Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.     

Transcriptional activation of cytochrome P450 CYP2C45 by drugs is mediated by the chicken xenobiotic receptor (CXR) interacting with a phenobarbital response enhancer unit

Cytochromes P450 (CYP)-2C enzymes fulfill an important role in xenobiotic metabolism and therefore have extensively been studied in rodents and humans. However, no CYP2C genes have been described in avian species to date. In this paper, we report the cloning, functional analysis, and regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with CYP2Cs in other species. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (LMH) led to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the mRNA level and in reporter gene assays using a construct containing 2.6 kb of its 5'-flanking region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression, respectively. A phenobarbital response enhancer unit (PBRU) of 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb fragment. Site-specific mutation of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The chicken xenobiotic receptor CXR interacted with the PBRU in electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.     

Impairment of human CYP1A2-mediated xenobiotic metabolism by Antley-Bixler syndrome variants of cytochrome P450 oxidoreductase

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR^null, POR^wt, POR^YH, and POR^VE, for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR^null, not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR^YH and POR^VE models than in POR^wt, indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.     

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of intestinal cytochrome P450 (CYP3A) by rifampicin in beagle dogs

Both male and female beagle dogs (four dogs/sex) were orally treated with rifampicin (Rif) at the dose of 10 mg/kg/day for 7 days and an additional eight dogs (four dogs/sex) were used as a control. The inducible effect of Rif on intestinal cytochrome P450, especially CYP3A enzyme, was investigated by measuring microsomal testosterone 6beta-hydroxylation (6beta-OHT) activity, immunoblot and ELISA analysis. In male dogs, microsomal 6beta-OHT activity in the duodenum, upper, middle and lower part of the jejunum and the ileum of the control was 229, 204, 194, 129 and 57 pmol/min/mg protein, while the activity of the Rif-treated dogs significantly increased to 456, 486, 430, 192 and 138 pmol/min/mg protein, respectively. The activity of intestinal 6beta-OHT in the control and Rif-treated female dogs showed almost similar levels to those observed in the corresponding male dogs. The activity of intestinal 6beta-OHT in both control and Rif-treated dogs was specifically inhibited by anti-CYP3A12 antiserum. The apparent K(m) value for 6beta-OHT activity in all sections of the small intestine was comparable with that in the liver, and no significant changes were observed in between control and Rif-treated dogs. In both control and Rif-treated dogs, immunoblotting of intestinal microsomes with anti-CYP3A12 antiserum produced a band indistinguishable from that of purified CYP3A12 or of immunoreactive CYP3A12 in liver microsomes. A significant increase in intestinal CYP3A content by Rif treatment was quantitatively verified by the ELISA analysis and the magnitude of its increase correlated well with that of 6beta-OHT activity elevation. Furthermore, the results of immunohistochemistry using the anti-CYP3A12 antiserum indicated that CYP3A protein was specifically distributed in epithelial cells throughout the small intestine and appeared to be predominant at the apical side of villus cells. These results demonstrate that Rif induces not only hepatic CYP3A12 but also intestinal CYP3A in dogs.     

Cloning and functional expression of a first inducible avian cytochrome P450 of the CYP3A subfamily (CYP3A37)

CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.     

Cloning and characterization of the rat cytochrome P450 4F5 (CYP4F5) gene

The analysis of a non-redundant set of human proteins, for which both the crystallographic structures and the corresponding gene sequences are available, show that bases at third codon position are non-uniformly distributed along the coding sequences. Significant compositional differences are found by comparing the gene regions corresponding to the different secondary structures of the proteins. Inter-and intra-structure differences were most pronounced in the GC-richest genes. These results are not compatible with any proposed hypotheses based on a neutral process of formation/maintenance of the high GC₃ levels of the genes localized in the GC-richest isochores of the human genome.     

Cocaine metabolism in human fetal and adult liver microsomes is related to cytochrome P450 3A expression

Cocaine N-demethylation to norcocaine was studied in human liver microsomes of different ages. Norcocaine was formed at a considerable rate in fetal (45.4+/-18.2 nmol/mg x hour, n = 8) and adult specimens (82.0+/-46.6 nmol/mg x hour, n = 15), p = 0.04 (Mann-Whitney). Furthermore, the apparent Km values in fetal specimens (0.57 and 0.48 mM, n = 2) showed a higher affinity compared with those of adults (mean value 2.7 (1.8-4.25) mM, n = 4). Estimated enzyme metabolic clearance with respect to P450 total content was higher in fetal than in adult liver microsomes (2.22 ml/nmol P450 x hour, and 0.18 (0.14-0.23) ml/nmol P450 x hour, respectively). Several drugs, known to be CYP3A substrates, were used as potential inhibitors of cocaine metabolism. Midazolam, ergotamine and erythromycin showed strong inhibition (approx. 70 %) when used at concentrations of 500 microM (midazolam, erythromycin) or 200 microM (ergotamine). The metabolism of 1 mM cocaine correlated strongly with immunodetected CYP3A protein determined by Western blotting in both fetal (r = 0.89, p = 0.19) and adult specimens (r = 0.82, p < 0.01) . These findings further support CYP3A as a major catalyst of norcocaine formation in human liver microsomes. These results are important given the potential risk of toxicity to the foetus of maternal cocaine abuse during pregnancy. Although the high Km values found in adult livers reduce the importance of this enzyme pathway in cocaine detoxication, this pathway would emerge as significant in circumstances of CYP3A induction and/or drug interactions leading to potential liver toxicity in chronic cocaine abusers.     

Peppermint (Mentha piperita L.) extract effectively inhibits cytochrome P450 3A4 (CYP3A4) mRNA induction in rifampicin-treated HepG2 cells

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (Mentha piperita L.). The structure of the compound was identified as that of pheophorbide a based on spectroscopic data. It suppressed the induction of CYP3A4 mRNA expression by rifampicin in a dose-dependent manner. Quantitative high-performance liquid chromatography showed that 2 g of dry leaves 0.43 mg in one cup of peppermint tea. These findings demonstrate that pheophorbide a suppresses the induction of CYP3A4 mRNA expression in rifampicin-treated HepG2 cells. Pheophorbide is known to cause photosensitivity. However, the effective dose of pheophorbide a that had a suppressive effect was very low, indicating a high safety margin.

Abbreviations: DAD: diode array detector; DMEM: Dulbecco’s modified Eagle's medium; ELISA: enzyme-linked immunosorbent assay; HPLC: high-performance liquid chromatography; PCR: polymerase chain reaction; PXR: pregnane X receptor; CAR: constitutive androstane receptor; AHR: aryl hydrocarbon receptor; TLC: thin-layer chromatography     

Substrate specificity of native and mutated cytochrome P450 (CYP102A3) from Bacillus subtilis

Within the Bacillus subtilis genome sequencing project, two monooxygenases (CYP102A2 and CYP102A3) were discovered which revealed a similarity of 76% to the well-known cytochrome P450 BM-3 (CYP102A1) of Bacillus megaterium. All enzymes are natural fusion proteins consisting of a heme domain and a reductase domain. We here report the cloning, expression and characterization of B. subtilis enzyme CYP102A3. The substrate specificity of this enzyme is similar to that of B. megaterium CYP102A1, which hydroxylates medium-chain fatty acids in subterminal positions. A double mutant was prepared that hydroxylates a number of other substrates, which do not bear any resemblance to the natural substrate of this enzyme family.     

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.     

Synthesis and evaluation of inhibitors of cytochrome P450 3A (CYP3A) for pharmacokinetic enhancement of drugs

The HIV protease inhibitor ritonavir (RTV) is also a potent inhibitor of the metabolizing enzyme cytochrome P450 3A (CYP3A) and is clinically useful in HIV therapy in its ability to enhance human plasma levels of other HIV protease inhibitors (PIs). A novel series of CYP3A inhibitors was designed around the structural elements of RTV believed to be important to CYP3A inhibition, with general design features being the attachment of groups that mimic the P2–P3 segment of RTV to a soluble core. Several analogs were found to strongly enhance plasma levels of lopinavir (LPV), including 8, which compares favorably with RTV in the same model. Interestingly, an inverse correlation between in vitro inhibition of CYP3A and elevation of LPV was observed. The compounds described in this study may be useful for enhancing the pharmacokinetics of drugs that are metabolized by CYP3A.     

Tissue distribution of cytochrome P450 3A (CYP3A) in brushtail possums (Trichosurus vulpecula) exposed to Eucalyptus terpenes

We evaluated the distribution pattern of a specific xenobiotic metabolizing enzyme, cytochrome P450 3A (CYP3A) in the common brushtail possum (Trichosurus vulpecula). Western blot studies using CYP3A antibodies were used to compare CYP3A levels in the intestine, liver, kidney, brain, testes and adrenal gland in possums fed diets with and without a mixture of terpenes. Possums appear to produce at least 3 different CYP3A-like isoforms that are differentially expressed in various tissues. The liver and duodenum produce all three isoforms (CYP3A P1, P2, P3), the jejunum only produces CYP3A P1, the ileum, kidney, testes and adrenal only produce CYP3A P2 and the brain only produces CYP3A P3. Terpene treatment did not alter relative levels of isoforms present in any tissue type. This study is the first to identify the presence and differential expression of several CYP3A-like isoforms in a variety of tissues of a wild mammalian herbivore. Data suggest that CYP3A-like enzymes are not induced by terpenes. However, the wide distribution of CYP3A-like isoforms in a variety of tissues, suggests that these enzymes are an important mechanism for metabolism in possums and may contribute to the high tolerance possums have to a wide range of xenobiotics.