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1.
Mol Pharmacol ; 89(1): 154-64, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26467040

RESUMO

Human cytochrome P450 (CYP) 2C enzymes metabolize ∼30% of clinically prescribed drugs and various environmental chemicals. CYP2C8, an important member of this subfamily, metabolizes the anticancer drug paclitaxel, certain antidiabetic drugs, and endogenous substrates, including arachidonic acid, to physiologically active epoxyeicosatrienoic acids. Previous studies from our laboratory showed that microRNA 107 (miR107) and microRNA 103 downregulate CYP2C8 post-transcriptionally. miR107 is located in intron 5 of the pantothenate kinase 1 (PANK1) gene. p53 has been reported to coregulate the induction of PANK1 and miR107. Here, we examine the possible downregulation of CYP2C8 by drugs capable of inducing miR107. Hypolipidemic drugs, such as bezafibrate, known activators of the peroxisome proliferator-activated receptor α (PPARα), induce both the PANK1 gene and miR107 (∼2.5-fold) in primary human hepatocytes. Surprisingly, CYP2C8 mRNA and protein levels were induced by bezafibrate. CYP2C8 promoter activity was increased by ectopic expression of PPARα in HepG2 cells, with a further increase after bezafibrate (∼18-fold), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (∼10-fold) treatment, or the antidiabetic drug rosiglitazone, all known PPAR activators. Promoter sequence analyses, deletion studies, mutagenesis studies, and electrophoretic mobility shift assays identified a PPARα response element located at position -2109 base pair relative to the translation start site of CYP2C8. Chromatin immunopreciptation assay analysis confirmed recruitment of PPARα to this PPARα response element after bezafibrate treatment of human hepatocytes. Thus, we show for the first time that CYP2C8 is transcriptionally regulated by PPARα, suggesting the potential for drug-drug interactions due to upregulation of CYP2C8 by PPAR activators.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2C8/metabolismo , Sistemas de Liberação de Medicamentos/tendências , Hipolipemiantes/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , PPAR alfa/metabolismo , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos
2.
J Biol Chem ; 290(4): 2264-78, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25391650

RESUMO

The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin-modifying enzymes and RNA polymerase II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9). MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 not only with the promoter region chromatin but with HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that were reliant on MED25, indicating that MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we showed evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9.


Assuntos
Citocromo P-450 CYP2C9/metabolismo , Epigênese Genética , Fator 4 Nuclear de Hepatócito/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Complexo Mediador/metabolismo , Animais , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Formaldeído/química , Inativação Gênica , Células Hep G2 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Complexo Mediador/genética , Camundongos , Microscopia Confocal , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica
3.
Mol Pharmacol ; 86(2): 125-37, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24830941

RESUMO

Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/genética , Xenobióticos/metabolismo
4.
Pharmacogenet Genomics ; 24(8): 381-6, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24945780

RESUMO

BACKGROUND/OBJECTIVES: The polymorphic hepatic enzyme CYP2C19 catalyzes the metabolism of clinically important drugs such as clopidogrel, proton-pump inhibitors, and others and clinical pharmacogenetic testing for clopidogrel is increasingly common. The CYP2C19*10 single-nucleotide polymorphism (SNP) is located 1 bp upstream the CYP2C19*2 SNP. Despite the low frequency of the CYP2C19*10 allele, its impact on metabolism of CYP2C19 substrates and CYP2C19*2 genotyping makes it an important SNP to consider for pharmacogenetic testing of CYP2C19. However, the effect of the CYP2C19*10 allele on clopidogrel metabolism has not been explored to date. METHODS: We measured the enzymatic activity of the CYP2C19.10 protein against clopidogrel. DNA samples from two clinical studies were genotyped for CYP2C19*2 and *10 by pyrosequencing genotyping method. RESULTS: The catalytic activity of CYP2C19.10 in the biotransformation of clopidogrel and 2-oxo-clopidogrel was significantly decreased relative to the wild-type CYP2C19.1B. We also reported that the CYP2C19*10 SNP interferes with the CYP2C19*2 TaqMan genotyping assay, resulting in miscalling of CYP2C19*10/*2 as CYP2C19*2/*2. CONCLUSIONS: Our data provide evidence that CYP2C19.10 variant partially metabolizes clopidogrel and 2-oxo-clopidogrel, and the presence of CYP2C19*10 allele affects the CY2C19*2 TaqMan genotyping assay and results in misclassification of CYP2C19*10/*2 as CYP2C19*2/*2.


Assuntos
Alelos , Hidrocarboneto de Aril Hidroxilases/genética , Ticlopidina/análogos & derivados , Catálise , Cromatografia Líquida , Clopidogrel , Citocromo P-450 CYP2C19 , Genótipo , Humanos , Cinética , Mefenitoína/metabolismo , Omeprazol/metabolismo , Farmacogenética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Ticlopidina/metabolismo
5.
Drug Metab Dispos ; 41(2): 398-405, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23160820

RESUMO

The pregnane X receptor (PXR, NR1I2) plays a pivotal role in the disposition and detoxification of numerous foreign and endogenous chemicals by increasing transcription of numerous target genes, including phase I and II drug-metabolizing enzymes and transporters. In the present study, yeast two-hybrid screening identified an E3 ubiquitin ligase, RBCK1 (Ring-B-box-coiled-coil protein interacting with protein kinase C-1), as a human pregnane X receptor (hPXR)-interacting protein. Coimmunoprecipitation studies confirmed the interaction between RBCK1 and hPXR when both were ectopically expressed in AD-293 cells. Domain mapping studies showed that the interaction between RBCK1 and hPXR involves all RBCK1 domains. We further demonstrate that RBCK1 ubiquitinates hPXR, and this may target hPXR for degradation by the ubiquitin-proteasome pathway. Simultaneous ectopic overexpression of RBCK1 and PXR decreased PXR levels in AD-293 cells, and this decrease was inhibited by the proteasomal inhibitor MG-132 (carbobenzoxy-Leu-Leu-leucinal). Furthermore, overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 cells. Of importance, ectopic overexpression and silencing of endogenous RBCK1 in primary human hepatocytes resulted in a decrease and increase, respectively, in endogenous PXR protein levels and in the induction of PXR target genes by rifampicin. These results suggest that RBCK1 is important for the ubiquitination of PXR and may play a role in its proteasomal degradation.


Assuntos
Hepatócitos/enzimologia , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Imunoprecipitação , Leupeptinas/farmacologia , Receptor de Pregnano X , Cultura Primária de Células , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Interferência de RNA , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases , Ubiquitinação
6.
Nucleic Acids Res ; 39(19): 8392-403, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21764778

RESUMO

Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100 bp sequence (-1000/-901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4α (HNF4α) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4α and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4α to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.


Assuntos
Cromatina/química , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Proteínas Repressoras/metabolismo , Sulfotransferases/genética , Células Cultivadas , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Ligantes , Receptor de Pregnano X
7.
Mol Pharmacol ; 82(3): 529-40, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22723340

RESUMO

The CYP2C genes are extensively regulated at the transcriptional stage. The present study shows for the first time that CYP2Cs are also regulated post-transcriptionally by microRNAs (miRNAs). By using online search engines, we found potential miRNA response elements (MREs) in the 3'-untranslated region (3'-UTR) of the CYP2C mRNAs. Among these were a MRE for the miRNAs miR-103 and miR-107 in the 3'-UTR of human CYP2C8. CYP2C8 protein levels (measured through immunoblot analyses) did not correlate with CYP2C8 mRNA levels (measured through quantitative polymerase chain reaction analyses) in human liver samples. The translation efficiency (protein/mRNA ratio) for CYP2C8 was inversely correlated with the expression of miR-103 and miR-107. When three copies of the putative MRE from CYP2C8 were inserted downstream from a luciferase expression reporter, transfection with precursors for miR-103 or miR-107 decreased luciferase activity in primary hepatocytes, whereas transfection with antisense oligonucleotides (AsOs) for miR-103/miR-107 increased luciferase activity. As expected, there was no effect of the precursors or AsOs when three copies of the putative MRE were inserted in the reverse orientation. When precursors for miR-103/miR-107 were transfected into primary human hepatocytes, CYP2C8 protein levels were decreased, whereas AsOs increased CYP2C8 protein levels. Neither precursors nor AsOs affected CYP2C8 mRNA levels, which indicated that the effect was post-transcriptional. Putative MRE motifs were also found in the 3'-UTRs of CYP2C9 and CYP2C19, which suggested that the same miRNAs could regulate translation of other members of the CYP2C family, although to a lesser degree than CYP2C8. These results clearly show that CYP2Cs are regulated post-transcriptionally by miR-103 and miR-107.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Fígado/fisiologia , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Regiões 3' não Traduzidas , Hidrocarboneto de Aril Hidroxilases/genética , Sobrevivência Celular/genética , Células Cultivadas , Citocromo P-450 CYP2C8 , Regulação Enzimológica da Expressão Gênica , Células Hep G2 , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Humanos , Fígado/enzimologia , Fígado/metabolismo , Luciferases/metabolismo , MicroRNAs/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transfecção/métodos
8.
Br J Clin Pharmacol ; 74(3): 445-55, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22380717

RESUMO

AIMS: Cyclophosphamide, the precursor to the active 4-hydroxycyclophosphamide, is used in active glomerulonephritis despite limited pharmacokinetics data. The pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide were evaluated. The influence of laboratory and pharmacogenomic covariates on pharmacokinetics was evaluated as a secondary aim. METHODS: Glomerulonephritis patients (n = 23) participated in a pharmacokinetic evaluation. Blood was serially collected and assayed for cyclophosphamide and 4-hydroxycyclophosphamide by LC/MS methods. Kidney function, serum albumin and polymorphisms in drug metabolism or transport genes were evaluated. Analyses included non-compartmental pharmacokinetics and parametric and non-parametric statistics. RESULTS: The mean area under the plasma concentration-time curve (AUC(0,∞)) data were 110,100 ± 42,900 ng ml(-1) h and 5388 ± 2841 ng ml(-1) h for cyclophosphamide and 4-hydroxycyclophosphamide, respectively. The mean metabolic ratio was 0.06 ± 0.04. A statistically significant relationship was found between increased serum albumin and increased half-life (0.584, P = 0.007, 95% CI 0.176, 0.820) and a borderline relationship with AUC(0,∞) (0.402, P = 0.079, 95% CI -0.064, 0.724) for 4-hydroxycyclophosphamide. Covariate relationships that trended toward significance for cyclophosphamide included decreased serum albumin and increased elimination rate constant (-0.427, P = 0.061, 95% CI 0.738, 0.034), increased urinary protein excretion and increased AUC(0,∞) (-0.392, P = 0.064, 95% CI -0.699 to 0.037), decreased C(max) (0.367, P = 0.085, 95% CI -0.067, 0.684) and decreased plasma clearance (-0.392, P = 0.064, 95% CI -0.699, 0.037). CYP2B6*9 variants vs. wildtype were found to have decreased elimination rate constant (P = 0.0005, 95% CI 0.033, 0.103), increased V(d) (P = 0.0271, 95% CI -57.5, -4.2) and decreased C(max) (P = 0.0176, 95% CI 0.696, 6179) for cyclophosphamide. ABCB1 C3435T variants had a borderline decrease in cyclophosphamide elimination rate constant (P = 0.0858; 95% CI -0.005, 0.102). CONCLUSIONS: Pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in patients with lupus nephritis and small vessel vasculitis are similar. Clinical and pharmacogenetic covariates alter disposition of cyclophosphamide and 4-hydroxycyclophosphamide. Clinical findings of worsened glomerulonephritis lead to increased exposure to cyclophosphamide vs. the active 4-hydroxycyclophosphamide, which could have relevance in terms of clinical efficacy. The CYP2B6*9 and ABCB1 C3435T polymorphisms alter the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide in glomerulonephritis.


Assuntos
Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacocinética , Glomerulonefrite/fisiopatologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Área Sob a Curva , Hidrocarboneto de Aril Hidroxilases/genética , Cromatografia Líquida , Citocromo P-450 CYP2B6 , Feminino , Glomerulonefrite/etiologia , Meia-Vida , Humanos , Lúpus Eritematoso Sistêmico/complicações , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Oxirredutases N-Desmetilantes/genética , Polimorfismo de Nucleotídeo Único , Albumina Sérica/metabolismo , Vasculite/complicações
9.
Pharmacol Res ; 63(5): 405-13, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21292004

RESUMO

CYP2Cs and CYP3A4 sub families of enzymes of the Cytochrome P450 super family metabolize clinically prescribed therapeutics. Constitutive and induced expressions of these enzymes are under the control of HNF4α and rifampicin activated PXR. In the present study, we show a mechanism for ligand dependent synergistic cross talk between PXR and HNF4α. Two-hybrid screening identified NCOA6 as a HNF4α interacting protein. NCOA6 was also found to interact with PXR through the first LXXLL motif in GST pull down and mammalian two hybrid assays. NCOA6 enhances the synergistic activation of CYP2C9 and CYP3A4 promoter activity by PXR and HNF4α in the presence of rifampicin. However silencing NCOA6 abrogated the synergistic activation and induction of CYP2C9 by PXR-HNF4α but not of CYP3A4. ChIP analysis revealed that NCOA6 could bridge HNF4α and PXR binding sites of the CYP2C9 promoter. Our results indicate that NCOA6 is responsible for the synergistic activation of CYP2C9 by HNF4α and PXR and NCOA6 differentially regulates CYP2C9 and CYP3A4 gene expression though both the genes are regulated by the same nuclear receptors.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP3A/genética , Regulação Enzimológica da Expressão Gênica , Coativadores de Receptor Nuclear/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Imunoprecipitação da Cromatina , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP3A/metabolismo , Células Hep G2 , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Coativadores de Receptor Nuclear/metabolismo , Receptor de Pregnano X , Mapeamento de Interação de Proteínas , Receptores de Esteroides/metabolismo , Técnicas do Sistema de Duplo-Híbrido
10.
Drug Metab Dispos ; 38(4): 591-9, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20086032

RESUMO

CYP2C enzymes are expressed constitutively and comprise approximately 20% of the total cytochrome P450 in human liver. However, the factors influencing the transcriptional regulation of the CYP2C subfamily have only been addressed recently. In the present study, we used primary cultures of human hepatocytes to investigate the role of HNF4alpha in the pregnane X receptor (PXR)/rifampicin-mediated up-regulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4alpha sites in the proximal CYP2C8 promoter [at -181 base pairs (bp) from the translation start site] and the CYP2C9 promoter (at -211 bp). Both sites bound HNF4alpha in gel shift assays. Thus, these and recent studies identified a total of three HNF4alpha sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4alpha sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4alpha abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4alpha reduced the constitutive expression of CYP2C8 (53%), CYP2C9 (55%), and CYP2C19 (43%) mRNAs and significantly decreased the magnitude of the rifampicin-mediated induction of CYP2C8 (6.6- versus 2.7-fold), CYP2C9 (3- versus 1.5-fold), and CYP2C19 (1.8- versus 1.1-fold). These results provide clear evidence that HNF4alpha contributes to the constitutive expression of the human CYP2C genes and is also important for up-regulation by the PXR agonist rifampicin.


Assuntos
Antibióticos Antituberculose/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/fisiologia , Hepatócitos/enzimologia , Rifampina/farmacologia , Adenoviridae/genética , Adulto , Idoso , Células Cultivadas , Receptor Constitutivo de Androstano , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Hepatócitos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Pregnano X , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores de Esteroides/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/efeitos dos fármacos , Transfecção
11.
Drug Metab Dispos ; 38(7): 1177-82, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20371638

RESUMO

Mouse CYP2C55 has been characterized as an enzyme that catalyzes synthesis of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid metabolite known to have important physiological functions such as regulation of renal vascular tone and ion transport. We have now found that CYP2C55 is induced by phenobarbital (PB) and pregnenolone 16alpha-carbonitrile (PCN) in both mouse kidney and liver. The nuclear xenobiotic receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) regulate these drug inductions: CYP2C55 mRNA was increased 25-fold in PB-treated Car(+/+) but not in Car(-/-) mice and was induced in Pxr(+/+) but not Pxr(-/-) mice after PCN treatment. Cell-based promoter analysis and gel shift assays identified the DNA sequence (-1679)TGAACCCAGTTGAACT(-1664) as a DR4 motif that regulates CAR- and PXR-mediated transcription of the Cyp2c55 gene. Chronic PB treatment increased hepatic microsomal CYP2C55 protein and serum 19-HETE levels. These findings indicate that CAR and PXR may play a role in regulation of drug-induced synthesis of 19-HETE in the mouse.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Ativação Transcricional/efeitos dos fármacos , Animais , Sequência de Bases , Receptor Constitutivo de Androstano , Sistema Enzimático do Citocromo P-450/biossíntese , Família 2 do Citocromo P450 , Ácidos Hidroxieicosatetraenoicos/sangue , Rim/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Fenobarbital/farmacologia , Receptor de Pregnano X , Carbonitrila de Pregnenolona/farmacologia , Distribuição Aleatória , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/agonistas , Receptores de Esteroides/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética
12.
J Am Soc Nephrol ; 20(4): 912-21, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19225037

RESUMO

Although management of warfarin is challenging for patients with chronic kidney disease (CKD), no prospective studies have compared response to warfarin among patients with minimal, moderate, and severe CKD. This secondary analysis of a prospective cohort of 578 patients evaluated the influence of kidney function on warfarin dosage, anticoagulation control, and risk for hemorrhagic complications. We adjusted all multivariable regression and proportional hazard analyses for clinical and genetic factors. Patients with severe CKD (estimated GFR <30 ml/min per 1.73 kg/m2) required significantly lower warfarin dosages (P = 0.0002), spent less time with their international normalized ratio within the target range (P = 0.049), and were at a higher risk for overanticoagulation (international normalized ratio >4; P = 0.052), compared with patients with no, mild, or moderate CKD. Patients with severe CKD had a risk for major hemorrhage more than double that of patients with lesser degrees of renal dysfunction (hazard ratio 2.4, 95% confidence interval 1.1 to 5.3). In conclusion, patients with reduced kidney function require lower dosages of warfarin, have poorer control of anticoagulation, and are at a higher risk for major hemorrhage. These observations suggest that warfarin may need to be initiated at a lower dosage and monitored more closely in patients with moderate or severe CKD compared with the general population. Diminished renal function may have implications for a larger proportion of warfarin users than previously estimated.


Assuntos
Hemorragia/epidemiologia , Falência Renal Crônica/tratamento farmacológico , Falência Renal Crônica/fisiopatologia , Varfarina/uso terapêutico , Adulto , Idoso , Anticoagulantes/efeitos adversos , Anticoagulantes/uso terapêutico , Nitrogênio da Ureia Sanguínea , Estudos de Coortes , Creatinina/sangue , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Hemorragia/prevenção & controle , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/classificação , Testes de Função Renal , Pessoa de Meia-Idade , Seleção de Pacientes , Modelos de Riscos Proporcionais , Análise de Regressão , Fatores de Risco , Índice de Gravidade de Doença , Varfarina/efeitos adversos
13.
J Pharmacol Exp Ther ; 329(1): 192-201, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19164466

RESUMO

Retinoid-related orphan nuclear receptors (RORs) alpha and gamma (NR1F1, -3) are highly expressed in liver, adipose tissue, thymus, and brain and are involved in many physiological processes, such as circadian rhythm and immune function. Enzymes in the cytochrome P450 2C subfamily metabolize many clinically important drugs and endogenous compounds, such as the anticancer drug paclitaxel and arachidonic acid, and are highly expressed in liver. Here, we present the first evidence that RORs regulate the transcription of human CYP2C8. Overexpression of RORalpha and RORgamma in HepG2 cells significantly enhanced the activity of the CYP2C8 promoter but not that of the CYP2C9 or CYP2C19 promoters. Computer analyses, promoter deletion studies, gel shift assays, and mutational analysis identified an essential ROR-responsive element at -2045 base pairs in the CYP2C8 promoter that mediates ROR transactivation. Adenoviral overexpression of RORalpha and -gamma significantly induced endogenous CYP2C8 transcripts in both HepG2 cells and human primary hepatocytes. Knockdown of endogenous RORalpha and -gamma expression in HepG2 cells by RNA interference decreased the expression of endogenous CYP2C8 mRNA by approximately 50%. These data indicate that RORs transcriptionally up-regulate CYP2C8 in human liver and, therefore, may be important modulators of the metabolism of drugs and physiologically active endogenous compounds by this enzyme in liver and possibly extrahepatic tissues where RORs are expressed.


Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/genética , Receptores dos Hormônios Tireóideos/genética , Transativadores/genética , Adenoviridae/genética , Hidrocarboneto de Aril Hidroxilases/fisiologia , Western Blotting , Linhagem Celular , Células Cultivadas , Citocromo P-450 CYP2C8 , DNA Complementar/biossíntese , DNA Complementar/genética , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Hepatócitos/fisiologia , Humanos , Fígado/enzimologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/farmacologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Receptores do Ácido Retinoico/fisiologia , Receptores dos Hormônios Tireóideos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/fisiologia , Ativação Transcricional , Transfecção
14.
Eur J Clin Pharmacol ; 65(9): 947-53, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19669737

RESUMO

BACKGROUND: Losartan is used for anti-proteinuric as well as blood pressure effects in chronic kidney disease (CKD). It is metabolized by cytochrome P450 (CYP) 2C9 to active E-3174. Single nucleotide polymorphisms in CYP2C9 that reduce catalytic activity could reduce clinical benefits. AIM: The study aims were to determine whether CYP2C9 variant alleles (*2 and *3) altered urinary protein excretion, glomerular filtration rate, and blood pressure in Caucasian patients prescribed losartan. METHODS: Differences between baseline and 6-month follow-up outcomes were compared by CYP2C9 genotypes in 59 patients using unpaired t test or Mann-Whitney U test. RESULTS: Primary renal disease patients had a trend toward less favorable antiproteinuric response (-31.7 +/- 156 vs. -125 +/- 323%; p = 0.123) when carrying variant alleles. Patients with secondary renal diseases had less favorable diastolic blood pressure (9.8 +/- 16.0 vs. -3.2 +/- 10.6 mmHg; p = 0.043) and systolic blood pressure (16.2 +/- 27.1 vs. -5.5 +/- 17.5 mmHg; p = 0.044) with CYP2C9 variants. CONCLUSION: These preliminary results suggest a possible influence of CYP2C9 genotype on proteinuria and blood pressure in Caucasian CKD patients treated with losartan.


Assuntos
Anti-Hipertensivos/farmacologia , Hidrocarboneto de Aril Hidroxilases/genética , Pressão Sanguínea/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Falência Renal Crônica/fisiopatologia , Losartan/farmacologia , Polimorfismo de Nucleotídeo Único , Proteinúria/prevenção & controle , Adulto , Idoso , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Citocromo P-450 CYP2C9 , Feminino , Frequência do Gene , Genótipo , Humanos , Falência Renal Crônica/etiologia , Masculino , Pessoa de Meia-Idade , População Branca/genética
15.
Mol Pharmacol ; 74(3): 913-23, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18552123

RESUMO

Nuclear receptor coactivator 6 (NCOA6) also known as PRIP/RAP250/ASC-2 anchors a steady-state complex of cofactors and function as a transcriptional coactivator for certain nuclear receptors. This is the first study to identify NCOA6 as a hepatic nuclear factor 4alpha (HNF4alpha)-interacting protein. CYP2C9 is an important enzyme that metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have shown previously that constitutive androstane receptor (CAR) (a xenobiotic-sensing receptor) up-regulates the CYP2C9 promoter through binding to a distal site, whereas HNF4alpha transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrate ligand-enhanced synergistic cross-talk between CAR and HNF4alpha. We identify NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4alpha-interacting protein in this study using a yeast two-hybrid screen and GST pull-down assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4alpha in HepG2 cells. Although the interaction of NCOA6 with CAR is specifically through the first LXXLL motif of NCOA6, both LXXLL motifs are involved in its interaction with HNF4alpha. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4alpha. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4alpha and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4alpha.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Hidrocarboneto de Aril Hidroxilases/genética , Linhagem Celular Tumoral , Clonagem Molecular , Receptor Constitutivo de Androstano , Citocromo P-450 CYP2C9 , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Fator 4 Nuclear de Hepatócito/genética , Humanos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Coativadores de Receptor Nuclear , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
16.
Mol Pharmacol ; 73(3): 1029-36, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18156313

RESUMO

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1'-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.


Assuntos
Ansiolíticos/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Midazolam/metabolismo , Animais , Citocromo P-450 CYP3A/genética , DNA Complementar/biossíntese , Interpretação Estatística de Dados , Camundongos , Camundongos Knockout , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Regulação para Cima/efeitos dos fármacos
17.
J Clin Pharmacol ; 46(5): 515-26, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16638735

RESUMO

The influence of diet and genetics was investigated in a healthy white person who had distinctly low methylprednisolone clearance. Pharmacokinetic and pharmacodynamic parameter values were similar on 2 occasions during the consumption of a low-carbohydrate diet and a Weight Watchers diet, indicating that the decreased clearance was unlikely attributable to a change in diet composition. Although the subject was found to be homozygous for CYP3A5*3, genetic findings were not significant for a number of other CYP3A4 and CYP3A5 allelic variants. Because of the high prevalence of CYP3A5*3/*3 in whites and because 5 of 7 white control subjects are also homozygous for CYP3A5*3, this genotype cannot fully explain the reduced metabolism of the drug. Other genetic or contributing factors might have been involved. New polymerase chain reaction-based genotyping methods for functionally defective CYP3A5*6, *8, *9, and *10 alleles were developed in this study. These assays will be useful for CYP3A5 genotype analysis in future clinical studies.


Assuntos
Metilprednisolona/farmacocinética , Adulto , Alelos , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Dieta , Histamina/sangue , Humanos , Hidrocortisona/sangue , Masculino , Taxa de Depuração Metabólica , Metilprednisolona/sangue , Metilprednisolona/farmacologia , Polimorfismo de Nucleotídeo Único
18.
Pharmacogenomics ; 6(4): 357-71, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16004554

RESUMO

Among the four cytochrome P450 (CYP)3A genes, CYP3A4 and CYP3A5 are the most abundantly expressed in the human liver. Eighty single nucleotide polymorphisms (SNPs) of CYP3A4/5 have been reported to the Human P450 Allele Nomenclature Committee. CYP3A4 alleles with minimal function compared with wild type include the CYP3A4*6 and CYP3A4*17. Alleles with moderately decreased or altered activity include: CYP3A4*2, *8, *11, *12, *13, *16, and *18. CYP3A5 alleles with minimal function include the splice variants CYP3A5*3, *5, *6 and CYP3A5* 10, as well as the null allele CYP3A5*7. Alleles with moderately decreased catalytic activity include CYP3A5*8 and CYP3A5*9. This report reviews the current progress in the functional characterization of CYP3A4 and CYP3A5 SNPs and provides genotyping tests for possible defective variants. A combination of genotyping tests for defective CYP3A4/CYP3A5 haplotypes will be necessary to understand the variations in the metabolism and clinical toxicity of a wide variety of clinical drugs, since these two CYP proteins have overlapping substrate specificities.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacogenética/métodos , Citocromo P-450 CYP3A , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único
19.
Pharmacogenetics ; 12(3): 251-63, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11927841

RESUMO

The discovery of six distinct polymorphisms in the genetic sequence encoding for the cytochrome P450 2C9 (CYP2C9) protein has stimulated numerous investigations in an attempt to characterize their population distribution and metabolic activity. Since the CYP2C9*1, *2 and *3 alleles were discovered first, they have undergone more thorough investigation than the recently identified *4, *5 and *6 alleles. Population distribution data suggest that the variant *2 and *3 alleles are present in approximately 35% of Caucasian individuals; however, these alleles are significantly less prevalent in African-American and Asian populations. In-vitro data have consistently demonstrated that the CYP2C9*2 and *3 alleles are associated with significant reductions in intrinsic clearance of a variety of 2C9 substrates compared with CYP2C9*1; however, the degree of these reductions appear to be highly substrate-dependent. In addition, multiple in-vivo investigations and clinical case reports have associated genotypes expressing the CYP2C9*2 and *3 alleles with significant reductions in both the metabolism and daily dose requirements of selected CYP2C9 substrates. Individuals expressing these variant genotypes also appear to be significantly more susceptible to adverse events with the narrow therapeutic index agents warfarin and phenytoin, particularly during the initiation of therapy. These findings have subsequently raised numerous questions regarding the potential clinical utility of genotyping for CYP2C9 prior to initiation of therapy with these agents. However, further clinical investigations evaluating the metabolic consequences in individuals expressing the CYP2C9*2, *3, *4, *5, or *6 alleles are required before large-scale clinical genotyping can be recommended.


Assuntos
Hidrocarboneto de Aril Hidroxilases , População Negra/genética , Sistema Enzimático do Citocromo P-450/genética , Polimorfismo Genético/genética , Esteroide 16-alfa-Hidroxilase , Esteroide Hidroxilases/genética , População Branca/genética , Alelos , Anticoagulantes/farmacologia , Anticonvulsivantes/farmacologia , Ásia/etnologia , Citocromo P-450 CYP2C9 , Genética Populacional , Genótipo , Humanos , Técnicas In Vitro , Fenitoína/farmacologia , Varfarina/farmacologia
20.
Pharmacogenetics ; 12(3): 221-5, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11927837

RESUMO

CYP2C19 is a polymorphically expressed cytochrome P450 responsible for the metabolism of several clinically used drugs, including some barbiturates, diazepam, proguanil, propranolol and several proton pump inhibitors. Genetic polymorphism of this enzyme shows marked interracial differences, with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 11-23% of Oriental populations. In the present study, CYP2C19 phenotype and genotype were investigated in 107 North-eastern Thai subjects using the omeprazole hydroxylation index (HI) and polymerase chain reaction-restriction fragment length polymorphism technique, respectively. It was found that the distribution of HI in these subjects was bimodal. Seven subjects [6.54%, 95% confidence (CI) 1.86-11.22%] were identified as PM, with an HI > 7. Analysis of CYP2C19 genotypes in these 107 Thai subjects revealed that the allele frequencies for CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 0.71 (95% CI 0.65-0.77), 0.27 (95% CI 0.21-0.33) and 0.02 (95% CI 0.01-0.05), respectively. The PM phenotype and the frequencies of CYP2C19 defective alleles in Thais, particularly CYP2C19*3, were lower than those observed in other Oriental populations. It is noteworthy that there was a case of nonaccordance between phenotype and genotype in one of the PMs. Whether this PM represents a novel defective allele requires further investigation.


Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Oxigenases de Função Mista/genética , Polimorfismo Genético/genética , Adulto , Alelos , Citocromo P-450 CYP2C19 , Feminino , Genética Populacional , Genótipo , Humanos , Masculino , Omeprazol/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Tailândia/etnologia
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