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1.
Drug Metab Pharmacokinet ; 28(6): 519-21, 2013.
Article in English | MEDLINE | ID: mdl-23648678

ABSTRACT

The diverse cultural and social habits of the Papua New Guinea (PNG) population include betel quid chewing and more recently smoking. The formation of DNA adducts from betel quid is mediated by the cytochrome P450 (CYP) enzymes, including CYP1A2. The tobacco smoke compounds can induce CYP1A2. The transcription factor AhR (aryl hydrocarbon receptor) is involved in the regulation of CYP1A2 expression. AhR activity is itself regulated by other transcription factors, including the aryl hydrocarbon receptor repressor (AhRR). The AhRR Pro185Ala (rs2292596; 565C>G) minor allele was recently associated with a lower AhR repressor activity, leading to a higher CYP1A2 inducibility. We investigated AhRR Pro185Ala SNP in the East Sepik populations in PNG and found a high frequency of 53.4% of the minor allele, significantly different from other Asian populations. We can hypothesize that a high frequency of the AhRR SNP can be a risk factor in the incidence of oral cancer and other neoplasias in PNG due to higher inducibility of CYP1A2. The potential role of AhRR pharmacogenetics in the risk of developing cancers associated with betel quid chewing and smoking should be addressed and clarified in future epidemiological studies in PNG.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Native Hawaiian or Other Pacific Islander/genetics , Repressor Proteins/genetics , Areca/adverse effects , Child , Child, Preschool , Gene Frequency , Humans , Incidence , Papua New Guinea , Pharmacogenetics , Polymorphism, Single Nucleotide , Receptors, Aryl Hydrocarbon/genetics , Smoking/adverse effects
3.
J Pharmacol Exp Ther ; 338(1): 302-9, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21493749

ABSTRACT

Cytochrome P4502C9 (CYP2C9) is an important drug-metabolizing enzyme responsible for the metabolism of approximately 16% of all clinically relevant drugs. It was shown previously that the activity of CYP2C9 in vivo is inhibited by oral contraceptives. The mechanisms of this effect have not been elucidated. We hypothesize that this may occur because of the sex steroid-dependent activation of estrogen receptor α (ERα) with further transactivation of the CYP2C9 gene. Here, we show that the CYP2C9 promoter indeed contains a functionally relevant estrogen responsive element (ERE) half-site at position -149/-145. Its ERα binding activity was tested by the luciferase gene reporter assay. Promoter constructs bearing this site were cotransfected with ERα into Huh7 hepatoma cells and treated with various ERα ligands including 4-hydroxytamoxifen (4-OHT), raloxifene (R), 17ß-estradiol (EE), and 17α-ethinylestradiol (ETE). The luciferase activity driven by the wild-type CYP2C9 promoter construct was up-regulated by 4-OHT and R and significantly or marginally suppressed by ETE and EE, respectively. An identical effect was observed in primary hepatocytes treated with these compounds. Mutations introduced into the ERE half-site abolished the observed effects in the Huh7 cells. Electrophoretic mobility-shift assay revealed sequence-specific binding of a nuclear protein to the oligonucleotide containing the ERE half-site, which was identified as ERα by antibody supershift analysis. In addition, the association of ERα with CYP2C9 promoter was strongly supported by chromatin immunoprecipitation data. Taken together, these results indicate that ERα and its ligands play an important role in the regulation of CYP2C9 expression.


Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Estrogen Receptor alpha/metabolism , Gene Expression Regulation, Enzymologic , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/physiology , Binding Sites/physiology , Cell Line, Tumor , Cells, Cultured , Cytochrome P-450 CYP2C9 , Estradiol/metabolism , Estrogen Receptor alpha/genetics , Hepatocytes/enzymology , Humans , Hydroxytestosterones/metabolism , Ligands
4.
Mol Pharmacol ; 78(5): 886-94, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20675569

ABSTRACT

Cytochrome P4502C19 (CYP2C19) is an important drug-metabolizing enzyme involved in the biotransformation of, for example, proton pump inhibitors and antidepressants. Several in vivo studies have shown that the CYP2C19 activity is inhibited by oral contraceptives, which can cause important drug interactions. The underlying molecular mechanism has been suggested to be competitive inhibition. However, the results presented here indicate that estradiol derivatives down-regulate CYP2C19 expression via estrogen receptor (ER) α, which interacts with the newly identified ER-binding half site [estrogen response element (ERE)] at the position -151/-147 in the CYP2C19 promoter. In gene reporter experiments in Huh-7 hepatoma cells, the activity of the luciferase construct carrying a 1.6-kb long CYP2C19 promoter fragment cotransfected with ERα was down-regulated upon treatment with 17ß-estradiol (EE) or 17α-ethinylestradiol (ETE) at half-maximum concentrations of 10(-7) and 10(-8) M, respectively. Mutations introduced into the ERE half site -151/-147 significantly inhibited these ligand-dependent effects. Electrophoretic mobility shift assays and quantitative chromatin immunoprecipitation experiments revealed that estrogen receptor α binds to this element. A significant suppression of CYP2C19 transcription by female sex steroids was confirmed by reverse transcription polymerase chain reaction after hormonal treatment of human hepatocytes. Inhibition experiments using a stable human embryonic kidney 293 CYP2C19 cell line revealed competitive inhibition at much higher concentrations of EE and ETE compared with those required for transcriptional inhibition. These results indicate that both EE and ETE inhibit CYP2C19 expression via an ERα-dependent regulatory pathway, thus providing a new insight into the molecular mechanism behind the inhibitory effect of oral contraceptives on CYP2C19 activity.


Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Contraceptives, Oral, Hormonal/pharmacology , Estradiol/pharmacology , Estrogen Receptor alpha/physiology , Ethinyl Estradiol/pharmacology , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Aryl Hydrocarbon Hydroxylases/genetics , Binding Sites , Cells, Cultured , Cytochrome P-450 CYP2C19 , Female , Gene Expression Regulation , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , Response Elements , Transcriptional Activation
5.
Life Sci ; 86(19-20): 699-706, 2010 May 08.
Article in English | MEDLINE | ID: mdl-20206639

ABSTRACT

AIMS: Cytochrome P4502C19 (CYP2C19) is an important enzyme involved in the metabolism of antiulcer drugs and antidepressants. However, despite the well documented drug-dependent variability of CYP2C19 expression, the mechanisms underlying the regulation of the enzyme remain unknown. In this study we investigated whether the transcription factor family GATA is involved in the regulation of CYP2C19 gene expression. MAIN METHODS: We identified a novel putative GATA binding site at position -165/-156 within the CYP2C19 gene promoter. 5'-Deletion fragments of the CYP2C19 promoter containing wild type or mutant variants of this GATA binding site were co-transfected with expression vectors encoding the transcription factors GATA-4 or GATA-2 and analyzed using dual luciferase gene reporter assays in HepG2 and Huh-7 hepatoma cells. Electrophoretic Mobility Shift Assay (EMSA) and Chromatin Immunoprecipitations (ChIP) were performed to proof a sequence-specific interaction of GATA proteins with the putative GATA binding site. KEY FINDINGS: The wild type fragments of CYP2C19 promoter were highly upregulated by GATA-4 and GATA-2 in luciferase gene reporter assay, whereas mutations introduced into the GATA binding sites caused a significant activity loss. Similar attenuation was observed upon co-transfection of GATA-4 with a known co-regulator of GATA activity, FOG-2. EMSA analysis revealed a sequence-specific binding of GATA-4 and GATA-6 to the wild type GATA binding site. In addition, the association of GATA-4 with the CYP2C19 promoter was confirmed by ChIP analysis. SIGNIFICANCE: These data indicate that GATA-4 plays an important role in the transcriptional regulation of CYP2C19 expression.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , GATA2 Transcription Factor/metabolism , GATA4 Transcription Factor/physiology , Gene Expression Regulation, Enzymologic , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Binding Sites , Cell Line, Tumor , Chromatin Immunoprecipitation , Cytochrome P-450 CYP2C19 , Electrophoretic Mobility Shift Assay , Hep G2 Cells , Humans , Luciferases/genetics , Mice , Mutation , Promoter Regions, Genetic , Transcription, Genetic
6.
Drug Metab Dispos ; 38(3): 415-21, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19995889

ABSTRACT

CYP2C9 is an important drug-metabolizing enzyme that metabolizes, e.g., warfarin, antidiabetics, and antiphlogistics. However, the endogenous regulation of this enzyme is largely unknown. In this study, we examined the role of GATA transcription factors in the gene expression of CYP2C9. We investigated four putative GATA binding sites within the first 200 base pairs of CYP2C9 promoter at the positions I: -173/-170, II: -167/-164, III: -118/-115, and IV: -106/-103. Luciferase activity driven by a wild-type CYP2C9 promoter construct was strongly up-regulated in Huh-7 cells upon cotransfection with expression plasmids for GATA-2 and GATA-4, whereas mutations introduced into GATA binding site III or I and II reduced this induction to a significant extent. Electrophoretic mobility shift assays revealed specific binding of GATA-4 and GATA-6 to the oligonucleotides containing GATA binding sites I and II. Furthermore, the association of GATA-4 with CYP2C9 promoter was confirmed by chromatin immunoprecipitation assays in HepG2 cells. Taken together, these data strongly suggest an involvement of liver-specific transcription factor GATA-4 in the transcriptional regulation of CYP2C9.


Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , GATA4 Transcription Factor/metabolism , Gene Expression Regulation, Enzymologic , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Binding Sites , Cell Line, Tumor , Chromatin Immunoprecipitation , Cytochrome P-450 CYP2C9 , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electrophoretic Mobility Shift Assay , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , GATA4 Transcription Factor/genetics , GATA6 Transcription Factor/genetics , GATA6 Transcription Factor/metabolism , Genes, Reporter , Hep G2 Cells , Hepatocytes/metabolism , Humans , Mice , Mutation , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection
7.
Ther Drug Monit ; 30(1): 10-5, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18223457

ABSTRACT

Artemisinin-based combination therapy is a main strategy for malaria control in Africa. Zanzibar introduced this new treatment policy in 2003. The authors have studied the prevalence of a number of functional single nucleotide polymorphisms (SNPs) in genes associated with the elimination of the artemisinin-based combination therapy compounds in use in Zanzibar to investigate the frequencies of subgroups potentially at higher drug exposure and therefore possible higher risk of toxicity. One hundred three unrelated children with uncomplicated malaria from the Unguja and Pemba islands of Zanzibar were enrolled. With use of polymerase chain reaction (PCR)-restriction fragment length polymorphism and real-time PCR-based allele discrimination methods, the CYP2B6 (G15631T), CYP3A4 (A-392G), CYP3A5 (A6986G, G14690A, 27131-132 insT, C3699T) SNPs and MDR1 SNPs C3435T, G2677T/A, and T-129C were analyzed. PCR product sequencing was applied to regulatory regions of MDR1, the CYP3A4 proximal promoter, and to exons 2 and 5 of PXR, a gene coding for a nuclear factor activated by artemisinin antimalarials and associated with the transcription induction of most of the studied genes. Homozygous subjects for alleles coding for low activity proteins were found at the following frequencies: 1) MDR1: 2.9%; 2) CYP2B6: 9.7%; 3) CYP3A5: 14.1%; and 4) CYP3A4: 49.5%. No functionally relevant allele was found in the analyzed regions of PXR. A new MDR1 SNP was found (T-158C), located in a putative antigen recognition element. Ten (10.1%) subjects were predicted to be low metabolizers simultaneously for CYP3A4 and CYP3A5. This fraction of the population is suggested to be under higher exposure to certain antimalarials, including lumefantrine and quinine.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antimalarials/pharmacokinetics , Cytochrome P-450 Enzyme System/genetics , Malaria/metabolism , Polymorphism, Single Nucleotide , Receptors, Steroid/genetics , ATP Binding Cassette Transporter, Subfamily B , Artemisinins/pharmacokinetics , Child, Preschool , Female , Humans , Malaria/drug therapy , Male , Polymerase Chain Reaction , Pregnane X Receptor , Tanzania
8.
Per Med ; 5(6): 627-639, 2008 Nov.
Article in English | MEDLINE | ID: mdl-29788622

ABSTRACT

Some of the largest therapeutic drug exposures in the planet involve drugs employed against malaria and TB, two main global infectious diseases. Amodiaquine for malaria and isoniazid for TB are two pivotal drugs in the management of these diseases. Both drugs have been associated with severe adverse events. Amodiaquine and isoniazid are metabolized polymorphically by CYP2C8 and N-acetyltransferase 2, respectively. The polymorphic genes coding for these enzymes presently represent the best candidates for the application of personal pharmacogenetics for these diseases. We review the main reasons for this view, while asking the pivotal question of whether it is presently possible for pharmacogenetic-based personalized medicine to be applied in the malaria and TB settings of the Developing World.

10.
Clin Chem Lab Med ; 44(2): 168-70, 2006.
Article in English | MEDLINE | ID: mdl-16475901

ABSTRACT

Cytochrome P450 2C8 (CYP2C8) is a polymorphic phase I drug-metabolising enzyme involved in the metabolism of a wide variety of xenobiotics, as well as a proposed player in the regulation of vascular tone. Polymorphisms in this gene may have an impact on the metabolism of therapeutic drugs such as paclitaxel and verapamil. In this report we have determined the frequencies of the main non-synonymous CYP2C8 alleles, 805A>T (CYP2C8*2), 416G>A/1196A>G (CYP2C8*3) and 792C>G (CYP2C8*4) in a sample representative of Portuguese Caucasians. The allelic frequencies determined were 1.2%, 19.8%, and 6.4% for CYP2C8*2, CYP2C8*3, and CYP2C*4, respectively. The observed CYP2C8*3 prevalence is significantly different from the frequencies previously reported in North European populations.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Polymorphism, Genetic , Alleles , Cytochrome P-450 CYP2C8 , Female , Gene Frequency , Humans , Male , Portugal/epidemiology , White People/genetics
11.
Clin Chem Lab Med ; 41(10): 1345-50, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14580164

ABSTRACT

Polymorphisms in cytochrome P450 CYP3A4 and multidrug resistance (MDR) 1 genes coding for the important drug-metabolising CYP3A4 and the ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) are poorly documented in the Portuguese population. In this study we have determined the frequencies of CYP3A4 and MDR1 alleles in Portuguese Caucasians. Both genes were simultaneously analysed as these genes are known to be frequently co-induced and their products to show a pronounced overlap of substrates. CYP3A4 A-392G (CYP3A4*1B), T673C (CYP3A4*2) and MDR1 T-129C, G2677T and C3435T single nucleotide polymorphisms (SNPs) were analysed in 100 individuals from the southern region of the country. We observed a frequency of 4.0% for CYP3A4*1B, not significantly different from that reported on other Caucasian European populations. CYP3A4*2 was found at an allele frequency of 4.5%, constituting the first report of the presence of this allele outside the Finnish population. Significant differences were found concerning the MDR1 C3435T SNP frequency (64.5%) compared with other European populations, while no differences were found concerning G2677T (47.5%) or T-129C (5%) SNPs. Linkage between the C3435T and G2677T SNPs was observed, although not as evidently as documented in other Caucasian populations. No preferential associations were detected between CYP3A4 and MDR1 alleles.


Subject(s)
Alleles , Cytochrome P-450 Enzyme System/genetics , Genes, MDR , Genetics, Population , Base Sequence , Cytochrome P-450 CYP3A , DNA Primers , Humans , Polymorphism, Single Nucleotide , Portugal
12.
Clin Chem Lab Med ; 41(4): 475-81, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12747589

ABSTRACT

In recent years there has been an increased interest in using biosensors for the recognition and monitoring of molecule interactions. DNA sensors and gene chips are particularly relevant for directly applying the information gathered from the genome projects. In this work electrochemical techniques are used to develop methodologies to detect DNA polymorphisms in human genes using cytochrome P450 3A4 (CYP3A4) as a model gene. CYP3A4*1B oligonucleotides were immobilized on the surface of a gold electrode and hybridized with fully complementary oligonucleotide sequences as well as with mismatched sequences corresponding to the CYP3A4*1A reference sequence. The methodology developed is based on double-stranded DNA's ability to transport charge along nucleotide stacking. The perturbation of the double helix pi-stack introduced by a mismatched nucleotide reduces electron flow and can be detected by measuring the attenuation of the charge transfer. The methodology developed could identify CYP3A4*1A homozygotes by the 5 microC charge attenuation observed when compared with DNA samples containing at least one CYP3A4*1B allele.


Subject(s)
Biosensing Techniques , Cytochrome P-450 Enzyme System/genetics , DNA/analysis , Electrochemistry/methods , Polymorphism, Single Nucleotide/genetics , Cytochrome P-450 CYP3A , DNA Primers/chemistry , DNA Probes , Electrodes , Genotype , Humans , Nucleic Acid Hybridization/methods , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length
13.
Clin Chem Lab Med ; 41(4): 606-9, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12747609

ABSTRACT

Single nucleotide polymorphisms were examined in the cytochrome 450 3A4 (CYP3A4) and N-acetyltransferase 2 (NAT2) genes, which code for major mediators of the metabolism of a wide variety of therapeutic drugs, as well as xenobiotics. We determined, in a population from Guinea-Bissau, the frequencies of CYP3A4 and NAT2 variants expected to be prevalent among Africans, due to the high frequency previously observed in African Americans. The observed frequencies were 72% for CYP3A4*1B and 19.2% for the NAT2 191 G>A variant. The high frequency found for these potentially function-altering polymorphisms suggests the possibility of impaired metabolism through CYP3A4 and NAT2 in this population. Strikingly, the frequency observed for the NAT2 191 G>A single nucleotide polymorphism (SNP), associated with the slow acetylator phenotype, was significantly higher than found in other African populations, suggesting the existence of a west to east gradient across Sub-Saharan Africa. The prevalence of these variants may be relevant with regard to therapeutic efficacy in African populations for it may potentially affect drug clearance and consequently, increase the incidence of side effects and drug-drug interactions.


Subject(s)
Arylamine N-Acetyltransferase/genetics , Black People/genetics , Cytochrome P-450 Enzyme System/genetics , Polymorphism, Genetic , Alleles , Cytochrome P-450 CYP3A , DNA/blood , DNA/genetics , DNA Primers/genetics , Female , Gene Frequency , Genotype , Heterozygote , Homozygote , Humans , Male , Polymerase Chain Reaction
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