selective, mechanism-based inhibitors of guinea pig pulmonary cytochrome P-450 in vitro

The potency and cytochrome P-450 (P-450) isozyme selectivity of 1-aminobenzotriazole (ABT) and three of its N-aralkylated analogues, N-benzyl-1-aminobenzotriazole (BBT), N-alpha-methylbenzyl-1-aminobenzotriazole (alpha MB), and the newly synthesized N-alpha-ethylbenzyl-1-aminobenzotriazole (alpha EB), as mechanism-based inhibitors were compared in pulmonary microsomes of untreated and beta-naphthoflavone (beta-NF)-induced guinea pigs. All four compounds were suicide substrates for pulmonary P-450, resulting in the loss of spectrally assayed hemoprotein (up to 50%). Monooxygenase activities were measured with isozyme-selective/specific substrates; the O-dealkylation of 7-pentoxyresorufin (PRF) for the guinea pig ortholog of rabbit P-450IIB4, the O-deethylation of 7-ethoxyresorufin for P-450IA1, and the N-hydroxylation of the aromatic amine 4-aminobiphenyl for P-450IVB1, BBT, alpha MB, and alpha EB were selective for the suicidal inhibition of P-450IIB4; for example, 1 microM alpha MB inactivated 95% of P-450IIB4-, and approximately 10% of P-450IA1- and IVB1-catalyzed, activity in microsomes from beta-NF-induced lungs. Isozyme selectivity was approximately the same for alpha EB and slightly lower for BBT, which inactivated relatively more P-450IA1. At low concentrations, 1 and 10 microM, respectively, ABT preferentially inactivated P-450IVB1, consistent with the efficient N-hydroxylation of aromatic amines by this form of P-450. alpha EB also was shown to efficiently inactivate P-450IIB4-catalyzed PRF activity in microsomes prepared from liver of phenobarbital-induced guinea pigs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex-related differences in rat hepatic cytochromes P450 expression following treatment with phenobarbital or 3-methylcholanthrene

The induction of hepatic cytochromes P450 and metabolic effects have been examined in male and female Sprague-Dawley rats following treatment with either phenobarbital or 3-methylcholanthrene. Hepatic cytochrome P450 levels were higher in males than in females by ≈40%. Treatment of male and female rats with phenobarbital or 3-methylcholanthrene resulted in an ≈1.6- and 2-fold increase, respectively, in heptic microsomal cytochrome P450 levels in both sexes, relative to untreated animals. Immunoblot analyses were performed to compare sex-related changes in P450 levels. Hepatic P450IIB1 levels in males were greater than those in females following phenobarbital treatment. 3-Methylcholanthrene-induced male hepatic microsomes exhibited greater levels of P450 IA1 and IA2 than female microsomes, whereas uninduced microsomes from males or females failed to exhibit a band. Mab PCN 2-13-1 against P450IIIA recognized an intense band in uninduced hepatic microsomes from males whereas no band was recognized in uninduced microsomes from female rats. The levels of P450IIIA in males were increased 2 to 3-fold following treatment with phenobarbital. while the increase of IIIA levels in females by phenobarbital was minimal, as monitored by immunoblot analysis. Solid phase radioimmunoassay using monoclonal antibodies supported the results of immunoblot analysis. Phenobarbital treatment caused a 6.5-fold increase in the monoclonal antibody binding to IIB1 in males, whereas treatment of females with phenobarbital resulted in a 12-fold increase of IIB1 binding, relative to respective controls. The relative increase of IA levels by 3-methylcholanthrene was also greater in females than in males (10-vs. 8-fold) although the levels of induced IA were comparable in both sexes, as assessed by radioimmunoassay. Radioimmunoassay also showed that hepatic IIE1 level was 1.5-fold higher in males than in females and that either phenobarbital or 3-methylcholanthrene treatment caused 80% to 40% decrease in IIE1 levels, relative to control, in both sexes. Sex-related metabolic activities were examined in hepatic microsomes. Hexobarbital hydroxylase activity was 2- to 3-fold higher in uninduced microsomes from males than that from females. This hydroxylase activity was increased 2- and 3-fold in males and females, respectively, following phenobarbital treatment, as compared to controls. Addition of anti-P450IIB1 antibody to phenobarbital-induced hepatic microsomes from males and females produced 64% and 84% inhibition of hexobarbital oxidation, respectively. Aryl hydrocarbon hydroxylase activity was increased ≈12- and 26-fold in males and females. respectively, following 3-methylcholanthrene treatment relative to controls. The anti-P450IA antibody inhibitable rate of aryl hydrocarbon hydroxylase activity was comparable in both sexes following 3-methylcholanthrene treatment (≈70%). These results demonstrate that levels of hepatic P450IIB1 or P450IA are greater in male than in female for untreated, phenobarbital- or 3-methylcholanthrene treated rats. In addition, the relative increase of P450IIB1 or IA by phenobarbital or 3-methylcholanthrene is more significant in females.     

Purification and characterization of three forms of microsomal cytochrome P-450 in liver from 3-methylcholanthrene-treated guinea pigs

One molecular form of cytochrome P-450IIA from liver microsomes of guinea pigs treated with 3-methylcholanthrene was purified to a specific content of 17.4 nmoles/mg of protein. The difference spectrum of reduced hemoprotein-carbon monoxide complex of this cytochrome exhibits an absorption maximum at 448 nm. The absolute absorption spectrum of the oxidized form of this hemoprotein suggests a high-spin state of heme iron. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein shows a single band of polypeptide stained with Coomassie brilliant blue at the position corresponding to Mr 54,000. On the other hand, the other two forms of cytochrome P-450, cytochrome P-450I and P-450IIB, were also separated and purified to specific contents of 8.7 and 5.2 nmoles/mg of protein, respectively. Both cytochrome P-450I and P-450IIB exhibit absorption maxima at 450 nm in the difference spectrum of reduced hemoprotein-carbon monoxide complex, and a low-spin state of ferric iron in the heme. The spectrophotometrical property of cytochrome P-450I and P-450IIB was clearly different from that of cytochrome P-450IIA. Molecular activities of a reconstituted aryl hydrocarbon hydroxylase (EC 1.14.14.1) containing, respectively, cytochrome P-450I, P-450IIA, and P-450IIB were 0.224, 0.250, and 0.395 (moles per minute per mole of cytochrome P-450), and were estimated to be one-tenth that of cytochrome P-448 induced in rat liver by 3-methylcholanthrene, indicating the presence of the low inducibility by 3-methylcholanthrene of aryl hydrocarbon hydroxylase in liver microsomes of guinea pigs.     

The increase in urinary excretion of 6 beta-hydroxycortisol as a marker of human hepatic cytochrome P450IIIA induction.

1. Urinary excretion of 6 beta-hydroxycortisol, hepatic microsomal cortisol 6 beta-hydroxylase and the specific content of several forms of cytochrome P450 were measured in 8 to 14 patients before and after treatment with rifampicin (600 mg orally per day for 4 days). 2. Rifampicin treatment produced an average five fold increase in daily excretion of urinary 6 beta-hydroxycortisol. 3. Cortisol 6 beta-hydroxylase activity increased from 15 +/- 6 pmol min-1 mg-1 in organ donors (considered as 'control subjects') to 87 +/- 31 pmol min-1 mg-1 in rifampicin treated patients. 4. Among three forms of human P450 (P450IA, IIC and IIIA), (1), (2), measured by Western blots, only P450IIIA was significantly induced by the antibiotic. 5. Only antibodies against P450IIIA selectively inhibited cortisol 6 beta-hydroxylase in human liver microsomes. 6. Cortisol 6 beta-hydroxylase was correlated with P450IIIA specific content. 7. The urinary level of 6 beta-hydroxycortisol correlated with liver microsomal cortisol 6 beta-hydroxylase and P450IIIA specific content. 8. We conclude that P450IIIA is predominantly responsible for cortisol 6 beta-hydroxylase activity in human liver microsomes and that urinary 6 beta-hydroxycortisol is a marker of the induction of this cytochrome P450.     

Metabolism of inhaled styrene in acetone-, phenobarbital- and 3-methylcholanthrene-pretreated rats: Stimulation and stereochemical effects by induction of cytochromes P450IIE1, P450IIB and P450IA

1. The effect of various cytochrome P-450 inducers, namely acetone, phenobarbital (PB) and 3-methylcholanthrene (MC), on the pharmacokinetics of styrene metabolism was studied.

2. Styrene metabolism in vivo was studied measuring phenylglyoxylic acid (PGA), the enantiomers of mandelic acid (MA), and total thioethers excreted in the urine during a 24 h period of airborne exposure to styrene at 500 cm³/m³ (2100 mg/m³). In acetone-pretreated rats, PGA and MA and thioether formation were elevated 30-50%. The R/S ratio of MA enantiomers was about two in all styrene-exposed groups except PB-pretreated rats, which showed a ratio of four.

3. Styrene metabolism in liver microsomes measured in vitro was increased by styrene 140%, acetone plus styrene by 190%, methylcholanthrene plus styrene by 180% and phenobarbital plus styrene by 250%.

4. N-Nitrosodimethylamine demethylation (NDMAD) and 7-pentoxyresorufin dealkylaticn (PROD) in liver microsomes were enhanced 100-150% by styrene inhalation. The metabolism of 7-ethoxyresorufin was not significantly enhanced.

5. Monoclonal antibodies to P-450 IA1, IA2, IIB1 and IIE1 were utilized to identify cytochrcme P-450s by Western blot analysis. These studies showed clearly that styrene inhalation induced principally cytochrome P450IE1, whereas styrene given by gavage at a high narcotic dosage induced both P450HE1 (NDMAD, 60%) and P450IIB (PROD, 3000%).

6. Our conclusions are that styrene metabolism in vivo is both autoinduced and induced by other foreign compounds, that cytochrome P450IIE1 induction has a major impact on styrene metabolism and that P450IIB1 induction yields an altered MA metabolite enantiomer ratio.     

Differential effects of human recombinant interleukin-1 beta on cytochrome P-450-dependent activities in cultured fetal rat hepatocytes.

The immunomodulator interleukin-1 beta (IL-1) is one of the major inflammatory mediators. In vivo, it has been reported to depress some rat liver cytochromes P-450 (cytochrome P-450). Our aim was to study those effects in vitro, using cultured fetal rat hepatocytes as a model. Testosterone 6 beta-hydroxylase (cytochrome P-450 IIIA family activity) was not depressed by IL-1 treatments, but its induction by dexamethasone was prevented. The effect was time- and dose-dependent. Ethoxyresorufine-O-deethylase (cytochrome P-450 IA1 activity) decreased after IL-1 treatment, and dexamethasone partially prevented this inhibition. Acute phase effects of IL-1 were assayed by albumin and transferrin secretions. The cell's sensitivity to glucocorticoids was determined by tyrosine-aminotransferase activity. Our data demonstrate that IL-1 was able to prevent the glucocorticoid induction of cytochrome P-450 IIIA involving at least two different mechanisms. This is in agreement with the theory suggesting that the induction of CYPIIIA family by glucocorticoids requires the presence of the glucocorticoid receptor and some other regulatory elements. Other cytochrome P-450-dependent activities (IIA1, IIB1/2, and IIC11) were inhibited by IL-1 treatments, depending on dose and time, but some were also protected by dexamethasone.     

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.