Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

Sexual differentiation and regulation of cytochrome P-450 CYP2C7.

The multigene family of proteins known as the cytochrome P-450-dependent monooxygenases play a central role in the metabolism of hormones and foreign compounds. As part of our studies into the function and regulation of these proteins we have isolated a little studied constitutively expressed isozyme CYP2C7 and have investigated its substrate specificity and mode of regulation. Interestingly the haem of this enzyme in its isolated form is almost 100% in the high spin state. The enzyme was active in the metabolism of a range of model resorufin substrates, but exhibits highest activity towards benzyloxyresorufin. Indeed, this isozyme appears to play a significant role in the metabolism of this substrate in microsomal samples from untreated male rats. Tissue distribution studies indicated that CYP2C7 was expressed in liver, kidney and possibly muscle tissue. Cytochrome P-450 CYP2C7 could not be significantly induced by any of a wide range of known modulators of cytochrome P-450 expression at the mRNA level, however some significant changes in protein expression were observed. Some of the agents used (e.g., diethylnitrosamine and carbon tetrachloride) caused a significant reduction in the expression of this protein. In agreement with other reports where mRNA levels were measured we found that the level of CYP2C7 protein expression was sexually differentiated. Female rats express two to three times the level found in males, the sex difference being reversible by hypophysectomy.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. II. Localization of some genes regulating cytochrome P-450 activity

The localization of some genes determining the capacity for some cytochrome P-450 -dependent reactions have been studied in adult Drosophila. Strains with genetically determined high or low enzyme activities were crossed with strains carrying recessive visible markers on the chromosomes, and enzyme activities were measured in microsomes from recombinant F2 progeny. A dominantly inherited high p-nitroanisole (PNA) demethylation and biphenyl 3-hydroxylation in insecticide-resistant strains were both shown to be located around 65 cM on the second chromosome, regulated by one gene or closely linked genes. This localizes these activities to the same region as the gene responsible for the cross resistance to several classes of insecticides and a high metabolism of vinyl chloride in resistant strains. The occurrence of a regulatory gene mutation as a basis for the insecticide resistance is proposed. Hydroxylation of benzo[a]pyrene (BP) and deethylation of 7-ethoxy-coumarin seems to be determined by two third chromosome genes, at approx. 51 and 58 cM, respectively. The capacity for biphenyl 4-hydroxylation was shown to be determined by two genes on the second chromosome, one at or to the left of the gene black (48 cM) responsible for a low metabolism in strain Berlin K, and one at about 63 cM giving high formation of this metabolite in Oregon R. The latter could not be separated from the gene in insecticide-resistant strains at c:a 65 cM discussed above on the basis of the genetic localization, but observations supporting the occurrence of two closely linked genes regulating these different activities are available. In conclusion, 4-5 genes determining the capacity for several reactions, being a part of the genetic regulation of the cytochrome P-450 system in Drosophila melanogaster were indicated.     

Genetic regulation of the cytochrome P-450 system in Drosophila melanogaster. I. Chromosomal determination of some cytochrome P-450-dependent reactions

The genetic regulation of some cytochrome P-450-dependent enzyme activities has been studied in adult Drosophila. Strains having genetically determined high or low enzyme activities were crossed with a marker strain and the metabolism was analyzed in microsomes from hybrids carrying different combinations of chromosomes from the strain under test. High p-nitroanisole (PNA) N-demethylation, biphenyl 3-hydroxylation and an increased amount of a protein with an apparent mol. wt. of 54 000, after SDS-gel electrophoresis of the microsomes in insecticide-resistant Drosophila strains, are inherited as dominant second chromosome traits. A low capacity for benzo[a]pyrene (BP) hydroxylation and 7-ethoxycoumarin O-deethylation in the Hikone R strain is semidominantly inherited in both cases and determined by gene(s) on the third chromosome. A semidominantly inherited high 4-hydroxylation of biphenyl and a high amount of a protein with an apparent mol. wt. of 56 000 in the Oregon R strain are also localized to the second chromosome. The results indicate that several other cytochrome P-450-dependent activities are not regulated by the genes mentioned above. In conclusion, at least three genes regulating the cytochrome P-450 system in Drosophila have been identified.     

Olfactory-specific cytochrome P-450 (P-450olf1; IIG1). Gene structure and developmental regulation

The olfactory neuroepithelium is the principal site of interaction for airborne molecules, mainly odorants, in the organism. The presence of an active cytochrome P-450-dependent oxidative metabolism in this tissue has not yet been studied as well as the hepatic cytochrome P-450-dependent oxidations. In this report, we describe cytochrome P-450olf1 (IIG1), a P-450 gene expressed at high levels uniquely in the olfactory epithelium. By Southern analysis and genomic DNA cloning, we demonstrate that a single copy of the P-450olf1 gene is present in the rat genome and contains 9 exons. We conclude that rat P-450IIG1 is a single gene subfamily. P-450olf1 gene expression was activated after birth in both male and female Sprague-Dawley rats and remained active in adult olfactory epithelium. A first maximum level of expression was reached around postnatal day 21. The coincidence between the temporal gene activation of P-450olf1 and the postnatal increase in the sensitivity of olfactory response to odorants is consistent with a potential role of this enzyme in olfactory function.     

Association of cytochrome P450 genes polymorphisms (CYP1A1 and CYP1A2) with the development of chronic obstructive pulmonary disease in Bashkortostan

To assess the role that polymorphisms of cytochrome P450 genes play in genetic predisposition to chronic obstructive pulmonary disease (COPD), the allele and genotype distributions of CYPIA1 (2455 A/G, 3801T/C) and CYP1A2 (-2464T/delT, -163C/A) genes were studied in Tatar and Russian COPD patients and in cases of healthy individuals (Russian, Tatar and Bashkir), residents of Bashkortostan. It was shown that the CYP1A1 and CYP1A2 genes haplotypes frequency distribution patterns do not differed between Tatars and Russians ethnic groups (chi2 = 0.973, df = 3, p = 1.00 and chi2 = 1.546, df = 3, p = 0.92, respectively). Analysis of the the CYP1A1 and CYP1A2 genes haplotypes revealed statistically significant differences in the haplotypes frequency distributions between Bashkirs versus Russians and Tatars (chi2 = 12.328, df= 3,p = 0.008; chi2 = 9.218, df=3, p = 0.034, respectively for CYP1A1 gene and (chi2 = 18.779, df=3, p = 0.0001, chi = 14.326, df=3, p = 0.003, respectively for CYP1A2 gene). The (-2467)delT allele and CYP1A2*1D haplotype of CYPIA2 gene was associated with higher risk of COPD in Tatar ethnic group (OR = 1.83, 95% CI 1.24-2.71, chi2 = 9.48, p = 0.003 and chi2 = 9.733, p = 0.0027, Pcor = 0.008; OR = 3.908, 95% CI 1.56-10.19, respectively). On the other hand the CYP1A2*1A haplotype had protective effect (chi2 = 6.319, p = 0.0127, Pcor = 0.038; OR = 0.6012, 95% CI 0.402-0.898). But at the same time we did not find any differences in the genotypes and haplotypes frequency distributions of the CYP1A2 gene within the patients and healthy groups in Russian ethnic group. We also did not find any association of CYP1A1 gene with COPD in ethnic groups of Bashkortostan.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Segmental homologies in the coding and 3' non-coding sequences of rat liver cytochrome P-450e and P-450b cDNAs and cytochrome P-450e-like genes

The nucleotide sequence of a cloned cDNA insert carried by pHDQ14 was determined and found to code for the 107 C-terminal amino acids of rat liver cytochrome P-450e. Comparison of the pHQ14 cDNA sequence with those of cloned cDNAs for cytochrome P-450b and of 2 P-450e-like genes revealed segmental homologies that may have resulted from gene conversion. These results suggest that gene conversion may generate sequence variants of genes for rat liver cytochrome P-450s.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Role of polymorphic variants of cytochrome P450 genes (CYP1A1, CYP2E1) and microsomal epoxide hydrolase (mEPHX) in pathogenesis of cystic fibrosis and chronic respiratory tract diseases

Frequencies of CYP1A1, CYP2E1, and mEPHX polymorphic variants were analyzed in cystic fibrosis, chronic obstructive lung disease, bronchiectatic disease, chronic nonobstructive bronchitis, and recurring bronchitis. Mutations in CYP1A1 and mEPHX were shown to modify the severity of respiratory disorders in cystic fibrosis, the combination of CYP1A1 genotype Val/Val with the "very slow" mEPHX phenotype being most unfavorable (odds ratio OR = 12.30). Heterozygosity at both CYP1A1 and CYP2E1 was associated with chronic obstructive lung disease and recurring bronchitis (OR = 4.08 and 11.72, respectively). The "very slow" phenotype of mEPHX was predisposing to chronic respiratory disorders regardless of the CYP1A1 or CYP2E1 alleles (OR = 4.06). Basing on the above correlations, a combination of the "very slow" mEPHX phenotype with elevated cytochrome P450 (CYP1A1 and CYP2E1) activities was assumed to expedite severe respiratory disorders.     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

Association of polymorphisms of the CYP1A1 and CYP1A2 cytochrome P450 genes with chronic obstructive pulmonary disease in Bashkortostan

The frequencies of polymorphisms of CYP1A1 (2455A/G, 3801T/C) and CYP1A2 (?2464T/delT, ?163C/A) were determined in healthy residents of Bashkortostan (Russians, Tatars, and Bashkirs) and tested for association with chronic obstructive pulmonary disease (COPD). Interethnic differences in the frequency distribution of the CYP1A1 and CYP1A2 polymorphisms were significant. In Tatars and Russians, the CYP1A1 and CYP1A2 haplotype frequencies were similar (χ² = 0.973, df = 3, P = 1.00 and χ² = 1.546, df = 3, P = 0.92, respectively). In Bashkirs, the CYP1A1 haplotype frequencies significantly differed from those in Russians and Tatars (χ² = 12.328, df = 3, P = 0.008 and χ² = 9.218, df = 3, P = 0.034, respectively) owing to a high frequency of CYP1A1*2B (10.17%). Similarly, Bashkirs differed from Russians and Tatars in the CYP1A2 haplotype frequencies (χ² = 18.779, df = 3, P = 0.0001 and χ² = 14.326, df = 3, P = 0.003, respectively). The frequency of the CYP1A2*1D haplotype in Bashkirs was 11.02% in contrast to 2.36% in Tatars and 1.61% in Russians. Allele *D of the CYP1A2 ?2467delT polymorphism was associated with COPD in Tatars (OR = 1.83, 95%CI 1.24–2.71, χ² = 9.48, P = 0.003). CYP1A2*1D was associated with an increased risk of COPD (8.65% vs. 2.36% in controls, χ² = 9.733, P = 0.0027, P _cor = 0.008, OR = 3.908, 95%CI 1.56–10.19). Haplotype CYP1A2*1A was significantly less frequent in patients with COPD (21.05% vs. 30.74%, χ² = 6.319, P = 0.0127, P _cor = 0.038, OR = 0.6012, 95%CI 0.402–0.898). The CYP1A1 polymorphisms were not associated with COPD in residents of Bashkortostan.     

Cloning and characterization of bovine cytochrome P-450(11 beta) genes

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.