Limitations on the metyrapone assay for the major phenobarbital inducible form of cytochrome P-450

Limitations on the determination of the concentration of the major phenobarbital inducible form of cytochrome P-450 (P-450b) in hepatic microsomes by the metyrapone assay of Luu-The $\text{et al.}$ (1) are reported. Compounds which bind to the Type I, II and IR binding sites, or convert cytochrome P-450 to P-420, decrease the apparent concentration of cytochrome P-450b by 20 to 100% in hepatic microsomes from untreated and pregnenolone-16α-carbonitrile or phenobarbital treated rats. It is calculated that errors of greater ca. 40% in the concentration of cytochrome P-450b can arise in the presence of appreciable quantities of the major pregnenolone-16α-carbonitrile or polycyclic hydrocarbon inducible forms of cytochrome P-450.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

A phenobarbital-inducible hepatic mitochondrial cytochrome P-450 immunochemically related to microsomal P-450b

We have purified and characterized a phenobarbital (PB)-inducible hepatic mitochondrial cytochrome P-450 (P-450), termed P-450mt4, which is distinctly different from the previously characterized mitochondrial isoforms. The level of induction of P-450mt4 by PB in the male livers is nearly 20-fold, as against a marginal induction in the female livers, suggesting that it may be a male predominant isoform. P-450mt4 shows a close resemblance to microsomal P-450b (the major PB-inducible form) with respect to electrophoretic migration (apparent molecular mass of 50 kDa) and immunological cross-reactivity, although it exhibits a distinct isoelectric pH (pI 6.9 vs 6.5 for P-450b), peptide fingerprint pattern, and amino acid composition. Further, the N-terminal sequence analysis shows over 90% positional identity (39 out of 42) between P-450mt4 and P-450b, suggesting that it is a close relative of the P-450 IIB gene family. In vitro reconstitution experiments show that P-450mt4 can metabolize a wide range of substrates such as benzphetamine, (dimethylamino)antipyrine, aflatoxin B1, and vitamin D3, exclusively in the presence of mitochondrial-specific ferredoxin and ferredoxin reductase as electron carriers. P-450mt4 is translated as a 53-kDa precursor, which is transported into mitochondria under in vitro conditions and processed into a mature 50-kDa protein. These results provide conclusive evidence for the occurrence of a male-specific P-450 belonging to the IIB gene family in rat liver mitochondria.     

Benzanthrone: a new substrate for hepatic microsomal cytochrome P-450

The metabolism of benzanthrone, a commonly used dy intermediate, by rat hepatic microsomes was investigated using thin layer chromatography (TLC) analysis. Incubation of benzanthrone with hepatic microsomes in the presence of NADPH generating system produced at least seven fluorescent metabolites on TLC plates. TLC spots numbered II, III, IV, V and VI were the major metabolites obtained from hepatic microsomes with the Rf values of 0.53, 0.45, 0.38, 0.33 and 0.26, respectively. Metabolites VII and VIII were faint bands with Rf values of 0.08 and 0.04, respectively. Preincubation of hepatic microsomes with either 1-benzyl-imidazole (10(-4)M) or SKF-525 A (10(-4)M) or metyrapone (10(-3)M) or flushing with carbon monoxide substantially inhibited the benzanthrone metabolism. alpha-Naphtho-flavone (10(-4)M) did not cause any change in hepatic microsomal metabolism of benzanthrone. Oral administration of benzanthrone to animals yielded at least six urinary metabolites. TLC spots numbered II, III, IV, V and VI in the urine were same as those of hepatic microsomal metabolites. However, one of the urinary metabolite numbered IX which stays at the origin of TLC plate with the Rf value of 0.05 may be a conjugate. Our results suggest that benzanthrone acts as a substrate for hepatic heme protein, cytochrome P-450 and that some of the metabolites are excreted in urine.     

On the glycosylation state of five rat hepatic microsomal cytochrome P-450 isozymes

The glycosylation states of five rat hepatic microsomal cytochrome P-450 isozymes (cytochromes P-450a, P-450b, P-450c, P-450d, and P-450e) were examined by quantitative carbohydrate analysis. Carbohydrate content of the purified enzymes as determined by acid hydrolysis, reduction, and gas chromatography of the alditol acetates revealed only trace amounts of neutral and amino hexoses in each of the five isozymes. Levels of mannose ranged from 0.3 to 1.7 mol/mol of cytochrome P-450 whereas levels of galactose were less than or equal to 0.2 mol/mol of cytochrome P-450 for the five hemoproteins. The amino sugars glucosamine and galactosamine were usually present at levels less than or equal to 0.2 mol/mol of cytochrome P-450, although one preparation of cytochrome P-450b had as much as 0.5 mol of glucosamine/mol of cytochrome P-450. Other carbohydrate residues (xylose and arabinose) were not detected in significant quantities. Since N- and O-glycosylation of proteins occurs primarily through N-acetylglucosaminyl and N-acetylgalactosaminyl residues, respectively, the lack of significant amounts of these amino sugars indicates that these five cytochrome P-450 isozymes are not normally glycosylated in the native state. Purified NADPH-cytochrome c reductase, which functions as an electron donor for microsomal cytochrome P-450, contained no detectable quantities of hexose sugars.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.     

Interactions of prostaglandins with hepatic microsomal cytochrome P-450

The spectral changes associated with the addition of prostaglandins (PGs) to hepatic microsomes from guinea pigs and rats were examined. PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α and PGF_2α when added to guinea pig liver microsomes exhibited type I spectra. The binding affinities as determined from spectral dissociation constants (K_s) were highest with PGA₁ and PGA₂. With liver microsomes from control or 3-methyl-cholanthrene (MC)-treated rats, PGs did not yield type I spectra; however, in this case a $\text{weak}$ spectrum, designated here as type “II” was at times observed, With microsomes from phenobarbital (Pb)-treated rats only PGA₁ and PGA₂ yielded type I spectra; again in absence of type I spectrum, a weak type “II” was occasionally observed. The addition of PGA₁ and PGA₂ to liver microsomes from Pb-treated rats inhibited the microcomal mediated hydroxylation of hexobarbital. The inhibition by PGA₁ was competitive; the K_i = 8.2 × 10^?4 M was found to be similar in magnitude to the K_s = 7.3 × 10^?4 M of PGA₁ observed with rat liver microsomes. These observations suggested that PGs particularly of the A series interact with the hepatic microsomal cytochrome P-450 monooxygenase system.     

Neonatal testosterone imprinting of hepatic microsomal drug metabolism and a male-specific form of cytochrome P-450 in the rat

The effects of neonatal castration and treatment with testosterone on sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, were studied. Neonatal castration of male rats resulted in a change in the population of forms of cytochrome P-450. Castration 1 day after birth abolished the synthesis of P-450-male and stimulated the synthesis of P-450-female. The decrease in the amount of P-450-male as well as the activities of drug metabolizing enzymes was partially reversed by administration of testosterone after castration.     

A regulatory polymorphism for rat hepatic cytochrome P-450g

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cis-acting.     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

Induction of the phenobarbital form of cytochrome P-450 by chemically unrelated compounds

The induction of the phenobarbital form of cytochrome P-450 by xenobiotics (phenobarbital, PB, hexachlorobenzene, HCB; hexachlorocyclohexane. HCCH, and aroclor 1016, Ar) was studied. It was demonstrated that administration of these compounds to animals is accompanied by an increase in the total cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine-N-demethylase and aldrin-epoxidase activities. Using monospecific antibodies against the cytochrome P-450 form isolated from PB-induced microsomes (PB-cytochrome P-450), a double immunodiffusion test revealed immunological identity of cytochrome P-450 forms induced by phenobarbital and other xenobiotics. The content of this form determined by rocket immunoelectrophoresis increased markedly and made up to 20-40% of the total cytochrome P-450 content. Antibodies against PB-cytochrome P-450 inhibited by 50-70% the benzphetamine-N-demethylase and aldrin-epoxidase activities, whereas the antibodies to methylcholanthrene-induced cytochrome P-450 were fairly ineffective. It was concluded that the chemically unrelated compounds induce in liver microsomes a cytochrome P-450 form, whose immunological properties and substrate specificity are close to the PB-form of cytochrome P-450.     

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.