EUToxRisk21

Institutions
  • FB Biologie
Publications
  Capinha, Liliana; Zhang, Yaran; Holzer, Anna-Katharina; Ückert, Anna-Katharina; Zana, Melinda; Carta, Giada; Murphy, Cormac; Baldovini, Jenna; Mazidi , Zahra; Leist, Marcel (2022): Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models Archives of Toxicology ; 2022. - Springer. - ISSN 0370-8497. - eISSN 1432-0738

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models

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Environmental 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.

Origin (projects)

  Klima, Stefanie; Brüll, Markus; Spreng, Anna-Sophie; Suciu, Ilinca; Falt, Tjalda; Schwamborn, Jens C.; Waldmann, Tanja; Karreman, Christiaan; Leist, Marcel (2021): A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling Archives of Toxicology ; 95 (2021), 5. - S. 1703-1722. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

A human stem cell-derived test system for agents modifying neuronal N-methyl-D-aspartate-type glutamate receptor Ca2+-signalling

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Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.

Origin (projects)

  Gupta, Rajinder; Schrooders, Yannick; Hauser, Duncan; van Herwijnen, Marcel; Albrecht, Wiebke; Ter Braak, Bas; Brecklinghaus, Tim; Castell, Jose V.; Leist, Marcel; Caiment, Florian (2021): Comparing in vitro human liver models to in vivo human liver using RNA-Seq Archives of Toxicology ; 95 (2021), 2. - S. 573-589. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

Comparing in vitro human liver models to in vivo human liver using RNA-Seq

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The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.

Origin (projects)

  Leist, Marcel (2021): Identifying, naming and documenting of test and tool compound stocks Alternatives to Animal Experimentation : ALTEX ; 38 (2021), 1. - S. 177-182. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

Identifying, naming and documenting of test and tool compound stocks

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Handling of chemicals is an often-neglected area of test descriptions. Some important aspects are highlighted here, using methyl-phenyl-tetrahydropyridine (MPTP), ferrous sulfate (FeSO4·xH2O) and ciguatoxin as example compounds. These are used to provide some background on aspects of acid-base equilibria, redox state, crystal water, natural compound mixtures, and chemical naming systems. Also, solvents and impurities are addressed, for instance concerning their often high (millimolar range) concentrations in assay buffers and cell culture media. The discussion of these aspects calls for a more standardized preparation of test solutions and a more extensive disclosure of the procedure in publications; it also suggests more flexibility in data mining, as compounds with clearly different identifiers may have been used to produce highly similar or fully identical test conditions. While this short overview is not intended as definitive guidance, it does demand more active involvement of all test developers and performers with these issues, and it calls for more transparent information disclosure concerning the preparation and use of test and control chemical solutions.

Origin (projects)

  Van der Stel, Wanda; Carta, Giada; Eakins, Julie; Delp, Johannes; Suciu, Ilinca; Forsby, Anna; Cediel-Ulloa, Andrea; Leist, Marcel; Hougaard Bennekou, Susanne; Van de Water, Bob (2021): New approach methods supporting read-across : Two neurotoxicity AOP-based IATA case studies Alternatives to Animal Experimentation : ALTEX ; 38 (2021), 4. - S. 615-635. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

New approach methods supporting read-across : Two neurotoxicity AOP-based IATA case studies

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Read-across approaches are considered key in moving away from in vivo animal testing towards addressing data-gaps using new approach methods (NAMs). Ample successful examples are still required to substantiate this strategy. Here we present and discuss the learnings from two OECD IATA endorsed read-across case studies. They involve two classes of pesticides -rotenoids and strobilurins- each having a defined mode-of-action that is assessed for its neurological hazard by means of an AOP-based testing strategy coupled to toxicokinetic simulations of human tissue concentrations. The endpoint in question is potential mitochondrial respiratory chain mediated neurotoxicity, specifically through inhibition of complex I or III. An AOP linking inhibition of mitochondrial respiratory chain complex I to the degeneration of dopaminergic neurons formed the basis for both cases, but was deployed in two different regulatory contexts. The two cases also exemplify several different read-across concepts: analogue versus category approach, consolidated versus putative AOP, positive versus negative prediction (i.e., neurotoxicity versus low potential for neurotoxicity), and structural versus biological similarity. We applied a range of NAMs to explore the toxicodynamic properties of the compounds, e.g., in silico docking as well as in vitro assays and readouts -including transcriptomics- in various cell systems, all anchored to the relevant AOPs. Interestingly, although some of the data addressing certain elements of the read-across were associated with high uncertainty, their impact on the overall read-across conclusion remained limited. Coupled to the elaborate regulatory review that the two cases underwent, we propose some generic learnings of AOP-based testing strategies supporting read-across.

Origin (projects)

  Golden, Emily; Macmillan, Donna S.; Dameron, Greg; Kern, Petra; Hartung, Thomas; Maertens, Alexandra (2021): Evaluation of the global performance of eight in silico skin sensitization models using human data Alternatives to Animal Experimentation : ALTEX ; 38 (2021), 1. - S. 33-48. - Springer Spektrum. - ISSN 0946-7785. - eISSN 1868-8551

Evaluation of the global performance of eight in silico skin sensitization models using human data

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Allergic contact dermatitis, or the clinical manifestation of skin sensitization, is a leading occupational hazard. Several testing approaches exist to assess skin sensitization, but in silico models are perhaps the most advantageous due to their high speed and low-cost results. Many in silico skin sensitization models exist, though many have only been tested against results from animal studies (e.g., LLNA); this creates uncertainty in human skin sensitization assessments in both a screening and regulatory context. This project’s aim was to evaluate the accuracy of eight in silico skin sensitization models against two human data sets: one highly curated (Basketter et al., 2014) and one screening level (HSDB). The binary skin sen­sitization status of each chemical in each of the two data sets was compared to the prediction from eight in silico skin sensitization tools (Toxtree, PredSkin, OECD’s QSAR Toolbox, UL’s REACHAcross™, Danish QSAR Database, TIMES-SS, and Lhasa Limited’s Derek Nexus). Models were assessed for coverage, accuracy, sensitivity, and specificity, as well as optimization features (e.g., probability of accuracy, applicability domain, etc.), if available. While there was a wide range of sensitivity and specificity, the models generally performed comparably to the LLNA in predicting human skin sensitization status (i.e., approximately 70-80% accuracy). Additionally, the models did not mispredict the same com­pounds, suggesting there might be an advantage in combining models. In silico skin sensitization models offer accurate and useful insights in a screening context; however, further improvements are necessary so these models may be con­sidered fully reliable for regulatory applications.

Origin (projects)

  Meisig, Johannes; Dreser, Nadine; Kapitza, Marion; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Hengstler, Jan G; Waldmann, Tanja; Leist, Marcel; Blüthgen, Nils (2020): Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation Nucleic Acids Research ; 48 (2020), 22. - S. 12577-12592. - Oxford University Press. - ISSN 0305-1048. - eISSN 1362-4962

Kinetic modeling of stem cell transcriptome dynamics to identify regulatory modules of normal and disturbed neuroectodermal differentiation

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Thousands of transcriptome data sets are available, but approaches for their use in dynamic cell response modelling are few, especially for processes affected simultaneously by two orthogonal influencing variables. We approached this problem for neuroepithelial development of human pluripotent stem cells (differentiation variable), in the presence or absence of valproic acid (signaling variable). Using few basic assumptions (sequential differentiation states of cells; discrete on/off states for individual genes in these states), and time-resolved transcriptome data, a comprehensive model of spontaneous and perturbed gene expression dynamics was developed. The model made reliable predictions (average correlation of 0.85 between predicted and subsequently tested expression values). Even regulations predicted to be non-monotonic were successfully validated by PCR in new sets of experiments. Transient patterns of gene regulation were identified from model predictions. They pointed towards activation of Wnt signaling as a candidate pathway leading to a redirection of differentiation away from neuroepithelial cells towards neural crest. Intervention experiments, using a Wnt/beta-catenin antagonist, led to a phenotypic rescue of this disturbed differentiation. Thus, our broadly applicable model allows the analysis of transcriptome changes in complex time/perturbation matrices.

Origin (projects)

  Jaklin, Manuela; Zhang, Jitao David; Barrow, Paul; Ebeling, Martin; Clemann, Nicole; Leist, Marcel; Kustermann, Stefan (2020): Focus on germ-layer markers : A human stem cell-based model for in vitro teratogenicity testing Reproductive Toxicology ; 98 (2020). - S. 286-298. - Elsevier. - ISSN 0890-6238. - eISSN 1873-1708

Focus on germ-layer markers : A human stem cell-based model for in vitro teratogenicity testing

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Human induced pluripotent stem cells (hiPSC) were used to develop an assay format that may deliver information on teratogenicity of drugs. A human pluripotent stem cell scorecard panel was used to monitor the expression of 96 marker genes that are indicative of the stem cell state or differentiation into the ectoderm, mesoderm and endoderm lineages. We selected a human episomal iPS cell line for the assay based on karyotype stability, initial pluripotency, differentiation capacity and overall gene expression variability. The assay is based on embryoid body formation and was developed to be simply automated. In this proof of concept study, we used eight reference compounds (valproic acid, all-trans-retinoic acid, thalidomide, methotrexate, hydroxyurea, ascorbic acid, penicillin G and ibuprofen) to test the technical performance of the assay (readout stability) in concentration-response and time-course experiments. We also found that each compound affected marker gene expression in a different way. Various forms of data analysis identified 19 out of 96 early developmental genes as potential predictive markers for teratogenicity. Machine-learning models were run to exemplify how the assay will be developed further. The preliminary results from these analyses suggest that the assay could be suitable for the pre-screening of candidate pharmaceutical compounds. The approach presented here points a way towards development of a human cell-based assay that could replace the murine EST currently used to screen for early indications of potential teratogenicity of drug candidates.

Origin (projects)

  Moné, Martijn J.; Pallocca, Giorgia; Escher, Sylvia E.; Exner, Thomas E.; Herzler, Matthias; Bennekou, Susanne Hougaard; Kamp, Hennicke; Kroese, E. Dinant; Leist, Marcel; Steger-Hartmann, Thomas (2020): Setting the stage for next-generation risk assessment with non-animal approaches : the EU-ToxRisk project experience Archives of Toxicology ; 94 (2020), 10. - S. 3581-3592. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

Setting the stage for next-generation risk assessment with non-animal approaches : the EU-ToxRisk project experience

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In 2016, the European Commission launched the EU-ToxRisk research project to develop and promote animal-free approaches in toxicology. The 36 partners of this consortium used in vitro and in silico methods in the context of case studies (CSs). These CSs included both compounds with a highly defined target (e.g. mitochondrial respiratory chain inhibitors) as well as compounds with poorly defined molecular initiation events (e.g. short-chain branched carboxylic acids). The initial project focus was on developing a science-based strategy for read-across (RAx) as an animal-free approach in chemical risk assessment. Moreover, seamless incorporation of new approach method (NAM) data into this process (= NAM-enhanced RAx) was explored. Here, the EU-ToxRisk consortium has collated its scientific and regulatory learnings from this particular project objective. For all CSs, a mechanistic hypothesis (in the form of an adverse outcome pathway) guided the safety evaluation. ADME data were generated from NAMs and used for comprehensive physiological-based kinetic modelling. Quality assurance and data management were optimized in parallel. Scientific and Regulatory Advisory Boards played a vital role in assessing the practical applicability of the new approaches. In a next step, external stakeholders evaluated the usefulness of NAMs in the context of RAx CSs for regulatory acceptance. For instance, the CSs were included in the OECD CS portfolio for the Integrated Approach to Testing and Assessment project. Feedback from regulators and other stakeholders was collected at several stages. Future chemical safety science projects can draw from this experience to implement systems toxicology-guided, animal-free next-generation risk assessment.

Origin (projects)

  Guth, Sabine; Roth, Angelika; Engeli, Barbara; Lachenmeier, Dirk W.; Cartus, Alexander T.; Hüser, Stephanie; Baum, Matthias; Diel, Patrick; Leist, Marcel; Zarn, Jürg (2020): Comparison of points of departure between subchronic and chronic toxicity studies on food additives, food contaminants and natural food constituents Food and Chemical Toxicology ; 146 (2020). - 111784. - Elsevier. - ISSN 0278-6915. - eISSN 1873-6351

Comparison of points of departure between subchronic and chronic toxicity studies on food additives, food contaminants and natural food constituents

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In the past, it was generally accepted as a default assumption that No-Observed-Adverse-Effect Levels (NOAELs) or Lowest-Observed-Adverse-Effect Levels (LOAELs) in long-term toxicity studies are lower than in short-term ones, i.e. the toxic potency increases with prolonged exposure duration. Recent studies on pesticides and industrial chemicals reported that subacute, subchronic or chronic NOAELs/LOAELs are similar when study design factors are appropriately considered. We investigated whether these findings also apply to certain food constituents. After reviewing subchronic and chronic toxicity studies on more than 100 compounds, a total of 32 compounds could be included in the analysis. Geometric mean (GM) values of subchronic vs. chronic NOAEL or LOAEL ratios ranged from 1.0 to 2.0, with a geometric standard deviation from 2.2 to 4.2, which is consistent with data reported in the literature. While for many of the investigated compounds the ratio is around 1 - suggesting that health-based guidance values could appropriately be derived from subchronic toxicity studies - our study also identified some substances with higher ratios leading to a GM of around 2. The EFSA Scientific Committee suggested to apply an uncertainty factor of 2 to extrapolate from subchronic to chronic studies and, as a precautionary approach, we concur with this suggestion.

Origin (projects)

  Gutbier, Simon; Kyriakou, Sotiris; Schildknecht, Stefan; Ückert, Anna-Katharina; Brüll, Markus; Lewis, Frank; Dickens, David; Pearson, Liam; Elson, Joanna L.; Leist, Marcel (2020): Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants Archives of Toxicology ; 94 (2020), 9. - S. 3105-3123. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

Design and evaluation of bi-functional iron chelators for protection of dopaminergic neurons from toxicants

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While the etiology of non-familial Parkinson’s disease (PD) remains unclear, there is evidence that increased levels of tissue iron may be a contributing factor. Moreover, exposure to some environmental toxicants is considered an additional risk factor. Therefore, brain-targeted iron chelators are of interest as antidotes for poisoning with dopaminergic toxicants, and as potential treatment of PD. We, therefore, designed a series of small molecules with high affinity for ferric iron and containing structural elements to allow their transport to the brain via the neutral amino acid transporter, LAT1 (SLC7A5). Five candidate molecules were synthesized and initially characterized for protection from ferroptosis in human neurons. The promising hydroxypyridinone SK4 was characterized further. Selective iron chelation within the physiological range of pH values and uptake by LAT1 were confirmed. Concentrations of 10–20 µM blocked neurite loss and cell demise triggered by the parkinsonian neurotoxicants, methyl-phenyl-pyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) in human dopaminergic neuronal cultures (LUHMES cells). Rescue was also observed when chelators were given after the toxicant. SK4 derivatives that either lacked LAT1 affinity or had reduced iron chelation potency showed altered activity in our assay panel, as expected. Thus, an iron chelator was developed that revealed neuroprotective properties, as assessed in several models. The data strongly support the role of iron in dopaminergic neurotoxicity and suggests further exploration of the proposed design strategy for improving brain iron chelation.

Origin (projects)

  van der Stel, Wanda; Carta, Giada; Eakins, Julie; Darici, Salihanur; Delp, Johannes; Forsby, Anna; Hougaard Bennekou, Susanne; Gardner, Iain; Leist, Marcel; Jennings, Paul (2020): Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals Archives of Toxicology ; 94 (2020), 8. - S. 2707-2729. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

Multiparametric assessment of mitochondrial respiratory inhibition in HepG2 and RPTEC/TERT1 cells using a panel of mitochondrial targeting agrochemicals

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Evidence is mounting for the central role of mitochondrial dysfunction in several pathologies including metabolic diseases, accelerated ageing, neurodegenerative diseases and in certain xenobiotic-induced organ toxicity. Assessing mitochondrial perturbations is not trivial and the outcomes of such investigations are dependent on the cell types used and assays employed. Here we systematically investigated the effect of electron transport chain (ETC) inhibitors on multiple mitochondrial-related parameters in two human cell types, HepG2 and RPTEC/TERT1. Cells were exposed to a broad range of concentrations of 20 ETC-inhibiting agrochemicals and capsaicin, consisting of inhibitors of NADH dehydrogenase (Complex I, CI), succinate dehydrogenase (Complex II, CII) and cytochrome bc1 complex (Complex III, CIII). A battery of tests was utilised, including viability assays, lactate production, mitochondrial membrane potential (MMP) and the Seahorse bioanalyser, which simultaneously measures extracellular acidification rate [ECAR] and oxygen consumption rate [OCR]. CI inhibitors caused a potent decrease in OCR, decreased mitochondrial membrane potential, increased ECAR and increased lactate production in both cell types. Twenty-fourhour exposure to CI inhibitors decreased viability of RPTEC/TERT1 cells and 3D spheroid-cultured HepG2 cells in the presence of glucose. CI inhibitors decreased 2D HepG2 viability only in the absence of glucose. CII inhibitors had no notable effects in intact cells up to 10 µM. CIII inhibitors had similar effects to the CI inhibitors. Antimycin A was the most potent CIII inhibitor, with activity in the nanomolar range. The proposed CIII inhibitor cyazofamid demonstrated a mitochondrial uncoupling signal in both cell types. The study presents a comprehensive example of a mitochondrial assessment workflow and establishes measurable key events of ETC inhibition.

Origin (projects)

  Krebs, Alice; van Vugt-Lussenburg, Barbara M. A.; Waldmann, Tanja; Busquet, Francois; Dolde, Xenia; Holzer, Anna-Katharina; Kisitu, Jaffar; Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2020): The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods Archives of Toxicology ; 94 (2020), 7. - S. 2435-2461. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods

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Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Origin (projects)

  Busquet, Francois; Hartung, Thomas; Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2020): Harnessing the power of novel animal-free test methods for the development of COVID-19 drugs and vaccines Archives of Toxicology ; 94 (2020), 6. - S. 2263-2272. - Springer. - ISSN 0340-5761. - eISSN 1432-0738

Harnessing the power of novel animal-free test methods for the development of COVID-19 drugs and vaccines

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The COVID-19-inducing virus, SARS-CoV2, is likely to remain a threat to human health unless efficient drugs or vaccines become available. Given the extent of the current pandemic (people in over one hundred countries infected) and its disastrous effect on world economy (associated with limitations of human rights), speedy drug discovery is critical. In this situation, past investments into the development of new (animal-free) approach methods (NAM) for drug safety, efficacy, and quality evaluation can be leveraged. For this, we provide an overview of repurposing ideas to shortcut drug development times. Animal-based testing would be too lengthy, and it largely fails, when a pathogen is species-specific or if the desired drug is based on specific features of human biology. Fortunately, industry has already largely shifted to NAM, and some public funding programs have advanced the development of animal-free technologies. For instance, NAM can predict genotoxicity (a major aspect of carcinogenicity) within days, human antibodies targeting virus epitopes can be generated in molecular biology laboratories within weeks, and various human cell-based organoids are available to test virus infectivity and the biological processes controlling them. The European Medicines Agency (EMA) has formed an expert group to pave the way for the use of such approaches for accelerated drug development. This situation illustrates the importance of diversification in drug discovery strategies and clearly shows the shortcomings of an approach that invests 95% of resources into a single technology (animal experimentation) in the face of challenges that require alternative approaches.

Origin (projects)

  Ripani, Paola; Delp, Johannes; Bode, Konstantin J.; Delgado, M. Eugenia; Dietrich, Lea; Betzler, Verena M.; von Scheven, Gudrun; Mayer, Thomas U.; Leist, Marcel; Brunner, Thomas (2020): Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain Oncogene ; 39 (2020), 11. - S. 2345-2357. - Springer Science and Business Media. - ISSN 0950-9232. - eISSN 1476-5594

Thiazolides promote G1 cell cycle arrest in colorectal cancer cells by targeting the mitochondrial respiratory chain

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Systemic toxicity and tumor cell resistance still limit the efficacy of chemotherapy in colorectal cancer. Therefore, alternative treatments are desperately needed. The thiazolide Nitazoxanide (NTZ) is an FDA-approved drug for the treatment of parasite-mediated infectious diarrhea with a favorable safety profile. Interestingly, NTZ and the thiazolide RM4819—its bromo-derivative lacking antibiotic activity—are also promising candidates for cancer treatment. Yet the exact anticancer mechanism(s) of these compounds still remains unclear. In this study, we systematically investigated RM4819 and NTZ in 2D and 3D colorectal cancer culture systems. Both compounds strongly inhibited proliferation of colon carcinoma cell lines by promoting G1 phase cell cycle arrest. Thiazolide-induced cell cycle arrest was independent of the p53/p21 axis, but was mediated by inhibition of protein translation via the mTOR/c-Myc/p27 pathway, likely caused by inhibition of mitochondrial respiration. While both thiazolides demonstrated mitochondrial uncoupling activity, only RM4819 inhibited the mitochondrial respiratory chain complex III. Interestingly, thiazolides also potently inhibited the growth of murine colonic tumoroids in a comparable manner with cisplatin, while in contrast to cisplatin thiazolides did not affect the growth of primary intestinal organoids. Thus, thiazolides appear to have a tumor-selective antiproliferative activity, which offers new perspectives in the treatment of colorectal cancer.

Origin (projects)

  Zhong, Xiali; Harris, Georgina; Smirnova, Lena; Zufferey, Valentin; de Cássia da Silveira e Sá, Rita; Baldino Russo, Fabiele; Baleeiro Beltrao Braga, Patricia Cristina; Chesnut, Megan; Hartung, Thomas; Pamies, David (2020): Antidepressant Paroxetine Exerts Developmental Neurotoxicity in an iPSC-Derived 3D Human Brain Model Frontiers in Cellular Neuroscience ; 14 (2020). - 25. - Frontiers Research Foundation. - eISSN 1662-5102

Antidepressant Paroxetine Exerts Developmental Neurotoxicity in an iPSC-Derived 3D Human Brain Model

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Selective serotonin reuptake inhibitors (SSRIs) are frequently used to treat depression during pregnancy. Various concerns have been raised about the possible effects of these drugs on fetal development. Current developmental neurotoxicity (DNT) testing conducted in rodents is expensive, time-consuming, and does not necessarily represent human pathophysiology. A human, in vitro testing battery to cover key events of brain development, could potentially overcome these challenges. In this study, we assess the DNT of paroxetine—a widely used SSRI which has shown contradictory evidence regarding effects on human brain development using a versatile, organotypic human induced pluripotent stem cell (iPSC)-derived brain model (BrainSpheres). At therapeutic blood concentrations, which lie between 20 and 60 ng/ml, Paroxetine led to an 80% decrease in the expression of synaptic markers, a 60% decrease in neurite outgrowth and a 40–75% decrease in the overall oligodendrocyte cell population, compared to controls. These results were consistently shown in two different iPSC lines and indicate that relevant therapeutic concentrations of Paroxetine induce brain cell development abnormalities which could lead to adverse effects.

Origin (projects)

  Chovancova, Petra; Karreman, Christiaan; Dold, Jeremias E.G.A.; Krebs, Alice; Funke, Melina; Holzer, Anna-Katharina; Klima, Stefanie; Nyffeler, Johanna; Helfrich, Stefan; Wittmann, Valentin; Leist, Marcel (2020): Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity Archives of Toxicology ; 94 (2020), 2. - S. 449-467. - Springer Science and Business Media. - ISSN 0340-5761. - eISSN 1432-0738

Time and space-resolved quantification of plasma membrane sialylation for measurements of cell function and neurotoxicity

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While there are many methods to quantify the synthesis, localization, and pool sizes of proteins and DNA during physiological responses and toxicological stress, only few approaches allow following the fate of carbohydrates. One of them is metabolic glycoengineering (MGE), which makes use of chemically modified sugars (CMS) that enter the cellular biosynthesis pathways leading to glycoproteins and glycolipids. The CMS can subsequently be coupled (via bio-orthogonal chemical reactions) to tags that are quantifiable by microscopic imaging. We asked here, whether MGE can be used in a quantitative and time-resolved way to study neuronal glycoprotein synthesis and its impairment. We focused on the detection of sialic acid (Sia), by feeding human neurons the biosynthetic precursor N-acetyl-mannosamine, modified by an azide tag. Using this system, we identified non-toxic conditions that allowed live cell labeling with high spatial and temporal resolution, as well as the quantification of cell surface Sia. Using combinations of immunostaining, chromatography, and western blotting, we quantified the percentage of cellular label incorporation and effects on glycoproteins such as polysialylated neural cell adhesion molecule. A specific imaging algorithm was used to quantify Sia incorporation into neuronal projections, as potential measure of complex cell function in toxicological studies. When various toxicants were studied, we identified a subgroup (mitochondrial respiration inhibitors) that affected neurite glycan levels several hours before any other viability parameter was affected. The MGE-based neurotoxicity assay, thus allowed the identification of subtle impairments of neurochemical function with very high sensitivity.

Origin (projects)

  Busquet, Francois; Kleensang, Andre; Rovida, Costanza; Herrmann, Kathrin; Leist, Marcel; Hartung, Thomas (2020): New European Union statistics on laboratory animal use : what really counts! Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 2. - S. 167-186. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

New European Union statistics on laboratory animal use : what really counts!

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Seven years after the last release, the European Commission has again collated and released data on laboratory animal use. The new report is the first to correspond to the requirements of the new Directive 2010/63/EU. Beside minor problems in reporting, the new reporting format is a major step forward, with additional new categories like severity allowing insight into animal use related questions that goes far beyond the previous reports. An in-depth analysis confirms a slight decrease in animal use from 2015 to 2017, but also compared to the 2005, 2008 and 2011 reports, though the new reporting scheme makes this comparison difficult. Notable success is evident for replacing rabbit pyrogen testing but, in general, the implementation of accepted alternative methods lags behind expec-tations. Beside the roughly 10 million animals per year covered in the report, about 8 million animals were identified that fall under the Directive but are not included in this number. Their omission downplays the impact of REACH on animal use. The report, second to none in its detail internationally, represents an important instrument for benchmarking and strategi-cally focusing activities in the 3Rs.

Origin (projects)

  Dreser, Nadine; Holzer, Anna-Katharina; Kapitza, Marion; Scholz, Christopher; Chovancova, Petra; Gutbier, Simon; Klima, Stefanie; Kolb, David; Dietz, Christian; Trefzer, Timo; Berthold, Michael R.; Waldmann, Tanja; Leist, Marcel (2020): Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances Archives of Toxicology ; 94 (2020), 1. - S. 151-171. - Springer Science and Business Media. - ISSN 0340-5761. - eISSN 1432-0738

Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances

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The first in vitro tests for developmental toxicity made use of rodent cells. Newer teratology tests, e.g. developed during the ESNATS project, use human cells and measure mechanistic endpoints (such as transcriptome changes). However, the toxicological implications of mechanistic parameters are hard to judge, without functional/morphological endpoints. To address this issue, we developed a new version of the human stem cell-based test STOP-tox(UKN). For this purpose, the capacity of the cells to self-organize to neural rosettes was assessed as functional endpoint: pluripotent stem cells were allowed to differentiate into neuroepithelial cells for 6 days in the presence or absence of toxicants. Then, both transcriptome changes were measured (standard STOP-tox(UKN)) and cells were allowed to form rosettes. After optimization of staining methods, an imaging algorithm for rosette quantification was implemented and used for an automated rosette formation assay (RoFA). Neural tube toxicants (like valproic acid), which are known to disturb human development at stages when rosette-forming cells are present, were used as positive controls. Established toxicants led to distinctly different tissue organization and differentiation stages. RoFA outcome and transcript changes largely correlated concerning (1) the concentration-dependence, (2) the time dependence, and (3) the set of positive hits identified amongst 24 potential toxicants. Using such comparative data, a prediction model for the RoFA was developed. The comparative analysis was also used to identify gene dysregulations that are particularly predictive for disturbed rosette formation. This ‘RoFA predictor gene set’ may be used for a simplified and less costly setup of the STOP-tox(UKN) assay.

Origin (projects)

  Marx, Uwe; Akabane, Takafumi; Andersson, Tommy B.; Baker, Elizabeth; Beilmann, Mario; Beken, Sonja; Brendler-Schwaab, Susanne; Hartung, Thomas; Leist, Marcel; Pallocca, Giorgia (2020): Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 3. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

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The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all.

Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Origin (projects)

  Kappenberg, Franziska; Brecklinghaus, Tim; Albrecht, Wiebke; Blum, Jonathan; van der Wurp, Carola; Leist, Marcel; Hengstler, Jan G.; Rahnenführer, Jörg (2020): Handling deviating control values in concentration-response curves Archives of Toxicology ; 94 (2020), 11. - S. 3787-3798. - Springer. - ISSN 0370-8497. - eISSN 1432-0738

Handling deviating control values in concentration-response curves

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In cell biology, pharmacology and toxicology dose-response and concentration-response curves are frequently fitted to data with statistical methods. Such fits are used to derive quantitative measures (e.g. EC[Formula: see text] values) describing the relationship between the concentration of a compound or the strength of an intervention applied to cells and its effect on viability or function of these cells. Often, a reference, called negative control (or solvent control), is used to normalize the data. The negative control data sometimes deviate from the values measured for low (ineffective) test compound concentrations. In such cases, normalization of the data with respect to control values leads to biased estimates of the parameters of the concentration-response curve. Low quality estimates of effective concentrations can be the consequence. In a literature study, we found that this problem occurs in a large percentage of toxicological publications. We propose different strategies to tackle the problem, including complete omission of the controls. Data from a controlled simulation study indicate the best-suited problem solution for different data structure scenarios. This was further exemplified by a real concentration-response study. We provide the following recommendations how to handle deviating controls: (1) The log-logistic 4pLL model is a good default option. (2) When there are at least two concentrations in the no-effect range, low variances of the replicate measurements, and deviating controls, control values should be omitted before fitting the model. (3) When data are missing in the no-effect range, the Brain-Cousens model sometimes leads to better results than the default model.

Origin (projects)

  Karreman, Christiaan; Klima, Stefanie; Holzer, Anna-Katharina; Leist, Marcel (2020): CaFFEE : A program for evaluating time courses of Ca2+ dependent signal changes of complex cells loaded with fluorescent indicator dyes Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 2. - S. 332-336. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

CaFFEE : A program for evaluating time courses of Ca2+ dependent signal changes of complex cells loaded with fluorescent indicator dyes

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Quantification of changes in intracellular free Ca2+ concentrations [Ca2+]i is fundamental to the understanding of the physiology of single cells in response to both environmental and endogenous stimuli. Here we present easy to use freeware that allows especially the evaluation of [Ca2+]i signals in complex and mixed cultures. The program CaFFEE (Calcium Fluorescent Flash Evaluating Engine) enables the user to evaluate the response of hundreds of cells to treat-ments that influence [Ca2+]i. CaFFEE processes large quantities of image data, automatically identifies individual cells in mixed, heterogeneous populations, and evaluates their fluorescence signal. All data are exported in spreadsheet format, and data on thousands of cells can be batch-processed. Moreover, the program optimizes the visual representation of time-lapse image data for user-guided data exploration (setting of parameters for semi-automated data processing). The freeware allows the standardized and transparent processing of imaging data independent of the platform used to generate the data.

Origin (projects)

  Nesterenko, Yevheniia; Dolde, Xenia; Leist, Marcel; Mayans, Olga (2020): Strategy to replace animal-derived ECM by a modular and highly defined matrix Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 3. - S. 482-489. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

Strategy to replace animal-derived ECM by a modular and highly defined matrix

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Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highly defined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed here ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study, whether ZT, modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as coating for neural crest cell (NCC)-based toxicity testing. Human NCC were generated from pluripotent stem cells and used in the automated version of a NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed for a high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.

Origin (projects)

  Rovida, Costanza; Barton-Maclaren, Tara; Benfenati, Emilio; Caloni, Francesca; Chandrasekera, Charu; Dietrich, Daniel R.; Kisitu, Jaffar; Leist, Marcel; Pallocca, Giorgia; Hartung, Thomas (2020): Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 4. - S. 579-606. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology

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Read-across (RAx) translates available information from well-characterized chemicals to the substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g. EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimisation to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target, in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular the RAx guidance, being worked out by the European Commission’s H2020 project EU-ToxRisk, together with many external partners with regulatory experience, is given.

Origin (projects)

  Krebs, Alice; Nyffeler, Johanna; Karreman, Christiaan; Schmidt, Béla Z; Kappenberg, Franziska; Mellert, Jan; Pallocca, Giorgia; Pastor, Manuel; Rahnenführer, Jörg; Leist, Marcel (2020): Determination of benchmark concentrations and their statistical uncertainty for cytotoxicity test data and functional in vitro assays Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 1. - ALTEX Edition. - ISSN 1868-596X. - eISSN 1868-596X

Determination of benchmark concentrations and their statistical uncertainty for cytotoxicity test data and functional in vitro assays

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Many toxicological test methods, including assays of cell viability and function, require an evaluation of concentration-response data. This often involves curve fitting, and the resulting mathematical functions are then used to determine the concentration at which a certain deviation from the control value occurs (e.g. a decrease of cell viability by 15%). Such a threshold is called the benchmark response (BMR). For a toxicological test, it is often of interest to determine the concentration of test compound at which a pre-defined BMR of e.g. 10, 25 or 50% is reached. The concentration at which the modelled curve crosses the BMR is called the benchmark concentration (BMC). We present a user-friendly, web-based tool (BMCeasy), designed for operators without programming skills and profound statistical background, to determine BMCs and their confidence intervals. BMCeasy allows simultaneous analysis of viability plus a functional test endpoint, and it yields absolute BMCs with confidence intervals for any BMR. Besides an explanation of the algorithm underlying BMCeasy, this article also gives multiple examples of data outputs. BMCeasy was used within the EU-ToxRisk project for preparing data packages that were submitted to regulatory authorities, demonstrating the real-life applicability of the tool.

Origin (projects)

  Sillé, Fenna C. M.; Karakitsios, Spyros; Kleensang, Andre; Koehler, Kirsten; Maertens, Alexandra; Miller, Gary W.; Prasse, Carsten; Quiros-Alcala, Lesliam; Ramachandran, Gurumurthy; Hartung, Thomas (2020): The exposome : a new approach for risk assessment Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 1. - S. 3-23. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

The exposome : a new approach for risk assessment

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Complementing the human genome with an exposome reflects the increasingly obvious impact of environmental exposure, which far exceeds the role of genetics, on human health. Considering the complexity of exposures and, in addition, the reactions of the body to exposures - i.e., the exposome - reverses classical exposure science where the precise measurement of single or few exposures is associated with specific health or environmental effects. The complete description of an individual's exposome is impossible; even less so is that of a population. We can, however, cast a wider net by foregoing some rigor in assessment and compensating with the statistical power of rich datasets. The advent of omics technologies enables a relatively cheap, high-content description of the biological effects of substances, especially in tissues and biofluids. They can be combined with many other rich data-streams, creating big data of exposure and effect. Computational methods increasingly allow data integration, discerning the signal from the noise and formulating hypotheses of exposure-effect relationships. These can be followed up in a targeted way. With a better exposure element in the risk equation, exposomics - new kid on the block of risk assessment - promises to identify novel exposure (interactions) and health/environment effect associations. This may also create opportunities to prioritize the more relevant chemicals for risk assessment, thereby lowering the burden on hazard assessment in an expo-sure-driven approach. Technological developments and synergies between approaches, quality assurance (ultimately as Good Exposome Practices), and the integration of mechanistic thinking will advance this approach.

Origin (projects)

  Brüll, Markus; Spreng, Anna-Sophie; Gutbier, Simon; Loser, Dominik; Krebs, Alice; Reich, Marvin; Kraushaar, Udo; Britschgi, Markus; Patsch, Christoph; Leist, Marcel (2020): Incorporation of stem cell-derived astrocytes into neuronal organoids to allow neuro-glial interactions in toxicological studies Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 3. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-596X

Incorporation of stem cell-derived astrocytes into neuronal organoids to allow neuro-glial interactions in toxicological studies

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Human cell-based neural organoids are increasingly being used for investigations of neurotoxicity, and to study the pathophysiology of neurodegenerative diseases. Here, we present a fast and robust method to generate 3D cultured human dopaminergic neurons (LUHMES) for toxicity testing and long-term culture. Moreover, a plating step was introduced to allow generation of neurite networks with defined 2D orientation and several mm length, while all cell bodies (somata) remained in a 3D, dome-like structure. These cultures, named here 2.5D (for 2.5 dimensional), offer new approaches to quantify toxicant effects on organoids by standard technology and high throughput. For instance, the system reacted to the parkinsonian model toxicants MPP+, rotenone, MG-132 and the ferroptosis-inducer erastin. Moreover, stable incorporation of human stem cell-derived astrocytes or microglia was possible. Added astrocytes stabilized the post mitotic state of the LUHMES neurons and thereby allowed the formation of a stable micro-physiological system. We observed neuroprotection against the proteasome inhibitor MG-132 and the ferroptosis-inducer erastin by such glia. This exemplifies the crucial protective role of astrocytes in neurodegeneration. The modularity of the system was further employed to incorporate microglia together with astrocytes into the organoids. Such ratio-defined, three cell type-based organoids will allow new approaches to study human pathophysiology and toxicology of the nervous system.

Origin (projects)

  Troger, Florentina; Delp, Johannes; Funke, Melina; van der Stel, Wanda; Colas, Claire; Leist, Marcel; van de Water, Bob; Ecker, Gerhard F. (2020): Identification of mitochondrial toxicants by combined in silico and in vitro studies : a structure-based view on the Adverse Outcome Pathway Computational Toxicology ; 14 (2020). - 100123. - Elsevier. - eISSN 2468-1113

Identification of mitochondrial toxicants by combined in silico and in vitro studies : a structure-based view on the Adverse Outcome Pathway

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Drugs that modulate mitochondrial function can cause severe adverse effects. Unfortunately, mitochondrial toxicity is often not detected in animal models, which stresses the need for predictive in silico approaches. In this study we present a model for predicting mitochondrial toxicity focusing on human mitochondrial respiratory complex I (CI) inhibition by combining structure-based methods with machine learning. The structure-based studies are based on CI inhibition by the pesticide rotenone, which is known to induce parkinsonian motor deficits, and its analogue deguelin. After predicting a common binding mode for these two compounds using induced-fit docking, two structure-based pharmacophore models were created and used for virtual screening of DrugBank and the Chemspace library. The hit list was further refined by three different machine learning models, and the top ranked compounds were selected for experimental testing. Using a tiered approach, the compounds were tested in three distinct in vitro assays, which led to the identification of three specific CI inhibitors. These results demonstrate that risk assessment and hazard analysis can benefit from combining structure-based methods and machine learning.

Origin (projects)

  Kisitu, Jaffar; Hollert, Henner; Fisher, Ciarán; Leist, Marcel (2020): Chemical concentrations in cell culture compartments (C5) : free concentrations Alternatives to Animal Experimentation : ALTEX ; 37 (2020), 4. - S. 693-708. - Springer Spektrum. - ISSN 1868-596X. - eISSN 1868-8551

Chemical concentrations in cell culture compartments (C5) : free concentrations

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In biological systems (cell culture media, cells, body fluids), drugs/toxicants are usually not freely dissolved but partially bound to biomolecules; only a fraction of the chemical is free/unbound (fu). To predict pharmacological effects and toxicity, it is important that the fu of the drug is known. As the differences between free and nominal concentrations are determined by test system parameters (e.g., the protein and lipid content, and the type of surface material), comparison of nominal concentrations between two different new approach methods (NAM) may lead to faulty conclusions. The same problem exists when in vitro concentrations are compared to those in human subjects. Therefore, the respective fu of a chemical in a test system needs to be determined for in vitro-to-in vivo extrapolations (IVIVE). Besides direct measurements, prediction models can help to obtain fu. Here we describe a simplified approach to approximate fu and provide background information on the underlying assumptions. Comparative predictions and measurements of fu of various drugs are shown to exemplify the approach. Basic input data, like protein and lipid concentrations, are also provided. Beyond such test systems data, the only required chemical-specific inputs are the lipophilicity of the candidate drug and its ionization state, as determined by the dissociation constants of its acidic or basic groups. This overview is intended to be used by any lab scientist without specific toxicokinetics training to obtain an estimate of fu in a given cell culture medium.

Origin (projects)

  Escher, Sylvia E.; Kamp, Hennicke; Bennekou, Susanne H.; Bitsch, Annette; Fisher, Ciarán; Graepel, Rabea; Hengstler, Jan G.; Herzler, Matthias; Leist, Marcel; van de Water, Bob (2019): Towards grouping concepts based on new approach methodologies in chemical hazard assessment : the read-across approach of the EU-ToxRisk project Archives of Toxicology ; 93 (2019), 12. - S. 3643-3667. - ISSN 0340-5761. - eISSN 1432-0738

Towards grouping concepts based on new approach methodologies in chemical hazard assessment : the read-across approach of the EU-ToxRisk project

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Read-across is one of the most frequently used alternative tools for hazard assessment, in particular for complex endpoints such as repeated dose or developmental and reproductive toxicity. Read-across extrapolates the outcome of a specific toxicological in vivo endpoint from tested (source) compounds to "similar" (target) compound(s). If appropriately applied, a read-across approach can be used instead of de novo animal testing. The read-across approach starts with structural/physicochemical similarity between target and source compounds, assuming that similar structural characteristics lead to similar human hazards. In addition, similarity also has to be shown for the toxicokinetic and toxicodynamic properties of the grouped compounds. To date, many read-across cases fail to demonstrate toxicokinetic and toxicodynamic similarities. New concepts, in vitro and in silico tools are needed to better characterise these properties, collectively called new approach methodologies (NAMs). This white paper outlines a general read-across assessment concept using NAMs to support hazard characterization of the grouped compounds by generating data on their dynamic and kinetic properties. Based on the overarching read-across hypothesis, the read-across workflow suggests targeted or untargeted NAM testing also outlining how mechanistic knowledge such as adverse outcome pathways (AOPs) can be utilized. Toxicokinetic models (biokinetic and PBPK), enriched by in vitro parameters such as plasma protein binding and hepatocellular clearance, are proposed to show (dis)similarity of target and source compound toxicokinetics. Furthermore, in vitro to in vivo extrapolation is proposed to predict a human equivalent dose, as potential point of departure for risk assessment. Finally, the generated NAM data are anchored to the existing in vivo data of source compounds to predict the hazard of the target compound in a qualitative and/or quantitative manner. To build this EU-ToxRisk read-across concept, case studies have been conducted and discussed with the regulatory community. These case studies are briefly outlined.

Origin (projects)

  Hartung, Thomas (2019): Predicting toxicity of chemicals : software beats animal testing EFSA Journal ; 17 (2019), S1. - e170710. - eISSN 1831-4732

Predicting toxicity of chemicals : software beats animal testing

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We created earlier a large machine‐readable database of 10,000 chemicals and 800,000 associated studies by natural language processing of the public parts of Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) registrations until December 2014. This database was used to assess the reproducibility of the six most frequently used Organisation for Economic Co‐operation and Development (OECD) guideline tests. These tests consume 55% of all animals in safety testing in Europe, i.e. about 600,000 animals. With 350–750 chemicals with multiple results per test, reproducibility (balanced accuracy) was 81% and 69% of toxic substances were found again in a repeat experiment (sensitivity 69%). Inspired by the increasingly used read‐across approach, we created a new type of QSAR, which is based on similarity of chemicals and not on chemical descriptors. A landscape of the chemical universe using 10 million structures was calculated, when based on Tanimoto indices similar chemicals are close and dissimilar chemicals far from each other. This allows placing any chemical of interest into the map and evaluating the information available for surrounding chemicals. In a data fusion approach, in which 74 different properties were taken into consideration, machine learning (random forest) allowed a fivefold cross‐validation for 190,000 (non‐) hazard labels of chemicals for which nine hazards were predicted. The balanced accuracy of this approach was 87% with a sensitivity of 89%. Each prediction comes with a certainty measure based on the homogeneity of data and distance of neighbours. Ongoing developments and future opportunities are discussed.

Origin (projects)

  Beilmann, Mario; Boonen, Harrie; Czich, Andreas; Dear, Gordon; Hewitt, Philip; Daneshian, Mardas; Hartung, Thomas; Leist, Marcel; Rovida, Costanza; Steger-Hartmann, Thomas (2019): Optimizing drug discovery by Investigative Toxicology : Current and future trends Alternatives to Animal Experimentation : ALTEX ; 36 (2019), 2. - S. 289-313. - ISSN 1868-596X. - eISSN 1868-8551

Optimizing drug discovery by Investigative Toxicology : Current and future trends

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Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives.

Origin (projects)

  Hirsch, Cordula; Schildknecht, Stefan (2019): In vitro research reproducibility : Keeping up high standards Frontiers in Pharmacology ; 10 (2019). - 1484. - eISSN 1663-9812

In vitro research reproducibility : Keeping up high standards

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Concern regarding the reproducibility of observations in life science research has emerged in recent years, particularly in view of unfavorable experiences with preclinical in vivo research. The use of cell-based systems has increasingly replaced in vivo research and the application of in vitro models enjoys an ever-growing popularity. To avoid repeating past mistakes, high standards of reproducibility and reliability must be established and maintained in the field of in vitro biomedical research. Detailed guidance documenting the appropriate handling of cells has been authored but was received with quite disparate perception by different branches in biomedical research. In that regard, we intend to raise awareness of the reproducibility issue among scientists in all branches of contemporary life science research and their individual responsibility in this matter. We have herein compiled a selection of the most susceptible steps of everyday in vitro cell culture routines that have the potential to influence cell quality and recommend practices to minimize the likelihood of poor cell quality impairing reproducibility with modest investment of time and resources.

Origin (projects)

  Kisitu, Jaffar; Hougaard Bennekou, Susanne; Leist, Marcel (2019): Chemical concentrations in cell culture compartments (C5) : concentration definitions Alternatives to Animal Experimentation : ALTEX ; 36 (2019), 1. - S. 154-160. - ISSN 1868-596X. - eISSN 1868-8551

Chemical concentrations in cell culture compartments (C5) : concentration definitions

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Some laboratory issues are taken for granted as they seem to be simple and not worth much thought. This applies to "concentrations of a chemical tested for bioactivity/toxicity". Can there be any issue about weighing a compound, diluting it in culture medium and calculating the final mass (or particle number)-to-volume ratio? We discuss here some basic concepts about concentrations and their units, addressing also differences between "dose" and "concentration". The problem of calculated nominal concentrations not necessarily corresponding to local concentrations (relevant for biological effects of a chemical) is highlighted. We present and exemplify different concentration measures, for instance those relying on weight, volume, or particle number of the test compound in a given volume; we also include normalizations to the mass, protein content, or cell number of the reference system. Interconversion is discussed as a major, often unresolved, issue. We put this into the context of the overall objective of defining concentrations, i.e., the determination of threshold values of bioactivity (e.g., an EC50). As standard approach for data display, the negative decadic logarithm of the molar concentrations (-log(M)) is recommended here, but arguments are also presented for exceptions from such a rule. These basic definitions are meant as a foundation for follow-up articles that examine the concepts of nominal, free, and intracellular concentrations to provide guidance on how to relate in vitro concentrations to in vivo doses by in vitro-to-in vivo extrapolation (IVIVE) in order to advance the use of new approach methods (NAM) in regulatory decision making.

Origin (projects)

  Krebs, Alice; Waldmann, Tanja; Rovida, Costanza; Pallocca, Giorgia; Hartung, Thomas; Gantner, Florian; Exner, Thomas E.; Dietrich, Daniel R.; Busquet, Francois; Leist, Marcel (2019): Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data Alternatives to Animal Experimentation : ALTEX ; 36 (2019), 4. - S. 682-699. - ISSN 1868-596X. - eISSN 1868-8551

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data

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Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.

Origin (projects)

  Karreman, Christiaan; Chovancova, Petra; Leist, Marcel (2019): SUIKER : Quantification of antigens in cell organelles, neurites and cellular sub-structures by imaging Alternatives to Animal Experimentation : ALTEX ; 36 (2019), 3. - S. 518-520. - ISSN 1868-596X. - eISSN 1868-8551

SUIKER : Quantification of antigens in cell organelles, neurites and cellular sub-structures by imaging

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Quantification of fluorescence colocalization and intensity of strongly overlapping cells, e.g., neuronal cultures, is challenging for programs that use image segmentation to identify cells as individual objects. Moreover, learning to use and apply one of the large imaging packages can be very time- and/or resource-demanding. Therefore, we developed the free and highly interactive image analysis program SUIKER (program for SUperImposing KEy Regions) that quantifies colocalization of different proteins or other features over an entire image field. The software allows definition of cellular subareas by subtraction ("punching out") of structures identified in one channel from structures in a second channel. This allows, e.g., definition of neurites without cell bodies. Moreover, normalization to live or total cell numbers is possible. Providing a detailed manual that contains image analysis examples, we demonstrate how the program uses a combination of colocalization information and fluorescence intensity to quantify carbohydrate-specific stains on neurites. SUIKER can import any multichannel histology or cell culture image, builds on user-guided threshold setting, batch processes large image stacks, and exports all data (including the settings, results and metadata) in flexible formats to be used in Excel.

Origin (projects)

  Gu, Xiaolong; Albrecht, Wiebke; Edlund, Karolina; Kappenberg, Franziska; Rahnenführer, Jörg; Leist, Marcel; Moritz, Wolfgang; Godoy, Patricio; Cadenas, Cristina; Hengstler, Jan G. (2018): Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes Archives of Toxicology ; 92 (2018), 12. - S. 3505-3515. - ISSN 0340-5761. - eISSN 1432-0738

Relevance of the incubation period in cytotoxicity testing with primary human hepatocytes

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Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity.

Origin (projects)

  Grinberg, Marianna; Stöber, Regina M.; Albrecht, Wiebke; Edlund, Karolina; Godoy, Patricio; Leist, Marcel; Gardner, Iain; Taboureau, Olivier; Rahnenführer, Jörg; Hengstler, Jan G. (2018): Toxicogenomics directory of rat hepatotoxicants in vivo and in cultivated hepatocytes Archives of toxicology ; 92 (2018), 12. - S. 3517-3533. - ISSN 0340-5761. - eISSN 1432-0738

Toxicogenomics directory of rat hepatotoxicants in vivo and in cultivated hepatocytes

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Transcriptomics is developing into an invaluable tool in toxicology. The aim of this study was, using a transcriptomics approach, to identify genes that respond similar to many different chemicals (including drugs and industrial compounds) in both rat liver in vivo and in cultivated hepatocytes. For this purpose, we analyzed Affymetrix microarray expression data from 162 compounds that were previously tested in a concentration-dependent manner in rat livers in vivo and in rat hepatocytes cultivated in sandwich culture. These data were obtained from the Japanese Toxicogenomics Project (TGP) and North Rhine-Westphalian (NRW) data sets, which represent 138 and 29 compounds, respectively, and have only 5 compounds in common between them. The in vitro gene expression data from the NRW data set were generated in the present study, while TGP is publicly available. For each of the data sets, the overlap between up- or down-regulated genes in vitro and in vivo was identified, and named in vitro-in vivo consensus genes. Interestingly, the in vivo-in vitro consensus genes overlapped to a remarkable extent between both data sets, and were 21-times (upregulated genes) or 12-times (down-regulated genes) enriched compared to random expectation. Finally, the genes in the TGP and NRW overlap were used to identify the upregulated genes with the highest compound coverage, resulting in a seven-gene set of Cyp1a1, Ugt2b1, Cdkn1a, Mdm2, Aldh1a1, Cyp4a3, and Ehhadh. This seven-gene set was then successfully tested with structural analogues of valproic acid that are not present in the TGP and NRW data sets. In conclusion, the seven-gene set identified in the present study responds similarly in vitro and in vivo to a wide range of different chemicals. Despite these promising results with the seven-gene set, transcriptomics with cultivated rat hepatocytes remains a challenge, because in general many genes are up- or downregulated by in vitro culture per se, respond differently to test compounds in vitro and in vivo, and/or show higher variability in the in vitro system compared to the corresponding in vivo data.

Origin (projects)

  Gutbier, Simon; May, Patrick; Berthelot, Sylvie; Krishna, Abhimanyu; Trefzer, Timo; Behbehani, Mehri; Efremova, Liudmila; Delp, Johannes; Gstraunthaler, Gerhard; Waldmann, Tanja; Leist, Marcel (2018): Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift Archives of Toxicology ; 92 (2018), 12. - S. 3487-3503. - ISSN 0340-5761. - eISSN 1432-0738

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift

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Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The "ATCC" cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while "UKN" cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells.

Origin (projects)

  Gutbier, Simon; Spreng, Anna-Sophie; Delp, Johannes; Schildknecht, Stefan; Karreman, Christiaan; Suciu, Ilinca; Brunner, Thomas; Gröttrup, Marcus; Leist, Marcel (2018): Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress Cell Death & Differentiation ; 25 (2018), 12. - S. 2101-2117. - ISSN 1350-9047. - eISSN 1476-5403

Prevention of neuronal apoptosis by astrocytes through thiol-mediated stress response modulation and accelerated recovery from proteotoxic stress

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The development of drugs directly interfering with neurodegeneration has proven to be astonishingly difficult. Alternative therapeutic approaches could result from a better understanding of the supportive function of glial cells for stressed neurons. Therefore, here, we investigated the mechanisms involved in the endogenous neuro-defensive activity of astrocytes. A well-established model of postmitotic human dopaminergic neurons (LUHMES cells) was used in the absence ('LUHMES' mono-culture) or presence ('co-culture') of astrocytes. Inhibition of the LUHMES proteasome led to proteotoxic (protein aggregates; ATF-4 induction) and oxidative (GSH-depletion; NRF-2 induction) stress, followed by neuronal apoptosis. The presence of astrocytes attenuated the neuronal stress response, and drastically reduced neurodegeneration. A similar difference between LUHMES mono- and co-cultures was observed, when proteotoxic and oxidative stress was triggered indirectly by inhibitors of mitochondrial function (rotenone, MPP+). Human and murine astrocytes continuously released glutathione (GSH) into the medium, and transfer of glia-conditioned medium was sufficient to rescue LUHMES, unless it was depleted for GSH. Also, direct addition of GSH to LUHMES rescued the neurons from inhibition of the proteasome. Both astrocytes and GSH blunted the neuronal ATF-4 response and similarly upregulated NRF-1/NFE2L1, a transcription factor counter-regulating neuronal proteotoxic stress. Astrocyte co-culture also helped to recover the neurons' ability to degrade aggregated poly-ubiquitinated proteins. Overexpression of NRF-1 attenuated the toxicity of proteasome inhibition, while knockdown increased toxicity. Thus, astrocytic thiol supply increased neuronal resilience to various proteotoxic stressors by simultaneously attenuating cell death-related stress responses, and enhancing the recovery from proteotoxic stress through upregulation of NRF-1.

Origin (projects)

  Luechtefeld, Thomas; Marsh, Dan; Rowlands, Craig; Hartung, Thomas (2018): Machine Learning of Toxicological Big Data Enables Read-Across Structure Activity Relationships (RASAR) Outperforming Animal Test Reproducibility Toxicological Sciences ; 165 (2018), 1. - S. 198-212. - ISSN 1096-6080. - eISSN 1096-0929

Machine Learning of Toxicological Big Data Enables Read-Across Structure Activity Relationships (RASAR) Outperforming Animal Test Reproducibility

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Earlier we created a chemical hazard database via natural language processing of dossiers submitted to the European Chemical Agency with approximately 10 000 chemicals. We identified repeat OECD guideline tests to establish reproducibility of acute oral and dermal toxicity, eye and skin irritation, mutagenicity and skin sensitization. Based on 350–700+ chemicals each, the probability that an OECD guideline animal test would output the same result in a repeat test was 78%–96% (sensitivity 50%–87%). An expanded database with more than 866 000 chemical properties/hazards was used as training data and to model health hazards and chemical properties. The constructed models automate and extend the read-across method of chemical classification. The novel models called RASARs (read-across structure activity relationship) use binary fingerprints and Jaccard distance to define chemical similarity. A large chemical similarity adjacency matrix is constructed from this similarity metric and is used to derive feature vectors for supervised learning. We show results on 9 health hazards from 2 kinds of RASARs—“Simple” and “Data Fusion”. The “Simple” RASAR seeks to duplicate the traditional read-across method, predicting hazard from chemical analogs with known hazard data. The “Data Fusion” RASAR extends this concept by creating large feature vectors from all available property data rather than only the modeled hazard. Simple RASAR models tested in cross-validation achieve 70%–80% balanced accuracies with constraints on tested compounds. Cross validation of data fusion RASARs show balanced accuracies in the 80%–95% range across 9 health hazards with no constraints on tested compounds.

Origin (projects)

  Fritsche, Ellen; Grandjean, Philippe; Crofton, Kevin M.; Aschner, Michael; Goldberg, Alan; Heinonen, Tuula; Hessel, Ellen V. S.; Hogberg, Helena T.; Leist, Marcel; Bal-Price, Anna (2018): Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes Toxicology and applied pharmacology ; 354 (2018). - S. 3-6. - ISSN 0041-008X. - eISSN 1096-0333

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes

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This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

Origin (projects)

  Delp, Johannes; Gutbier, Simon; Cerff, Martin; Hartung, Thomas; Borlinghaus, Hanna; Schreiber, Falk; Waldmann, Tanja; Kempa, Stefan; Nöh, Katharina; Leist, Marcel (2018): Stage-specific metabolic features of differentiating neurons : Implications for toxicant sensitivity Toxicology and Applied Pharmacology ; 354 (2018). - S. 64-80. - ISSN 0041-008X. - eISSN 1096-0333

Stage-specific metabolic features of differentiating neurons : Implications for toxicant sensitivity

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Developmental neurotoxicity (DNT) may be induced when chemicals disturb a key neurodevelopmental process, and many tests focus on this type of toxicity. Alternatively, DNT may occur when chemicals are cytotoxic only during a specific neurodevelopmental stage. The toxicant sensitivity is affected by the expression of toxicant targets and by resilience factors. Although cellular metabolism plays an important role, little is known how it changes during human neurogenesis, and how potential alterations affect toxicant sensitivity of mature vs. immature neurons. We used immature (d0) and mature (d6) LUHMES cells (dopaminergic human neurons) to provide initial answers to these questions. Transcriptome profiling and characterization of energy metabolism suggested a switch from predominantly glycolytic energy generation to a more pronounced contribution of the tricarboxylic acid cycle (TCA) during neuronal maturation. Therefore, we used pulsed stable isotope-resolved metabolomics (pSIRM) to determine intracellular metabolite pool sizes (concentrations), and isotopically non-stationary 13C-metabolic flux analysis (INST 13C-MFA) to calculate metabolic fluxes. We found that d0 cells mainly use glutamine to fuel the TCA. Furthermore, they rely on extracellular pyruvate to allow continuous growth. This metabolic situation does not allow for mitochondrial or glycolytic spare capacity, i.e. the ability to adapt energy generation to altered needs. Accordingly, neuronal precursor cells displayed a higher sensitivity to several mitochondrial toxicants than mature neurons differentiated from them. In summary, this study shows that precursor cells lose their glutamine dependency during differentiation while they gain flexibility of energy generation and thereby increase their resistance to low concentrations of mitochondrial toxicants.

Origin (projects)

  Nyffeler, Johanna; Chovancova, Petra; Dolde, Xenia; Holzer, Anna-Katharina; Purvanov, Vladimir; Kindinger, Ilona; Kerins, Anna; Higton, David; Legler, Daniel; Leist, Marcel (2018): A structure-activity relationship linking non-planar PCBs to functional deficits of neural crest cells : new roles for connexins Archives of toxicology ; 92 (2018), 3. - S. 1225-1247. - ISSN 0340-5761. - eISSN 1432-0738

A structure-activity relationship linking non-planar PCBs to functional deficits of neural crest cells : new roles for connexins

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Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the 'migration-inhibition of NCC (cMINC)' assay, a large number of toxicity mechanisms and protein targets can be covered. However, the key events that lead to the adverse effects of a given chemical or group of related compounds are hard to elucidate. To address this issue, we explored here, whether the establishment of two overlapping structure-activity relationships (SAR)-linking chemical structure on the one hand to a phenotypic test outcome, and on the other hand to a mechanistic endpoint-was useful as strategy to identify relevant toxicity mechanisms. For this purpose, we chose polychlorinated biphenyls (PCB) as a large group of related, but still toxicologically and physicochemically diverse structures. We obtained concentration-dependent data for 26 PCBs in the cMINC assay. Moreover, the test chemicals were evaluated by a new high-content imaging method for their effect on cellular re-distribution of connexin43 and for their capacity to inhibit gap junctions. Non-planar PCBs inhibited NCC migration. The potency (1-10 µM) correlated with the number of ortho-chlorine substituents; non-ortho-chloro (planar) PCBs were non-toxic. The toxicity to NCC partially correlated with gap junction inhibition, while it fully correlated (p < 0.0004) with connexin43 cellular re-distribution. Thus, our double-SAR strategy revealed a mechanistic step tightly linked to NCC toxicity of PCBs. Connexin43 patterns in NCC may be explored as a new endpoint relevant to developmental toxicity screening.

Origin (projects)

  Delp, Johannes; Gutbier, Simon; Klima, Stefanie; Hoelting, Lisa; Pinto-Gil, Kevin; Hsieh, Jui-Hua; Aichem, Michael; Klein, Karsten; Schreiber, Falk; Leist, Marcel (2018): A high-throughput approach to identify specific neurotoxicants/ developmental toxicants in human neuronal cell function assays Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 2. - S. 235-253. - ISSN 1868-596X. - eISSN 1868-8551

A high-throughput approach to identify specific neurotoxicants/ developmental toxicants in human neuronal cell function assays

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The (developmental) neurotoxicity hazard is still unknown for most chemicals. Establishing a test battery covering most of the relevant adverse outcome pathways may close this gap, without requiring a huge animal experimentation program. Ideally, each of the assays would cover multiple mechanisms of toxicity. One candidate test is the human LUHMES cell-based NeuriTox test. To evaluate its readiness for larger-scale testing, a proof of concept library assembled by the U.S. National Toxicology Program (NTP) was screened. Of the 75 unique compounds, seven were defined as specifically neurotoxic after the hit-confirmation phase and additional ten compounds were generally cytotoxic within the concentration range of up to 20 micromolar. As complementary approach, the library was screened in the PeriTox test, which identifies toxicants affecting the human peripheral nervous system. Of the eight PeriTox hits, five were similar to the NeuriTox hits: rotenone, colchicine, diethylstilbestrol, berberine chloride, and valinomycin. The unique NeuriTox hit, methyl-phenylpyridinium (MPP+) is known from in vivo studies to affect only dopaminergic neurons (which LUHMES cells are). Conversely, the known peripheral neurotoxicant acrylamide was picked up in the PeriTox, but not in the NeuriTox assay. All of the five common hits had also been identified in the published neural crest migration (cMINC) assay, while none of them emerged as cardiotoxicant in a previous screen using the same library. These comparative data suggest that complementary in vitro tests can pick up a broad range of toxicants, and that multiple test results might help to predict organ specificity patterns.

Origin (projects)

  Pamies, David; Bal-Price, Anna K.; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Hartung, Thomas; Leist, Marcel; Daneshian, Mardas (2018): Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 3. - S. 353-378. - ISSN 1868-596X. - eISSN 1868-8551

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems

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A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Origin (projects)

  Krebs, Alice; Nyffeler, Johanna; Rahnenführer, Jörg; Leist, Marcel (2018): Normalization of data for viability and relative cell function curves Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 2. - S. 268-271. - ISSN 1868-596X. - eISSN 1868-8551

Normalization of data for viability and relative cell function curves

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Many types of assays in cell biology, pharmacology and toxicology generate data in which a parameter is measured in a reference system (negative control) and then also under conditions of increasing stress or drug exposure. To make such data easily comparable, they are normalized, i.e., the initial value of the system (e.g., viability or transport function) is set to 100%, and all data are indicated relative to this value. Then, curves are fitted through the data points and summary data of the system behavior are determined. For this, a benchmark response (BMR) is given (e.g., a curve drop by 15 or 50%), and the corresponding benchmark concentration (BMC15 or BMC50) is determined. Especially for low BMRs, this procedure is not very robust and often results in incorrect summary data. It is often neglected that a second normalization (re-normalization) is necessary to make the data suitable for curve fitting. It is also frequently overlooked that this requires knowledge of the system behavior at very low stress conditions. Here, good in vitro practice guidance for the re-normalization procedure is provided so that data of higher fidelity can be generated and presented.

Origin (projects)

  Terron, Andrea; Bal-Price, Anna; Paini, Alicia; Monnet-Tschudi, Florianne; Bennekou, Susanne Hougaard; Leist, Marcel; Schildknecht, Stefan (2018): An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition Archives of Toxicology ; 92 (2018), 1. - S. 41-82. - ISSN 0340-5761. - eISSN 1432-0738

An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition

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Epidemiological studies have observed an association between pesticide exposure and the development of Parkinson's disease, but have not established causality. The concept of an adverse outcome pathway (AOP) has been developed as a framework for the organization of available information linking the modulation of a molecular target [molecular initiating event (MIE)], via a sequence of essential biological key events (KEs), with an adverse outcome (AO). Here, we present an AOP covering the toxicological pathways that link the binding of an inhibitor to mitochondrial complex I (i.e., the MIE) with the onset of parkinsonian motor deficits (i.e., the AO). This AOP was developed according to the Organisation for Economic Co-operation and Development guidelines and uploaded to the AOP database. The KEs linking complex I inhibition to parkinsonian motor deficits are mitochondrial dysfunction, impaired proteostasis, neuroinflammation, and the degeneration of dopaminergic neurons of the substantia nigra. These KEs, by convention, were linearly organized. However, there was also evidence of additional feed-forward connections and shortcuts between the KEs, possibly depending on the intensity of the insult and the model system applied. The present AOP demonstrates mechanistic plausibility for epidemiological observations on a relationship between pesticide exposure and an elevated risk for Parkinson's disease development.

Origin (projects)

  Smirnova, Lena; Kleinstreuer, Nicole; Corvi, Raffaella; Levchenko, Andre; Fitzpatrick, Suzanne C; Hartung, Thomas (2018): 3S - Systematic, systemic, and systems biology and toxicology Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 2. - S. 139-162. - ISSN 1868-596X. - eISSN 1868-8551

3S - Systematic, systemic, and systems biology and toxicology

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A biological system is more than the sum of its parts – it accomplishes many functions via synergy. Deconstructing the system down to the molecular mechanism level necessitates the complement of reconstructing functions on all levels, i.e., in our conceptualization of biology and its perturbations, our experimental models and computer modelling.

Toxicology contains the somewhat arbitrary subclass “systemic toxicities”; however, there is no relevant toxic insult or general disease that is not systemic. At least inflammation and repair are involved that require coordinated signaling mechanisms across the organism. However, the more body components involved, the greater the challenge to reca­pitulate such toxicities using non-animal models. Here, the shortcomings of current systemic testing and the development of alternative approaches are summarized.

We argue that we need a systematic approach to integrating existing knowledge as exemplified by systematic reviews and other evidence-based approaches. Such knowledge can guide us in modelling these systems using bioengineering and virtual computer models, i.e., via systems biology or systems toxicology approaches. Experimental multi-organ-on-chip and microphysiological systems (MPS) provide a more physiological view of the organism, facilitating more comprehensive coverage of systemic toxicities, i.e., the perturbation on organism level, without using substitute organisms (animals). The next challenge is to establish disease models, i.e., micropathophysiological systems (MPPS), to expand their utility to encompass biomedicine. Combining computational and experimental systems approaches and the chal­lenges of validating them are discussed. The suggested 3S approach promises to leverage 21st century technology and systematic thinking to achieve a paradigm change in studying systemic effects.

Origin (projects)

  Bal-Price, Anna K.; Hogberg, Helena T.; Crofton, Kevin M.; Daneshian, Mardas; FitzGerald, Rex E.; Fritsche, Ellen; Heinonen, Tuula; Klima, Stefanie; Waldmann, Tanja; Leist, Marcel (2018): Recommendation on test readiness criteria for new approach methods in toxicology : Exemplified for developmental neurotoxicity Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 3. - S. 306-352. - ISSN 1868-596X. - eISSN 1868-8551

Recommendation on test readiness criteria for new approach methods in toxicology : Exemplified for developmental neurotoxicity

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Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

Origin (projects)

  Luechtefeld, Thomas; Rowlands, Craig; Hartung, Thomas (2018): Big-Data and Machine Learning to Revamp Computational Toxicology and its Use in Risk Assessment Toxicology Research ; 7 (2018), 5. - S. 732-744. - ISSN 2045-452X. - eISSN 2045-4538

Big-Data and Machine Learning to Revamp Computational Toxicology and its Use in Risk Assessment

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The creation of large toxicological databases and advances in machine-learning techniques have empowered computational approaches in toxicology. Work with these large databases based on regulatory data has allowed reproducibility assessment of animal models, which highlight weaknesses in traditional in vivo methods. This should lower the bars for the introduction of new approaches and represents a benchmark what is achievable for any alternative method validated against these methods. Quantitative Structure Activity Relationships (QSAR) models for skin sensitization, eye irritation, and other human health hazards based on these big databases, however, also have made apparent some of the challenges facing computational modeling, including validation challenges, model interpretation issues, and model selection issues. A first implementation of machine learning-based predictions termed REACHacross achieved unprecedented sensitivities of >80% with specificities >70% in predicting the six most common acute and topical hazards covering about two thirds of the chemical universe. While this is awaiting formal validation, it demonstrates the new quality introduced by big data and modern data-mining technologies. The rapid increase in the diversity and number of computational models, as well as the data they are based on, create challenges and opportunities for the use of computational methods.

Origin (projects)

  Leist, Marcel; Hengstler, Jan G. (2018): Essential components of methods papers Alternatives to Animal Experimentation : ALTEX ; 35 (2018), 3. - S. 429-432. - ISSN 1868-596X. - eISSN 1868-8551

Essential components of methods papers

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Methods papers are important for the progress of biomedical research, as they provide the essential tools to explore new questions and help to better answer old ones. However, it is often not clear how a methods paper differs from a methods protocol. Confusion between these two very different types of publication is widespread. The resultant misunderstanding contributes to a relatively poor reputation of methods research in biology despite the fact that many Nobel prizes have been awarded specifically for method development. Here, the key components of a methods paper are summarized: (i) methods description, (ii) performance standards, (iii) applicability domains, (iv) evidence for advances compared to the state-of-the-art, (v) exemplification of the method by practical application. In addition, information domains are discussed that are desirable but may be provided on a case-by-case basis or over the course of a series of papers: (vi) method robustness, (vii) accuracy and (viii) precision measures, including various quantifications of method performance, and (ix) measures of uncertainty, including a sensitivity analysis. Finally, elements of the overall framing of the method description are highlighted. These include the scientific, technical and, e.g., toxicological rationale for the method, and also the prediction model, i.e., the procedure used to transform primary data into new information.

Origin (projects)

  Leist, Marcel; Ghallab, Ahmed; Graepel, Rabea; Marchan, Rosemarie; Hassan, Reham; Bennekou, Susanne Hougaard; Schildknecht, Stefan; Waldmann, Tanja; Hartung, Thomas; Hengstler, Jan G. (2017): Adverse outcome pathways : opportunities, limitations and open questions Archives of toxicology ; 91 (2017), 11. - S. 3477-3505. - ISSN 0340-5761. - eISSN 1432-0738

Adverse outcome pathways : opportunities, limitations and open questions

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Adverse outcome pathways (AOPs) are a recent toxicological construct that connects, in a formalized, transparent and quality-controlled way, mechanistic information to apical endpoints for regulatory purposes. AOP links a molecular initiating event (MIE) to the adverse outcome (AO) via key events (KE), in a way specified by key event relationships (KER). Although this approach to formalize mechanistic toxicological information only started in 2010, over 200 AOPs have already been established. At this stage, new requirements arise, such as the need for harmonization and re-assessment, for continuous updating, as well as for alerting about pitfalls, misuses and limits of applicability. In this review, the history of the AOP concept and its most prominent strengths are discussed, including the advantages of a formalized approach, the systematic collection of weight of evidence, the linkage of mechanisms to apical end points, the examination of the plausibility of epidemiological data, the identification of critical knowledge gaps and the design of mechanistic test methods. To prepare the ground for a broadened and appropriate use of AOPs, some widespread misconceptions are explained. Moreover, potential weaknesses and shortcomings of the current AOP rule set are addressed (1) to facilitate the discussion on its further evolution and (2) to better define appropriate vs. less suitable application areas. Exemplary toxicological studies are presented to discuss the linearity assumptions of AOP, the management of event modifiers and compensatory mechanisms, and whether a separation of toxicodynamics from toxicokinetics including metabolism is possible in the framework of pathway plasticity. Suggestions on how to compromise between different needs of AOP stakeholders have been added. A clear definition of open questions and limitations is provided to encourage further progress in the field.

Origin (projects)

  Nyffeler, Johanna; Dolde, Xenia; Krebs, Alice; Pinto-Gil, Kevin; Pastor, Manuel; Behl, Mamta; Waldmann, Tanja; Leist, Marcel (2017): Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library Archives of Toxicology ; 91 (2017), 11. - S. 3613-3632. - ISSN 0340-5761. - eISSN 1432-0738

Combination of multiple neural crest migration assays to identify environmental toxicants from a proof-of-concept chemical library

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Many in vitro tests have been developed to screen for potential neurotoxicity. However, only few cell function-based tests have been used for comparative screening, and thus experience is scarce on how to confirm and evaluate screening hits. We addressed these questions for the neural crest cell migration test (cMINC). After an initial screen, a hit follow-up strategy was devised. A library of 75 compounds plus internal controls (NTP80-list), assembled by the National Toxicology Program of the USA (NTP) was used. It contained some known classes of (developmental) neurotoxic compounds. The primary screen yielded 23 confirmed hits, which comprised ten flame retardants, seven pesticides and six drug-like compounds. Comparison of concentration-response curves for migration and viability showed that all hits were specific. The extent to which migration was inhibited was 25-90%, and two organochlorine pesticides (DDT, heptachlor) were most efficient. In the second part of this study, (1) the cMINC assay was repeated under conditions that prevent proliferation; (2) a transwell migration assay was used as a different type of migration assay; (3) cells were traced to assess cell speed. Some toxicants had largely varying effects between assays, but each hit was confirmed in at least one additional test. This comparative study allows an estimate on how confidently the primary hits from a cell function-based screen can be considered as toxicants disturbing a key neurodevelopmental process. Testing of the NTP80-list in more assays will be highly interesting to assemble a test battery and to build prediction models for developmental toxicity.

Origin (projects)

  Pallocca, Giorgia; Nyffeler, Johanna; Dolde, Xenia; Grinberg, Marianna; Gstraunthaler, Gerhard; Waldmann, Tanja; Rahnenführer, Jörg; Sachinidis, Agapios; Leist, Marcel (2017): Impairment of human neural crest cell migration by prolonged exposure to interferon-beta Archives of Toxicology ; 91 (2017), 10. - S. 3385-3402. - ISSN 0340-5761. - eISSN 1432-0738

Impairment of human neural crest cell migration by prolonged exposure to interferon-beta

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Human cell-based toxicological assays have been used successfully to detect known toxicants, and to distinguish them from negative controls. However, there is at present little experience on how to deal with hits from screens of compounds with yet unknown hazard. As a case study to this issue, we characterized human interferon-beta (IFNβ) as potential developmental toxicant affecting neural crest cells (NCC). The protein was identified as a hit during a screen of clinically used drugs in the 'migration inhibition of neural crest' (MINC) assay. Concentration-response studies in the MINC combined with immunocytochemistry and mRNA quantification of cellular markers showed that IFNβ inhibited NCC migration at concentrations as low as 20 pM. The effective concentrations found here correspond to levels found in human plasma, and they were neither cytostatic nor cytotoxic nor did they did they affect the differentiation state and overall phenotype of NCC. Data from two other migration assays confirmed that picomolar concentration of IFNβ reduced the motility of NCC, while other interferons were less potent. The activation of JAK kinase by IFNβ, as suggested by bioinformatics analysis of the transcriptome changes, was confirmed by biochemical methods. The degree and duration of pathway activation correlated with the extent of migration inhibition, and pharmacological block of this signaling pathway before, or up to 6 h after exposure to the cytokine prevented the effects of IFNβ on migration. Thus, the reduction of vital functions of human NCC is a hitherto unknown potential hazard of endogenous or pharmacologically applied interferons.

Origin (projects)

  Schildknecht, Stefan; Di Monte, Donato A.; Pape, Regina; Tieu, Kim; Leist, Marcel (2017): Tipping Points and Endogenous Determinants of Nigrostriatal Degeneration by MPTP Trends in Pharmacological Sciences ; 38 (2017), 6. - S. 541-555. - ISSN 0165-6147. - eISSN 1873-3735

Tipping Points and Endogenous Determinants of Nigrostriatal Degeneration by MPTP

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The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a Parkinson's disease (PD)-like syndrome by inducing degeneration of nigrostriatal dopaminergic neurons. Studies of the MPTP model have revealed the pathomechanisms underlying dopaminergic neurodegeneration and facilitated the development of drug treatments for PD. In this review, we provide an update on MPTP bioactivation and biodistribution, reconcile the distinct views on energetic failure versus reactive oxygen species (ROS) formation as main drivers of MPTP-induced neurodegeneration, and describe recently identified intrinsic features of the nigrostriatal system that make it particularly vulnerable to MPTP. We discuss these new perspectives on the endogenous tipping points of tissue homeostasis and the drivers responsible for vicious cycles in relation to their relevance for the development of novel intervention strategies for PD.

Origin (projects)

  Waldmann, Tanja; Grinberg, Marianna; König, André; Rempel, Eugen; Schildknecht, Stefan; Henry, Margit; Holzer, Anna-Katharina; Dreser, Nadine; Shinde, Vaibhav; Sachinidis, Agapios; Rahnenführer, Jörg; Hengstler, Jan G; Leist, Marcel (2017): Stem Cell transcriptome responses and corresponding biomarkers that indicate the transition from adaptive responses to cytotoxicity Chemical research in toxicology ; 30 (2017), 4. - S. 905-922. - ISSN 0893-228X. - eISSN 1520-5010

Stem Cell transcriptome responses and corresponding biomarkers that indicate the transition from adaptive responses to cytotoxicity

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Analysis of transcriptome changes has become an established method to characterize the reaction of cells to toxicants. Such experiments are mostly performed at compound concentrations close to the cytotoxicity threshold. At present, little information is available on concentration-dependent features of transcriptome changes, in particular, at the transition from noncytotoxic concentrations to conditions that are associated with cell death. Thus, it is unclear in how far cell death confounds the results of transcriptome studies. To explore this gap of knowledge, we treated pluripotent stem cells differentiating to human neuroepithelial cells (UKN1 assay) for short periods (48 h) with increasing concentrations of valproic acid (VPA) and methyl mercury (MeHg), two compounds with vastly different modes of action. We developed various visualization tools to describe cellular responses, and the overall response was classified as "tolerance" (minor transcriptome changes), "functional adaptation" (moderate/strong transcriptome responses, but no cytotoxicity), and "degeneration". The latter two conditions were compared, using various statistical approaches. We identified (i) genes regulated at cytotoxic, but not at noncytotoxic, concentrations and (ii) KEGG pathways, gene ontology term groups, and superordinate biological processes that were only regulated at cytotoxic concentrations. The consensus markers and processes found after 48 h treatment were then overlaid with those found after prolonged (6 days) treatment. The study highlights the importance of careful concentration selection and of controlling viability for transcriptome studies. Moreover, it allowed identification of 39 candidate "biomarkers of cytotoxicity". These could serve to provide alerts that data sets of interest may have been affected by cell death in the model system studied.

Origin (projects)

  Ockleford, Colin; Adriaanse, Paulien; Berny, Philippe; Brock, Theodorus; Duquesne, Sabine; Grilli, Sandro; Hernandez-Jerez, Antonio F; Bennekou, Susanne Hougaard; Klein, Michael; Leist, Marcel (2017): Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia EFSA Journal ; 15 (2017), 3. - e04691. - eISSN 1831-4732

Investigation into experimental toxicological properties of plant protection products having a potential link to Parkinson's disease and childhood leukaemia

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In 2013, EFSA published a literature review on epidemiological studies linking exposure to pesticides and human health outcome. As a follow up, the EFSA Panel on Plant Protection Products and their residues (PPR Panel) was requested to investigate the plausible involvement of pesticide exposure as a risk factor for Parkinson's disease (PD) and childhood leukaemia (CHL). A systematic literature review on PD and CHL and mode of actions for pesticides was published by EFSA in 2016 and used as background documentation. The Panel used the Adverse Outcome Pathway (AOP) conceptual framework to define the biological plausibility in relation to epidemiological studies by means of identification of specific symptoms of the diseases as AO. The AOP combines multiple information and provides knowledge of biological pathways, highlights species differences and similarities, identifies research needs and supports regulatory decisions. In this context, the AOP approach could help in organising the available experimental knowledge to assess biological plausibility by describing the link between a molecular initiating event (MIE) and the AO through a series of biologically plausible and essential key events (KEs). As the AOP is chemically agnostic, tool chemical compounds were selected to empirically support the response and temporal concordance of the key event relationships (KERs). Three qualitative and one putative AOP were developed by the Panel using the results obtained. The Panel supports the use of the AOP framework to scientifically and transparently explore the biological plausibility of the association between pesticide exposure and human health outcomes, identify data gaps, define a tailored testing strategy and suggests an AOP's informed Integrated Approach for Testing and Assessment (IATA).

Origin (projects)

  Efremova, Liudmila; Chovancova, Petra; Adam, Martina; Gutbier, Simon; Schildknecht, Stefan; Leist, Marcel (2017): Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation Archives of Toxicology ; 91 (2017), 1. - S. 231-246. - ISSN 0003-9446. - eISSN 1432-0738

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

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Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.

Origin (projects)

  Hirsch, Cordula; Striegl, Britta; Mathes, Stephanie; Adlhart, Christian; Krebs, Alice; Nyffeler, Johanna; Pape, Regina; Bürkle, Alexander; Leist, Marcel; Schildknecht, Stefan (2017): Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants Archives of Toxicology ; 91 (2017), 1. - S. 407-425. - ISSN 0003-9446. - eISSN 1432-0738

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants

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Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.

Origin (projects)

  Schmidt, Béla Z; Lehmann, Martin; Gutbier, Simon; Nembo, Erastus; Noel, Sabrina; Smirnova, Lena; Forsby, Anna; Hescheler, Jürgen; Avci, Hasan X; Hartung, Thomas; Leist, Marcel; Kobolák, Julianna; Dinnyés, András (2017): In vitro acute and developmental neurotoxicity screening : an overview of cellular platforms and high-throughput technical possibilities Archives of toxicology ; 91 (2017), 1. - S. 1-33. - ISSN 0003-9446. - eISSN 1432-0738

In vitro acute and developmental neurotoxicity screening : an overview of cellular platforms and high-throughput technical possibilities

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Neurotoxicity and developmental neurotoxicity are important issues of chemical hazard assessment. Since the interpretation of animal data and their extrapolation to man is challenging, and the amount of substances with information gaps exceeds present animal testing capacities, there is a big demand for in vitro tests to provide initial information and to prioritize for further evaluation. During the last decade, many in vitro tests emerged. These are based on animal cells, human tumour cell lines, primary cells, immortalized cell lines, embryonic stem cells, or induced pluripotent stem cells. They differ in their read-outs and range from simple viability assays to complex functional endpoints such as neural crest cell migration. Monitoring of toxicological effects on differentiation often requires multiomics approaches, while the acute disturbance of neuronal functions may be analysed by assessing electrophysiological features. Extrapolation from in vitro data to humans requires a deep understanding of the test system biology, of the endpoints used, and of the applicability domains of the tests. Moreover, it is important that these be combined in the right way to assess toxicity. Therefore, knowledge on the advantages and disadvantages of all cellular platforms, endpoints, and analytical methods is essential when establishing in vitro test systems for different aspects of neurotoxicity. The elements of a test, and their evaluation, are discussed here in the context of comprehensive prediction of potential hazardous effects of a compound. We summarize the main cellular characteristics underlying neurotoxicity, present an overview of cellular platforms and read-out combinations assessing distinct parts of acute and developmental neurotoxicology, and highlight especially the use of stem cell-based test systems to close gaps in the available battery of tests.

Origin (projects)

  Shinde, Vaibhav; Hoelting, Lisa; Srinivasan, Sureshkumar Perumal; Meisig, Johannes; Meganathan, Kesavan; Jagtap, Smita; Schildknecht, Stefan; Waldmann, Tanja; Leist, Marcel; Sachinidis, Agapios (2017): Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Toxukn and STOP-Toxukk tests Archives of Toxicology ; 91 (2017), 2. - S. 839-864. - ISSN 0003-9446. - eISSN 1432-0738

Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Toxukn and STOP-Toxukk tests

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Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as 'developmental genes'. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds ('mercurials') and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of 'diagnostic genes' appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, 'developmental potency' (D p) and 'developmental index' (D i), whereby D p is the fraction of all developmental genes that are up- or down-regulated by a test compound, and D i is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of D i makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low D p but a high D i. In conclusion, the concept based on the indices D p and D i offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.

Origin (projects)

  Leist, Marcel (2017): New Animal-free Concepts and Test Methods for Developmental Toxicity and Peripheral Neurotoxicity Alternatives to Laboratory Animals : ATLA ; 45 (2017), 5. - S. 253-260. - ISSN 0261-1929

New Animal-free Concepts and Test Methods for Developmental Toxicity and Peripheral Neurotoxicity

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The complex toxicological fields of repeat dose organ toxicity (RDT) and developmental and reproductive toxicity (DART) still require new concepts and approaches to achieve a fully animal-free safety assessment of chemicals. One novel approach is the generation of relevant human cell types from pluripotent stem cells, and the use of such cells for the establishment of phenotypic test methods. Due to their broad endpoints, such tests capture multiple types of toxicants, i.e. they are a readout for the activation of many adverse outcome pathways (AOPs). The 2016 Lush Science Prize was awarded for the development of one such assay, the PeriTox test, which uses human peripheral neurons generated from stem cells. The assay endpoints measure various cell functions, and these give information on the potential neurotoxicity and developmental neurotoxicity hazard of test compounds. The PeriTox test method has a high predictivity and sensitivity for peripheral neurotoxicants, and thus addresses the inherent challenges in pesticide testing and drug development. Data from the test can be obtained quickly and at a relatively high-throughput, and thus, the assay has the potential to replace animal-based safety assessment during early product development or for screening potential environmental toxicants.

Origin (projects)

  Aschner, Michael; Ceccatelli, Sandra; Daneshian, Mardas; Fritsche, Ellen; Hasiwa, Nina; Hartung, Thomas; Hogberg, Helena T.; Leist, Marcel; Zimmer, Bastian; Lein, Pamela J. (2017): Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals : example lists and criteria for their selection and use Alternatives to animal experimentation : ALTEX ; 34 (2017), 1. - S. 49-74. - ISSN 0946-7785. - eISSN 1868-8551

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals : example lists and criteria for their selection and use

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There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e. alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of >50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicantsis proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the >100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.

Origin (projects)

  Nyffeler, Johanna; Karreman, Christiaan; Leisner, Heidrun; Kim, Yong Jun; Lee, Gabsang; Waldmann, Tanja; Leist, Marcel (2017): Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants Alternatives to Animal Experimentation : ALTEX ; 34 (2017), 1. - S. 75-94. - ISSN 0946-7785. - eISSN 1868-8551

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants

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Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise, if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Therefore, a new test format was established here. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of this barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of automated image processing to obtain data on viability and migration was addressed by development of a software made generally available for downloading. To optimize the biological system, data on cell proliferation were obtained by labelling of replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as experimental confounder was tested experimentally by performance of the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allowed classification of toxicants as either being inactive, leading to unspecific cytotoxicity or specifically inhibiting NC migration at non-cytotoxic concentrations.

Origin (projects)

  Sá, João V.; Kirner, Susanne; Brito, Catarina; Sonnewald, Ursula; Leist, Marcel; Teixeira, Ana P.; Alves, Paula M. (2017): Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis Neurochemical Research ; 42 (2017), 1. - S. 244-253. - ISSN 0364-3190. - eISSN 1573-6903

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

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Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-(13)C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained (13)C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis ((13)C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.

Origin (projects)

  Fritsche, Ellen; Crofton, Kevin M; Hernandez, Antonio F; Hougaard Bennekou, Susanne; Leist, Marcel; Bal-Price, Anna; Reaves, Elissa; Wilks, Martin F; Terron, Andrea; Gourmelon, Anne (2017): OECD/EFSA workshop on developmental neurotoxicity (DNT) : The use of non-animal test methods for regulatory purposes ALTEX ; 34 (2017), 2. - S. 311-315. - ISSN 1868-596X. - eISSN 1868-8551

OECD/EFSA workshop on developmental neurotoxicity (DNT) : The use of non-animal test methods for regulatory purposes

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dc.title:


dc.contributor.author: Fritsche, Ellen; Crofton, Kevin M; Hernandez, Antonio F; Hougaard Bennekou, Susanne; Leist, Marcel; Bal-Price, Anna; Reaves, Elissa; Wilks, Martin F; Terron, Andrea; Gourmelon, Anne

Origin (projects)

  Pamies, David; Bal-Price, Anna; Simeonov, Anton; Tagle, Danilo; Allen, Dave; Gerhold, David; Yin, Dezhong; Pistollato, Francesca; Inutsuka, Takashi; Sullivan, Kristie; Stacey, Glyn; Salem, Harry; Leist, Marcel; Daneshian, Mardas; Vemuri, Mohan C; McFarland, Richard; Coecke, Sandra; Fitzpatrick, Suzanne C; Lakshmipathy, Uma; Mack, Amanda; Wang, Wen Bo; Daiju, Yamazaki; Sekino, Yuko; Kanda, Yasunari; Smirnova, Lena; Hartung, Thomas (2017): Good cell culture practice for stem cells and stem-cell-derived models Alternatives to Animal Experimentation : ALTEX ; 34 (2017), 1. - S. 95-132. - ISSN 0946-7785. - eISSN 1868-8551

Good cell culture practice for stem cells and stem-cell-derived models

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The first guidance on Good Cell Culture Practice dates back to 2005. This document expands this to aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice which can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance, which will facilitate the generation of reliable data from cell culture systems, but is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered as a first step toward a revised GCCP 2.0.

Origin (projects)

  Hoelting, Lisa; Klima, Stefanie; Karreman, Christiaan; Grinberg, Marianna; Meisig, Johannes; Henry, Margit; Rotshteyn, Tamara; Rahnenführer, Jörg; Waldmann, Tanja; Leist, Marcel (2016): Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants Stem Cells Translational Medicine ; 5 (2016), 4. - S. 476-487. - ISSN 2157-6564. - eISSN 2157-6580

Stem Cell-Derived Immature Human Dorsal Root Ganglia Neurons to Identify Peripheral Neurotoxicants

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Safety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates.

Origin (projects)

  Daneshian, Mardas; Kamp, Hennicke; Hengstler, Jan; Leist, Marcel; van de Water, Bob (2016): Highlight report : Launch of a large integrated European in vitro toxicology project: EU-ToxRisk Archives of Toxicology ; 90 (2016), 5. - S. 1021-1024. - ISSN 0003-9446. - eISSN 1432-0738

Highlight report : Launch of a large integrated European in vitro toxicology project: EU-ToxRisk

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The integrated European project, EU-ToxRisk, proudly sees itself as "flagship" exploring new alternative-to-animal approaches to chemical safety evaluation. It promotes mechanism-based toxicity testing and risk assessment according to the principles laid down for toxicology for the twenty-first century. The project was officially launched in January 2016 with a kickoff meeting in Egmond aan Zee, the Netherlands. Over 100 scientists representing academia and industry as well as regulatory authorities attended the inaugural meeting. The project will integrate advances in in vitro and in silico toxicology, read-across methods, and adverse outcome pathways. EU-ToxRisk will continue to make use of the case study strategy deployed in SEURAT-1, a FP7 initiative ended in December 2015. Even though the development of new non-animal methods is one target of EU-ToxRisk, the project puts special emphasis on their acceptance and implementation in regulatory contexts. This €30 million Horizon 2020 project involves 38 European partners and one from the USA. EU-ToxRisk aims at the "development of a new way of risk assessment."

Origin (projects)

  Luechtefeld, Thomas; Maertens, Alexandra; Russo, Daniel P.; Rovida, Costanza; Zhu, Hao; Hartung, Thomas (2016): Global analysis of publicly available safety data for 9,801 substances registered under REACH from 2008-2014 Alternatives to Animal Experimentation : ALTEX ; 33 (2016), 2. - S. 95-109. - ISSN 0946-7785. - eISSN 1868-8551

Global analysis of publicly available safety data for 9,801 substances registered under REACH from 2008-2014

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The European Chemicals Agency (ECHA) warehouses the largest public dataset of in vivo and in vitro toxicity tests. In December 2014 this data was converted into a structured, machine readable and searchable database using linguistic search engines. It contains data for 9,801 unique substances, 3,609 unique study descriptions and 816,048 study documents.This allows exploring toxicological data on a scale far larger than previously available. Substance similarity analysis was used to determine clustering of substances for hazards by mapping to PubChem. Similarity was measured using PubChem 2D conformational substructure fingerprints, which were compared via the Tanimoto metric. Following K-Core filtration, the Blondel et al.(2008) module recognition algorithm was used to identify chemical modules showing clusters of substances in use within the chemical universe. Global Harmonized System of Classification and Labelling provides a valuable information source for hazard analysis. The most prevalent hazards are H317 "May cause an allergic skin reaction" with 20% and H318 "Causes serious eye damage" with 17% positive substances. Such prevalences obtained for all hazards here are key for the design of integrated testing strategies. The data allowed estimation of animal use. ECHA cover about 20% of substances in the high-throughput biological assay database Tox21 (1,737 substances) and have a 917 substance overlap with the Comparative Toxicogenomics Database (~7% of CTD). The biological data available in these datasets combined with ECHA in vivo endpoints have enormous modeling potential. A case is made that REACH should systematically open regulatory data for research purposes.

Origin (projects)

  Meiser, Johannes; Delcambre, Sylvie; Wegner, André; Jäger, Christian; Ghelfi, Jenny; Fouquier d'Herouel, Aymeric; Dong, Xiangyi; Schildknecht, Stefan; Leist, Marcel; Hiller, Karsten (2016): Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism Neurobiology of Disease ; 89 (2016). - S. 112-125. - ISSN 0969-9961. - eISSN 1095-953X

Loss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism

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The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches.

Origin (projects)

  Ball, Nicholas; Cronin, Mark T. D.; Shen, Jie; Blackburn, Karen; Booth, Ewan D.; Bouhifd, Mounir; Donley, Elizabeth; Egnash, Laura; Hastings, Charles; Juberg, Daland R.; Hartung, Thomas (2016): Toward Good Read-Across Practice (GRAP) guidance ALTEX ; 33 (2016), 2. - S. 149-166. - ISSN 0946-7785. - eISSN 1868-8551

Toward Good Read-Across Practice (GRAP) guidance

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Grouping of substances and utilizing read-across of data within those groups represents an important data gap filling technique for chemical safety assessments. Categories/analogue groups are typically developed based on structural similarity and, increasingly often, also on mechanistic (biological) similarity. While read-across can play a key role in complying with legislations such as the European REACH regulation, the lack of consensus regarding the extent and type of evidence necessary to support it often hampers its successful application and acceptance by regulatory authorities. Despite a potentially broad user community, expertise is still concentrated across a handful of organizations and individuals. In order to facilitate the effective use of read-across, this document aims to summarize the state-of-the-art, summarizes insights learned from reviewing ECHA published decisions as far as the relative successes/pitfalls surrounding read-across under REACH and compile the relevant activities and guidance documents. Special emphasis is given to the available existing tools and approaches, an analysis of ECHA's published final decisions associated with all levels of compliance checks and testing proposals, the consideration and expression of uncertainty, the use of biological support data and the impact of the ECHA Read-Across Assessment Framework (RAAF) published in 2015.

Origin (projects)

  Luechtefeld, Thomas; Maertens, Alexandra; Russo, Daniel P.; Rovida, Costanza; Zhu, Hao; Hartung, Thomas (2016): Analysis of public oral toxicity data from REACH registrations 2008-2014 Alternatives to Animal Experimentation : ALTEX ; 33 (2016), 2. - S. 111-122. - ISSN 0946-7785. - eISSN 1868-8551

Analysis of public oral toxicity data from REACH registrations 2008-2014

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The European Chemicals Agency, ECHA, made available a total of 13,832 oral toxicity studies for 8,568 substances up to December 2014. 75% of studies were from the retired OECD Test Guideline 401 (11% TG 420, 11% TG 423 and 1.5% TG 425). Concordance across guidelines, evaluated by comparing LD50 values ≥ 2000 or < 2000 mg/kg body weight from chemicals tested multiple times between different guidelines, was at least 75% and for their own repetition more than 90%. In 2009, Bulgheroni et al. created a simple model for predicting acute oral toxicity using no observed adverse effect levels (NOAEL) from 28-day repeated dose toxicity studies in rats. This was reproduced here for 1,625 substances. In 2014, Taylor et al. suggested no added value of the 90-day repeated dose oral toxicity test given the availability of a low 28-day study with some constraints. We confirm that the 28-day NOAEL is predictive (albeit imperfectly) of 90-day NOAELs, however, the suggested constraints did not affect predictivity. 1,059 substances with acute oral toxicity data (268 positives, 791 negatives, all Klimisch score 1) were used for modeling: The Chemical Development Kit was used to generate 27 molecular descriptors and a similarity-informed multilayer perceptron showing 71% sensitivity and 72% specificity. Additionally, the k-nearest neighbors (KNN) algorithm indicated that similarity-based approaches alone may be poor predictors of acute oral toxicity, but can be used to inform the multilayer perceptron model, where this was the feature with highest information value.

Origin (projects)

  Shinde, Vaibhav; Perumal Srinivasan, Sureshkumar; Henry, Margit; Rotshteyn, Tamara; Hescheler, Jürgen; Rahnenführer, Jörg; Grinberg, Marianna; Meisig, Johannes; Blüthgen, Nils; Waldmann, Tanja; Leist, Marcel; Hengstler, Jan Georg; Sachinidis, Agapios (2016): Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells Stem cell research & therapy ; 7 (2016), 1. - 190. - eISSN 1757-6512

Comparison of a teratogenic transcriptome-based predictive test based on human embryonic versus inducible pluripotent stem cells

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Human embryonic stem cells (hESCs) partially recapitulate early embryonic three germ layer development, allowing testing of potential teratogenic hazards. Because use of hESCs is ethically debated, we investigated the potential for human induced pluripotent stem cells (hiPSCs) to replace hESCs in such tests.

Origin (projects)

  Luechtefeld, Thomas; Maertens, Alexandra; Russo, Daniel P.; Rovida, Costanza; Zhu, Hao; Hartung, Thomas (2016): Analysis of Draize eye irritation testing and its prediction by mining publicly available 2008-2014 REACH data Alternatives to Animal Experimentation : ALTEX ; 33 (2016), 2. - S. 123-134. - ISSN 0946-7785. - eISSN 1868-8551

Analysis of Draize eye irritation testing and its prediction by mining publicly available 2008-2014 REACH data

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Public data from ECHA online dossiers on 9,801 substances encompassing 326,749 experimental key studies and additional information on classification and labeling were made computable. Eye irritation hazard, for which the rabbit Draize eye test still represents the reference method, was analyzed. Dossiers contained 9,782 Draize eye studies on 3,420 unique substances, indicating frequent retesting of substances. This allowed assessment of the test's reproducibility test based on all substances tested more than once. There was a 10% chance of a non-irritant evaluation given after a prior severe-irritant result as given by UN GHS classification criteria. The most reproducible outcomes were the results negative (94% reproducible) and severe eye irritant (73% reproducible). To evaluate whether other GHS categorizations predict eye irritation we built a dataset of 5,629 substances (1,931 'irritant' and 3,698 'non-irritant'). The two best decision trees with up to three other GHS classifications resulted in balanced accuracies of 68% and 73%, i.e., in the rank order of the Draize rabbit eye test itself, but both use inhalation toxicity data ("May cause respiratory irritation"), which is not typically available. Next, a dataset of 929 substances with at least one Draize study was mapped to PubChem to compute chemical similarity using 2D conformational fingerprints and Tanimoto similarity. Using a minimum similarity of 0.7 and simple classification by the closest chemical neighbor resulted in balanced accuracy from 73% over 737 substances to 100% at a threshold of 0.975 over 41 substances. This represents a strong support of read-across and (Q)SAR approaches in this area.

Origin (projects)

  Luechtefeld, Thomas; Maertens, Alexandra; Russo, Daniel P.; Rovida, Costanza; Zhu, Hao; Hartung, Thomas (2016): Analysis of publically available skin sensitization data from REACH registrations 2008-2014 Alternatives to Animal Experimentation : ALTEX ; 33 (2016), 2. - S. 135-148. - ISSN 0946-7785. - eISSN 1868-8551

Analysis of publically available skin sensitization data from REACH registrations 2008-2014

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The public data on skin sensitization from REACH registrations already included 19,111 studies on skin sensitization in December 2014, making it the largest repository of such data so far (1,470 substances with mouse LLNA, 2,787 with GPMT, 762 with both in vivo and in vitro and 139 with only in vitro data). 21% were classified as sensitizers. The extracted skin sensitization data was analyzed to identify relationships in skin sensitization guidelines, visualize structural relationships of sensitizers, and build models to predict sensitization. A chemical with molecular weight > 500 Da is generally considered non-sensitizing owing to low bioavailability, but 49 sensitizing chemicals with a molecular weight > 500 Da were found. A chemical similarity map was produced using PubChem's 2D Tanimoto similarity metric and Gephi force layout visualization. Nine clusters of chemicals were identified by Blondel's module recognition algorithm revealing wide module-dependent variation. Approximately 31% of mapped chemicals are Michaell's acceptors but alone this does not imply skin sensitization. A simple sensitization model using molecular weight and five ToxTree structural alerts showed a balanced accuracy of 65.8% (specificity 80.4%, sensitivity 51.4%), demonstrating that structural alerts have information value. A simple variant of k-nearest neighbors outperformed the ToxTree approach even at 75% similarity threshold (82% balanced accuracy at 0.95 threshold). At higher thresholds, the balanced accuracy increased. Lower similarity thresholds decrease sensitivity faster than specificity. This analysis scopes the landscape of chemical skin sensitization, demonstrating the value of large public datasets for health hazard prediction.

Origin (projects)

  Zhu, Hao; Bouhifd, Mounir; Donley, Elizabeth; Egnash, Laura; Kleinstreuer, Nicole; Kroese, E. Dinant; Liu, Zhichao; Luechtefeld, Thomas; Palmer, Jessica; Pamies, David; Shen, Jie; Strauss, Volker; Wu, Shengde; Hartung, Thomas (2016): Supporting read-across using biological data ALTEX ; 33 (2016), 2. - S. 167-182. - ISSN 1868-596X. - eISSN 1868-596X

Supporting read-across using biological data

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Read-across, i.e. filling toxicological data gaps by relating to similar chemicals, for which test data are available, is usually done based on chemical similarity. Besides structure and physico-chemical properties, however, biological similarity based on biological data adds extra strength to this process. In the context of developing Good Read-Across Practice guidance, a number of case studies were evaluated to demonstrate the use of biological data to enrich read-across. In the simplest case, chemically similar substances also show similar test results in relevant in vitro assays. This is a well-established method for the read-across of e.g. genotoxicity assays. Larger datasets of biological and toxicological properties of hundreds and thousands of substances become increasingly available enabling big data approaches in read-across studies. Several case studies using various big data sources are described in this paper. An example is given for the US EPA's ToxCast dataset allowing read-across for high quality uterotrophic assays for estrogenic endocrine disruption. Similarly, an example for REACH registration data enhancing read-across for acute toxicity studies is given. A different approach is taken using omics data to establish biological similarity: Examples are given for stem cell models in vitro and short-term repeated dose studies in rats in vivo to support read-across and category formation. These preliminary biological data-driven read-across studies highlight the road to the new generation of read-across approaches that can be applied in chemical safety assessment.

Origin (projects)

  Chandrasekaran, Abinaya; Avci, Hasan X; Leist, Marcel; Kobolák, Julianna; Dinnyés, Andras (2016): Astrocyte differentiation of human pluripotent stem cells: new tools for neurological disorder research Frontiers in cellular neuroscience ; 10 (2016). - 215. - eISSN 1662-5102

Astrocyte differentiation of human pluripotent stem cells: new tools for neurological disorder research

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Astrocytes have a central role in brain development and function, and so have gained increasing attention over the past two decades. Consequently, our knowledge about their origin, differentiation and function has increased significantly, with new research showing that astrocytes cultured alone or co-cultured with neurons have the potential to improve our understanding of various central nervous system diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, or Alexander disease. The generation of astrocytes derived from pluripotent stem cells (PSCs) opens up a new area for studying neurologic diseases in vitro; these models could be exploited to identify and validate potential drugs by detecting adverse effects in the early stages of drug development. However, as it is now known that a range of astrocyte populations exist in the brain, it will be important in vitro to develop standardized protocols for the in vitro generation of astrocyte subsets with defined maturity status and phenotypic properties. This will then open new possibilities for co-cultures with neurons and the generation of neural organoids for research purposes. The aim of this review article is to compare and summarize the currently available protocols and their strategies to generate human astrocytes from PSCs. Furthermore, we discuss the potential role of human-induced PSCs derived astrocytes in disease modeling.

Origin (projects)

Funding sources
Name Project no. Description Period
Europäische Union758/15
Further information
Period: 01.01.2016 – 31.12.2021