Phenobarbital-inducible Form Of Cytochrome P-450 Research Articles

High activity of cytochrome P-450-linked aminopyrine N-demethylase in mouse brain microsomes, and associated sex-related difference

The presence of cytochrome P-450 and associated mono-oxygenase activities was examined in brain microsomes from male and female mice. Although the cytochrome P-450 level in male mouse brain was very low as compared with mouse liver, the aminopyrine N-demethylase and morphine N-demethylase specific activities in male mouse brain were much higher than those observed in mouse liver. Ethoxycoumarin O-de-ethylase and aniline hydroxylase activities were, however, not detected in mouse brain. Sex-related differences were observed in both the cytochrome P-450 levels and aminopyrine N-demethylase activity in mouse brain, the levels of both being higher in male mouse brain as compared with female mouse brain. Aminopyrine N-demethylase activity in mouse brain microsomes was dependent on the presence of oxygen and NADPH and could be inhibited by piperonyl butoxide, N-octyl imidazole and carbon monoxide. Antiserum raised to the phenobarbital-inducible form of rat liver cytochrome P-450 [P-450(b+e)] inhibited mouse brain aminopyrine N-demethylase activity by around 80+ mouse brain microsomal protein exhibited cross-reactivity against this antiserum when examined by Ouchterlony double diffusion and immunoblotting. The present results indicate the presence of a phenobarbital-inducible form of cytochrome P-450 (or a form of cytochrome P-450 that is similar immunologically) in mouse brain microsomes, which is associated with a sex-related difference.

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. IX. A comparative study with enantiomeric 2-nitroso-1-phenylpropane in microsomes and a reconstituted cytochrome P-450 system from rat liver

Formation of cytochrome P-455 nm complexes was investigated with enantiomeric 2-nitroso-1-phenylpropane — the C-nitroso analogue of amphetamine — and optically active N-hydroxyamphetamine, in the presence of NADPH. For comparative reasons, three different drug-metabolizing enzyme systems were used, namely microsomes from control and phenobarbital-treated rats, and a reconstituted system containing the main phenobarbital-induced form of cytochrome P-450 from rat liver. In microsomes obtained from phenobarbital-treated rats, pronounced differences in the kinetics of complex formation were observed between the enantiomeric C-nitroso compounds, but not between the isomers of N-hydroxyamphetamine. In the reconstituted enzyme system the S-nitroso compound formed the P-455 nm chromophore at the highest initial rate, while the R analogue was devoid of complexing activity. The rates of complex formation from the N-hydroxylamine enantiomers were high and equal.

The site of cyclic AMP-dependent protein kinase catalyzed phosphorylation of cytochrome P-450 LM2

The phenobarbital-inducible form of cytochrome P-450 purified from rabbit liver microsomes is phosphorylated by cAMP-dependent protein kinase at a single site, the serine residue in position 128 of the amino acid sequence. The serine is located in a characteristic recognition sequence for cAMP-dependent protein kinase and is part of a primary structure which is conserved during evolution, present also in phenobarbitalinducible rat cytochrome and cytochrome P-450 CAM from Pseudomonas putida. The contribution of these findings to our understanding of the structure and membrane topology of cytochrome P-450 LM2 and its turnover regulated by phosphorylation is discussed.

Cytochrome P450 of small intestinal epithelial cells: Immunochemical characterization of the increase in cytochrome P450 caused by phenobarbital

We have studied total cytochrome P450 and the major form of cytochrome P450 increased by phenobarbital in small intestinal epithelial cells and livers of male Sprague-Dawley rats. Using an improved method for preparing microsomes from intestinal epithelial cells, we find that concentrations of total cytochrome P450 in intestinal cell microsomes are 10% of those in liver microsomes, and that this percentage is unchanged after phenobarbital treatment. In untreated rats, < 5% of total cytochrome P450 of liver or intestinal epithelium is the form induced by phenobarbital, as measured by rocket immunoelectrophoresis. In phenobarbital-treated rats, the major phenobarbital-induced form accounts for ~50% of the total in both organs. In the small intestine of phenobarbital-treated rats, the concentrations of total cytochrome P450 and of the major phenobarbital-induced form increase concurrently as epithelial cells mature from crypt to upper villus. Concentrations of total cytochrome P450 and of the major phenobarbital-induced form in the proximal two-thirds of the rat small intestine are twofold higher than in the distal third. Immunoblotting performed with a monoclonal antibody to the major phenobarbital-induced form of cytochrome P450 from rat liver revealed a subtle difference between this form in liver and intestine.

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl 4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.

Dinemorphan N-demethylation by mouse liver microsomes.

Dinemorphan, an antitussive drug, is N-demethylated in vitro by mouse liver microsomes with biphasic kinetics showing two apparent Km and Vmax. Moreover, dinemorphan N-demethylation is inhibited by CO, SKF-525A, metyrapone and it is specifically catalyzed by a phenobarbital-inducible form of cytochrome P-450.

Direct in vitro effects of bis (tri- n-butyltin)oxide on hepatic cytochrome P-450

Bis(tri- n-butyltin)oxide, an agriculturally important biocidal agent, when added in vitro to liver microsomes containing the phenobarbital-induced form of cytochrome P-450, produced a typical type I binding spectrum (an absorption maximum at 390 nm; an absorption minimum at 420 nm). Studies with microsomal preparations containing cytochrome P-448, induced by 3-methylcholanthrene or β-naphthoflavone, revealed that this hemeprotein was more susceptible to direct degradation by bis (tri- n-butyltin)oxide than was the uninduced or phenobarbital-induced forms of cytochrome P-450. The disappearance of spectrally detectable cytochrome P-450 was accompanied by an increase in cytochrome P-420. The formation of cytochrome P-420 was both time and temperature dependent, and it also occurred to a greater extent in microsomal preparations containing cytochrome P-448 than in microsomes containing the phenobarbital-induced form of cytochrome P-450. In all cases, the decreases in spectrally detectable cytochrome P-450 produced by the organotin were not accompanied by decreases in microsomal heme or cytochrome b 5 content. The findings provide evidence for the direct interaction followed by conversion of cytochrome P-450 to cytochrome P-420 produced by a trialkyltin compound in vitro, and indicate that different susceptibilities to degradation exist within the various subspecies of this hemeprotein.

A cytochrome P-450 multigene family. Characterization of a gene activated by phenobarbital administration.

The capacity of the liver microsomal mixed function oxidase system to metabolize a great variety of exogenous as well as endogenous compounds is thought to reflect the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. In order to approach the questions of the genetic basis for the existence of multiple cytochrome P-450 isozymes and the molecular mechanisms of the induction process, we have used a cloned cDNA for a major phenobarbital-induced form of cytochrome P-450 to identify and characterize thirteen distinct rat genomic clones containing segments of six different genes. Only three clones, representing overlapping segments of a single gene, hybridized to the cloned cDNA or to phenobarbital-induced mRNA under conditions of high stringency. In vitro transcription studies with isolated rat liver nuclei indicated that only this gene, but not the genes represented by the other genomic clones, appears to be markedly activated by phenobarbital treatment of rats. Although there are a small number of differences, DNA sequence analysis of the eight exons of the gene present in these genomic clones indicates that they encode residues 58 to 491 (the COOH terminus) of cytochrome P-450e, a major phenobarbital-induced isozyme. It appears that the other cloned genes may contain only a small region of very strong homology to the cytochrome P-450e gene, a region which includes the exon encoding a tridecapeptide which is also present in two dissimilar forms of rabbit liver cytochrome P-450, one constitutive and one induced by phenobarbital. Southern blotting analysis of rat liver DNA indicates that the rat genome may contain two additional genes which are very closely related to the cytochrome P-450e gene but which we have not yet isolated from the genomic library. One of these genes is likely to encode cytochrome P-450b, the major phenobarbital induced form of cytochrome P-450 whose mRNA is greater than 95% homologous to that encoding cytochrome P-450e.

Comparison of trans-stilbene oxide, phenobarbital and 3-methylcholanthrene as inducers of steroid metabolism by the rat liver microsomal cytochrome P-450 system

The metabolism of testosterone and androstenedione by liver microsomes was investigated after treatment of rats with trans-stilbene oxide, phenobarbital, or 3-methylcholanthrene. Conditions for linearity of the assay with time and amount of cytochrome P-450, as well as saturating substrate concentrations, were established. The metabolites were separated by thin-layer chromatography and quantitated by scintillation counting. The rates of formation of different testosterone and androstenedione metabolites after induction with trans-stilbene oxide or phenobarbital were similar, indicating that these xenobiotics induce the same isozyme of cytochrome P-450. This conclusion was further supported using inhibitors of cytochrome P-450 (SKF-525A, metyrapone and α-naphthoflavone) and with immunoinhibition by antibodies directed towards the phenobarbital-inducible form of cytochrome P-450. After treatment with trans-stilbene oxide or phenobarbital, the specific rates of formation of the 6β- and/or 2β-hydroxy metabolites and of 17β-hydroxy-4-androstene-3,16-dione were increased. In contrast, administration of 3-methylcholanthrene led to decreases in the specific rates of formation of almost all testosterone and androstenedione metabolites investigated. However, all three of these inducers cause increases in the total liver metabolism of testosterone and androstenedione. These increases are 2–30-fold in the case of trans-stilbene oxide, 3–46-fold for phenobarbital and 1–4-fold after treatment with 3-methylcholanthrene. The possible physiological significance of these effects is as yet unknown.

Stimulation of de novo synthesis of cytochrome P-450 by phenobarbital in primary nonproliferating cultures of adult rat hepatocytes.

Primary monolayer cultures of nonproliferating parenchymal cells prepared from adult rat liver and maintained in serum-free medium responded to additions of phenobarbital with concentration-dependent increases in synthesis and accumulation of a cytochrome P-450 protein immunochemically and catalytically indistinguishable from that found in the livers of adult rats treated with phenobarbital. Maximal stimulation of the rate of synthesis of this cytochrome protein by phenobarbital, as much as 20-fold higher than in control cultures (1.01% of the rate of synthesis of total cellular protein), could be achieved when the drug was first added to cultures no older than 24 hr and then was maintained in the medium for 96 hr. In addition to phenobarbital, chemicals classified as "phenobarbital-like" inducers in vivo (mephenytoin, mirex, 2,2',4,4',5,5'-hexabromobiphenyl) induced synthesis in culture of this same immunoreactive protein. Supplementation of the medium with 0.1 microM H2SeO3 plus phenobarbital produced an average 2-fold enhancement in the rate of synthesis of this inducible cytochrome protein as compared to that in cultures receiving phenobarbital alone. Inasmuch as there was a decline in selenium content and in the activity of the seleno-enzyme glutathione peroxidase in hepatocyte cultures maintained in standard culture medium for more than 24 hr, the added selenium appears to correct a spontaneously acquired cellular deficiency in selenium. Contrary to the concept that liver cells placed in culture promptly dedifferentiate with general loss of specialized functions such as cytochrome P-450, our data demonstrate that expression of the phenobarbital-inducible form of cytochrome P-450 is not extinguished in culture, but rather it is masked transiently and is attenuated as the cells adapt to the imperfect conditions of the culture environment.

Interindividual variation in benzo(a)pyrene metabolism and composition of isoenzymes of cytochrome P-450 as revealed by SDS-gel electrophoresis of human liver microsomal fractions.

Human liver microsomal fractions from 13 different individuals were characterized with respect to SDS-polyacrylamide gel electrophoretic profiles and regiospecificity in the metabolism of the polyaromatic hydrocarbon benzo(a)pyrene. Pronounced interindividual differences in the composition of microsomal proteins in the molecular weight region of Mr=49,000-60,000 were found. Furthermore, gel electrophoresis combined with staining for peroxidase activity indicated most of the variations among the profiles of microsomal proteins being attributed to interindividual differences in the composition of isoenzymes of cytochrome P-450. One type of human liver microsomal fraction was selectively induced in specimens obtained from patients with known regular drug intake before death and correlated well in molecular weight to the phenobarbital-inducible form of cytochrome P-450 in the rabbit. Large variations among the human liver microsomal samples were also seen in the benzo(a)pyrene metabolism. The results indicate the presence of 7-8 different froms of cytochrome P-450 in human liver microsomes and that the interindividual variations seen in drug metabolism may at least in part be explained by interindividual variations in the distribution of these isoenzymes.

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant ( K s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding ( ΔA max) was increased only following phenobarbital induction. The K s values ( ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant ( K m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO 2 and H 2O 2, but not NaIO 4, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b 5 or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.

A simple and rapid procedure for the purification of phenobarbitalinducible cytochrome P-450 from rat liver microsomes

A rapid and simple procedure has been developed for the purification of a phenobarbital-inducible form of cytochrome P-450 from the liver microsomes of phenobarbitalpretreated rats. Within 2 days approximately 1000–1500 nmol of highly purified cytochrome P-450 with a specific content of 16 nmol/mg protein can be recovered from 4 g of microsomal protein. The procedure consists of solubilization of microsomal protein with sodium cholate, fractionation with polyethylene glycol, and column chromatography at room temperature on DEAE-cellulose. The resulting DEAE-cellulose fraction electrophoreses on polyacrylamide gels in the presence of sodium dodecyl sulfate as a major protein band with a minimum molecular weight of 52,000 and a few faint bands. Further chromatography on QAE Sephadex A-25 essentially removes these faint bands and increases the specific content slightly to 17 nmol/mg protein. Relatively low amounts of this form of cytochrome P-450 appear to be present in microsomes of untreated rats since less than 1% can be recovered as the DEAE-cellulose fraction by this procedure. An identical form is inducible by phenobarbital in rats of different ages and sex. In a reconstituted system under optimal assay conditions, this form of cytochrome P-450 catalyses the N-demethylation of benzphetamine with a turnover number greater than 100 and hydroxylates testosterone at the 16α position but not at the 6β or 7α position.