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ABSTRACT: PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, while its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706 could be activated by CYP2W1 into potent antitumor agents. Experimental design: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on SCID mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism demonstrated in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that utilizes a locoregional activation of systemically inactive prodrug by the tumor specific activator enzyme CYP2W1.
Clinical Cancer Research 04/2013; · 7.74 Impact Factor
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ABSTRACT: Women recover faster from propofol anaesthesia and have been described to have a higher incidence of awareness during surgery, compared to men; an effect that may be inherent in sex differences in propofol metabolism.
Basic & Clinical Pharmacology & Toxicology 03/2013; · 2.18 Impact Factor
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Reha Yildirimman,
Gabriella Brolén,
Mireia Vilardell,
Gustav Eriksson,
Jane Synnergren,
Hans Gmuender,
Atanas Kamburov, Magnus Ingelman-Sundberg,
José Castell,
Agustin Lahoz,
Jos Kleinjans,
Joost van Delft,
Petter Björquist,
Ralf Herwig
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Reha Yildirimman,
Gabriella Brolén,
Mireia Vilardell,
Gustav Eriksson,
Jane Synnergren,
Hans Gmuender,
Atanas Kamburov, Magnus Ingelman-Sundberg,
José Castell,
Agustin Lahoz,
Jos Kleinjans,
Joost van Delft,
Petter Björquist,
Ralf Herwig
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ABSTRACT: Drug induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities (2) expression of liver-specific proteins and (3) responses to 3 drugs (acetaminophen, troglitazone and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1 and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose as well as CYP2E1 activity were significantly higher in the 3D versus the 2D cultures. Toxicity studies show the organotypic cultures are more sensitive to acetaminophen and rosiglitazone induced toxicity, but less sensitive to troglitazone induced toxicity than the 2D cultures. Furthermore, the EC(50) value (2.7 mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG cultures is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug induced hepatotoxicity.
Toxicological Sciences 02/2013; · 4.65 Impact Factor
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ABSTRACT: DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina's HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.
Nucleic Acids Research 01/2013; · 8.03 Impact Factor
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Fred Nagelkerke,
Greetje Elaut,
Andre Guillouzo,
Olavi Pelkonen,
Thomas Hartung,
Michael L Cunningham,
Jessica Ponti,
Sandra Coecke,
Pilar Prieto,
Hans Ahr, [......],
Brighitta Eletti,
Annarita Meneguz,
Alessandra Gennari,
Andreas Freidig,
Robert Combes,
Mario Monshouwer,
Jean-François Ghersi-Egea,
Raffaella Corvi,
Siegfried Morath,
Charles L Crespi
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ABSTRACT: Interindividual variability in xenobiotic metabolism and drug response is extensive and genetic factors play an important role in this variation. A majority of clinically used drugs are substrates for the cytochrome P450 (CYP) enzyme system and interindividual variability in expression and function of these enzymes is a major factor for explaining individual susceptibility for adverse drug reactions and drug response. Because of the existence of many polymorphic CYP genes, for many of which the number of allelic variants is continually increasing, a universal and official nomenclature system is important. Since 1999, all functionally relevant polymorphic CYP alleles are named and published on the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Web site (http://www.cypalleles.ki.se). Currently, the database covers nomenclature of more than 660 alleles in a total of 30 genes that includes 29 CYPs as well as the cytochrome P450 oxidoreductase (POR) gene. On the CYP-allele Web site, each gene has its own Webpage, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications identifying or characterizing the alleles. CYP2D6, CYP2C9, CYP2C19, and CYP3A4 are the most important CYPs in terms of drug metabolism, which is also reflected in their corresponding highest number of Webpage hits at the CYP-allele Web site.The main advantage of the CYP-allele database is that it offers a rapid online publication of CYP-alleles and their effects and provides an overview of peer-reviewed data to the scientific community. Here, we provide an update of the CYP-allele database and the associated nomenclature.
Methods in molecular biology (Clifton, N.J.) 01/2013; 987:251-9.
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ABSTRACT: We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein and activity levels. Here, the mechanisms behind were investigated and a transcriptome analysis revealed significant up-regulation of liver specific functions, whereas pathways related to proliferation and cell cycle were down-regulated in the confluent cells. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving PXR. PXR expression was increased and PXR protein accumulated in the nuclei during confluent growth. The PXR ligand rifampicin further increased the expression of CYP3A4 and siRNA mediated knock-down of PXR in confluent cells resulted in decreased CYP3A4 expression. CDK2, a known modulator of the cell cycle and a negative regulator of PXR, was higher expressed in proliferating control cells. Trypsinization of the confluent cells and replating them subconfluent resulted in a decrease in CYP3A4 and PXR expression back to levels observed in subconfluent control cells whereas the CDK2 levels increased. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. A phosphorylation-deficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. In conclusion, the data strongly suggest that the increased CYP3A4 expression in confluent Huh7 cells is caused by the endogenous induction of PXR as a result of cell-cell contact inhibited proliferation and subsequent decreased CDK2 activities indicating an endogenous, non-ligand dependent regulation of PXR and CYP3A4 possibly of physiological and pharmacological significance.
Molecular pharmacology 12/2012; · 4.53 Impact Factor
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ABSTRACT: Research in pharmacogenomics has been intensive at Karolinska Institutet (KI) for approximately 25 years. Initial initiatives were focused on the identification and characterization of novel CYP2D6 alleles causing ultrarapid or defective drug metabolism. Such discoveries were possible owing to the early implementation of therapeutic drug monitoring and the access to individuals phenotyped with respect to drug metabolism. The translational work at KI has been of utmost importance for successful research, including functional characterization and clinical validation of allelic variants in drug metabolism, as well as discoveries of novel polymorphisms, recent examples being the CYP2C19 and UGT2B17 genes. The clinical pharmacology laboratory at KI campus Huddinge is one of the leading sites for therapeutic drug monitoring in northern Europe and obtains an increasing number of clinical requests, also important for pharmacogenetic research. Furthermore, the recently opened Center for Hematology and Regenerative Medicine, with a clear translational emphasis, offers an opportunity for studying drug metabolism and toxicity in vitro by use of human hepatocytes.
Pharmacogenomics 12/2012; 13(16):1887-1891. · 3.97 Impact Factor
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ABSTRACT: Drug induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC towards the hepatic lineage, mostly using 2D cultures. Although the results are encouraging these cells lack important functionality. Here we investigate if hepatic differentiation of hESC can be improved by using a 3D bioreactor system. Human ESCs were differentiated towards the hepatic lineage using the same cells in either the 3D or the 2D system. A global transcriptional analysis identified important differences between the two differentiation regimes and we identified 10 pathways, highly related to liver functions, which were significantly up-regulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems.
Stem cells and development 09/2012; · 4.15 Impact Factor
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ABSTRACT: The pharmacokinetics of a drug are subject to large interindividual variability, which can result in lack of response or adverse drug reactions. In addition to genetic polymorphisms and drug interactions, key genes involved in the metabolism and transport of drugs are demonstrated to have epigenetic influences that can potentially affect interindividual variability in drug response. Emerging studies have focused on the importance of DNA methylation for ADME gene expression and for drug action and resistance, particularly in cancer. However, the epigenetic and ncRNA-dependent regulation of these genes, as well as the pharmacological consequences, is in need of greater attention. In the current review we provide an update of epigenetic and ncRNA-dependent regulation of ADME genes.
Pharmacogenomics 09/2012; 13(12):1373-85. · 3.97 Impact Factor
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ABSTRACT: Aim: The enzyme Cytochrome P450 2W1 (CYP2W1) is found in fetal colon tissue and is also detected in colorectal cancer but not in non-transformed tissue. In a pilot study, we reported that the immunohistochemically-detected expression of CYP2W1 might be of prognostic value since high expression of CYP2W1 was indicative of a worse prognosis. The aim of this study was to validate the pilot study's results using a larger, independent group of patients with colon cancer.
Immunohistochemical detection of CYP2W1 in 235 malignant colon tumors of stage II and III, was carried out using a polyclonal antibody. Grading of staining was carried out by two independent readers. The highest grade that involved more than 5% of the tumor area on each slide was used for the classification of CYP2W1 expression.
CYP2W1 was expressed at high levels in 30% of the tumors. In the entire colon cancer group it was an independent prognostic factor in multivariate analysis (p=0.04), where high expression (grade 3) correlated with worse outcome. CYP2W1 expression was an independent prognostic factor in the subgroup of patients with colon cancer stage III (p=0.003), but not for those with stage II. In 107 cases, two slices from different areas of the same tumor were available, and no significant difference in CYP2W1 expression between the slices was observed (r=0.53, p<0.001).
The results of the current study were in agreement with those of the previous pilot study and show that higher expression of CYP2W1 seems to be of prognostic value in colon cancer. Furthermore, we found equal expression in slices from two different areas of the same tumor. Since the CYP2W1 enzyme has been shown to catalytically activate compounds to cytotoxic products, the enzyme might be used as a novel drug target for the treatment of colon cancer.
Anticancer research 09/2012; 32(9):3869-74. · 1.73 Impact Factor
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ABSTRACT: The CYP3A4 gene, encoding the major drug metabolizing enzyme in humans, exhibits a high interindividual variation in hepatic expression that can lead to interindividual differences in drug metabolism and associated adverse drug effects. Much of the interindividual variability in CYP3A4 remains unexplained. In the present study we investigated the role of DNA methylation in influencing the interindividual CYP3A4 expression. Individual CpG methylation within the ∼12 kb CYP3A4 regulatory region was investigated in 72 adult as well as in 7 fetal human livers using bisulfite sequencing. We identified highly variable CpG methylation sites in adult livers, which correspond to important CYP3A4 transcription factor binding sites including the proximal promoter, XREM and CLEM4 as well as in separate C/EBP and HNF4α binding regions. CpG hypermethylation within these regulatory regions was observed in fetal livers when compared to adult livers. This data suggests that dynamic DNA methylation elements are likely associated with key regulatory CYP3A4 promoter regions and may potentially contribute to the commonly observed interindividual expression of CYP3A4 as well as the hepatic developmental shift in its expression. The findings provide novel insight to CYP3A4 regulation with possible implications for understanding interindividual differences in drug response.
Biochimie 07/2012; 94(11):2338-44. · 3.02 Impact Factor
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ABSTRACT: Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase).
Journal of Biological Chemistry 12/2011; 287(9):6307-17. · 4.77 Impact Factor
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ABSTRACT: To develop a limited sampling strategy (LSS) to predict area under the concentration-time curve (AUC) ratios of omeprazole (AUC(OPZ)) to its metabolites 5-hydroxyomeprazole (AUC(5OH)) and omeprazole sulfone (AUC(SUL)) as phenotyping parameters for cytochrome P450 (CYP) 2C19 and 3A.
Data were obtained from 37 (4 women) Caucasian, Chinese, and Korean healthy adults from three published studies. The AUC(OPZ), AUC(5OH), and AUC(SUL) were calculated via noncompartmental analysis. Observed AUC(OPZ, OBS)/AUC(5OH, OBS) and AUC(OPZ, OBS)/AUC(SUL, OBS) were determined. Plasma concentrations of omeprazole, 5-hydroxyomeprazole, and omeprazole sulfone at 1, 1.5, 2, 3, 4, 6, and 8 h post-dose were used to generate limited sampling strategy (LSS) models to predict AUC(OPZ,PRE)/AUC(5OH,PRE) and AUC(OPZ,PRE/)AUC(SUL,PRE). Bias and precision were assessed via percentage mean prediction error (%MPE) and percentage mean absolute error (%MAE), with acceptable limits being <15%.
For CYP2C19, the AUC(OPZ,OBS)/AUC(5OH,OBS) was [mean ± standard deviation (SD)] 2.10 ± 1.63. Five LSS models of AUC(OPZ,PRE)/AUC(5OH,PRE) were generated, but none met the bias or precision criteria. Upon stratification by CYP2C19 genotype and ethnicity, a three-timepoint (at 1, 2, and 4 h) LSS model accurately predicted AUC(OPZ)/AUC(5OH) in Caucasian CYP2C19*1/*1 subjects. For CYP3A, AUC(OPZ,OBS)/AUC(SUL,OBS) (mean ± SD) was 1.79 ± 0.67. All LSS models had unacceptable %MAE, even when stratified by CYP2C19 genotype and ethnicity.
A LSS model to predict AUC(OPZ)/AUC(5OH), and thus CYP2C19 activity, was generated for Caucasian CYP2C19*1/*1 subjects. However, additional model validation is needed prior to general use. LSS models to predict AUC(OPZ)/AUC(SUL), and thus CYP3A activity, were not possible, even upon stratification by CYP2C19 genotype and ethnicity.
European Journal of Clinical Pharmacology 10/2011; 68(4):407-13. · 2.85 Impact Factor
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ABSTRACT: The basis of high intersubject variability of propofol metabolism is unclear. Therefore, we examined the influence of genetic polymorphisms of the key metabolizing enzymes cytochrome P450 2B6 (CYP2B6) and uridine diphosphate (UDP)-glucuronosyltransferase 1A9 (UGT1A9), age, and sex on propofol biotransformation in vitro and in vivo.
Plasma concentrations of propofol, 4-hydroxypropofol, and their glucuronides were measured over 20 min in 105 patients after a single intravenous bolus of propofol. Propofol 4-hydroxylation activity, genotypes, and content of CYP2B6 protein in 68 human livers were determined. The common single nucleotide polymorphisms (SNPs) for the CYP2B6 and UGT1A9 genes were analyzed by polymerase chain reaction (PCR).
Plasma levels of propofol metabolites showed high interindividual variability (range of coefficient of variation 89-128%). This was supported by in vitro data showing similar variability of propofol 4-hydroxylation in liver microsomes and 1.9-fold higher CYP2B6 protein content in the livers from women. No significant relationships were revealed between the SNPs studied and propofol metabolism. However, patients' sex had a pronounced effect on propofol metabolism. Thus, women had higher amounts of propofol glucuronide (1.25-fold; p = 0.03), 4-hydroxypropofol-1-glucuronide (2.1-fold; p = 0.0009), and 4-hydroxypropofol-4-glucuronide (1.7-fold; p = 0.02) as shown by the weight-corrected area under the time-plasma concentration curve of metabolites. Additionally, the sexual dimorphism in 4-hydroxypropofol glucuronidation was prominent in the 35- to 64-year-old subgroup.
No significant effects of CYP2B6 and UGT1A9 SNPs or age on propofol metabolism were revealed in this pilot study, but there was a pronounced effect of sex, a finding that indicates an important factor for the previously described sex difference in systemic clearance of propofol seen.
European Journal of Clinical Pharmacology 10/2011; 68(4):397-406. · 2.85 Impact Factor
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Patricia Huezo-Diaz,
Nader Perroud,
Edgar P Spencer,
Rebecca Smith,
Sarah Sim,
Susanne Virding,
Rudolf Uher,
Cerisse Gunasinghe,
Jo Gray,
Desmond Campbell, [......],
Marcella Rietschel,
Jorge Perez,
Caterina Giovannini,
Ole Mors,
Julien Mendlewicz,
Peter McGuffin,
Anne E Farmer, Magnus Ingelman-Sundberg,
Ian W Craig,
Katherine J Aitchison
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ABSTRACT: In vitro work shows CYP2C19 and CYP2D6 contribute to the metabolism of escitalopram to its primary metabolite, N-desmethylescitalopram. We report the effect of CYP2C19 and CYP2D6 genotypes on steady state morning concentrations of escitalopram and N-desmethylescitalopram and the ratio of this metabolite to the parent drug in 196 adult patients with depression in GENDEP, a clinical pharmacogenomic trial. Subjects who had one CYP2D6 allele associated with intermediate metabolizer phenotype and one associated with poor metabolizer (i.e. IM/PM genotypic category) had a higher mean logarithm escitalopram concentration than CYP2D6 extensive metabolizers (EMs) (p = 0.004). Older age was also associated with higher concentrations of escitalopram. Covarying for CYP2D6 and age, we found those homozygous for the CYP2C19*17 allele associated with ultrarapid metabolizer (UM) phenotype had a significantly lower mean escitalopram concentration (2-fold, p = 0.0001) and a higher mean metabolic ratio (p = 0.0003) than EMs, while those homozygous for alleles conferring the PM phenotype had a higher mean escitalopram concentration than EMs (1.55-fold, p = 0.008). There was a significant overall association between CYP2C19 genotypic category and escitalopram concentration (p = 0.0003; p = 0.0012 Bonferroni corrected). In conclusion, we have demonstrated an association between CYP2C19 genotype, including the CYP2C19*17 allele, and steady state escitalopram concentration.
Journal of Psychopharmacology 09/2011; 26(3):398-407. · 3.04 Impact Factor
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Reha Yildirimman,
Gabriella Brolén,
Mireia Vilardell,
Gustav Eriksson,
Jane Synnergren,
Hans Gmuender,
Atanas Kamburov, Magnus Ingelman-Sundberg,
José Castell,
Agustin Lahoz,
Jos Kleinjans,
Joost van Delft,
Petter Björquist,
Ralf Herwig
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ABSTRACT: Hepatocyte-like cells derived from the differentiation of human embryonic stem cells (hES-Hep) have potential to provide a human relevant in vitro test system in which to evaluate the carcinogenic hazard of chemicals. In this study, we have investigated this potential using a panel of 15 chemicals classified as noncarcinogens, genotoxic carcinogens, and nongenotoxic carcinogens and measured whole-genome transcriptome responses with gene expression microarrays. We applied an ANOVA model that identified 592 genes highly discriminative for the panel of chemicals. Supervised classification with these genes achieved a cross-validation accuracy of > 95%. Moreover, the expression of the response genes in hES-Hep was strongly correlated with that in human primary hepatocytes cultured in vitro. In order to infer mechanistic information on the consequences of chemical exposure in hES-Hep, we developed a computational method that measures the responses of biochemical pathways to the panel of treatments and showed that these responses were discriminative for the three toxicity classes and linked to carcinogenesis through p53, mitogen-activated protein kinases, and apoptosis pathway modules. It could further be shown that the discrimination of toxicity classes was improved when analyzing the microarray data at the pathway level. In summary, our results demonstrate, for the first time, the potential of human embryonic stem cell--derived hepatic cells as an in vitro model for hazard assessment of chemical carcinogenesis, although it should be noted that more compounds are needed to test the robustness of the assay.
Toxicological Sciences 08/2011; 124(2):278-90. · 4.65 Impact Factor
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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), 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.
Journal of Pharmacology and Experimental Therapeutics 07/2011; 338(1):302-9. · 3.83 Impact Factor
