Netherlands Toxicogenomics Center

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Katika M.R.,Wageningen University | Katika M.R.,Maastricht University | Katika M.R.,Netherlands Toxicogenomics Center | Hendriksen P.J.M.,Wageningen University | And 6 more authors.
Toxicology and Applied Pharmacology | Year: 2011

Tributyltin oxide (TBTO) is an organotin compound that is widely used as a biocide in agriculture and as an antifouling agent in paints. TBTO is toxic for many cell types, particularly immune cells. The present study aimed to identify the effects of TBTO on the human T lymphocyte cell line Jurkat. Cells were treated with 0.2 and 0.5. μM TBTO for 3, 6, 12 and 24 h and then subjected to whole genome gene expression microarray analysis. The biological interpretation of the gene expression profiles revealed that endoplasmic reticulum (ER) stress is among the earliest effects of TBTO. Simultaneously or shortly thereafter, oxidative stress, activation of NFKB and NFAT, T cell activation, and apoptosis are induced. The effects of TBTO on genes involved in ER stress, NFAT pathway, T cell activation and apoptosis were confirmed by qRT-PCR. Activation and nuclear translocation of NFATC1 and the oxidative stress response proteins NRF2 and KEAP1 were confirmed by immunocytology. Taking advantage of previously published microarray data, we demonstrated that the induction of ER stress, oxidative stress, T cell activation and apoptosis by TBTO is not unique for Jurkat cells but does also occur in mouse thymocytes both ex vivo and in vivo and rat thymocytes ex vivo. We propose that the induction of ER stress leading to a T cell activation response is a major factor in the higher sensitivity of immune cells above other types of cells for TBTO. © 2011 Elsevier Inc.


Van Den Hof W.F.P.M.,Maastricht University | Van Den Hof W.F.P.M.,Netherlands Toxicogenomics Center | Coonen M.L.J.,Maastricht University | Coonen M.L.J.,Netherlands Toxicogenomics Center | And 8 more authors.
Chemical Research in Toxicology | Year: 2014

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. © 2014 American Chemical Society.


van Dartel D.A.M.,National Health Research Institute | van Dartel D.A.M.,Maastricht University | van Dartel D.A.M.,Wageningen University | Piersma A.H.,National Health Research Institute | And 2 more authors.
Reproductive Toxicology | Year: 2011

One of the most studied in vitro alternative testing methods for identification of developmental toxicity is the embryonic stem cell test (EST). Although the EST has been formally validated, the applicability domain as well as the predictability of the model needs further study to allow successful implementation of the EST as an alternative testing method in regulatory toxicity testing. Genomics technologies have already provided a proof of principle of their value in identification of toxicants such as carcinogenic compounds. Also within the EST, gene expression profiling has shown its value in the identification of developmental toxicity and in the evaluation of factors critical for risk assessment, such as dose and time responses. It is expected that the implementation of genomics into the EST will provide a more detailed end point evaluation as compared to the classical morphological scoring of differentiation cultures. Therefore, genomics may contribute to improvement of the EST, both in terms of definition of its applicability domain as well as its predictive capacity. In the present review, we present the progress that has been made with regard to the prediction of developmental toxicity using the EST combined with transcriptomics. Furthermore, we discuss the developments of additional aspects required for further optimization of the EST, including kinetics, the use of human embryonic stem cells (ESC) and computational toxicology. Finally, the current and future use of the EST model for prediction of developmental toxicity in testing strategies and in regulatory toxicity evaluations is discussed. © 2011 Elsevier Inc.


Van Summeren A.,Maastricht University | Van Summeren A.,Netherlands Toxicogenomics Center | Renes J.,Maastricht University | Renes J.,Netherlands Toxicogenomics Center | And 6 more authors.
Toxicology in Vitro | Year: 2012

The safety assessment for pharmaceuticals includes in vivo repeated dose toxicity tests in laboratory animals. These in vivo studies often generate false negative results and unexpected toxicity. The appearance of this unexpected toxicity is one of the major reasons for the drawback of a drug from the market. The liver is often a target organ in toxicology since it is responsible for the metabolism and elimination of chemical compounds. Therefore, there is need for new screening methods which classify hepatotoxic compounds earlier in development. This will lead to safer drugs and a more efficient drug discovery process. Furthermore, these new screening methods are preferably in vitro test systems, aiming at reducing the use of laboratory animals. In this review the possibilities of proteomics and its promising results for improving current predictive and mechanistic toxicological studies are described. Biomarkers or protein panels for hepatotoxic mechanisms, which reflect the in vivo situation, need to be identified to allow a better toxicity screening. Therefore, in vivo studies and in vitro cell models are discussed and evaluated with regard to the protein expression of their metabolic enzymes, their similarities with liver, their use for analyzing toxicological mechanisms and hepatotoxicity screening. Studies in which proteomics are combined with other omics-technologies are also presented. The results from these integrated data analyses can be used for the development of improved panels of biomarkers for toxicity screening. © 2012 Elsevier Ltd.


von Stechow L.,Leiden University | von Stechow L.,Netherlands Toxicogenomics Center | Ruiz-Aracama A.,RIKILT Institute of Food Safety | Ruiz-Aracama A.,Netherlands Toxicogenomics Center | And 4 more authors.
PLoS ONE | Year: 2013

The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells. © 2013 von Stechow et al.


Magkoufopoulou C.,Maastricht University | Magkoufopoulou C.,Netherlands Toxicogenomics Center | Claessen S.M.H.,Maastricht University | Tsamou M.,Maastricht University | And 6 more authors.
Carcinogenesis | Year: 2012

The lack of accurate in vitro assays for predicting in vivo toxicity of chemicals together with new legislations demanding replacement and reduction of animal testing has triggered the development of alternative methods. This study aimed at developing a transcriptomics-based in vitro prediction assay for in vivo genotoxicity. Transcriptomics changes induced in the human liver cell line HepG2 by 34 compounds after treatment for 12, 24, and 48h were used for the selection of gene-sets that are capable of discriminating between in vivo genotoxins (GTX) and in vivo nongenotoxins (NGTX). By combining transcriptomics with publicly available results for these chemicals from standard in vitro genotoxicity studies, we developed several prediction models. These models were validated by using an additional set of 28 chemicals. The best prediction was achieved after stratification of chemicals according to results from the Ames bacterial gene mutation assay prior to transcriptomics evaluation after 24h of treatment. A total of 33 genes were selected for discriminating GTX from NGTX for Ames-positive chemicals and 22 for Ames-negative chemicals. Overall, this method resulted in 89% accuracy and 91% specificity, thereby clearly outperforming the standard in vitro test battery. Transcription factor network analysis revealed HNF3a, HNF4a, HNF6, androgen receptor, and SP1 as main factors regulating the expression of classifiers for Ames-positive chemicals. Thus, the classical bacterial gene mutation assay in combination with in vitro transcriptomics in HepG2 is proposed as an upgraded in vitro approach for predicting in vivo genotoxicity of chemicals holding a great promise for reducing animal experimentations on genotoxicity. © The Author 2012. Published by Oxford University Press. All rights reserved.


Ruiz-Aracama A.,Wageningen University | Ruiz-Aracama A.,Netherlands Toxicogenomics Center | Peijnenburg A.,Wageningen University | Peijnenburg A.,Netherlands Toxicogenomics Center | And 9 more authors.
BMC Genomics | Year: 2011

Background: In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively.Results: The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD.Conclusions: Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work. © 2011 Ruiz-Aracama et al; licensee BioMed Central Ltd.


Jennen D.G.J.,Maastricht University | Jennen D.G.J.,Netherlands Toxicogenomics Center | Magkoufopoulou C.,Maastricht University | Magkoufopoulou C.,Netherlands Toxicogenomics Center | And 7 more authors.
Toxicological Sciences | Year: 2010

Direct comparison of the hepatoma cell lines HepG2 and HepaRG has previously been performed by only evaluating a limited set of genes or proteins. In this study, we examined the whole-genome gene expression of both cell lines before and after exposure to the genotoxic (GTX) carcinogens aflatoxin B1 and benzo[a]pyrene and the nongenotoxic (NGTX) carcinogens cyclosporin A, 17β-estradiol, and 2,3,7,8-tetrachlorodibenzo-paradioxin for 12 and 48 h. Before exposure, this analysis revealed an extensive network of genes and pathways, which were regulated differentially for each cell line. The comparison of the basal gene expression between HepG2, HepaRG, primary human hepatocytes (PHH), and liver clearly showed that HepaRG resembles PHH and liver the most. After exposure to the GTX and NGTX carcinogens, for both cell lines, common pathways were found that are important in carcinogenesis, for example, cell cycle regulation and apoptosis. However, also clear differences between exposed HepG2 and HepaRG were observed, and these are related to common metabolic processes, immune response, and transcription processes. Furthermore, HepG2 performs better in discriminating between GTX and NGTX carcinogens. In conclusion, these results have shown that HepaRG is a more suited in vitro liver model for biological interpretations of the effects of exposure to chemicals, whereas HepG2 is a more promising in vitro liver model for classification studies using the toxicogenomics approach. Although, it should be noted that only five carcinogens were used in this study. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.


Puigvert J.C.,Leiden University | Von Stechow L.,Leiden University | Von Stechow L.,Netherlands Toxicogenomics Center | Siddappa R.,Leiden University | And 11 more authors.
Science Signaling | Year: 2013

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.


Puigvert J.C.,Leiden University | Puigvert J.C.,Netherlands Toxicogenomics Center | De Bont H.,Leiden University | Van De Water B.,Leiden University | Danen E.H.J.,Leiden University
Current Protocols in Cell Biology | Year: 2010

Apoptosis is important for embryonic development, tissue homeostasis, and removal of cells with (potentially transforming) DNA lesions or other types of injuries. Functional genomics screens performed to unravel apoptotic signaling cascades in the context of toxicant-induced cell injury commonly use apoptosis as an end-point. Here, a method to detect the accumulation of apoptotic cells in real time that is well suited for high-throughput screens is described. The method uses automated microscopy in a 96-well format setting to visualize binding of fluorescent annexin V to the outer membrane leaflet of apoptotic cells. The automated image acquisition is followed by quantitative analysis using bioinformatics software. A protocol for each of the steps in this kinetic method is described, which includes the caspase-dependent apoptotic response to toxic compounds in multiple cell types and demonstrates that RNAi-based gene silencing of candidate apoptotic regulators affects the apoptosis kinetics as expected. This protocol will be useful for functional genomics as well as chemical (drug) screens. © 2010 John Wiley & Sons, Inc.

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