Cytochrome P450IIB1 Research Articles

Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes

We present data showing that the major phenobarbital inducible cytochromes P-450 (cytochrome P-450IIB1 and cytochrome P-450IIB2) were phosphorylated in intact hepatocytes. This phosphorylation was greatly increased by the cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP mediated by a cAMP-dependent protein kinase. Most importantly the phosphorylation status of cytochromes P-450 was shown to change in the hepatocytes after treatment with glucagon, which is known to increase the level of cAMP in hepatocytes. The observed impact of the hormone glucagon on the phosphorylation of distinct cytochrome P-450 forms in intact hepatocytes reveals the possibility that the enzyme activity of cytochromes P-450 could be rapidly and differentially regulated by their phosphorylation and therefore dependent on the hormonal status of the organism.

The expression of cytochrome P450IIB1 in Saccharomyces cerevisia results in an increased mutation frequency when exposed to cyclophosphamide

A recombinant plasmid containing a full length cDNA encoding the rat cytochrome P450IIB1 under the control of the Saccharomyces cerevisiae ADC1 promoter was constructed and transformed into the yeast strain KY118. The encoded P450IIB1 protein was produced at a level of between 0.1 and 0.2% of total yeast cellular protein (0.068 nmol/mg total cellular protein). This protein was localized in the microsomal fraction and had activity towards the substrate benzyloxyresorufin, the activity being 0.16 nmol resorufin produced/min/mg microsomal protein. When exposed to the anticancer drug cyclophosphamide the mutation frequency, as determined by the development of resistance to the arginine analogue canavanine, increased in a dose-dependent manner over a control strain and was up to 16-fold higher at the highest doses used.

Close linkage of the human cytochrome P450IIA and P450IIB gene subfamilies: implications for the assignment of substrate specificity

We have isolated from human liver libraries two cytochrome P450 cDNA clones (lambda MP14 and lambda MP3) which are highly similar (83% over the coding region) to mouse testosterone 15 alpha hydroxylase and are therefore part of the cytochrome P450IIA gene subfamily. The P450IIA (CYP2A) gene subfamily was found to be closely linked to the P450IIB (CYP2B) subfamily and their chromosomal location could not be distinguished using somatic cell hybrids containing fragments of chromosome 19 between 19q12 and 19q13.2. Pulsed field gel electrophoresis indicates that both gene subfamilies are contained within 350-kb genomic DNA fragments, but were separated using various restriction enzymes. Northern blot analysis identified three P450IIA mRNAs each showing a wide inter-individual variation in their levels in the liver. High levels of P450IIA transcript were associated with high levels of P450IIB transcript suggesting that common factors may influence the expression of genes within these subfamilies. Genetic analysis has suggested previously that a member of the P450IIB subfamily is responsible for coumarin hydroxylase activity in the mouse. We discuss the possibility, based on our findings of tight linkage of the human P450IIA and IIB subfamilies, that a member of the IIA subfamily is a better candidate for this enzyme activity.

Effect of a Prudhoe Bay crude oil on hepatic and renal peroxisomal beta-oxidation and mixed-function oxidase activities in rats.

The effect of oral administration of a Prudhoe Bay crude oil (PBCO) to male rats (PBCO, 2.6 g/kg body weight, daily) for 5-12 days on hepatic and renal microsomal monooxygenase activities and peroxisomal beta-oxidation has been investigated. PBCO administration leads to liver enlargement. This is associated with induction of microsomal cytochrome P-450 levels (1.6- to 2.0-fold) and dependent mixed-function oxidase activities (7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-depentylase, representing cytochrome P-450I and cytochrome P-450IIB isoenzymes respectively, 9- to 15-fold; omega-oxidation of lauric acid representing the cytochrome P-450IVA1 isoenzyme, 1.4- to 1.5-fold) along with peroxisomal beta-oxidation (palmitoyl CoA oxidation, 2- to 5-fold). It was observed that rats exposed to PBCO showed an increase in renal microsomal cytochrome P-450 content (1.6- to 2.3-fold), cytochrome P-450I activity (5- to 8-fold) and omega-oxidation activity (1.3- to 1.4-fold). However, renal peroxisomal beta-oxidation was unaltered. Serum total triglycerides were lowered by 41-46% after PBCO exposure. These results suggest that induction of peroxisomal beta-oxidation and possibly mono-oxygenases may be related to the carcinogenic/tumorigenic potential of crude oil.

Induction of the rat hepatic microsomal mixed-function oxidases by two aza-arenes: A comparison with their non-heterocyclic analogues

The ability of the aza-aromatic polycyclic aromatic hydrocarbons 10-azobenz( a)pyrene and benz( a) acridine to induce the rat hepatic microsomal mixed-function oxidases was compared to that of their non-heterocyclic analogues benz( a) pyrene and benz( a) anthracene respectively. All four hydro-carbons markedly increased the O-deethylations of ethoxyresorufln and ethoxycoumarin, the non-heterocyclic analogues being the more potent. A more modest increase was seen in the O-dealkylation of pentoxyresorufin. All four hydrocarbons induced proteins recognised by antibodies to cytochrome P-450aIAI but no increase was seen when antibodies to cytochrome P-450IIB1 were employed. The metabolic activation of benz( a)pyrene and Glu-P-1 to mutagenic intermediates in the Ames test was enhanced by all pretreatments. It is concluded that the aza-aromatic polycyclic hydrocarbons, like their non-heterocyclic analogues, selectively induce the cytochrome P-450I family of proteins.

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme.

Induction of the rat hepatic microsomal mixed-function oxidases by 3 imidazole-containing antifungal agents: selectivity for the cytochrome P-450IIB and P-450III families of cytochromes P-450

Administration of the imidazole antifungal agents ketoconazole, miconazole and clotrimazole gave rise to increases in the microsomal cytochrome P-450 levels and the NADPH-dependent reduction of cytochrome c. Clotrimazole, and to a much lesser extent miconazole and ketoconazole, stimulated the dealkylation of pentoxyresorufin. All 3 agents gave rise to small, but significant increases in the O-deethylations of ethoxycoumarin and ethoxyresorufin. The antifungal-induced O-deethylation of ethoxycoumarin was much more sensitive to inhibition by metyrapone rather than by α-naphthoflavone. The binding of metyrapone to reduced microsomes was enhanced by treatment of animals with the 3 antifungal agents, clotrimazole being clearly the most potent. Immunoquantitation of cytochrome P-450 proteins using an ELISA procedure and employing anti-cytochrome P-450c ( P-450IA1, P-448 low spin) and P-450b ( P-450IIB1) antisera revealed that clotrimazole and miconazole, but not ketoconazole, induced the levels of phenobarbital-induced cytochromes P-450, while none of the antifungal agents increased the levels of cytochrome of P-448 proteins. Similar results were obtained using Western blots employing the above antibodies. On SDS-polyacrylamide gel electrophoresis microsomes derived from animals pretreated with clotrimazole showed intensification of a band at 51 kDa which was identified by Western blotting as the PCN-inducible form of cytochrome P-450 (cytochrome P-450p, P-450III family). Similar, but less pronounced intensification was seen with microsomes from animals pretreated with miconazole and ketoconazole. Furthermore, microsomes from clotrimazole- and ketoconazole-treated animals interacted with erythromycin to yield type I spectra. It is concluded that the imidazole-containing agents clotrimazole and miconazole, and to a much lesser extent ketoconazole, are potent inducers of the rat hepatic microsomal mixed-function oxidases, displaying selectivity towards the P-450IIB (phenobarbital-inducible) and P-450III (PCN-inducible) families of cytochrome P-450 proteins.

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.

A constitutive member of the rat cytochrome P450IIB subfamily: full-length coding sequence of the P450IIB3 cDNA.

The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, have long been the subjects of intense interest, in part because they are highly inducible by phenobarbital (PB). We have previously cloned and sequenced an 858-bp cDNA fragment (the PB24 insert) that encodes the carboxy-terminal portion of a P450b/P450e-like protein, henceforth referred to as P450IIB3. A Bam HI-Eco RI fragment of the PB24 insert hybridizes with a 1.9-kb mRNA present constitutively in rat liver and not inducible by PB (Affolter et al., 1986). We have now obtained, from a lambda gt11 rat liver cDNA library, cDNA inserts corresponding to the complete coding sequence of the IIB3 mRNA. The 491-amino acid IIB3 protein sequence, deduced from the cDNA sequence, is 77% identical to that of P450b. Northern blot analysis, with a IIB3-specific oligonucleotide probe, confirmed the constitutive presence of the polyadenylated 1.9-kb IIB3 mRNA in male rat liver. The IIB3 mRNA was undetectable in the lung, kidney, and prostate. It was constitutively present, and not inducible by PB, in female rat liver. Our results demonstrate unequivocally the existence of a constitutive member of the rat P450IIB subfamily. The remarkable inducibility of the P450b/P450e genes is in striking contrast to the absence of a PB effect on IIB3 gene expression. The molecular basis for this difference remains to be revealed.

Comparison of primary structures deduced from cDNA nucleotide sequences for various forms of liver microsomal cytochrome P-450 from phenobarbital-treated rabbits.

cDNA clones, termed pHP2, b32-3, b43, and b43-1, encoding cytochromes P-450 that are expressed in the liver of phenobarbital- (PB-) treated rabbits were isolated, and their nucleotide sequences were determined. pHP2 cDNA contains an open reading frame for a 490-residue protein and is a full-length counterpart of pP-450PBc2 [Leighton, J. K., Debrunner-Vossbrinck, B. A., & Kemper, B. (1984) Biochemistry 23, 204-210]. The b32-3 insert has a sequence for a protein whose primary structure is 91% similar to that of progesterone 21-hydroxylase P-450 1, though this cDNA lacks the sequence encoding the amino-terminal 110 residues. The overlapping clones b43 and b43-1 together encode an ethanol-inducible form of cytochrome P-450, though the amino-terminal five or more residues are missing in the composite b43/b43-1 sequence. Northern blot analysis showed that the b43/b43-1 protein is more strongly inducible by polycyclic aromatic hydrocarbons and isosafrole than by PB, in contrast to the case of the HP2 and b32-3 proteins. A comparison of the primary structures of eight forms of cytochrome P-450, including the HP2, b32-3, and b43/b43-1 proteins, that are expressed in the liver of PB-treated rabbits showed that 149 out of 487-492 amino acid residues are conserved in these cytochromes P-450. The eight forms can be assigned to three rabbit cytochrome P-450 gene subfamilies, P450IIB, P450IIC, and P450IIE. It was also shown that the members of the rabbit P450IIC subfamily can be further classified into three subgroups on the basis of their sequence similarity.