BACKGROUND\nOBJECTIVES\nMETHODS\nRESULTS\nDISCUSSION\nUnderstanding the variability across the human population with respect to toxicodynamic responses after exposure to chemicals, such as... Show moreBACKGROUND\nOBJECTIVES\nMETHODS\nRESULTS\nDISCUSSION\nUnderstanding the variability across the human population with respect to toxicodynamic responses after exposure to chemicals, such as environmental toxicants or drugs, is essential to define safety factors for risk assessment to protect the entire population. Activation of cellular stress response pathways are early adverse outcome pathway (AOP) key events of chemical-induced toxicity and would elucidate the estimation of population variability of toxicodynamic responses.\nWe aimed to map the variability in cellular stress response activation in a large panel of primary human hepatocyte (PHH) donors to aid in the quantification of toxicodynamic interindividual variability to derive safety uncertainty factors.\n signaling. Using a population mixed-effect framework, the distribution of benchmark concentrations (BMCs) and maximum fold change were modeled to evaluate the influence of PHH donor panel size on the correct estimation of interindividual variability for the various stimuli.\n signaling-related genes, respectively. Population modeling revealed that small PHH panel sizes systematically underestimated the variance and gave low probabilities in estimating the correct human population variance. Estimated toxicodynamic variability factors of stress response activation in PHHs based on this dataset ranged between 1.6 and 6.3.\nOverall, by combining high-throughput transcriptomics and population modeling, improved understanding of interindividual variability in chemical-induced activation of toxicity relevant stress pathways across the human population using a large panel of plated cryopreserved PHHs was established, thereby contributing toward increasing the confidence of in vitro-based prediction of adverse responses, in particular hepatotoxicity. https://doi.org/10.1289/EHP11891. Show less
Vlasveld, M.P.; Callegaro, G.; Fisher, C.; Eakins, J.; Walker, P.; Lok, S.; ... ; Water, B. van de 2024
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
Bergonzini, C.; Gregori, A.; Hagens, T.M.S.; Noord, V.E. van der; Water, B. van de; Zweemer, A.J.M.; ... ; Danen, E.H.J. 2024
BACKGROUND\nMETHODS\nRESULTS\nCONCLUSION\nPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted... Show moreBACKGROUND\nMETHODS\nRESULTS\nCONCLUSION\nPancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and the chemotherapies such as gemcitabine/nab-paclitaxel are confronted with intrinsic or acquired resistance. The aim of this study was to investigate mechanisms underlying paclitaxel resistance in PDAC and explore strategies to overcome it.\nThree paclitaxel (PR) and gemcitabine resistant (GR) PDAC models were established. Transcriptomics and proteomics were used to identify conserved mechanisms of drug resistance. Genetic and pharmacological approaches were used to overcome paclitaxel resistance.\nUpregulation of ABCB1 through locus amplification was identified as a conserved feature unique to PR cells. ABCB1 was not affected in any of the GR models and no cross resistance was observed. The ABCB1 inhibitor verapamil or siRNA-mediated ABCB1 depletion sensitized PR cells to paclitaxel and prevented efflux of ABCB1 substrates in all models. ABCB1 expression was associated with a trend towards shorter survival in patients who had received gemcitabine/nab-paclitaxel treatment. A pharmacological screen identified known and novel kinase inhibitors that attenuate efflux of ABCB1 substrates and sensitize PR PDAC cells to paclitaxel.\nUpregulation of ABCB1 through locus amplification represents a novel, conserved mechanism of PDAC paclitaxel resistance. Kinase inhibitors identified in this study can be further (pre) clinically explored as therapeutic strategies to overcome paclitaxel resistance in PDAC. Show less
Audebert, M.; Assmann, A.S.; Azqueta, A.; Babica, P.; Benfenati, E.; Bortoli, S.; ... ; Oelgeschläger, M. 2023
Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in... Show moreCarcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens. Show less
Holzer, A.K.; Dreser, N.; Pallocca, G.; Mangerich, A.; Stacey, G.; Dipalo, M.; ... ; Leist, M. 2023
Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects,... Show moreEvery test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method's objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series. 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
Oxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as... Show moreOxidative stress is the consequence of an abnormal increase of reactive oxygen species (ROS). ROS are generated mainly during the metabolism in both normal and pathological conditions as well as from exposure to xenobiotics. Xenobiotics can, on the one hand, disrupt molecular machinery involved in redox processes and, on the other hand, reduce the effectiveness of the antioxidant activity. Such dysregulation may lead to oxidative damage when combined with oxidative stress overpassing the cell capacity to detoxify ROS. In this work, a green fluorescent protein (GFP)-tagged nuclear factor erythroid 2-related factor 2 (NRF2)-regulated sulfiredoxin reporter (Srxn1-GFP) was used to measure the antioxidant response of HepG2 cells to a large series of drug and drug-like compounds (2230 compounds). These compounds were then classified as positive or negative depending on cellular response and distributed among different modeling groups to establish structure-activity relationship (SAR) models. A selection of models was used to prospectively predict oxidative stress induced by a new set of compounds subsequently experimentally tested to validate the model predictions. Altogether, this exercise exemplifies the different challenges of developing SAR models of a phenotypic cellular readout, model combination, chemical space selection, and results interpretation. Show less
The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for... Show moreThe predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA). Show less
Hosseini, R.; Vlasveld, M.P.; Willemse, J.J.; Water, B. van de; Le Dévédec, S.E.; Wolstencroft, K.J. 2023
The Minimum Information for High Content Screening Microscopy Experiments (MIHCSME) is a metadata model and reusable tabular template for sharing and integrating high content imaging data. It has... Show moreThe Minimum Information for High Content Screening Microscopy Experiments (MIHCSME) is a metadata model and reusable tabular template for sharing and integrating high content imaging data. It has been developed by combining the ISA (Investigations, Studies, Assays) metadata standard with a semantically enriched instantiation of REMBI (Recommended Metadata for Biological Images). The tabular template provides an easy-to-use practical implementation of REMBI, specifically for High Content Screening (HCS) data. In addition, ISA compliance enables broader integration with other types of experimental data, paving the way for visual omics and multi-Omics integration. We show the utility of MIHCSME for HCS data using multiple examples from the Leiden FAIR Cell Observatory, a Euro-Bioimaging flagship node for high content screening and the pilot node for implementing Findable, Accessible, Interoperable and Reusable (FAIR) bioimaging data throughout the Netherlands Bioimaging network. Show less
Carta, G.; Stel, W. van der; Scuric, E.W.J.; Capinha, L.; Delp, J.; Bennekou, S.H.; ... ; Jennings, P. 2023
Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and... Show moreAnalysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound's toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies. Show less
Nongenotoxic (NGTX) carcinogens induce cancer via other mechanisms than direct DNA damage. A recognized mode of action for NGTX carcinogens is induction of oxidative stress, a state in which the... Show moreNongenotoxic (NGTX) carcinogens induce cancer via other mechanisms than direct DNA damage. A recognized mode of action for NGTX carcinogens is induction of oxidative stress, a state in which the amount of oxidants in a cell exceeds its antioxidant capacity, leading to regenerative proliferation. Currently, carcinogenicity assessment of environmental chemicals primarily relies on genetic toxicity end points. Since NGTX carcinogens lack genotoxic potential, these chemicals may remain undetected in such evaluations. To enhance the predictivity of test strategies for carcinogenicity assessment, a shift toward mechanism-based approaches is required. Here, we present an adverse outcome pathway (AOP) network for chemically induced oxidative stress leading to (NGTX) carcinogenesis. To develop this AOP network, we first investigated the role of oxidative stress in the various cancer hallmarks. Next, possible mechanisms for chemical induction of oxidative stress and the biological effects of oxidative damage to macromolecules were considered. This resulted in an AOP network, of which associated uncertainties were explored. Ultimately, development of AOP networks relevant for carcinogenesis in humans will aid the transition to a mechanism-based, human relevant carcinogenicity assessment that involves a substantially lower number of laboratory animals. Show less
Noord, V.E. van der; Stel, W. van der; Louwerens, G.; Verhoeven, D.; Kuiken, H.J.; Lieftink, C.; ... ; Water, B. van de 2023
Triple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough... Show moreTriple-negative breast cancer (TNBC) is a subtype of breast cancer with limited treatment options and poor clinical prognosis. Inhibitors of transcriptional CDKs are currently under thorough investigation for application in the treatment of multiple cancer types, including breast cancer. These studies have raised interest in combining these inhibitors, including CDK12/13 inhibitor THZ531, with a variety of other anti-cancer agents. However, the full scope of these potential synergistic interactions of transcriptional CDK inhibitors with kinase inhibitors has not been systematically investigated. Moreover, the mechanisms behind these previously described synergistic interactions remain largely elusive.\nKinase inhibitor combination screenings were performed to identify kinase inhibitors that synergize with CDK7 inhibitor THZ1 and CDK12/13 inhibitor THZ531 in TNBC cell lines. CRISPR-Cas9 knockout screening and transcriptomic evaluation of resistant versus sensitive cell lines were performed to identify genes critical for THZ531 resistance. RNA sequencing analysis after treatment with individual and combined synergistic treatments was performed to gain further insights into the mechanism of this synergy. Kinase inhibitor screening in combination with visualization of ABCG2-substrate pheophorbide A was used to identify kinase inhibitors that inhibit ABCG2. Multiple transcriptional CDK inhibitors were evaluated to extend the significance of the found mechanism to other transcriptional CDK inhibitors.\nWe show that a very high number of tyrosine kinase inhibitors synergize with the CDK12/13 inhibitor THZ531. Yet, we identified the multidrug transporter ABCG2 as key determinant of THZ531 resistance in TNBC cells. Mechanistically, we demonstrate that most synergistic kinase inhibitors block ABCG2 function, thereby sensitizing cells to transcriptional CDK inhibitors, including THZ531. Accordingly, these kinase inhibitors potentiate the effects of THZ531, disrupting gene expression and increasing intronic polyadenylation.\nOverall, this study demonstrates the critical role of ABCG2 in limiting the efficacy of transcriptional CDK inhibitors and identifies multiple kinase inhibitors that disrupt ABCG2 transporter function and thereby synergize with these CDK inhibitors. These findings therefore further facilitate the development of new (combination) therapies targeting transcriptional CDKs and highlight the importance of evaluating the role of ABC transporters in synergistic drug-drug interactions in general. Show less
Callegaro, G.; Schimming, J.P.; Piñero González, J.; Kunnen, S.J.; Wijaya, L.S.; Trairatphisan, P.; ... ; 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
Liu, Q.; Liu, N.S.; Noord, V.E. van der; Stel, W. van der; Water, B. van de; Danen, E.H.J.; Le Dévédec, S.E. 2023
Hypoxia is linked to disease progression and poor prognosis in several cancers, including breast cancer. Cancer cells can encounter acute, chronic, and/or intermittent periods of oxygen deprivation... Show moreHypoxia is linked to disease progression and poor prognosis in several cancers, including breast cancer. Cancer cells can encounter acute, chronic, and/or intermittent periods of oxygen deprivation and it is poorly understood how the different breast cancer subtypes respond to such hypoxia regimes. Here, we assessed the response of representative cell lines for the luminal and basal A subtype to acute (24 h) and chronic hypoxia (5 days). High throughput targeted transcriptomics analysis showed that HIF-related pathways are significantly activated in both subtypes. Indeed, HIF1⍺ nuclear accumulation and activation of the HIF1⍺ target gene CA9 were comparable. Based on the number of differentially expressed genes: (i) 5 days of exposure to hypoxia induced a more profound transcriptional reprogramming than 24 h, and (ii) basal A cells were less affected by acute and chronic hypoxia as compared to luminal cells. Hypoxia-regulated gene networks were identified of which hub genes were associated with worse survival in breast cancer patients. Notably, while chronic hypoxia altered the regulation of the cell cycle in both cell lines, it induced two distinct adaptation programs in these subtypes. Mainly genes controlling central carbon metabolism were affected in the luminal cells whereas genes controlling the cytoskeleton were affected in the basal A cells. In agreement, in response to chronic hypoxia, lactate secretion was more prominently increased in the luminal cell lines which were associated with the upregulation of the GAPDH glycolytic enzyme. This was not observed in the basal A cell lines. In contrast, basal A cells displayed enhanced cell migration associated with more F-actin stress fibers whereas luminal cells did not. Altogether, these data show distinct responses to acute and chronic hypoxia that differ considerably between luminal and basal A cells. This differential adaptation is expected to play a role in the progression of these different breast cancer subtypes. Show less
Béquignon, O.J.M.; Bongers, B.J.; Jespers, W.; IJzerman, A.P.; Water, B. van de; Westen, G.J.P. van 2023
With the ongoing rapid growth of publicly available ligand-protein bioactivity data, there is a trove of valuable data that can be used to train a plethora of machine-learning algorithms. However,... Show moreWith the ongoing rapid growth of publicly available ligand-protein bioactivity data, there is a trove of valuable data that can be used to train a plethora of machine-learning algorithms. However, not all data is equal in terms of size and quality and a significant portion of researchers' time is needed to adapt the data to their needs. On top of that, finding the right data for a research question can often be a challenge on its own. To meet these challenges, we have constructed the Papyrus dataset. Papyrus is comprised of around 60 million data points. This dataset contains multiple large publicly available datasets such as ChEMBL and ExCAPE-DB combined with several smaller datasets containing high-quality data. The aggregated data has been standardised and normalised in a manner that is suitable for machine learning. We show how data can be filtered in a variety of ways and also perform some examples of quantitative structure-activity relationship analyses and proteochemometric modelling. Our ambition is that this pruned data collection constitutes a benchmark set that can be used for constructing predictive models, while also providing an accessible data source for research. Show less
To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based... Show moreTo reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 μg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints. Show less
Capinha, L.; Zhang, Y.; Holzer, A.K.; Ückert, A.K.; Zana, M.; Carta, G.; ... ; Jennings, P. 2022
Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation... Show moreEnvironmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies. Show less
