FOR HUMAN
SAFETY
FOR HUMAN
SAFETY
FOR HUMAN
SAFETY
Novel testing strategies for endocrine disruptors in the context of developmental neurotoxicity (ENDPOINTS)
As a member of the ENDpoiNTs H2020 research consortium, we develop innovative, human NPC-based models to identify chemicals that disrupt brain development by interfering with human endocrine processes. The ENDpoiNTs project is divided into two parts: The first deals with the development of a testing battery for chemical screening and with the acquisition of specialist knowledge about the molecular connections between the developmental neurotoxicological observations and the underlying disorders of the endocrine system. In the second part, we combine our studies with the and data of the other ENDpoiNTs partners and evaluate our results in an epidemiological context in order to guarantee the human relevance of our test methods. The test methods developed by ENDpoiNTs should ultimately be incorporated into the European and international regulatory framework.
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Funding European Commission (Horizon2020)
hiPSC-derived dopaminergic neurons in green, nuclei in blue
Cells migrate radially out of a sphere (lower left corner)
In vitro cardiomyocytes
The human induced pluripotent stem cell test (hiPS-test) is based on the ability of hiPSCs to differentiate into beating cardiomyocytes, thereby representing approximately the third week of pregnancy. We are developing standardized protocols to use this test in the future for the identification of embryotoxic substances. The assay currently includes the following endpoints: gene and protein expression of cardiac maturation markers, statement “does not beat / beats”, and analysis of the beating frequency of the cardiomyocytes – in each case according to substance exposure and with established positive and negative controls. In the near future, we plan to add metabolomics as another measure.
In 3D differentiated neurosphere. Green: cytoskeleton, red: neurons, blue: nuclei
Automated image analysis and biostatistics
Automated image analysis is carried out in close cooperation with the bioinformatics group (Prof. Axel Mosig) based at Ruhr University Bochum. Neuronal networks are trained with fluorescence microscopic images of neurons and glial cells in order to be able to perform artificial intelligence-based automatic evaluations of the neurotoxic analyses. This enables us to expand our in-house analysis software Omnisphero (Schmuck et al., 2017). We carry out the biostatistical evaluations of such high-content imaging analysis (HCA) data sets with the help of internationally recognized biostatistical methods. CERST collaborates with the United States' National Toxicology Program at the National Institute of Environmental Health Sciences and with Brunel University, London.
In 3D differentiated neurosphere. Green: cytoskeleton, red: neurons, blue: nuclei
Automated image analysis and biostatistics
Automated image analysis is carried out in close cooperation with the bioinformatics group (Prof. Axel Mosig) based at Ruhr University Bochum. Neuronal networks are trained with fluorescence microscopic images of neurons and glial cells in order to be able to perform artificial intelligence-based automatic evaluations of the neurotoxic analyses. This enables us to expand our in-house analysis software Omnisphero (Schmuck et al., 2017). We carry out the biostatistical evaluations of such high-content imaging analysis (HCA) data sets with the help of internationally recognized biostatistical methods. CERST collaborates with the United States' National Toxicology Program at the National Institute of Environmental Health Sciences and with Brunel University, London.
An in vitro testing battery for the assessment of developmental neurotoxicity
Developmental neurotoxicity (DNT) is of great social relevance since most of the chemicals currently in circulation have not been tested for their DNT potential. This is mainly due to the animal-based standard method, which is very resource-intensive and therefore not suitable for testing large amounts of substances. We are part of an international consortium (University of Konstanz, United States Environmental Protection Agency, European Food Safety Authority (EFSA)) that has assembled, established, and scientifically validated an test battery for predicting the influence of substances on the developing nervous system of children more quickly and cost effectively without the need for animal testing. This data forms the basis of an OECD guidance on testing for DNT, which is currently in preparation.
A differentiated neurosphere after 5 days in culture. Red: neurons, green: oligodendrocytes, blue: nuclei
EFSA case study on flame retardants
Flame retardants prevent or delay the spread of fire and can therefore be found in many everyday items and furnishings. In recent decades, the substance class of polyhalogenated flame retardants – such as polychlorinated biphenyls and polybrominated diphenyl ethers – has been classified as harmful to human health and therefore banned from the market. These were subsequently replaced by less persistent, alternative flame retardants such as organophosphates, but without knowing their full toxicological profile. In this case study, we use the EFSA DNT test battery assays developed at the IUF (see “An testing battery for the assessment of developmental neurotoxicity”) to assess the toxicological potential of flame retardants for basic brain development processes. In collaboration with the Biological Medical Research Center of Heinrich Heine University Düsseldorf (Prof. Köhrer) and the core facility of the IUF (Dr. Rossi), these are also analyzed on a molecular level.
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Funding European Food Safety Authority (EFSA)
Representative immunocytochemical image of differentiated hNPCs on day 5 of differentiation.
Blue: nuclei, pink: oligodendrocytes
Representative immunocytochemical image of differentiated hNPCs on day 5 of differentiation.
Blue: nuclei , pink: oligodendrocytes
The video shows the electrical activity of NeuCyte cells on microelectrode arrays (MEAs)
Representative spike raster plot
From (screen) hit to DNT toxicant
In recent years, great progress has been made in the development of animal-free test methods that map certain key processes in brain development (see “An in vitro testing battery for the assessment of developmental neurotoxicity”). Based on the test methods of such an in vitro test battery, the developmental neurotoxic (DNT) hazard potential of chemicals is assessed. To improve the prediction of these alternative test methods, we are establishing an hiPSC-based “neural network formation assay” and testing the ability of pesticides to interfere with neural network formation. Together with the University of Konstanz (Prof. Marcel Leist) we are carrying out an interlaboratory comparison of the test methods of the DNT in vitro battery in order to increase the regulatory acceptance and applicability of the DNT test methods.
Funding Danish Environmental Protection Agency (DK-EPA) – Pesticide Research Program
Respresentative confocal images of differentiated LUHMES cells on day 6 of differentiation.
Blue: nuclei, red: cytoskeleton, green: neurons.
The establishment of an AOP-based in vitro test battery for identifying compounds including Parkinsonian motor deficits
Parkinson’s disease (PD) is the second most common neurodegenerative disease in the elderly and is significantly more common in Egypt than in the rest of the world. Egypt is an agricultural country with a high level of occupational exposure to pesticides. Numerous epidemiological studies support the hypothesis that exposure to pesticides could increase the risk of PD. For this reason, the European Food Safety Authority (EFSA) has re-evaluated the hypothesis that pesticides could be the cause of Parkinsonian motor deficits (PMDs). To this end, adverse outcome pathways (AOPs) were generated. In this project, a human testing battery based on these AOPs is being established to determine the hazard potential of substances that might cause PMDs.
Funding Deutscher Akademischer Austauschdienst (DAAD), Bayer AG
Development of organ-specific in vitro models under consideration of physiological and pathophysiological characteristics
Within the EU Horizon 2020-funded PATROLS project (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment) representatives of academia, industry and governmental agencies collaborate to develop innovative laboratory- and computer-based methods for the safety assessment of nanomaterials. These developments shall contribute to minimise – and ideally eliminate – the necessity to perform animal studies for the evaluation of nanomaterials. In the context of in vitro model development, our focus is on the intestine as entry organ for nanomaterials following oral uptake.
Our aim is to mimic the physiological complexity of the organ as well as the impact of the digestive processes on the physico-chemical properties of the nanomaterials as closely as possible to the in vivo situation. Special consideration is given to the emulation of organ-specific pathological processes, e.g. as present during chronic inflammatory conditions of the intestine. The quality and transferability of the in vitro results will be analysed by targeted comparison with findings from animal studies.
Funding European Commission (Horizon 2020)
Epithelial cell layer of the intestinal co-culture (Caco-2/HT29-MTX-E12) after 21 days of differentiation.
Blue: nuclei; red: tight junction-associated protein (ZO-1); green: cytoskeleton
