Differential regulation of human CYP4A genes by peroxisome proliferators and dexamethasone

HepG2 cells that stably overexpress PPARalpha were used to examine the regulation of the two known human CYP4A genes by Wy14643. Specific PCR amplification across intron 5 and restriction endonuclease analysis indicated that HepG2 cells possess genes corresponding to both the CYP4A11 cDNA and a more recently characterized gene, CYP4A22, that exhibits 95% identity to CYP4A11 in the coding region. These are unlikely to represent alleles because both genes were present in DNA samples from 100 of 100 individuals. Quantitative real-time PCR determined that CYP4A22 mRNA is expressed at significantly lower levels than CYP4A11 mRNA in human liver samples. The PPARalpha agonist Wy14643 induced CYP4A11 mRNA in confluent cultures of HepG2 cells stably expressing the murine PPARalpha-E282G mutant. This mutant exhibits a significantly decreased ligand-independent trans-activation and can be activated by Wy14643 to a level similar to that of wild-type PPARalpha. Dexamethasone induced CYP4A11 mRNA in both control and PPARalpha- E282G-expressing HepG2 cells, indicating that the induction of CYP4A11 by dexamethasone is independent of elevated PPARalpha expression. Wy14643 or dexamethasone induction of CYP4A22 mRNA was not evident in either control or PPARalpha -E282G-expressing HepG2 cells. The results indicate that CYP4A11 expression can be induced by glucocorticoids and peroxisome proliferators.     

Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius

Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C_10:0 to C_18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, K_m, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined.     

Localization of CYP4B1 in the rat nasal cavity and analysis of CYPs as secreted proteins

CYP4B1 is highly expressed in rat nasal respiratory mucosa, and to a lesser extent in olfactory mucosa. Examination of high-power photomicrographs suggests that CYP4B1 may be a secreted protein, based on the fact that immunoreactivity appears to be present in the lumens of ducts of Bowman's glands (rather than intracellular localization, as we observed with an antibody recognizing CYP2F4) and in secretory granules in respiratory mucosa. Furthermore, anti-CYP4B1 immunoreactivity is present on the surface of both respiratory and olfactory mucosa. We used SignalP 3.0 analysis to ascertain the likelihood that rat CYP4B1 is a secreted protein. While this analysis does not suggest that rat CYP4B1 is a secreted protein, several other cytochrome P450 enzymes were predicted to be secreted proteins. The observation that multiple human cytochrome P450s appear to be secreted proteins helps to explain the appearance of anti-cytochrome P450 antigens in cases of human autoimmune liver diseases.     

Leukotriene B4 omega-side chain hydroxylation by CYP4F5 and CYP4F6

Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs.     

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

Differential expression of CYP102 in Bacillus megaterium by 17-beta-estradiol and 4-sec-butylphenol

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.     

Inhibition of cytochrome P450 enzymes participating in p-nitrophenol hydroxylation by drugs known as CYP2E1 inhibitors

p-Nitrophenol hydroxylation is widely used as a probe for microsomal CYP2E1. Several drugs are known as CYP2E1 inhibitors because of their capability to inhibit p-nitrophenol hydroxylation. Our results suggest further participation of CYP2A6 and CYP2C19 enzymes in p-nitrophenol hydroxylation. Moreover, CYP2A6 and CYP2C19 may be considered as the primary catalysts, whereas CYP2E1 can also contribute to the hydroxylation of p-nitrophenol. Further aim of our study was to evaluate the selectivity of p-nitrophenol hydroxylase inhibitors towards cytochrome P450 enzymes. The effects of antifungals: bifonazole, econazole, clotrimazole, ketoconazole, miconazole; CNS-active drugs: chlorpromazine, desipramine, fluphenazine, thioridazine; and the non-steroidal anti-inflammatory drug: diclofenac were investigated on the enzyme activities selective for CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. None of the drugs could be considered as a potent inhibitor of CYP2E1. Strong inhibition was observed for CYP3A4 by antifungals with IC(50) values in submicromolar range. However, ketoconazole was the only imidazole derivative that could be considered as a selective inhibitor of CYP3A4. The CNS-active drugs investigated were found to be weak inhibitors of CYP2A6, CYP2C9, CYP2C19, CYP2E1 and CYP3A4. Diclofenac efficiently inhibited CYP2C9 and to a less extent CYP3A4 enzyme.     

Evolution of the CYP2ABFGST gene cluster in rat, and a fine-scale comparison among rodent and primate species

The evolution of gene families can be best understood by studying the modern organization and functions of family members, and by comparing parallel families in different species. In this study, the CYP2ABFGST gene cluster has been characterized in rat and compared to the syntenic clusters in mouse and human, providing an interesting example of gene family evolution. In the rat, 18 loci from six subfamilies have been identified by specifically amplifying and sequencing gene fragments from cloned DNA, and have been exactly placed on chromosome 1. The overall organization of the gene cluster in rat is relatively simple, with genes from each subfamily in tandem, and is more similar to the mouse than to the human cluster. We have reconstructed the probable structure of the CYP2ABFGST cluster in the common ancestor of primates and rodents, and inferred a model of the evolution of this gene cluster in the three species. Numerous nontandem and block duplications, inversions, and translocations have occurred entirely inside the cluster, indicating that pairing between duplicate genes is keeping the rearrangements within the cluster region. The initial tandem duplication of a CYP2 gene in an early mammalian ancestor has made this region particularly subject to such localized rearrangements. Even if duplicated genes do not have a large-scale effect on chromosomal rearrangements, on a local level clustered gene families may have contributed significantly to the genomic complexity of modern mammals.     

CYP35: xenobiotically induced gene expression in the nematode Caenorhabditis elegans

Although over 80 cytochrome P450 (CYP) encoding genes have been identified in the genome of the nematode Caenorhabditis elegans very little is known about their involvement in biotransformation. This paper demonstrates a concentration-dependent relationship of C. elegans CYP35A1, A2, A5, and C1 gene expression in response to four organic xenobiotics, namely atrazine, PCB52, fluoranthene, and lansoprazole. The toxicity of these xenobiotics was determined using a reproduction assay. CYP-specific messenger RNA expression was analyzed by semi-quantitative RT-PCR resulting in a strongly increasing, concentration-dependent induction well below the EC50 for reproduction. For PCB52, approximately 0.5% of the EC50 induces a 2-fold increase of CYP35 gene expression. Using a double mutant and multiple RNAi of CYP35A/C it was possible to diminish the reproduction decline caused by PCB52 and fluoranthene.     

Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Characterisation of two novel CYP4 genes from the marine polychaete Nereis virens and their involvement in pyrene hydroxylase activity

Cytochrome P450 enzymes (CYP enzymes) catalyse the initial step in biotransformation of xenobiotics like polycyclic aromatic hydrocarbons (PAHs). The marine polychaete Nereis virens has a high capacity for biotransformation of PAHs. In the present study, the complete cDNA sequences of two novel CYP genes isolated from N. virens gut tissue are reported. One named CYP342A1, the first member of a new family and the other named CYP4BB1, the first member of a new subfamily. This is the first investigation of specific CYP enzymes from marine polychaetes in which catalytic activity has been determined. Both CYP enzymes had monooxygenase activity and catalysed hydroxylation of pyrene to 1-hydroxypyrene. Based on the present results it is likely that both CYP4BB1 and CYP342A1 are involved in xenobiotic biotransformation. Furthermore, site-directed mutagenesis of the conserved cysteine residue of the heme binding domain resulted in complete loss of monooxygenase activity of both CYP enzymes, indicating that this cysteine residue is indispensable for monooxygenase activity of invertebrate CYP enzymes, as has been well documented in vertebrates. Considering the important role of CYP enzymes in biotransformation of xenobiotics and the presence of N. virens in estuarine environments that accumulates organic xenobiotics, our results are important in understanding the molecular mechanism of biotransformation in marine polychaetes.     

Renal localization, expression, and developmental regulation of P450 4F cytochromes in three substrains of spontaneously hypertensive rats

Cytochrome P450 4F isoforms have been shown to metabolize arachidonic acid to generate 20-hydroxyeicosatetraenoic acid (20-HETE), a potent eicosanoid that modulates vascular tone and renal tubular function. 20-HETE production in the kidney is implicated in the development of essential hypertension in the spontaneously hypertensive rat (SHR). In this study, we determined CYP4F mRNA localization and distribution in rat liver and kidney by in situ hybridization and real time quantitative PCR. CYP4Fs are regionally distributed in the kidney with CYP4F1, 4F4, and 4F5 being expressed more in the renal cortex than medulla while CYP4F6 shows higher medullary expression. We investigated developmental CYP4F gene expression in three different substrains of SHR. Distinct age-dependent patterns of expression were seen for individual CYP4F isoforms in Wistar-Kyoto (WKY) and three SHR substrains (B2, C, and A3). A steady increase in CYP4F1 expression with age was seen in each of the three substrains which correlate well with increased 20-HETE levels and elevated blood pressure seen in these animals. CYP4F4 expression increased significantly at 8 weeks followed by a precipitous fall in WKY and A3 strains at 12 weeks of age. In strains B2 and C, CYP4F4 levels started declining as early as 8 weeks of age. CYP4F5 and 4F6 levels fluctuated with age in a biphasic manner with a different profile for each sub-strain. Based on the expression profile and catalytic activity, CYP4F1 seems to be the most critical 4F isoform involved in the production of 20-HETE in the SHR kidney.     

Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.