Major Phenobarbital-inducible Cytochrome P-450 Research Articles

Mechanistic Studies of 9-Ethynylphenanthrene-Inactivated Cytochrome P450 2B1

The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization–mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995)Arch. Biochem. Biophys.323, 000–000]. The inactivation by 9EPh resulted in a 90–95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen–iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation.

Modulation of the control of mutagenic metabolites derived from cyclophosphamide and ifosfamide by stimulation of protein kinase A

The phosphorylation of the 2 major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in intact hepatocytes by the action of the membrane-permeating cAMP derivative N 6,O 2′-dibutyryl-cAMP. Under these conditions cyclophosphamide and ifosfamide (which are known to be activated by cytochrome P450 IIB1) were investigated for mutagenicity in Salmonella typhimurium TA1535 and TA100 an for cytotoxicity in TA1535. Cyclophosphamide nd ifosfamide were transformed to mutagenic and cytotoxic metabolites by the hepatocytes. The activation of both drugs to mutagens was markedly reduced after pretreatment of the hepatocytes with the membrane-permeating cAMP derivative N 6,O 2′-dibutyryl-cAMP. Cyclophosphamide and ifosfamide activation were reduced to 51% and 38% of unstimulated controls respectively, when hepatocytes were incubated for 1 h with N 6,O 2′-dibutyryl-cAMP in the presence of the phosphodiesterase inhibitor theophylline, and Salmonella typhimuriumm TA1535 was used. A marked reduction inmutagenicity of cyclophosphamide (35% compared with unstimulated controls) was also observed under different experimental conditions, namely after pretreatment of the hepatocytes with N 6,O 2′-dibutyryl-caMP for 1.5 h without theophylline and using Salmonella typhimurium TA100 as target strain. Continued presence of the cytochrome P450 IIB1 and P450 IIB2 inducer phenobarbital in the stimulation medium increased the mutagenicity of cyclophosphamide and led to an even more marked reduction of mutagenicity by preatreatment of the hepatocytes with N 6,O 2′-dibutyryl-cAMP and theophylline. In order to investigate whether the observed changes were metabolism-related the ifosfamide metabolite ifosfamide mustard which does not require metabolic activation by cytochrome P450 was studied under the same conditions. Its mutagenicity was indistinguishable after incubation with N 6,O 2′-dibutyryl-cAMP-treated or with unstimulated hepatocytes. Also the metabolic formation of cytotoxic metabolites from cyclophosphamide and ifosfamide but not that of ifosfamide mustard was markedly decreased by pretreatment of the hepatocytes with N 6,O 2′-dibutyryl-cAMP and theophylline. Thus the stimulation of protein kinase A in intact cells has important consequences for the control of genotoxic and cytotoxic metabolites and represents a fast and short-term regulation of it.

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regio- and stereoselective regulation of monooxygenase activities by isoenzyme-selective phosphorylation of cytochrome P450

The phosphorylation of the two major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in hepatocytes by the action of the membrane permeating cAMP derivatives N 6-dibutyryl-cAMP and 8-thiomethyl-cAMP. Under these conditions the dealkylation of 7-pentoxyresorufin, a selective substrate of cytochrome P450IIB1 and P450IIB2 was markedly reduced. 16β-Hydroxylation of testosterone which is catalyzed specifically only by cytochrome P450IIB1 and IIB2 was strongly reduced; for 16α-hydroxylation which is also catalyzed by cytochrome P450IIB1 and IIB2 but additionally by 3 further cytochrome P450 isoenzymes, this reduction was less pronounced; for the oxidation of the 17β-hydroxyl group which besides cytochromes P450IIB1 and IIB2 is additionally catalyzed not only by other cytochromes P450 but also by 17β-hydroxysteroid dehydrogenase there was a clear tendency of reduction which, however, no longer reached statistical significance. Hydroxylation at other positions of testosterone which are catalyzed by other cytochrome P450 isoenzymes were not significantly changed. Hence isoenzyme-selective phosphorylation of cytochrome P450 leads to a corresponding isoenzyme-selective modulation of monooxygenase activity which holds promise to be especially important as a fast regulation of the control of genotoxic metabolites.

CDNA cloning, sequence, and regulation of a major female-specific and growth hormone-inducible rat liver cytochrome P-450 active in 15 beta-hydroxylation of steroid sulfates.

cDNA clones encompassing the complete coding sequence for the female-specific rat liver microsomal 15 beta-hydroxylase have been isolated and sequenced. This cytochrome P-450 species (P-450 15 beta) has a total of 490 amino acid residues, and its deduced amino acid sequence as well as direct sequencing of the amino-terminal portion of the purified protein revealed its identity with P-450 i. P-450 15 beta was found to be 66% similar to the male-specific rat liver microsomal 16 alpha-hydroxylase, P-450 16 alpha. Furthermore, P-450 15 beta is 47% similar to the major phenobarbital-inducible cytochrome P-450 in rat liver, P-450 b, while its structural similarity to P-450 c, P-450 pregnenolone-16a-carbonitrile, and P-450 lauric acid omega-hydroxylase is less than 30%. However, the developmentally regulated cytochromes P-450 f and P-450 PB-1 both have 68% similarity to P-450 15 beta. It is therefore concluded that P-450 15 beta belongs to the same gene subfamily (P450IIC) that also includes cytochromes P-450 f, PB-1, and 16 alpha. The plasma pattern of growth hormone (GH) is known to be a major determinant for the sex-specific expression of P-450 15 beta and P-450 16 alpha. Continuous infusion of GH to hypophysectomized male rats causes an increase in P-450 15 beta mRNA after 6 days of treatment, while intermittent injections have no effect. On the other hand, no effect on P-450 16 alpha mRNA is observed after continuous administration of GH, while intermittent injections cause an increase after 2 days of treatment. This relatively long period before P-450 15 beta and P-450 16 alpha mRNA induction is seen might indicate that protein factors mediating GH action are involved.

The major phenobarbital‐inducible cytochrome P‐450 gene subfamily (P450IIB) mapped to the long arm of human chromosome 19

We have recently isolated a cloned cDNA that codes for a human orthologue of the major phenobarbital-inducible cytochrome P450IIB subfamily of rodents. The cloned human cDNA was used to analyse, by Southern blot hybridization, DNA extracted from a panel of 9 independent human-rodent somatic cell hybrids. The results indicate that all members of the P450IIB gene subfamily of man are located on chromosome 19. Evidence from hybrids containing different regions of human chromosome 19 localizes this cytochrome P-450 gene subfamily further to the long arm of chromosome 19 in the region cen-q13.3. We propose the designation CYP2B for this locus.

Cytochrome P-450 related to P-4504 from phenobarbital-treated rabbit liver: molecular cloning of cDNA and characterization of cytochrome P-450 obtained by its expression in yeast cells.

cDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library. The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids. The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3. Two expression plasmids, pAHF3 and pAH delta N3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase 1 (ADH1) promoter and terminator regions. pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2nd to the 3rd amino acid) were deleted in pAH delta N3. On introduction of the constructed plasmids into Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3. On the other hand, cytochrome P-450 could not be detected spectrophotometrically in any subcellular fractions from the yeast cells carrying pAH delta N3, although the transcript of pHP3 insert was detected in RNA blot analysis. These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells. Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein). In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450. The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm. The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase. Cytochrome P-450 (pHP3) catalyzed N-demethylation of benzphetamine and aminopyrine and denitrification of 1-nitropropane. Addition of cytochrome b5 to the reconstituted system resulted in stimulation of the N-demethylation activities but inhibition of the denitrification activity. Neither 7-ethoxycoumarin O-deethylation activity nor acetanilide p-hydroxylation activity was detected, either in the presence or absence of cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation between the metabolism of hexobarbital and aminopyrine in vivo in rats.

Two model substrates for oxidative hepatic enzyme activity, namely hexobarbital and aminopyrine, were simultaneously orally administered to rats, and blood concentrations of the substrates measured by g.l.c. The apparent intrinsic clearances of hexobarbital (Cl*int.HB) and of aminopyrine (Cl*int,AM) were correlated in untreated rats, and in rats pretreated with phenobarbital, 3-methylcholanthrene, polychlorinated biphenyls or carbon tetrachloride. Cl*int,HB and Cl*int,AM were both increased by phenobarbital and polychlorinated biphenyl pretreatment. Pretreatment with 3-methylcholanthrene had hardly any effect, and carbon tetrachloride caused a strong diminution of Cl*int.HB and Cl*int.AM. When the dose of aminopyrine was decreased, both Cl*int,HB and Cl*int,AM increased. This indicated that the primary metabolite of aminopyrine, monomethylaminopyrine, inhibits cytochrome P-450. The correlation coefficient for all clearance data was 0.92 (N = 36). It was concluded that both hexobarbital and aminopyrine are metabolized in vivo by the same or closely related cytochrome P-450 isozymes, and both may be used as model substrates in vivo for metabolic conversions primarily mediated by the major phenobarbital-inducible cytochrome P-450 subspecies.

A cytochrome P-450 gene family mapped to human chromosome 19.

We have recently isolated a cloned cDNA coding for a cytochrome P-450 of human liver microsomal membranes, which corresponds to a major phenobarbital-inducible cytochrome P-450 of rat liver. This human cytochrome P-450 is encoded by a member of a multigene family. DNA extracted from a panel of 12 independent human-rodent somatic cell hybrids was analysed by Southern blot hybridization with the cloned cDNA. The results indicate that all components of this cytochrome P-450 gene family are located on chromosome 19. Evidence from hybrids derived from an individual carrying a balanced translocation suggests a regional localization of 19p13.2----qter. Analysis of human metaphase chromosomes by in situ hybridization localizes this cytochrome P-450 gene family further to the long arm of chromosome 19 in the region q13.1----qter. We propose the designation P450PB for this locus.

Induction of cytochrome P-450 by phenobarbital is mediated at the level of transcription

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

Genetic polymorphisms for a phenobarbital-inducible cytochrome P-450 map to the Coh locus in mice.

Southern blot analysis suggests that multiple sequences homologous to a phenobarbital-inducible cytochrome P-450 cDNA are present in the rat and mouse genomes. A cDNA (pP-450b-5) to a major phenobarbital-inducible cytochrome P-450 mRNA species in the rat detected 6 polymorphic DNA fragments when hybridized to DNA from C57BL/6J and DBA/2J mice restricted with endonucleases EcoRI, BamHI, and PvuII. Using the BXD recombinant inbred strains, five of these polymorphisms were mapped to the Coh (coumarin hydroxylase) locus on chromosome 7 of the mouse. The Coh locus has previously been shown to code for a phenobarbital-inducible enzyme, believed to be a cytochrome P-450, which catalyzes the conversion of coumarin to 7-hydroxycoumarin (umbelliferone). The DNA polymorphisms appear to reflect changes in either cytochrome P-450 genes or pseudogenes that are very closely linked to the gene responsible for differential coumarin hydroxylase in mice or it may represent a change(s) in the Coh gene itself. The region of the Coh locus on chromosome 7 may be the site of a cluster of cytochrome P-450 genes.

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.

Isolation and characterization of a constitutive form of rabbit liver microsomal cytochrome P-450.

A heretofore unrecognized form of cytochrome P-450 was purified from rabbit liver microsomes with an average yield and purity similar to that of other highly purified forms of cytochrome P-450. Several properties of this cytochrome are contrasted with those of form 2, the major phenobarbital-inducible cytochrome P-450, form 4, the major 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome, and form 6, a cytochrome that is selectively induced in liver microsomes by 2,3,7,8-tetrachlorodibenzo-p-dioxin during the perinatal period. Thes four forms can be distinguished by virtue of their molecular weights as determined using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, by their respective peptide fingerprints, and by the monospecificity of their antisera. Since the enumerated properties are thought to reflect the primary structure of the cytochromes and since the observed differences are extensive, we suggest that these four forms are not derived from a common protein precursor.