EUToxRisk21

Institutions
  • FB Biologie
Publications
  Dreser, Nadine; Holzer, Anna-Katharina; Kapitza, Marion; Scholz, Christopher; Kranaster, Petra; Gutbier, Simon; Klima, Stefanie; Kolb, David; Dietz, Christian; Trefzer, Timo; Berthold, Michael R.; Waldmann, Tanja; Leist, Marcel(2019): Development of a neural rosette formation assay (RoFA) to identify neurodevelopmental toxicants and to characterize their transcriptome disturbances Archives of Toxicology ; 2019. - 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<sub>(UKN)</sub>. 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<sub>(UKN)</sub> assay.

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)

    Karreman, Christiaan; Kranaster, 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)

    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)

    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; 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)

 31.12.19  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)

    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 EC<sub>50</sub> 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 EC<sub>50</sub> 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 EC<sub>50</sub> 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 EC<sub>50</sub> 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)

    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 <sup>13</sup>C-metabolic flux analysis (INST <sup>13</sup>C-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)

    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)

    Nyffeler, Johanna; Chovancova, Petra; Dolde, Xenia; Holzer, Anna-Katharina; Purvanov, Vladimir; Kindinger, Ilona; Kerins, Anna; Higton, David; Legler, Daniel F.; 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)

    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.<br /><br />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.<br /><br />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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

  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)

    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)

    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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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)

    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)

    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-Tox<sub>ukn</sub> and STOP-Tox<sub>ukk</sub> 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)

    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)

    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)

    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)

    Sá, João V.; Kleiderman, 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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

    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)

Funding sources
NameProject no.DescriptionPeriod
Europäische Union758/15no information
Further information
Period: 01.01.2016 – 31.12.2021