RESUMEN
Drug-induced liver injury remains the most common cause of acute liver failure and a frequently indicated reason for withdrawal of drugs. For the purpose of evaluating the relevance of liver cell models for assessing hepatotoxic risks in intact humans, we here aimed to benchmark 'omics-derived mechanistic data from three in vitro models for parenchymal liver function, intended for the investigation of drug-induced cholestasis, against 'omics data from cholestatic patients. Transcriptomic changes in HepG2 cells, primary mouse hepatocytes and primary human hepatocytes exposed to known cholestatic compounds were analyzed using microarrays. Some of the differentially expressed genes in HepG2 cells were also differentially expressed into the same direction in human cholestasis. The overlap between drug-induced transcriptomic responses in primary mouse hepatocytes and primary human hepatocytes appeared limited and no genes overlapping with in vivo cholestasis were found. Thereupon, a pathway for drug-induced cholestasis was used to map the drug-induced transcriptomic modifications involved in bile salt homeostasis. Indications of an adaptive response to prevent and reduce intracellular bile salt accumulation were observed in vivo as well as in the in vitro liver models. Furthermore, drug-specific changes were found, which may be indicative for their cholestatic properties. Furthermore, connectivity mapping was applied in order to investigate the predictive value of the in vitro models for in vivo cholestasis. This analysis resulted in a positive connection score for most compounds, which may indicate that for identifying cholestatic compounds the focus should be on gene expression signatures rather than on differentially expressed genes.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Clorpromazina/toxicidad , Colestasis/genética , Ciclosporina/toxicidad , Etinilestradiol/toxicidad , Transcriptoma/efectos de los fármacos , Animales , Ácidos y Sales Biliares/metabolismo , Células Cultivadas , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , RatonesRESUMEN
The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t-test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses.
Asunto(s)
Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , MicroARNs/efectos de los fármacos , Proteínas/efectos de los fármacos , Toxicogenética/métodos , Transcriptoma , Animales , Carcinógenos/farmacología , Hepatocitos/metabolismo , Masculino , Ratones , MicroARNs/genética , Pruebas de Mutagenicidad/métodos , Proteínas/genética , ARN Mensajero/genética , Sensibilidad y EspecificidadRESUMEN
Chemical carcinogenesis can be induced by genotoxic (GTX) or non-genotoxic (NGTX) carcinogens. GTX carcinogens have a well-described mode of action. However, the complex mechanisms by which NGTX carcinogens act are less clear and may result in conflicting results between species [e.g. Wy-14,643 (Wy)]. We hypothesise that common microRNA response pathways exist for each class of carcinogenic agents. Therefore, this study compares and integrates mRNA and microRNA expression profiles following short term acute exposure (24 and 48h) to three GTX [aflatoxin B1 (AFB1), benzo[a]pyrene (BaP) and cisplatin (CisPl)] or three NGTX (2,3,7,8-tetrachloordibenzodioxine (TCDD), cyclosporine A (CsA) and Wy) carcinogens in primary mouse hepatocytes. Discriminative gene sets, microRNAs (not for 24h) and processes were identified following 24 and 48h of exposure. From the three discriminative microRNAs found following 48h of exposure, mmu-miR-503-5p revealed to have an interaction with mRNA target gene cyclin D2 (Ccnd2 - 12444) which was involved in the discriminative process of p53 signalling and metabolism. Following exposure to NGTX carcinogens Mmu-miR-503-5p may have an oncogenic function by stimulating Ccnd2 possibly leading to a tumourigenic cell cycle progression. By contrast, after GTX carcinogen exposure it may have a tumour-suppressive function (repressing Ccnd2) leading to cell cycle arrest and to increased DNA repair activities. In addition, compound-specific microRNA-mRNA interactions [mmu-miR-301b-3p-Papss2 (for AFB1), as well as mmu-miR-29b-3p-Col4a2 and mmu-miR-24-3p-Flna (for BaP)] were found to contribute to a better understanding of microRNAs in cell cycle arrest and the impairment of the DNA damage repair, an important hallmark of GTX-induced carcinogenesis. Overall, our results indicate that microRNAs represent yet another relevant intracellular regulatory level in chemical carcinogenesis.
Asunto(s)
Carcinógenos/toxicidad , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , MicroARNs/genética , Transcriptoma , Animales , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Mensajero/genética , Transducción de SeñalRESUMEN
Acetaminophen (APAP) overdosage results in hepatotoxicity, but the underlying molecular mechanisms are still not completely understood. In the current study, we focused on mitochondrial-specific oxidative liver injury induced by APAP exposure. Owning to genetic polymorphisms in the CYP2E1 gene or varying inducibility by xenobiotics, the CYP2E1 mRNA level and protein activity vary extensively among individuals. As CYP2E1 is a known ROS generating enzyme, we chose HepG2 to minimize CYP2E1-induced ROS formation, which will help us better understand the APAP induced mitochondrial-specific hepatotoxicity in a subpopulation with low CYP2E1 activity. HepG2 cells were exposed to a low and toxic dose (0.5 and 10mM) of APAP and analyzed at four time points for genome-wide gene expression. Mitochondria were isolated and electron spin resonance spectroscopy was performed to measure the formation of mitochondrial ROS. The yield of ATP was measured to confirm the impact of the toxic dose of APAP on cellular energy production. Our results indicate that 10mM APAP significantly influences the expression of mitochondrial protein-encoding genes in association with an increase in mitochondrial ROS formation. Additionally, 10mM APAP affects the expression of genes encoding the subunits of electron transport chain (ETC) complexes, which may alter normal mitochondrial functions by disrupting the assembly, stability, and structural integrity of ETC complexes, leading to a measurable depletion of ATP, and cell death. The expression of mitochondrium-specific antioxidant enzyme, SOD2, is reduced which may limit the ROS scavenging ability and cause imbalance of the mitochondrial ROS homeostasis. Overall, transcriptome analysis reveals the molecular processes involved in the observed APAP-induced increase of mitochondrial ROS formation and the associated APAP-induced oxidative stress.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Hepatocitos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Adenosina Trifosfato/metabolismo , Muerte Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Metabolismo Energético/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Mitocondrias Hepáticas/metabolismo , Mitocondrias Hepáticas/patología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Toxicogenética/métodosRESUMEN
In order to improve attrition rates of candidate-drugs there is a need for a better understanding of the mechanisms underlying drug-induced hepatotoxicity. We aim to further unravel the toxicological response of hepatocytes to a prototypical cholestatic compound by integrating transcriptomic and metabonomic profiling of HepG2 cells exposed to Cyclosporin A. Cyclosporin A exposure induced intracellular cholesterol accumulation and diminished intracellular bile acid levels. Performing pathway analyses of significant mRNAs and metabolites separately and integrated, resulted in more relevant pathways for the latter. Integrated analyses showed pathways involved in cell cycle and cellular metabolism to be significantly changed. Moreover, pathways involved in protein processing of the endoplasmic reticulum, bile acid biosynthesis and cholesterol metabolism were significantly affected. Our findings indicate that an integrated approach combining metabonomics and transcriptomics data derived from representative in vitro models, with bioinformatics can improve our understanding of the mechanisms of action underlying drug-induced hepatotoxicity. Furthermore, we showed that integrating multiple omics and thereby analyzing genes, microRNAs and metabolites of the opposed model for drug-induced cholestasis can give valuable information about mechanisms of drug-induced cholestasis in vitro and therefore could be used in toxicity screening of new drug candidates at an early stage of drug discovery.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Ciclosporina/toxicidad , Inmunosupresores/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Técnicas In Vitro , Metabolómica , MicroARNs/biosíntesis , ARN Mensajero/biosíntesis , TranscriptomaRESUMEN
Arsenic is an established human carcinogen, but the mechanisms through which it contributes to for instance lung cancer development are still unclear. As arsenic is methylated during its metabolism, it may interfere with the DNA methylation process, and is therefore considered to be an epigenetic carcinogen. In the present study, we hypothesize that arsenic is able to induce DNA methylation changes, which lead to changes in specific gene expression, in pathways associated with lung cancer promotion and progression. A549 human adenocarcinoma lung cells were exposed to a low (0.08 µM), intermediate (0.4 µM) and high (2 µM) concentration of sodium arsenite for 1, 2 and 8 weeks. DNA was isolated for whole-genome DNA methylation analyses using NimbleGen 2.1 M deluxe promoter arrays. In addition, RNA was isolated for whole-genome transcriptomic analysis using Affymetrix microarrays. Arsenic modulated DNA methylation and expression levels of hundreds of genes in a dose-dependent and time-dependent manner. By combining whole-genome DNA methylation and gene expression data with possibly involved transcription factors, a large molecular interaction network was created based on transcription factor-target gene pairs, consisting of 216 genes. A tumor protein p53 (TP53) subnetwork was identified, showing the interactions of TP53 with other genes affected by arsenic. Furthermore, multiple other new genes were discovered showing altered DNA methylation and gene expression. In particular, arsenic modulated genes which function as transcription factor, thereby affecting target genes which are known to play a role in lung cancer promotion and progression.
Asunto(s)
Adenocarcinoma/inducido químicamente , Arsenitos/toxicidad , Carcinógenos/toxicidad , Neoplasias Pulmonares/inducido químicamente , Compuestos de Sodio/toxicidad , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Arsenitos/administración & dosificación , Carcinógenos/administración & dosificación , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Compuestos de Sodio/administración & dosificación , Factores de Tiempo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites. Integrative analysis of transcriptomics and proteomics showed that protein disulfide isomerase family A, member 4 was up-regulated on both the protein level and mRNA level. This protein is involved in protein folding and secretion in the endoplasmic reticulum. Furthermore, the microRNA mmu-miR-182-5p which is predicted to interact with the mRNA of this protein, was also differentially expressed, further emphasizing endoplasmic reticulum stress as important event in drug-induced toxicity. To further investigate the interaction between the significantly expressed proteins, a network was created including genes and microRNAs known to interact with these proteins and this network was used to visualize the experimental data. In total 6 clusters could be distinguished which appeared to be involved in several toxicity related processes, including alteration of protein folding and secretion in the endoplasmic reticulum. Metabonomic analyses resulted in 5 differentially expressed metabolites, indicative of an altered glucose, lipid and cholesterol homeostasis which can be related to cholestasis. Single and integrative analyses of transcriptomics, proteomics and metabonomics reveal mechanisms underlying cyclosporin A-induced cholestasis demonstrating that endoplasmic reticulum stress and the unfolded protein response are important processes in drug-induced liver toxicity.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Ciclosporina/toxicidad , Genómica , Hepatocitos/efectos de los fármacos , Inmunosupresores/toxicidad , Hígado/efectos de los fármacos , Metabolómica , Animales , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Marcadores Genéticos , Genómica/métodos , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Metabolómica/métodos , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Cultivo Primario de Células , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Proteómica , ARN Mensajero/metabolismo , Medición de Riesgo , Biología de SistemasRESUMEN
In the context of environmental health research, biobank blood samples have recently been identified as suitable for high-throughput omics analyses enabling the identification of new biomarkers of exposure and disease. However, blood samples containing the anti-coagulant heparin could complicate transcriptomic analysis because heparin may inhibit RNA polymerase causing inefficient cRNA synthesis and fluorophore labelling. We investigated the inhibitory effect of heparin and the influence of storage conditions (0 or 3 hr bench times, storage at room temperature or -80°C) on fluorophore labelling in heparinized fresh human buffy coat and whole blood biobank samples during the mRNA work-up protocol for microarray analysis. Subsequently, we removed heparin by lithium chloride (LiCl) treatment and performed a quality control analysis of LiCl-treated biobank sample microarrays to prove their suitability for downstream data analysis. Both fresh and biobank samples experienced varying degrees of heparin-induced inhibition of fluorophore labelling, making most samples unusable for microarray analysis. RNA derived from EDTA and citrate blood was not inhibited. No effect of bench time was observed but room temperature storage gave slightly better results. Strong correlations were observed between original blood sample RNA yield and the amount of synthesized cRNA. LiCl treatment restored sample quality to normal standards in both fresh and biobank samples and the previously identified correlations disappeared. Microarrays hybridized with LiCl-treated biobank samples were of excellent quality with no identifiable influence of heparin. We conclude that, to obtain high quality results, in most cases heparin removal is essential in blood-derived RNA samples intended for microarray analysis.
Asunto(s)
Almacenamiento de Sangre/métodos , Análisis Químico de la Sangre/métodos , Heparina/análisis , Análisis por Micromatrices/métodos , ARN/sangre , Colorantes Fluorescentes/metabolismo , Heparina/metabolismo , Humanos , Cloruro de Litio/farmacología , Control de Calidad , TemperaturaRESUMEN
With the number of new drug candidates increasing every year, there is a need for high-throughput human toxicity screenings. As the liver is the most important organ in drug metabolism and thus capable of generating relatively high levels of toxic metabolites, it is important to find a reliable strategy to screen for drug-induced hepatotoxicity. Microarray-based transcriptomics is a well-established technique in toxicogenomics research and is an ideal approach to screen for drug-induced injury at an early stage. The aim of this study was to prove the principle of classifying known hepatotoxicants and nonhepatotoxicants using their distinctive gene expression profiles in vitro in HepG2 cells. Furthermore, we undertook to subclassify the hepatotoxic compounds by investigating the subclass of cholestatic compounds. Prediction analysis for microarrays was used for classification of hepatotoxicants and nonhepatotoxicants, which resulted in an accuracy of 92% on the training set and 91% on the validation set, using 36 genes. A second model was set up with the goal of finding classifiers for cholestasis, resulting in 12 genes that appeared capable of correctly classifying 8 of the 9 cholestatic compounds, resulting in an accuracy of 93%. We were able to prove the principle that transcriptomic analyses of HepG2 cells can indeed be used to classify chemical entities for hepatotoxicity. Genes selected for classification of hepatotoxicity and cholestasis indicate that endoplasmic reticulum stress and the unfolded protein response may be important cellular effects of drug-induced liver injury. However, the number of compounds in both the training set and the validation set should be increased to improve the reliability of the prediction.
Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Antiinfecciosos/química , Antiinfecciosos/toxicidad , Antiinflamatorios/química , Antiinflamatorios/toxicidad , Anticonvulsivantes/química , Anticonvulsivantes/toxicidad , Antineoplásicos/química , Antineoplásicos/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Perfilación de la Expresión Génica , Células Hep G2 , Humanos , Modelos Teóricos , Análisis de Secuencia por Matrices de Oligonucleótidos , Preparaciones Farmacéuticas/clasificación , Toxicogenética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The toxic mechanisms of cisplatin have been frequently studied in many species and in vitro cell models. The Netherlands Toxicogenomics Centre focuses on developing in vitro alternatives using genomics technologies for animal-based assays on, e.g. genotoxic hazards. Models such as human hepatocellular carcinoma cell line (HepG2) cells, mouse primary hepatocytes (PMH) and mouse embryonic stem cells (mESC) are used. Our aim was to identify possibly robust conserved mechanisms between these models using cisplatin as model genotoxic agent. Transcriptomic data newly generated from HepG2 cells and PMH exposed to 7 µM cisplatin for 12, 24 and 48h and 24 and 48h, respectively, were compared with published data from mESC exposed to 5 µM cisplatin for 2-24h. Due to differences in response time between models and marginal changes after shorter exposure periods, we focused on 24 and 48h. At gene level, 44 conserved differentially expressed genes (DEG), involved in processes such as apoptosis, cell cycle, DNA damage response and DNA repair, were found. Functional analysis shows that limited numbers of pathways are conserved. Transcription factor (TF) network analysis indicates 12 common TF networks responding among all models and time points. Four TF, HNF4-α, SP1, c-MYC and p53, capable of regulating ±50% of all DEG, seem of equal importance in all models and exposure periods. Here we showed that transcriptomic responses across several in vitro cell models following exposure to cisplatin are mainly determined by a conserved complex network of 4 TFs. These conserved responses are hypothesised to provide most relevant information for human toxicity prediction and may form the basis for new in vitro alternatives of risk assessment.
Asunto(s)
Cisplatino/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Neoplasias Hepáticas/genética , Factores de Transcripción/genética , Transcriptoma/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Whole-genome transcriptome measurements are pivotal for characterizing molecular mechanisms of chemicals and predicting toxic classes, such as genotoxicity and carcinogenicity, from in vitro and in vivo assays. In recent years, deep sequencing technologies have been developed that hold the promise of measuring the transcriptome in a more complete and unbiased manner than DNA microarrays. Here, we applied this RNA-seq technology for the characterization of the transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging human carcinogen. Based on EnsEMBL genes, we demonstrate that RNA-seq detects ca 20% more genes than microarray-based technology but almost threefold more significantly differentially expressed genes. Functional enrichment analyses show that RNA-seq yields more insight into the biology and mechanisms related to the toxic effects caused by BaP, i.e., two- to fivefold more affected pathways and biological processes. Additionally, we demonstrate that RNA-seq allows detecting alternative isoform expression in many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Moreover, potentially novel isoforms were found, such as fragments of known transcripts, transcripts with additional exons, intron retention or exon-skipping events. The biological function(s) of these isoforms remain for the time being unknown. Finally, we demonstrate that RNA-seq enables the investigation of allele-specific gene expression, although no changes could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology, which, compared with microarrays, is capable of generating novel and valuable information at the transcriptome level for characterizing deleterious effects caused by chemicals.
Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Hepáticas/genética , Análisis de Secuencia de ARN , Transcriptoma/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Toxicological studies assessing the safety of compounds for humans frequently use in vitro systems to characterize toxic responses in combination with transcriptomic analyses. Thus far, changes have mostly been investigated at the mRNA level. Recently, microRNAs have attracted attention because they are powerful negative regulators of mRNA levels and, thus, may be responsible for the modulation of important mRNA networks implicated in toxicity. This study aimed to identify possible microRNA-mRNA networks as novel interactions on the gene expression level after a genotoxic insult. We used benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon, as a model genotoxic/carcinogenic compound. We analyzed time-dependent effects on mRNA and microRNA profiles in HepG2 cells, a widely used human liver cell line that expresses active p53 and is competent for the biotransformation of BaP. Changes in microRNA expression in response to BaP, in combination with multiple alterations of mRNA levels, were observed. Many of these altered mRNAs are targets of altered microRNAs. Using pathway analysis, we evaluated the relevance of such microRNA deregulations to genotoxicity. This revealed eight microRNAs that appear to participate in specific BaP-responsive pathways relevant to genotoxicity, such as apoptotic signaling, cell cycle arrest, DNA damage response, and DNA damage repair. Our results particularly highlight the potential of microRNA-29b, microRNA-26a-1*, and microRNA-122* as novel players in the BaP response. Therefore, this study demonstrates the added value of an integrated microRNA-mRNA approach for identifying molecular mechanisms induced by BaP in an in vitro human model.
Asunto(s)
Benzo(a)pireno/toxicidad , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Apoptosis/efectos de los fármacos , Benzo(a)pireno/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Células Hep G2 , Humanos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
N-nitroso compounds (NOCs) may represent a carcinogenic risk to humans following endogenous colonic nitrosation processes. We used the colon adenocarcinoma cell line Caco-2 to investigate transcriptomic changes at three time points (1, 6, 24 h) following exposure to genotoxic concentrations of six different NOCs (two nitrosamides, four nitrosamines) with the purpose of identifying biological processes that may play a part in the carcinogenicity of these compounds. This is especially important for nitrosamide exposure where, in light of their high reactivity, important gene expression modifications may take place early in the exposure. We also analyzed NOC-induced O(6)-methylguanine adducts in relation to transcriptomics since these adducts may influence the expression of genes pivotal in NOC-associated carcinogenicity. Many modified pathways appeared related to DNA damage, cell cycle, apoptosis, growth factor signaling and differentiation, which are linked with carcinogenicity. Nitrosamides showed the strongest response at 1h of exposure, while nitrosamines had the strongest effect at 6 and 24 h. Additionally, methylation was strongly associated with processes that may contribute to the carcinogenic risk. In summary, we have found that NOC-induced gene expression changes vary over time and that many of the modified pathways and processes indicate a carcinogenic risk associated with NOC exposure.
Asunto(s)
Carcinógenos/toxicidad , Colon/efectos de los fármacos , Perfilación de la Expresión Génica , Nitrosaminas/toxicidad , Compuestos Nitrosos/toxicidad , Apoptosis/efectos de los fármacos , Células CACO-2/efectos de los fármacos , Células CACO-2/metabolismo , Ciclo Celular/efectos de los fármacos , Colon/citología , Dietilnitrosamina/toxicidad , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metilnitronitrosoguanidina/toxicidad , Metilnitrosourea/toxicidad , N-Nitrosopirrolidina/toxicidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de TiempoRESUMEN
The murine embryonic stem cell test (EST) is designed to evaluate developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation into cardiomyocytes. The addition of transcriptomic evaluation within the EST may result in enhanced predictability and improved characterization of the applicability domain, therefore improving usage of the EST for regulatory testing strategies. Transcriptomic analyses assessing factors critical for risk assessment (i.e. dose) are needed to determine the value of transcriptomic evaluation in the EST. Here, using the developmentally toxic compound, flusilazole, we investigated the effect of compound concentration on gene expression regulation and toxicity prediction in ESC differentiation cultures. Cultures were exposed for 24 h to multiple concentrations of flusilazole (0.54-54 µM) and RNA was isolated. In addition, we sampled control cultures 0, 24, and 48 h to evaluate the transcriptomic status of the cultures across differentiation. Transcriptomic profiling identified a higher sensitivity of development-related processes as compared to cell division-related processes in flusilazole-exposed differentiation cultures. Furthermore, the sterol synthesis-related mode of action of flusilazole toxicity was detected. Principal component analysis using gene sets related to normal ESC differentiation was used to describe the dynamics of ESC differentiation, defined as the 'differentiation track'. The concentration-dependent effects on development were reflected in the significance of deviation of flusilazole-exposed cultures from this transcriptomic-based differentiation track. Thus, the detection of developmental toxicity in EST using transcriptomics was shown to be compound concentration-dependent. This study provides further insight into the possible application of transcriptomics in the EST as an improved alternative model system for developmental toxicity testing.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Silanos/toxicidad , Triazoles/toxicidad , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Madre Embrionarias/fisiología , Genes del Desarrollo/efectos de los fármacos , Ratones , Silanos/administración & dosificación , Triazoles/administración & dosificaciónRESUMEN
The murine embryonic stem cell test (EST) is an alternative testing method designed to assess potential developmental toxicity of compounds. The implementation of transcriptomics in the EST has been shown to reduce the culture duration and improve endpoint evaluation and is expected to result in an enhanced predictability and definition of the applicability domain. We evaluated the identification of developmental toxicity in the EST using two gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes") defined in our earlier studies. Nonexposed embryonic stem cells (ESC) differentiation cultures were sampled 0, 24, and 48 h after initiation of differentiation. Additionally, cultures exposed to 12 diverse well-characterized positive and negative developmental toxicants were isolated 24 h after the onset of exposure. Inhibition of ESC differentiation was evaluated in parallel by morphological scoring on culture day 10. Transcriptomics analysis was conducted using the Affymetrix Gene Chips platform. We applied principal component analysis on the basis of the two predefined gene sets to define the "differentiation track" that represents ESC differentiation. The significance of derivations in the gene expression-based differentiation track because of compound exposures were evaluated to determine developmental toxicity of tested compounds. We successfully predicted developmental toxicity using transcriptomics for 83% (10/12) and 67% (8/12) of the compounds, respectively, using the two predefined gene sets ("Van_Dartel_heartdiff_24h" and "EST biomarker genes"). Our study suggests that the application of transcriptomics may improve the applicability of the EST for the prediction of the developmental toxicity of chemicals.
Asunto(s)
Alternativas a las Pruebas en Animales , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Perfilación de la Expresión Génica , Expresión Génica/efectos de los fármacos , Teratógenos/toxicidad , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Supervivencia Celular/efectos de los fármacos , Ratones , Miocitos Cardíacos/efectos de los fármacos , Valor Predictivo de las Pruebas , Pruebas de Toxicidad/métodos , ToxicogenéticaRESUMEN
Chemical carcinogens may cause a multitude of effects inside cells, thereby affecting transcript levels of genes by direct activation of transcription factors (TF) or indirectly through the formation of DNA damage. As the temporal profiles of these responses may be profoundly different, examining time-dependent changes may provide new insights in TF networks related to cellular responses to chemical carcinogens. Therefore, we investigated in human hepatoma cells gene expression changes caused by benzo[a]pyrene at 12 time points after exposure, in relation to DNA adduct and cell cycle. Temporal profiles for functional gene sets demonstrate both early and late effects in up- and downregulation of relevant gene sets involved in cell cycle, apoptosis, DNA repair, and metabolism of amino acids and lipids. Many significant transcription regulation networks appeared to be around TF that are proto-oncogenes or tumor suppressor genes. The time series analysis tool Short Time-series Expression Miner (STEM) was used to identify time-dependent correlation of pathways, gene sets, TF networks, and biological parameters. Most correlations are with DNA adduct levels, which is an early response, and less with the later responses on G1 and S phase cells. The majority of the modulated genes in the Reactome pathways can be regulated by several of these TF, e.g., 73% by nuclear factor-kappa B and 34-42% by c-MYC, SRF, AP1, and E2F1. All these TF can also regulate one or more of the others. Our data indicate that a complex network of a few TF is responsible for the majority of the transcriptional changes induced by BaP. This network hardly changes over time, despite that the transcriptional profiles clearly alter, suggesting that also other regulatory mechanisms are involved.
Asunto(s)
Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Transcripción Genética/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Aductos de ADN/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Factores de TiempoRESUMEN
The nuclear enzyme poly(ADP-ribose) polymerse-1 (PARP-1) has previously been reported to play an important role in lipopolysaccharide (LPS)-induced pulmonary inflammation and is highly activated in COPD patients. In the present study, the anti-inflammatory efficacy of a previously identified poly(ADP-ribose) polymerase-1 (PARP-1) inhibiting caffeine metabolite, 1,7-dimethylxanthine, was both in vivo as well as ex vivo evaluated. Orally administered 1,7-dimethylxanthine significantly attenuated lung myeloperoxidase-levels, transcription of IL-6, TNF-alpha, MIP1alpha and MIP2 genes as well as PAR-polymer formation in a mouse model of intratracheally LPS-induced acute pulmonary inflammation. Serum amyloid P component and plasma IL-6 were also lowered in 1,7-dimethylxanthine treated mice, indicating a reduced systemic inflammatory response. In addition, at 24h after LPS administration anti-inflammatory effects of 1,7-dimethylxanthine appeared more pronounced than those of the orally administered PARP-1 inhibitor 3-aminobenzamide. In the second model, in blood of COPD-patients and healthy controls ex vivo pre-incubated with a physiological concentration of 1,7-dimethylxanthine (10microM), LPS-induced production of the cytokines IL-6 and TNF-alpha was significantly suppressed. 1,7-Dimethylxanthine exerts anti-inflammatory effects, both in vivo mouse as well as ex vivo human. These results suggest that the PARP-1 inhibiting caffeine metabolite 1,7-dimethylxanthine may have therapeutic potential in pulmonary inflammatory diseases such as COPD.
Asunto(s)
Antiinflamatorios/farmacología , Inhibidores Enzimáticos/farmacología , Neumonía/tratamiento farmacológico , Teofilina/farmacología , Administración Oral , Anciano , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/uso terapéutico , Benzamidas/farmacología , Estudios de Casos y Controles , Citocinas/sangre , Citocinas/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Interleucina-6/sangre , Lipopolisacáridos/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Peroxidasa/metabolismo , Neumonía/metabolismo , Neumonía/patología , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/biosíntesis , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Poli(ADP-Ribosa) Polimerasas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Enfermedad Pulmonar Obstructiva Crónica/inducido químicamente , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Teofilina/administración & dosificación , Teofilina/uso terapéutico , Transcripción Genética/efectos de los fármacosRESUMEN
Assessing the potential carcinogenicity of chemicals for humans represents an ongoing challenge. Chronic rodent bioassays predict human cancer risk at only limited reliability and are simultaneously expensive and long lasting. In order to seek for alternatives, the ability of a transcriptomics-based primary mouse hepatocyte model to classify carcinogens by their modes of action was evaluated. As it is obvious that exposure will induce a cascade of gene expression modifications, in particular, the influence of exposure time in vitro on discriminating genotoxic (GTX) carcinogens from nongenotoxic (NGTX) carcinogens class discrimination was investigated. Primary mouse hepatocytes from male C57Bl6 mice were treated for 12, 24, 36, and 48 h with two GTX and two NGTX carcinogens. For validation, two additional GTX compounds were studied at 24 and 48 h. Immunostaining of gammaH2AX foci was applied in order to phenotypically verify DNA damage. It confirmed significant induction of DNA damage after treatment with GTX compounds but not with NGTX compounds. Whole-genome gene expression modifications were analyzed by means of Affymetrix microarrays. When using differentially expressed genes from data sets normalized by Robust Multi-array Average, the two classes and various compounds were better separated from each other by hierarchical clustering when increasing the treatment period. Discrimination of GTX and NGTX carcinogens by Prediction Analysis of Microarray improved with time and resulted in correct classification of the validation compounds. The present study shows that gene expression profiling in primary mouse hepatocytes is promising for discriminating GTX from NGTX compounds and that this discrimination improves with increasing treatment period.
Asunto(s)
Carcinógenos/toxicidad , Perfilación de la Expresión Génica , Hepatocitos/efectos de los fármacos , Mutágenos/toxicidad , Animales , Células Cultivadas , Análisis por Conglomerados , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
In the present study, the anti-inflammatory effects of the flavonoids flavone, fisetin and tricetin were evaluated in a mouse model of LPS-induced acute pulmonary inflammation. The flavonoid fisetin significantly reduced lung myeloperoxidase-levels and gene-expression of inflammatory mediators such as IL-6, TNF-alpha, IL-1beta, MIP-1alpha and MIP-2. The LPS-induced gene transcription of HO-1 and SOD2 was also significantly reduced by fisetin. Overall, the anti-inflammatory effects of fisetin in this in vivo model were much more pronounced as compared to the observed effects of flavone or tricetin and the anti-inflammatory glucocorticoid dexamethasone. The results of this study indicate that flavonoids such as fisetin might be potential candidates as pharmaceuticals or nutraceuticals in the treatment of pulmonary inflammatory diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Cromonas/uso terapéutico , Flavonoides/uso terapéutico , Neumonía/tratamiento farmacológico , Animales , Flavonas , Flavonoles , Expresión Génica , Hemo-Oxigenasa 1/genética , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Interleucina-6/sangre , Lipopolisacáridos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Peroxidasa/antagonistas & inhibidores , Neumonía/inmunología , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Superóxido Dismutasa/genéticaRESUMEN
Primary human and rat hepatocyte cultures are well established in vitro systems used in toxicological studies. However, whereas transgenic mouse models provide an opportunity for studying mechanisms of toxicity, mouse primary hepatocyte cultures are less well described. The potential usefulness of a mouse hepatocyte-based in vitro model was assessed in this study by investigating time-dependent competence for xenobiotic metabolism and gene expression profiles. Primary mouse hepatocytes, isolated using two-step collagenase perfusion, were cultured in a collagen sandwich configuration. Gene expression profiles and the activities of various cytochrome P450 (P450) enzymes were determined after 0, 42, and 90 h in culture. Principal component analysis of gene expression profiles shows that replicates per time point are similar. Gene expression levels of most phase I biotransformation enzymes decrease to approximately 69 and 57% of the original levels at 42 and 90 h, respectively, whereas enzyme activities for most of the studied P450s decrease to 59 and 34%. The decrease for phase II gene expression is only to 96 and 92% of the original levels at 42 and 90 h, respectively. Pathway analysis reveals initial effects at the level of proteins, external signaling pathways, and energy production. Later effects are observed for transcription, translation, membranes, and cell cycle-related gene sets. These results indicate that the sandwich-cultured primary mouse hepatocyte system is robust and seems to maintain its metabolic competence better than that of the rat hepatocyte system.