Expression Of Cytochrome P450 2C9 Research Articles

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.

CYP2C9 inhibits the invasion and migration of esophageal squamous cell carcinoma via downregulation of HDAC.

Cytochrome P450 2C9 (CYP2C9) is involved in the metabolism of cancer drugs and exogenous carcinogens. In our study, CYP2C9 was downregulated in multiple cohorts of human esophageal squamous cell carcinoma (ESCC). Until now, its role and epigenetic regulation of CYP2C9 repression in ESCC remain poorly understood. CYP2C9 repression in collected ESCC patient tumor tissues was demonstrated by RT-qPCR and Western blot. The histone acetylation level was carried out by the treatment of histone deacetylase inhibitor TSA and RNA interference. Epigenetic analysis revealed that the increased expression of CYP2C9 in KYSE-150 and TE1 cells was characterized by inhibition of HDAC8 and HDAC1, respectively. TSA decreased the levels of HDAC occupancy around CYP2C9 promoter region greatly. Overexpression of CYP2C9 reduced the invasion and migration of ESCC cells.

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%–17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS‐7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus‐based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild‐type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.

Expression of cytochrome P450 2C9 (CYP2C9) in Escherichia coli and its functional characterization

This study aimed to express the major human hepatic drug metabolizing cytochrome P450 (CYP), CYP2C9, together with NADPH cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate its catalytic activities. Co-expression of CYP2C9 and OxR was achieved by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates valsartan and tolbutamide. Results from immunoblotting demonstrated the presence of CYP2C9 protein in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to previously published data. Kinetic parameters, Km and Vmax values determined from the valsartan and tolbutamide hydroxylase assays, were also concordant with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP2C9 suitable for drug metabolism and interaction studies.

The label-free detection and distinction of CYP2C9-expressing and non-expressing cells by surface-enhanced Raman scattering substrates based on bimetallic AuNPs-AgNWs.

Cytochrome P450 2C9 (CYP2C9) is capable of catalyzing the biotransformation of endogenous compounds in cells, indicating that this enzyme could change the intracellular environment and is related to the pathogenesis of diseases. Currently, it is still a challenge to study the differences in cellular components between CYP2C9-expressing and non-expressing cells. In this study, employing a Au nanoparticles–Ag nanowires (AuNPs–AgNWs) decorated silicon wafer as a novel non-destructive and label-free tool, we applied surface-enhanced Raman scattering (SERS) spectroscopy to detect and distinguish the cellular composition of CYP2C9-expressing cells (293T-Mig-2C9) and non-expressing cells (293T-Mig-R1). AgNWs with high surface roughness were formed by modification of AuNPs onto their surface by electrostatic interactions, which enabled them to exhibit greatly enhanced SERS ability. Then, they were employed to fabricate SERS substrates via an electrostatically assisted 3-aminopropyltriethoxysilane (APTES)-functionalized surface-assembly method. The SERS substrates exhibited high sensitivity with a detection limit of 1 × 10−9 M for 4-mercaptobenzoic acid (4-MBA). Meanwhile, the SERS substrates exhibited good uniformity and reproducibility. The cytotoxicity assay demonstrated that the SERS substrates displayed good biocompatibility with 293T cells. Before SERS measurements, CYP2C9 constantly expressed cells (293T-Mig-2C9 cells) and control cells (293T-Mig-R1 cells) were constructed. The expression of CYP2C9 and the catalytic activity in the cells were checked. Using the AuNPs–AgNWs substrates as a high-performance in vitro sensing platform allowed us to obtain fingerprint spectra of 293T-Mig-R1 and 293T-Mig-2C9 cells. The difference spectra between the two cell lines were studied to interpret the spectral differences and gain insight into the biochemical variations. Finally, principal component analysis (PCA) score plots of the SERS spectra were also used to better view the differences between the two cell lines. SERS detection based on the AuNPs–AgNWs substrates provides a sensitive, non-destructive and label-free method for differentiation between 293T-Mig-R1 and 293T-Mig-2C9 cells.

MicroRNA hsa-miR-29a-3p modulates CYP2C19 in human liver cells

Cytochrome P450 2C19 (CYP2C19) is involved in the metabolism of many drugs. Extensive studies have demonstrated that genetic variants and endogenous and environmental factors play important roles in the expression of CYP2C19. However, the role of microRNAs (miRNAs) in controlling CYP2C19 expression has not been investigated completely. In the present study, we performed in silico analysis to rank putative miRNA/CYP2C19 hybrids with regards to the predicted stabilities of their duplexes and then we applied a series of biochemical and molecular assays to elucidate the underlying functional mechanisms for the regulation of CYP2C19 by miRNAs. In silico analysis indicated that hsa-miR-23a-3p and hsa-miR-29a-3p target the coding region of CYP2C19 with hybrid stabilities of –27.5kcal/mol and –23.3kcal/mol, respectively. RNA electrophoresis mobility shift assays showed that both hsa-miR-23a-3p and hsa-miR-29a-3p miRNAs were able to bind directly to their cognate targets in the CYP2C19 transcript. Further, a significant inverse correlation was found between chemically-induced up-regulation of hsa-miR-29a-3p and CYP2C19 expression in HepaRG cells. In addition, inverse correlations were also observed in human liver tissue samples between the level of CYP2C19 mRNA expression and both hsa-miR-23a-3p and hsa-miR-29a-3p levels. All these results demonstrated the suppressing role of hsa-miR-29a-3p on CYP2C19 expression.

Inhibition of cytochrome P450 2C9 expression and activity in vitro by allyl isothiocyanate.

The growing interest in the use of natural herbal products and dietary supplements to treat and prevent diseases raises the question of medicinal drug safety. Allyl isothiocyanate, a hydrolysis product of a glucosinolate, sinigrin, has multiple beneficial properties, and based on this fact, allyl isothiocyanate-containing dietary supplements have been developed. To date, no studies of the effects of this compound on the cytochrome P450 2C9 have been reported. In this study, we found that allyl isothiocyanate reduced catalytic activity, messenger ribonucleic acid, and protein expression of cytochrome P450 2C9 in HepaRG cells. An investigation of the transcriptional activity of the pregnane X receptor and the constitutive androstane receptor revealed that allyl isothiocyanate disrupted the transcriptional coregulation effects of the pregnane X receptor/constitutive androstane receptor with several important coregulators and interfered with the assembly of transcriptional complexes of the cytochrome P450 2C9 pregnane X receptor/constitutive androstane receptor-response element. The decrease of cytochrome P450 2C9 expression and activity mediated by allyl isothiocyanate suggested that this agent could alter the metabolism of drugs metabolized by cytochrome P450 2C9. This may cause food/dietary supplement-drug interactions or alter the therapeutic effects, and even the toxicity of drugs coadministered with allyl isothiocyanate. Since the consumption of allyl isothiocyanate-containing food/dietary supplements continues to increase, it is important to predict and ultimately avoid interactions with concomitant drugs. It is required that these possible pharmacokinetic interactions be characterized and the recommendations available to patients and healthcare professionals be improved.

Renal drug metabolism in humans: the potential for drug–endobiotic interactions involving cytochrome P450 (CYP) and UDP‐glucuronosyltransferase (UGT)

Although knowledge of human renal cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and their role in xenobiotic and endobiotic metabolism is limited compared with hepatic drug and chemical metabolism, accumulating evidence indicates that human kidney has significant metabolic capacity. Of the drug metabolizing P450s in families 1 to 3, there is definitive evidence for only CYP 2B6 and 3A5 expression in human kidney. CYP 1A1, 1A2, 1B1, 2A6, 2C19, 2D6 and 2E1 are not expressed in human kidney, while data for CYP 2C8, 2C9 and 3A4 expression are equivocal. It is further known that several P450 enzymes involved in the metabolism of arachidonic acid and eicosanoids are expressed in human kidney, CYP 4A11, 4F2, 4F8, 4F11 and 4F12. With the current limited evidence of drug substrates for human renal P450s drug-endobiotic interactions arising from inhibition of renal P450s, particularly effects on arachidonic acid metabolism, appear unlikely. With respect to the UGTs, 1A5, 1A6, 1A7, 1A9, 2B4, 2B7 and 2B17 are expressed in human kidney, whereas UGT 1A1, 1A3, 1A4, 1A8, 1A10, 2B10, 2B11 and 2B15 are not. The most abundantly expressed renal UGTs are 1A9 and 2B7, which play a significant role in the glucuronidation of drugs, arachidonic acid, prostaglandins, leukotrienes and P450 derived arachidonic acid metabolites. Modulation by drug substrates (e.g. NSAIDs) of the intrarenal activity of UGT1A9 and UGT2B7 has the potential to perturb the metabolism of renal mediators including aldosterone, prostaglandins and 20-hydroxyeicosatetraenoic acid, thus disrupting renal homeostasis.

Decreased hippocampal volume and increased anxiety in a transgenic mouse model expressing the human CYP2C19 gene

Selective serotonin reuptake inhibitors, tricyclic antidepressants, various psychoactive drugs, as well as endogenous steroids and cannabinoid-like compounds are metabolized by the polymorphic cytochrome P450 2C19 (CYP2C19). Absence of this enzyme has been recently shown to associate with lower levels of depressive symptoms in human subjects. To investigate endogenous functions of CYP2C19 and its potential role in brain function, we have used a transgenic mouse model carrying the human CYP2C19 gene. Here, CYP2C19 was expressed in the developing fetal, but not adult brain and was associated with altered fetal brain morphology, where mice homozygous for the CYP2C19 transgenic insert had severely underdeveloped hippocampus and complete callosal agenesis and high neonatal lethality. CYP2C19 expression was also found in human fetal brain. In adult hemizygous mice we observed besides decreased hippocampal volume, an altered neuronal composition in the hippocampal dentate gyrus. Reduced hippocampal volumes have been reported in several psychiatric disorders, supporting the relevance of this model. Here we found that adult hemizygous CYP2C19 transgenic mice demonstrate behavior indicative of increased stress and anxiety based on four different tests. We hypothesize that expression of the CYP2C19 enzyme prenatally may affect brain development by metabolizing endogenous compounds influencing this development. Furthermore, CYP2C19 polymorphism may have a role in interindividual susceptibility for psychiatric disorders.

CYP2C9 promoter region single-nucleotide polymorphisms linked to the R150H polymorphism are functional suggesting their role in CYP2C9*8-mediated effects

Cytochrome P450 2C9 (CYP2C9) c.449G>A (*8) is common in African Americans and is associated with decreased warfarin clearance. We examined the effect of promoter region variants inherited with 449G>A on warfarin clearance, dose requirements, and CYP2C9 expression. In an African American cohort, 449G>A was in linkage disequilibrium with c.-1766T>C (r(2) = 0.89) and c.-1188T>C (D' = 1). The combination of the -1766C and 449A alleles with the -1188CC genotype was associated with lower S-warfarin clearance (0.86 ± 0.22 vs. 1.66 ± 0.75 ml/min/m(2); n = 48; P < 0.01) and dose requirements [33 (25-49) vs. 43 (35-56) mg/week; n = 243; P = 0.03] compared with other genotypes. In liver tissue, alleles with the -1766C/-1188C/449A haplotype showed two-fold decreased mRNA expression compared with reference alleles. In a promoter reporter assay, the -1766C/-1188C haplotype decreased CYP2C9 promoter activity. These data suggest that promoter region polymorphisms inherited with 449G>A decrease CYP2C9 expression and contribute to CYP2C9*8 effects on warfarin clearance and dose requirements.

The Ligands of Estrogen Receptor α Regulate Cytochrome P4502C9 (CYP2C9) Expression

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), 17β-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.

Esophageal cancer proliferation is mediated by cytochrome P450 2C9 (CYP2C9)

Cytochrome P450 epoxygenases (CYP450) have been recently shown to promote malignant progression. Here we investigated the mRNA and protein expression and potential clinical relevance of CYP2C9 in esophageal cancer. Highest expression was detected in esophageal adenocarcinoma (EAC; n = 78) and adjacent esophageal mucosa (NEM; n = 79). Levels of CYP2C9 in EAC and NEM were significantly higher compared to esophageal squamous cell carcinoma (ESCC; n = 105). Early tumor stages and well-differentiated tumors showed a significantly higher CYP2C9 expression compared to progressed tumors. Moreover, CYP2C9 expression was correlated to high Ki-67 labeling indices in EAC and Ki-67 positive tumor cells in EAC and ESCC. Selective inhibition of CYP2C9 decreased tumor cell proliferation (KYSE30, PT1590 and OE19) in vitro, which was abolished by 11,12-epoxyeicosatrienoic acid (11,12-EET). Cell-cycle analysis using FACS revealed that inhibition of CYP2C9 leads to a G0/G1 phase cell-cycle arrest. CYP2C9 seems to be relevant for early esophageal cancer development by promoting tumor cell proliferation. Pharmacological inhibition of CYP2C9 might contribute to a more efficient therapy in CYP2C9 highly expressing esophageal cancers.

Regulation of CYP2C19 Expression by Estrogen Receptor α: Implications for Estrogen-Dependent Inhibition of Drug Metabolism

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 17β-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.

The transcription factor GATA-4 regulates cytochrome P4502C19 gene expression

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.

Involvement of CYP 2C9 in Mediating the Proinflammatory Effects of Linoleic Acid in Vascular Endothelial Cells

Objective: Polyunsaturated fatty acids such as linoleic acid are well known dietary lipids that may be atherogenic by activating vascular endothelial cells. In the liver, fatty acids can be metabolized by cytochrome P450 (CYP) enzymes, but little is known about the role of these enzymes in the vascular endothelium. CYP 2C9 is involved in linoleic acid epoxygenation, and the major product of this reaction is leukotoxin (LTX). We investigated the role of CYP-mediated mechanisms of linoleic acid metabolism in endothelial cell activation by examining the effects of linoleic acid or its oxidized metabolites such as LTX and leukotoxin diol (LTD).Methods: The effect of linoleic acid on CYP 2C9 gene expression was studied by RT-PCR. Oxidative stress was monitored by measuring DCF fluorescence and intracellular glutathione levels, and electrophoretic mobility shift assay was carried out to study the activation of oxidative stress sensitive transcription factors. Analysis of oxidized lipids was carried out by liquid chromatography/mass spectrometry.Results: Linoleic acid treatment for six hours increased the expression of CYP 2C9 in endothelial cells. Linoleic acid-mediated increase in oxidative stress and activation of AP-1 were blocked by sulfaphenazole, a specific inhibitor of CYP 2C9. The linoleic acid metabolites LTX and LTD increased oxidative stress and activation of transcription factors only at high concentrations.Conclusion: Our data show that CYP 2C9 plays a key role in linoleic acid-induced oxidative stress and subsequent proinflammatory events in vascular endothelial cells by possibly causing superoxide generation through uncoupling processes.

Transcriptional Regulation of CYP2C9 Gene: ROLE OF GLUCOCORTICOID RECEPTOR AND CONSTITUTIVE ANDROSTANE RECEPTOR

Although cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9 is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9. Deletional analysis of CYP2C9 regulatory region (+21 to -2088) in the presence of various hormone nuclear receptors suggested the presence of two functional response elements, a glucocorticoid receptor-responsive element (-1648/-1684) and a constitutive androstane receptor-responsive element (CAR, -1783/-1856). Each of these were characterized by co-transfection experiments, directed mutagenesis, gel shift assays, and response to specific antagonists RU486 and androstanol. By these experiments we located a glucocorticoid-responsive element imperfect palindrome at -1662/-1676, and a DR4 motif at -1803/-1818 recognized and transactivated by human glucocorticoid receptor and by hCAR and pregnane X receptor, respectively. Identification of these functional elements provides rational mechanistic basis for CYP2C9 induction by dexamethasone (submicromolar concentrations), and by phenobarbital and rifampicin, respectively. CYP2C9 appears therefore to be a primary glucocorticoid-responsive gene, which in addition, may be induced by xenobiotics through CAR/pregnane X receptor activation.

Genetic association between sensitivity to warfarin and expression of CYP2C9*3.

Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.