BACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSIONS\nDrug-induced liver injury (DILI) is one of the most frequent reasons for failure of drugs in clinical trials or market withdrawal. Early... Show moreBACKGROUND AND AIMS\nMETHODS\nRESULTS\nCONCLUSIONS\nDrug-induced liver injury (DILI) is one of the most frequent reasons for failure of drugs in clinical trials or market withdrawal. Early assessment of DILI risk remains a major challenge during drug development. Here, we present a mechanism-based weight-of-evidence approach able to identify certain candidate compounds with DILI liabilities due to mitochondrial toxicity.\nA total of 1587 FDA-approved drugs and 378 kinase inhibitors were screened for cellular stress response activation associated with DILI using an imaging-based HepG2 BAC-GFP reporter platform including the integrated stress response (CHOP), DNA damage response (P21) and oxidative stress response (SRXN1).\nIn total 389, 219 and 104 drugs were able to induce CHOP-GFP, P21-GFP and SRXN1-GFP expression at 50 μM respectively. Concentration response analysis identified 154 FDA-approved drugs as critical CHOP-GFP inducers. Based on predicted and observed (pre-)clinical DILI liabilities of these drugs, nine antimycotic drugs (e.g. butoconazole, miconazole, tioconazole) and 13 central nervous system (CNS) agents (e.g. duloxetine, fluoxetine) were selected for transcriptomic evaluation using whole-genome RNA-sequencing of primary human hepatocytes. Gene network analysis uncovered mitochondrial processes, NRF2 signalling and xenobiotic metabolism as most affected by the antimycotic drugs and CNS agents. Both the selected antimycotics and CNS agents caused impairment of mitochondrial oxygen consumption in both HepG2 and primary human hepatocytes.\nTogether, the results suggest that early pre-clinical screening for CHOP expression could indicate liability of mitochondrial toxicity in the context of DILI, and, therefore, could serve as an important warning signal to consider during decision-making in drug development. Show less
Wijaya, L.S.; Gabor, A.; Pot, I.E.; Have, L. van de; Saez-Rodriguez, J.; Stevens, J.L.; ... ; Water, B. van de 2023
Drug-induced liver injury (DILI) remains the main reason for drug development attritions largely due to poor mechanistic understanding. Toxicogenomic to interrogate the mechanism of DILI has been... Show moreDrug-induced liver injury (DILI) remains the main reason for drug development attritions largely due to poor mechanistic understanding. Toxicogenomic to interrogate the mechanism of DILI has been broadly performed. Gene co-regulation network-based transcriptome analysis is a bioinformatics approach that potentially contributes to improve mechanistic interpretation of toxicogenomic data. Here we performed an extensive concentration time course response-toxicogenomic study in the HepG2 cell line exposed to 20 DILI compounds, 7 reference compounds for stress response pathways, and 10 agonists for cytokines and growth factor receptors. We performed whole transcriptome targeted RNA sequencing to more than 500 conditions to and applied weighted gene co-regulated network analysis (WGCNA) to the transcriptomics data followed by identification of gene co-regulated networks (modules) that were strongly modulated upon the exposure of DILI compounds. Preservation analysis on the module responses of HepG2 and PHH demonstrated highly preserved adaptive stress response gene co-regulated networks. We correlated gene co-regulated networks with cell death onset and causal relationships of 67 critical target genes of these modules with onset of cell death was evaluated using RNA interference screening. We identified GTPBP2, HSPA1B, IRF1, SIRT1 and TSC22D3 as essential modulators of DILI compound-induced cell death. These genes were also induced by DILI compounds in PHH. Altogether, we demonstrate the application of large transcriptome datasets combined with network-based analysis and biological validation to uncover the candidate determinants of DILI. Show less
Danilyuk, T.Y.; Niemeijer, M.C.; Wijaya, L.S.; Snijders, K.; Berk, L. van der; Braak, S.J. ter; ... ; Water, B. van de 2023
Animal testing is the current standard for drug and chemicals safety assessment, but hazards translation to human is uncertain. Human in vitro models can address the species translation but might... Show moreAnimal testing is the current standard for drug and chemicals safety assessment, but hazards translation to human is uncertain. Human in vitro models can address the species translation but might not replicate in vivo complexity. Herein, we propose a network-based method addressing these translational multiscale problems that derives in vivo liver injury biomarkers applicable to in vitro human early safety screening. We applied weighted correlation network analysis (WGCNA) to a large rat liver transcriptomic dataset to obtain co-regulated gene clusters (modules). We identified modules statistically associated with liver pathologies, including a module enriched for ATF4-regulated genes as associated with the occurrence of hepatocellular single-cell necrosis, and as preserved in human liver in vitro models. Within the module, we identified TRIB3 and MTHFD2 as a novel candidate stress biomarkers, and developed and used BAC-eGFPHepG2 reporters in a compound screening, identifying compounds showing ATF4-dependent stress response and potential early safety signals. Show less
Vrijenhoek, N.G.; Wehr, M.M.; Kunnen, S.J.; Wijaya, L.S.; Callegaro, G.; Moné, M.J.; ... ; Water, B. van de 2022
Chemical read-across is commonly evaluated without particular knowledge of the biological mechanisms leading to observed adverse outcomes in vivo. Integrating data that indicate shared modes of... Show moreChemical read-across is commonly evaluated without particular knowledge of the biological mechanisms leading to observed adverse outcomes in vivo. Integrating data that indicate shared modes of action in humans will strengthen read-across cases. Here we studied transcriptomic responses of primary human hepatocytes (PHH) to a large panel of carboxylic acids to include detailed mode-of-action data as a proof-of-concept for read-across in risk assessment. In rodents, some carboxylic acids, including valproic acid (VPA), are known to cause hepatic steatosis, whereas others do not. We investigated transcriptomics responses of PHHs stimulated for 24 h by 18 structurally different VPA analogues in a concentration range to determine biological similarity in relation to in vivo steatotic potential. Using a targeted high-throughput screening assay we assessed the differential expression of ~3,000 genes covering relevant biological pathways. Differentially expressed gene analysis revealed differences in potency of carboxylic acids and expression patterns were highly similar for structurally similar compounds. Strong clustering occurred for steatosis-positive versus steatosis-negative carboxylic acids. To quantitatively define biological read-across, we combined pathway analysis and weighted gene co-expression network analysis. Active carboxylic acids displayed high similarity in gene network modulation. Importantly, free fatty acid synthesis modulation and stress pathway responses are affected by active carboxylic acids, providing coherent mechanistic underpinning for our findings. Our work shows that transcriptomic analysis of cultured human hepatocytes can reinforce the prediction of liver injury outcome based on quantitative and mechanistic biological data and support the application in read-across. Show less
Stel, W. van der; Yang, H.; Vrijenhoek, N.G.; Schimming, J.P.; Callegaro, G.; Carta, G.; ... ; Danen, E.H.J. 2021
Mitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII... Show moreMitochondrial perturbation is a key event in chemical-induced organ toxicities that is incompletely understood. Here, we studied how electron transport chain (ETC) complex I, II, or III (CI, CII and CIII) inhibitors affect mitochondrial functionality, stress response activation, and cell viability using a combination of high-content imaging and TempO-Seq in HepG2 hepatocyte cells. CI and CIII inhibitors perturbed mitochondrial membrane potential (MMP) and mitochondrial and cellular ATP levels in a concentration- and time-dependent fashion and, under conditions preventing a switch to glycolysis attenuated cell viability, whereas CII inhibitors had no effect. TempO-Seq analysis of changes in mRNA expression pointed to a shared cellular response to CI and CIII inhibition. First, to define specific ETC inhibition responses, a gene set responsive toward ETC inhibition (and not to genotoxic, oxidative, or endoplasmic reticulum stress) was identified using targeted TempO-Seq in HepG2. Silencing of one of these genes, NOS3, exacerbated the impact of CI and CIII inhibitors on cell viability, indicating its functional implication in cellular responses to mitochondrial stress. Then by monitoring dynamic responses to ETC inhibition using a HepG2 GFP reporter panel for different classes of stress response pathways and applying pathway and gene network analysis to TempO-Seq data, we looked for downstream cellular events of ETC inhibition and identified the amino acid response (AAR) as being triggered in HepG2 by ETC inhibition. Through in silico approaches we provide evidence indicating that a similar AAR is associated with exposure to mitochondrial toxicants in primary human hepatocytes. Altogether, we (i) unravel quantitative, time- and concentration-resolved cellular responses to mitochondrial perturbation, (ii) identify a gene set associated with adaptation to exposure to active ETC inhibitors, and (iii) show that ER stress and an AAR accompany ETC inhibition in HepG2 and primary hepatocytes. Show less
Callegaro, G.; Kunnen, S.J.; Trairatphisan, P.; Grosdidier, S.; Niemeijer, M.; Hollander, W. den; ... ; Water, B. van de 2021
Mechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising... Show moreMechanism-based risk assessment is urged to advance and fully permeate into current safety assessment practices, possibly at early phases of drug safety testing. Toxicogenomics is a promising source of mechanisms-revealing data, but interpretative analysis tools specific for the testing systems (e.g. hepatocytes) are lacking. In this study, we present the TXG-MAPr webtool (available at https://txg-mapr.eu/WGCNA_PHH/TGGATEs_PHH/ ), an R-Shiny-based implementation of weighted gene co-expression network analysis (WGCNA) obtained from the Primary Human Hepatocytes (PHH) TG-GATEs dataset. The 398 gene co-expression networks (modules) were annotated with functional information (pathway enrichment, transcription factor) to reveal their mechanistic interpretation. Several well-known stress response pathways were captured in the modules, were perturbed by specific stressors and showed preservation in rat systems (rat primary hepatocytes and rat in vivo liver), with the exception of DNA damage and oxidative stress responses. A subset of 87 well-annotated and preserved modules was used to evaluate mechanisms of toxicity of endoplasmic reticulum (ER) stress and oxidative stress inducers, including cyclosporine A, tunicamycin and acetaminophen. In addition, module responses can be calculated from external datasets obtained with different hepatocyte cells and platforms, including targeted RNA-seq data, therefore, imputing biological responses from a limited gene set. As another application, donors' sensitivity towards tunicamycin was investigated with the TXG-MAPr, identifying higher basal level of intrinsic immune response in donors with pre-existing liver pathology. In conclusion, we demonstrated that gene co-expression analysis coupled to an interactive visualization environment, the TXG-MAPr, is a promising approach to achieve mechanistic relevant, cross-species and cross-platform evaluation of toxicogenomic data. Show less
eTRANSAFE is a research project funded within the Innovative Medicines Initiative (IMI), which aims at developing integrated databases and computational tools (the eTRANSAFE ToxHub) that support... Show moreeTRANSAFE is a research project funded within the Innovative Medicines Initiative (IMI), which aims at developing integrated databases and computational tools (the eTRANSAFE ToxHub) that support the translational safety assessment of new drugs by using legacy data provided by the pharmaceutical companies that participate in the project. The project objectives include the development of databases containing preclinical and clinical data, computational systems for translational analysis including tools for data query, analysis and visualization, as well as computational models to explain and predict drug safety events. Show less
Hiemstra, S.; Ramaiahgari, S.C.; Wink, S.; Callegaro, G.; Coonen, M.; Meerman, J.; ... ; Water, B. van de 2019
Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2... Show moreAdaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI. Show less
Hiemstra, S.; Ramaiahgari, S.C.; Wink, S.; Callegaro, G.; Coonen, M.; Meerman, J.; ... ; Water, B. van de 2019
Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2... Show moreAdaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI. Show less
Copple, I.M.; Hollander, W. den; Callegaro, G.; Mutter, F.E.; Maggs, J.L.; Schofield, A.L.; ... ; Park, B.K. 2018
The transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical... Show moreThe transcription factor NRF2, governed by its repressor KEAP1, protects cells against oxidative stress. There is interest in modelling the NRF2 response to improve the prediction of clinical toxicities such as drug-induced liver injury (DILI). However, very little is known about the makeup of the NRF2 transcriptional network and its response to chemical perturbation in primary human hepatocytes (PHH), which are often used as a translational model for investigating DILI. Here, microarray analysis identified 108 transcripts (including several putative novel NRF2-regulated genes) that were both downregulated by siRNA targeting NRF2 and upregulated by siRNA targeting KEAP1 in PHH. Applying weighted gene co-expression network analysis (WGCNA) to transcriptomic data from the Open TG-GATES toxicogenomics repository (representing PHH exposed to 158 compounds) revealed four co-expressed gene sets or 'modules' enriched for these and other NRF2-associated genes. By classifying the 158 TG-GATES compounds based on published evidence, and employing the four modules as network perturbation metrics, we found that the activation of NRF2 is a very good indicator of the intrinsic biochemical reactivity of a compound (i.e. its propensity to cause direct chemical stress), with relatively high sensitivity, specificity, accuracy and positive/negative predictive values. We also found that NRF2 activation has lower sensitivity for the prediction of clinical DILI risk, although relatively high specificity and positive predictive values indicate that false positive detection rates are likely to be low in this setting. Underpinned by our comprehensive analysis, activation of the NRF2 network is one of several mechanism-based components that can be incorporated into holistic systems toxicology models to improve mechanistic understanding and preclinical prediction of DILI in man. Show less
