RESUMEN
We present a generic workflow combining physiology-based computational modeling and in vitro data to assess the clinical cholestatic risk of different drugs systematically. Changes in expression levels of genes involved in the enterohepatic circulation of bile acids were obtained from an in vitro assay mimicking 14 days of repeated drug administration for 10 marketed drugs. These changes in gene expression over time were contextualized in a physiology-based bile acid model of glycochenodeoxycholic acid. The simulated drug-induced response in bile acid concentrations was then scaled with the applied drug doses to calculate the cholestatic potential for each compound. A ranking of the cholestatic potential correlated very well with the clinical cholestasis risk obtained from medical literature. The proposed workflow allows benchmarking the cholestatic risk of novel drug candidates. We expect the application of our workflow to significantly contribute to the stratification of the cholestatic potential of new drugs and to support animal-free testing in future drug development.
Asunto(s)
Benchmarking/métodos , Colestasis/inducido químicamente , Colestasis/metabolismo , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Modelos Biológicos , Flujo de Trabajo , Adulto , Animales , Colestasis/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Femenino , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Preparaciones Farmacéuticas , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Adulto JovenRESUMEN
Anthracyclines, including doxorubicin, idarubicin, and epirubicin, are common antitumor drugs as well as well-known cardiotoxic agents. This study analyzed the proteomics alteration in cardiac tissues caused by these 3 anthracyclines analogs. The in vitro human cardiac microtissues were exposed to drugs in 2 weeks; the proteomic data were measured at 7 time points. The heart biopsy data were collected from heart failure patients, in which some patients underwent anthracycline treatment. The anthracyclines-affected proteins were separately identified in the in vitro and in vivo dataset using the WGCNA method. These proteins engage in different cellular pathways including translation, metabolism, mitochondrial function, muscle contraction, and signaling pathways. From proteins detected in 2 datasets, a protein-protein network was established with 4 hub proteins, and 7 weighted proteins from both cardiac microtissue and human biopsies data. These 11 proteins, which involve in mitochondrial functions and the NF-κB signaling pathway, could provide insights into the anthracycline toxic mechanism. Some of them, such as HSPA5, BAG3, and SH3BGRL, are cardiac therapy targets or cardiotoxicity biomarkers. Other proteins, such as ATP5F1B and EEF1D, showed similar responses in both the in vitro and in vivo data. This suggests that the in vitro outcomes could link to clinical phenomena in proteomic analysis.
RESUMEN
Uncovering cellular responses from heterogeneous genomic data is crucial for molecular medicine in particular for drug safety. This can be realized by integrating the molecular activities in networks of interacting proteins. As proof-of-concept we challenge network modeling with time-resolved proteome, transcriptome and methylome measurements in iPSC-derived human 3D cardiac microtissues to elucidate adverse mechanisms of anthracycline cardiotoxicity measured with four different drugs (doxorubicin, epirubicin, idarubicin and daunorubicin). Dynamic molecular analysis at in vivo drug exposure levels reveal a network of 175 disease-associated proteins and identify common modules of anthracycline cardiotoxicity in vitro, related to mitochondrial and sarcomere function as well as remodeling of extracellular matrix. These in vitro-identified modules are transferable and are evaluated with biopsies of cardiomyopathy patients. This to our knowledge most comprehensive study on anthracycline cardiotoxicity demonstrates a reproducible workflow for molecular medicine and serves as a template for detecting adverse drug responses from complex omics data.
Asunto(s)
Metaboloma , Modelos Biológicos , Proteoma , Transcriptoma , Epigénesis Genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Metabolómica/métodos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteómica/métodos , Sarcómeros/genética , Sarcómeros/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Epidemiologic evidence indicates common risk factors, including air pollution exposure, for respiratory and cardiovascular diseases, suggesting the involvement of common altered molecular pathways. OBJECTIVES: The goal was to find intermediate metabolites or metabolic pathways that could be associated with both air pollutants and health outcomes ("meeting-in-the-middle"), thus shedding light on mechanisms and reinforcing causality. METHODS: We applied a statistical approach named 'meet-in-the-middle' to untargeted metabolomics in two independent case-control studies nested in cohorts on adult-onset asthma (AOA) and cardio-cerebrovascular diseases (CCVD). We compared the results to identify both common and disease-specific altered metabolic pathways. RESULTS: A novel finding was a strong association of AOA with ultrafine particles (UFP; odds ratio 1.80 [1.26, 2.55] per increase by 5000â¯particles/cm3). Further, we have identified several metabolic pathways that potentially mediate the effect of air pollution on health outcomes. Among those, perturbation of Linoleate metabolism pathway was associated with air pollution exposure, AOA and CCVD. CONCLUSIONS: Our results suggest common pathway perturbations may occur as a consequence of chronic exposure to air pollution leading to increased risk for both AOA and CCVD.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Asma/epidemiología , Enfermedades Cardiovasculares/epidemiología , Exposición a Riesgos Ambientales , Redes y Vías Metabólicas/efectos de los fármacos , Adulto , Estudios de Casos y Controles , Exposición a Riesgos Ambientales/análisis , Exposición a Riesgos Ambientales/estadística & datos numéricos , HumanosRESUMEN
Doxorubicin (DOX) is a chemotherapeutic agent of which the medical use is limited due to cardiotoxicity. While acute cardiotoxicity is reversible, chronic cardiotoxicity is persistent or progressive, dose-dependent and irreversible. While DOX mechanisms of action are not fully understood yet, 3 toxicity processes are known to occur in vivo: cardiomyocyte dysfunction, mitochondrial dysfunction and cell death. We present an in vitro experimental design aimed at detecting DOX-induced cardiotoxicity by obtaining a global view of the induced molecular mechanisms through RNA-sequencing. To better reflect the in vivo situation, human 3D cardiac microtissues were exposed to physiologically-based pharmacokinetic (PBPK) relevant doses of DOX for 2 weeks. We analysed a therapeutic and a toxic dosing profile. Transcriptomics analysis revealed significant gene expression changes in pathways related to "striated muscle contraction" and "respiratory electron transport", thus suggesting mitochondrial dysfunction as an underlying mechanism for cardiotoxicity. Furthermore, expression changes in mitochondrial processes differed significantly between the doses. Therapeutic dose reflects processes resembling the phenotype of delayed chronic cardiotoxicity, while toxic doses resembled acute cardiotoxicity. Overall, these results demonstrate the capability of our innovative in vitro approach to detect the three known mechanisms of DOX leading to toxicity, thus suggesting its potential relevance for reflecting the patient situation. Our study also demonstrated the importance of applying physiologically relevant doses during toxicological research, since mechanisms of acute and chronic toxicity differ.
Asunto(s)
Cardiotoxinas/efectos adversos , Doxorrubicina/efectos adversos , Ventrículos Cardíacos/efectos de los fármacos , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Inhibidores de Topoisomerasa II/efectos adversos , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/metabolismo , Cardiotoxinas/metabolismo , Células Cultivadas , Doxorrubicina/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Metabolómica/métodos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Concentración Osmolar , Análisis de Secuencia de ARN , Esferoides Celulares/citología , Esferoides Celulares/metabolismo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Inhibidores de Topoisomerasa II/metabolismo , Pruebas de Toxicidad Aguda/métodos , Pruebas de Toxicidad Crónica/métodosRESUMEN
Current studies of gene × air pollution interaction typically seek to identify unknown heritability of common complex illnesses arising from variability in the host's susceptibility to environmental pollutants of interest. Accordingly, a single component generalized linear models are often used to model the risk posed by an environmental exposure variable of interest in relation to a priori determined DNA variants. However, reducing the phenotypic heterogeneity may further optimize such approach, primarily represented by the modeled DNA variants. Here, we reduce phenotypic heterogeneity of asthma severity, and also identify single nucleotide polymorphisms (SNP) associated with phenotype subgroups. Specifically, we first apply an unsupervised learning algorithm method and a non-parametric regression to find a biclustering structure of children according to their allergy and asthma severity. We then identify a set of SNPs most closely correlated with each sub-group. We subsequently fit a logistic regression model for each group against the healthy controls using benzo[a]pyrene (B[a]P) as a representative airborne carcinogen. Application of such approach in a case-control data set shows that SNP clustering may help to partly explain heterogeneity in children's asthma susceptibility in relation to ambient B[a]P concentration with greater efficiency.
Asunto(s)
Asma/inducido químicamente , Asma/genética , Benzo(a)pireno/toxicidad , Predisposición Genética a la Enfermedad , Herencia Multifactorial , Contaminantes Atmosféricos/toxicidad , Contaminación del Aire/efectos adversos , Algoritmos , Estudios de Casos y Controles , Niño , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Interacción Gen-Ambiente , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Estadística como Asunto , Aprendizaje Automático no SupervisadoAsunto(s)
Evaluación Preclínica de Medicamentos/métodos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Técnicas de Cultivo de Órganos/métodos , Pruebas de Toxicidad Aguda/métodos , Animales , Relación Dosis-Respuesta a Droga , Corazón/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/sangre , FarmacocinéticaRESUMEN
BACKGROUND: Within fossil- and solid-fuel dependent geographic locations, mechanisms of air pollution-induced asthma remains unknown. In particular, sources of greater genetic susceptibility to airborne carcinogen, namely, benzo[a]pyrene (B[a]P) has never been investigated beyond that of a few well known genes. OBJECTIVES: To deepen our understanding on how the genotypic variations within the candidate genes contribute to the variability in the children's susceptibility to ambient B[a]P on doctor-diagnosed asthma. METHODS: Clinically confirmed asthmatic versus healthy control children (aged, 7-15) were enrolled from historically polluted and rural background regions in Czech Republic. Contemporaneous ambient B[a]P concentration was obtained from the routine monitoring network. The sputum DNA was genotyped for 95 genes. B[a]P interaction with SNPs was studied by two-stage, semi-agnostic screening of 621 SNPs. RESULTS: The median B[a]P within the highly polluted urban center was 8-times higher than that in the background region (7.8 vs. 1.1 ng/m3) during the period of investigation. Within the baseline model, which considered B[a]P exposure-only, the second tertile range was associated with a significantly reduced odds (aOR = 0.28) of asthma (95% CI, 0.16 to 0.50) compared to those at the lowest range. However, the highest range of B[a]P was associated with 3.18-times greater odds of the outcome (95% CI, 1.77 to 5.71). Within the gene-environment interaction models, joint occurrence of a high B[a]P exposure range and having a high-risk genotype at CTLA4 gene (rs11571316) was associated with 9-times greater odds (95% CI, 4.56-18.36) of the asthma diagnosis. Similarly, rs11571319 at CTLA4 and a high B[a]P exposure range was associated with a 8-times greater odds (95% CI, 3.95-14.27) of asthma diagnosis. Furthermore, having TG + GG genotypes on rs1031509 near STAT4 was associated with 5-times (95% CI, 3.03-8.55) greater odds of asthma diagnosis at the highest B[a]P range, compared to the odds at the reference range. Also CYP2E1 AT + TT genotypes (rs2070673) was associated with 5-times (95% CI, 3.1-8.8) greater odds of asthma diagnosis at the highest B[a]P exposure. CONCLUSIONS: The children, who jointly experience a high B[a]P exposure (6.3-8.5 ng/m3) as well as susceptible genotypes in CTLA4 (rs11571316 and rs11571319), STAT4 (rs1031509), and CYP2E1 (rs2070673), respectively, are associated with a significantly greater odds of having doctor-diagnosed asthma, compared to those with neither risk factors.
Asunto(s)
Contaminación del Aire/análisis , Asma/genética , Benzo(a)pireno/análisis , Antígeno CTLA-4/genética , Citocromo P-450 CYP2E1/genética , Predisposición Genética a la Enfermedad , Factor de Transcripción STAT4/genética , Asma/inducido químicamente , Estudios de Casos y Controles , Niño , República Checa , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales/análisis , Femenino , Interacción Gen-Ambiente , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Población Rural , Población UrbanaRESUMEN
Differentiated human bronchial epithelial cells in air liquid interface cultures (ALI-PBEC) represent a promising alternative for inhalation studies with rodents as these 3D airway epithelial tissue cultures recapitulate the human airway in multiple aspects, including morphology, cell type composition, gene expression and xenobiotic metabolism. We performed a detailed longitudinal gene expression analysis during the differentiation of submerged primary human bronchial epithelial cells into ALI-PBEC to assess the reproducibility and inter-individual variability of changes in transcriptional activity during this process. We generated ALI-PBEC cultures from four donors and focussed our analysis on the expression levels of 362 genes involved in biotransformation, which are of primary importance for toxicological studies. Expression of various of these genes (e.g., GSTA1, ADH1C, ALDH1A1, CYP2B6, CYP2F1, CYP4B1, CYP4X1 and CYP4Z1) was elevated following the mucociliary differentiation of airway epithelial cells into a pseudo-stratified epithelial layer. Although a substantial number of genes were differentially expressed between donors, the differences in fold changes were generally small. Metabolic activity measurements applying a variety of different cytochrome p450 substrates indicated that epithelial cultures at the early stages of differentiation are incapable of biotransformation. In contrast, mature ALI-PBEC cultures were proficient in the metabolic conversion of a variety of substrates albeit with considerable variation between donors. In summary, our data indicate a distinct increase in biotransformation capacity during differentiation of PBECs at the air-liquid interface and that the generation of biotransformation competent ALI-PBEC cultures is a reproducible process with little variability between cultures derived from four different donors.
Asunto(s)
Bronquios/citología , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Xenobióticos/farmacocinética , Benzo(a)Antracenos/farmacocinética , Benzo(a)pireno/farmacocinética , Biotransformación/genética , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Citocromos/genética , Citocromos/metabolismo , Enzimas/genética , Células Epiteliales/metabolismo , Humanos , Dibenzodioxinas Policloradas/farmacocinética , Reproducibilidad de los Resultados , Xenobióticos/metabolismoRESUMEN
Women persistently infected with human papillomavirus (HPV) type 16 are at high risk for development of cervical intraepithelial neoplasia grade 3 or cervical cancer (CIN3+). We aimed to identify biomarkers for progression to CIN3+ in women with persistent HPV16 infection. In this prospective study, 11,088 women aged 20-29 years were enrolled during 1991-1993, and re-invited for a second visit two years later. Cervical cytology samples obtained at both visits were tested for HPV DNA by Hybrid Capture 2 (HC2), and HC2-positive samples were genotyped by INNO-LiPA. The cohort was followed for up to 19 years via a national pathology register. To identify markers for progression to CIN3+, we performed microarray analysis on RNA extracted from cervical swabs of 30 women with persistent HPV16-infection and 11 HPV-negative women. Six genes were selected and validated by quantitative PCR. Three genes were subsequently validated within a different and large group of women from the same cohort. Secondly, Kaplan-Meier and Cox-regression analyses were used to investigate whether expression levels of those three genes predict progression to CIN3+. We found that high transcript levels of TMEM45A, SERPINB5 and p16INK4a at baseline were associated with increased risk of CIN3+ during follow-up. The hazard ratios of CIN3+ per 10-fold increase in baseline expression level were 1.6 (95% CI: 1.1-2.3) for TMEM45A, 1.6 (95% CI: 1.1-2.5) for p16INK4a, and 1.8 (95% CI: 1.2-2.7) for SERPINB5. In conclusion, high mRNA expression levels of TMEM45A, SERPINB5 and p16INK4a were associated with increased risk of CIN3+ in persistently HPV16-infected women.
RESUMEN
We analyzed the ability of particulate matter (PM) and chemicals adsorbed onto it to induce diverse gene expression profiles in subjects living in two regions of the Czech Republic differing in levels and sources of the air pollution. A total of 312 samples from polluted Ostrava region and 154 control samples from Prague were collected in winter 2009, summer 2009 and winter 2010. The highest concentrations of air pollutants were detected in winter 2010 when the subjects were exposed to: PM of aerodynamic diameter <2.5µm (PM2.5) (70 vs. 44.9µg/m(3)); benzo[a]pyrene (9.02 vs. 2.56ng/m(3)) and benzene (10.2 vs. 5.5µg/m(3)) in Ostrava and Prague, respectively. Global gene expression analysis of total RNA extracted from leukocytes was performed using Illumina Expression BeadChips microarrays. The expression of selected genes was verified by quantitative real-time PCR (qRT-PCR). Gene expression profiles differed by locations and seasons. Despite lower concentrations of air pollutants a higher number of differentially expressed genes and affected KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways was found in subjects from Prague. In both locations immune response pathways were affected, in Prague also neurodegenerative diseases-related pathways. Over-representation of the latter pathways was associated with the exposure to PM2.5. The qRT-PCR analysis showed a significant decrease in expression of APEX, ATM, FAS, GSTM1, IL1B and RAD21 in subjects from Ostrava, in a comparison of winter 2010 and summer 2009. In Prague, an increase in gene expression was observed for GADD45A and PTGS2. In conclusion, high concentrations of pollutants in Ostrava were not associated with higher number of differentially expressed genes, affected KEGG pathways and expression levels of selected genes. This observation suggests that chronic exposure to air pollution may result in reduced gene expression response with possible negative health consequences.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Exposición a Riesgos Ambientales/efectos adversos , Regulación de la Expresión Génica , Leucocitos/metabolismo , Estaciones del Año , Adulto , República Checa , Perfilación de la Expresión Génica , Humanos , Leucocitos/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
MOTIVATION: The field of toxicogenomics (the application of '-omics' technologies to risk assessment of compound toxicities) has expanded in the last decade, partly driven by new legislation, aimed at reducing animal testing in chemical risk assessment but mainly as a result of a paradigm change in toxicology towards the use and integration of genome wide data. Many research groups worldwide have generated large amounts of such toxicogenomics data. However, there is no centralized repository for archiving and making these data and associated tools for their analysis easily available. RESULTS: The Data Infrastructure for Chemical Safety Assessment (diXa) is a robust and sustainable infrastructure storing toxicogenomics data. A central data warehouse is connected to a portal with links to chemical information and molecular and phenotype data. diXa is publicly available through a user-friendly web interface. New data can be readily deposited into diXa using guidelines and templates available online. Analysis descriptions and tools for interrogating the data are available via the diXa portal. AVAILABILITY AND IMPLEMENTATION: http://www.dixa-fp7.eu CONTACT: d.hendrickx@maastrichtuniversity.nl; info@dixa-fp7.eu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Bases de Datos de Compuestos Químicos , Toxicogenética , Animales , Perfilación de la Expresión Génica , Humanos , Metabolómica , Proteómica , RatasRESUMEN
BACKGROUND: Leukemia incidence has increased in recent decades among European children, suggesting that early-life environmental exposures play an important role in disease development. OBJECTIVES: We investigated the hypothesis that childhood susceptibility may increase as a result of in utero exposure to carcinogens and hormonally acting factors. Using cord blood samples from the NewGeneris cohort, we examined associations between a range of biomarkers of carcinogen exposure and hormonally acting factors with micronuclei (MN) frequency as a proxy measure of cancer risk. Associations with gene expression and genotype were also explored. METHODS: DNA and protein adducts, gene expression profiles, circulating hormonally acting factors, and GWAS (genome-wide association study) data were investigated in relation to genomic damage measured by MN frequency in lymphocytes from 623 newborns enrolled between 2006 and 2010 across Europe. RESULTS: Malondialdehyde DNA adducts (M1dG) were associated with increased MN frequency in binucleated lymphocytes (MNBN), and exposure to androgenic, estrogenic, and dioxin-like compounds was associated with MN frequency in mononucleated lymphocytes (MNMONO), although no monotonic exposure-outcome relationship was observed. Lower frequencies of MNBN were associated with a 1-unit increase expression of PDCD11, LATS2, TRIM13, CD28, SMC1A, IL7R, and NIPBL genes. Gene expression was significantly higher in association with the highest versus lowest category of bulky and M1dG-DNA adducts for five and six genes, respectively. Gene expression levels were significantly lower for 11 genes in association with the highest versus lowest category of plasma AR CALUX® (chemically activated luciferase expression for androgens) (8 genes), ERα CALUX® (for estrogens) (2 genes), and DR CALUX® (for dioxins). Several SNPs (single-nucleotide polymorphisms) on chromosome 11 near FOLH1 significantly modified associations between androgen activity and MNBN frequency. Polymorphisms in EPHX1/2 and CYP2E1 were associated with MNBN. CONCLUSION: We measured in utero exposure to selected environmental carcinogens and circulating hormonally acting factors and detected associations with MN frequency in newborns circulating T lymphocytes. The results highlight mechanisms that may contribute to carcinogen-induced leukemia and require further research.
Asunto(s)
Biomarcadores/análisis , Carcinógenos/análisis , Sangre Fetal/citología , Hormonas/análisis , Leucemia/epidemiología , Efectos Tardíos de la Exposición Prenatal/epidemiología , Linfocitos T/química , Carcinógenos/toxicidad , Niño , Estudios de Cohortes , Aductos de ADN/efectos adversos , Aductos de ADN/análisis , Europa (Continente)/epidemiología , Femenino , Sangre Fetal/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Hormonas/efectos adversos , Humanos , Leucemia/inducido químicamente , Malondialdehído/efectos adversos , Malondialdehído/análisis , Pruebas de Micronúcleos , Embarazo , Efectos Tardíos de la Exposición Prenatal/inducido químicamente , Linfocitos T/efectos de los fármacosRESUMEN
The EU FP6 project carcinoGENOMICS explored the combination of toxicogenomics and in vitro cell culture models for identifying organotypical genotoxic- and non-genotoxic carcinogen-specific gene signatures. Here the performance of its gene classifier, derived from exposure of metabolically competent human HepaRG cells to prototypical non-carcinogens (10 compounds) and hepatocarcinogens (20 compounds), is reported. Analysis of the data at the gene and the pathway level by using independent biostatistical approaches showed a distinct separation of genotoxic from non-genotoxic hepatocarcinogens and non-carcinogens (up to 88 % correct prediction). The most characteristic pathway responding to genotoxic exposure was DNA damage. Interlaboratory reproducibility was assessed by blindly testing of three compounds, from the set of 30 compounds, by three independent laboratories. Subsequent classification of these compounds resulted in correct prediction of the genotoxicants. As expected, results on the non-genotoxic carcinogens and the non-carcinogens were less predictive. In conclusion, the combination of transcriptomics with the HepaRG in vitro cell model provides a potential weight of evidence approach for the evaluation of the genotoxic potential of chemical substances.
RESUMEN
Gene expression levels are significantly regulated by DNA methylation. Differences in gene expression profiles in the populations from various locations with different environmental conditions were repeatedly observed. In this study we compare the methylation profiles in 200 blood samples of children (aged 7-15 years) with and without bronchial asthma from two regions in the Czech Republic with different levels of air pollution (a highly polluted Ostrava region and a control Prachatice region). Samples were collected in March 2010 when the mean concentrations of benzo[a]pyrene (B[a]P) measured by stationary monitoring were 10.1±2.4ng/m(3) in Ostrava Bartovice (5.6 times higher than in the control region). Significantly higher concentrations of other pollutants (benzene, NO2, respirable air particles and metals) were also found in Ostrava. We applied the Infinium Methylation Assay, using the Human Methylation 27K BeadChip with 27,578 CpG loci for identification of the DNA methylation pattern in studied groups. Results demonstrate a significant impact of different environmental conditions on the DNA methylation patterns of children from the two regions. We found 9916 CpG sites with significantly different methylation (beta value) between children from Ostrava vs. Prachatice from which 58 CpG sites had differences >10%. The methylation of all these 58 CpG sites was lower in children from polluted Ostrava, which indicates a higher gene expression in comparison with the control Prachatice region. We did not find a difference in DNA methylation patterns between children with and without bronchial asthma in individual locations, but patterns in both asthmatics and healthy children differed between Ostrava and Prachatice. Further, we show differences in DNA methylation pattern depending on gender and urinary cotinine levels. Other factors including length of gestation, birth weight and length of full breastfeeding are suggested as possible factors that can impact the DNA methylation pattern in future life.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Asma/etiología , Biomarcadores/análisis , Metilación de ADN , Adolescente , Contaminantes Atmosféricos/análisis , Asma/diagnóstico , Estudios de Casos y Controles , Niño , Cotinina/orina , República Checa , Femenino , Humanos , Masculino , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.
Asunto(s)
Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Mutágenos/toxicidad , Transcriptoma/efectos de los fármacos , Animales , Carcinógenos/farmacología , Línea Celular Tumoral , Células Madre Embrionarias/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Neoplasias Hepáticas , Mutágenos/farmacología , Ratas , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/efectos de los fármacosRESUMEN
BACKGROUND: It has been suggested that fetal carcinogenic exposure might lead to predisposition to develop cancer during childhood or in later life possibly through modulation of the fetal transcriptome. Because gender effects in the incidence of childhood cancers have been described, we hypothesized differences at the transcriptomic level in cord blood between male and female newborns as a consequence of fetal carcinogenic exposure. The objective was to investigate whether transcriptomic responses to dietary genotoxic and nongenotoxic carcinogens show gender-specific mechanisms-of-action relevant for chemical carcinogenesis. METHODS: Global gene expression was applied in umbilical cord blood samples, the CALUX-assay was used for measuring dioxin(-like), androgen(-like), and estrogen(-like) internal exposure, and acrylamide-hemoglobin adduct levels were determined by mass spectrometry adduct-FIRE-procedure(TM). To link gene expression to an established phenotypic biomarker of cancer risk, micronuclei frequencies were investigated. RESULTS: While exposure levels did not differ between sexes at birth, important gender-specific differences were observed in gene expressions associated with these exposures linked with cell cycle, the immune system and more general cellular processes such as posttranslation. Moreover, oppositely correlating leukemia/lymphoma genes between male and female newborns were identified in relation to the different biomarkers of exposure that might be relevant to male-specific predisposition to develop these cancers in childhood. CONCLUSIONS/IMPACT: This study reveals different transcriptomic responses to environmental carcinogens between the sexes. In particular, male-specific TNF-alpha-NF-kB signaling upon dioxin exposure and activation of the Wnt-pathway in boys upon acrylamide exposure might represent possible mechanistic explanations for gender specificity in the incidence of childhood leukemia.
Asunto(s)
Carcinógenos/toxicidad , Sangre Fetal/metabolismo , Feto/efectos de los fármacos , Perfilación de la Expresión Génica , Acrilamida/metabolismo , Adulto , Biomarcadores , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Masculino , Micronúcleos con Defecto Cromosómico/estadística & datos numéricos , Embarazo , Receptores Androgénicos/genética , Caracteres Sexuales , Transducción de SeñalRESUMEN
The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line. Using both scanning electron microscopy and immunofluorescent labeling of the ciliary markers acetylated tubulin and Arl13b, we confirmed that RPTEC/TERT1 cells express primary cilium upon reaching confluence. Treatment with the carcinogens ochratoxin A (OTA) and potassium bromate (KBrO(3)) caused a significant reduction in the number of ciliated cells, while exposure to nifedipine, a noncarcinogenic renal toxin, had no effect on primary cilium expression. Flow cytometric analysis of the effects of all three compounds on the cell cycle revealed that only KBrO(3) resulted in an increase in the proportion of cells entering the cell cycle. Microarray analysis revealed dysregulation of multiple pathways affecting ciliogenesis and ciliary maintenance following OTA and KBrO(3) exposure, which were unaffected by nifedipine exposure. The primary cilium represents a unique physical checkpoint with relevance to carcinogenesis. We have shown that the renal carcinogens OTA and KBrO(3) cause significant deciliation in a model of the proximal tubule. With KBrO(3), this was followed by reentry into the cell cycle; however, deciliation was not found to be associated with reentry into the cell cycle following OTA exposure. Transcriptomic analysis identified dysregulation of Wnt signaling and ciliary trafficking in response to OTA and KBrO(3) exposure.
Asunto(s)
Bromatos/toxicidad , Carcinógenos/toxicidad , Ciclo Celular/efectos de los fármacos , Túbulos Renales Proximales/efectos de los fármacos , Ocratoxinas/toxicidad , Factores de Ribosilacion-ADP/análisis , Línea Celular , Cilios/efectos de los fármacos , Cilios/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Túbulos Renales Proximales/ultraestructura , Nifedipino/toxicidad , Transcriptoma/efectos de los fármacos , Tubulina (Proteína)/análisis , Tubulina (Proteína)/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
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.
Asunto(s)
Pruebas de Carcinogenicidad/métodos , Carcinógenos/toxicidad , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Sustancias Peligrosas/toxicidad , Hepatocitos/efectos de los fármacos , Análisis de Varianza , Técnicas de Cultivo de Célula , Diferenciación Celular , Biología Computacional , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/enzimología , Humanos , Inmunohistoquímica , Análisis por Micromatrices , Microscopía de Contraste de Fase , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The DNA gyrase inhibitor cyclothialidine had been shown to be a valuable lead structure for the discovery of new antibacterial classes able to overcome bacterial resistance to clinically used drugs. Bicyclic lactone derivatives containing in their 12-14-membered ring a thioamide functionality were reported previously to exhibit potent antibacterial activity against gram-positive bacteria. Moderate in vivo efficacy, however, was demonstrated only for derivatives bearing hydrophilic substituents, which were found to have a favorable impact on pharmcokinetics, and to reduce metabolic degradation, in particular glucuronidation. The incorporation of an additional amide unit into the 14-membered monolactam-lactone scaffold of cyclothialidine analogues provided a new "dilactam" subclass of DNA gyrase inhibitors of inherently higher polarity. After adjusting their lipophilicity by methyl-halogen exchange at the benzene ring, compounds of this series did not require the thioamide functionality to exert a decent antibacterial potency and consequently exhibited improved pharmacokinetic properties resulting in a pronounced in vivo efficacy in a mouse septicaemia infection model.