Anke Tukker – In vitro assessment of human neurotoxicity using hiPSCs

Exposure to (environmental) chemicals may result in severe neurological effects and it is therefore of utmost importance to identify and understand the health risks associated with these exposures. Current test methods strongly rely on large-scale animal use, mainly rodents. Importantly, since rodents are not little humans, these tests are not truly predictive. Consequently, there is an urgent need for more predictive, human-based test methods, and to limit or even eliminate the use of animals for human neurotoxic hazard and risk assessment.

In this project, different hIPSC-derived neurons grown both in 2D and in 3D will be used to investigate the (neurotoxic) effects of selected reference compounds on neuronal function for comparison with existing in vitro (rodent) and in vivo (rodent and human) data. Therefore, effects of selected reference compounds on neuronal (electrical) activity and calcium homeostasis will be investigated with state of the art multi-electrode array recordings and single cell fluoresce microscopy, respectively. The overarching aim is the large-scale implementation of animal-free testing methods upon successful characterization and validation of truly predictive (2D or 3D) hIPSC-derived neurons for human neurotoxicity assessment.

Jonelle Meijer – Neurotoxic potential of micro- and nanoparticles

Given the global abundance and environmental persistence, humans are continuously exposed to micro- and nanoplastics (MNPs). Despite their abundance, even in consumer products, drinking water and the food chain, the effects of MNPs on human health have not been extensively studied. However, current evidence indicates that MNPs can be taken up by mammals and can reach the brain. Nevertheless, to date, there is no solid hazard characterisation of MNPs with respect to neurotoxicity. As the brain is amongst the most sensitive organs of our body, the aim of this project is to determine the potential neurotoxic effects of exposure to (environmentally relevant) micro- and nanoplastics (MNPs). This project on the potential neurotoxic effects of small plastic particles is part of the ZonMW research programme.

This project is focused on two central questions: Can MNPs reach the brain, and are MNPs able to affect neuronal development and function. To investigate if MNPs can reach the brain, an in vitro human BBB model grown on inserts will be exposed to different sizes of plastic particles, including fluorescent polystyrene reference particles as well as environmentally relevant polyethylene particles. The ability of plastic particles to cross the BBB and thus reach the brain will be assessed by collecting and analyzing the medium for the presence of MNPs using flow cytometry and (fluorescence) microscopy.

In parallel, functional neurotoxicity will be determined using multiwell microelectrode array recordings (mwMEA). The MEA provides a real-time recording of the activity of neurons and can be used as a measure to determine adverse effects. Potential effects of MNP exposure will be assessed following short-term acute exposures as well as following chronic exposures during the development of the neuronal culture. In the final stage of the project, efforts will focus on functional integration of the BBB model with MEA recordings of neuronal cultures.

Anne Zwartsen – Neurotoxicity of drug of abuse

Currently, over 400 New Psychoactive Substances (NPS) that may pose a health threat are monitored by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). The use and number of NPS is constantly increasing, and the lifetime prevalence of NPS in young adults increased to nearly 10% in the EU. Similarly, the reported number of intoxications and deaths in Europe related to NPS exposure has increased as well, with NPS accounting for nearly 10% of all drug-related Emergency Department visits in 2014. The negative health effects related to NPS use can be severe, including neurological, psychological and cardiovascular effects. However, the modes of action of most NPS are not well studied. Therefore, it has become an urgent matter to advance the understanding of the mechanisms underlying the neurotoxic potential of NPS. Within this project, specific expertise on cellular neurophysiological mechanisms and methodologies will therefore be combined with current state of the art (human) in vitro models to identify potential novel modes of action of NPS and to investigate the associated health risks.

In this project, mechanistic in vitro studies will focus on effects of NPS on neuronal activity as well as novel modes of action. Therefore, effects of NPS on electrical activity will be investigated with state of the art multi-electrode array recordings of rat primary cortical neurons as well as human ‘induced pluripotent stem cell’ (hIPSC)-derived neurons. Additionally, effects of NPS on specific modes of action will be assessed, including neurotransmitter transporters as well as clinically relevant neurotransmitter receptors.

Marieke Meijer / Milou Dingemans – DENAMIC: Screening of low-dose chemical mixtures

This research takes place within the multi-disciplinary EU collaborative project DENAMIC (Developmental Neurotoxicity Assessment of Mixtures in Children). The aim of this project is to evaluate and develop screening methods for chemical-induced developmental neurotoxicity and to improve assessment of exposures and effects. To this aim a dual approach is followed by the DENAMIC consortium consisting of 13 partners (universities, research institutes and SMEs). Hazard characterization for developmental neurotoxicity by chemicals and low-dose mixtures using newly developed experimental approaches (in vitro and in vivo studies) will be combined with the study of associations between neurodevelopment and exposure to these chemicals in large EU epidemiological cohorts. In both the experimental and epidemiological part of DENAMIC, biomarkers for developmental neurotoxicity will be developed. There is particular attention for the exposure to environmental pollutants and possible neurotoxic effects at low doses and in mixtures. The Neurotoxicology Research Group at IRAS will focus on the effects of single chemicals and mixtures in in vitro screening tools for developmental neurotoxicity.

A reference set of chemicals will be used for the evaluation of in vitro screens for developmental neurotoxicity. The reference set consists of known (developmentally) neurotoxic compounds as well as emerging chemicals for which the (developmentally) neurotoxic potential is suspected or unknown. Effects of the reference chemicals on cell viability of neuronotypic cell lines will be performed for concentration range-finding and as initial screening of toxicity. Based on these results, chemicals will be selected for additional screening for inter- and intracellular neurotoxicity and for effect assessment of in vitro developmental processes using fluorescent imaging approaches. Binary mixtures and human milk extracts are included to mimic real-life exposure situations and to investigate mixture toxicity.In addition to neuronotypic cell lines, neural progenitor cells will be used as an in vitro model for the developing brain. Furthermore, for evaluation of the in vitro tools, synaptic plasticity will be investigated ex vivo in brain tissues from developmentally exposed mice.

Martje de Groot – Neurotoxicity of low-frequency electromagnetic fields

Exposure to ELF-EMFs (50 Hz), such as those generated by power lines and domestic electric appliances in homes, has increased considerably in recent years. Early epidemiological studies suggesting a small but significant association of ELF-EMF exposure with the development of childhood leukemia, raised scientific and public concern regarding the potential effects of ELF-EMFs on human health. These concerns regard in particular effects on human neuronal development and function. However, despite ongoing research, it is unclear whether ELF-EMF exposure is associated with adverse human health effects other than childhood leukemia. Additionally, the basic interaction mechanisms between these relatively weak fields and living matter are presently unclear, even though a large number of reports have been published regarding biological effects caused by 50/60 Hz EMFs during the last few years. To identify the underlying associated mechanisms and enable risk identification and assessment for ELF-EMF exposure, it is important to establish dose-response relationships and no-observed-effect-levels of ELF-EMF exposure with respect to cellular processes, including calcium homeostasis and intracellular levels of anti-oxidants.

In this project, several in vitro models will be used to investigate the possible neurotoxic potential of ELF-EMF exposure. Using both naïve and differentiated PC12 cells, as well as developing and mature neurospheres we try to mimic different (and presumably more or less susceptible) stages of brain development. The effects of acute, developmental and/or chronic exposure to 50Hz ELF-EMF exposure will be examined for intensities ranging from 0.01 uT (well below background exposure) to 1000 uT (10.000 times higher than typical human exposure). Using single cell microscopy and biochemical techniques, effects of ELF-EMF exposure on neuronal viability, morphology and function, with focus on calcium homeostasis, will be determined. The collected data will be used to determine if a causal relation between ELF-EMF exposure and effects on neuronal function or development exists and to determine so-called dose-effect curves and no-observed-effect-levels.

Hester Hendriks – ENFIRO: Neurotoxicity of alternative flame retardents

The wide application of flame retardants in our living environment and the increasing environmental burden of these compounds has raised toxicological and ecological concern. Therefore, brominated flame retardants (BFRs), including polybrominated diphenylethers (PBDEs), must be replaced by safe and less persistent alternatives. However, the toxic potential of the proposed (AFRs) is currently unknown. Previous research identified the nervous system as being the most sensitive target organ for flame retardants. BFRs are reported to cause lasting neurobehavioral changes in rodents as well as alterations in synaptic plasticity in brain slices. On a cellular level, BFRs have been reported to alter calcium homeostasis and neurotransmitter release. It is therefore essential to assess the neurotoxic potential of AFRs before these compounds are globally used on a large scale.

The potential cytotoxic effects of AFRs will be assessed in PC12 cells using a MTT assay. Amperometry and calcium-imaging techniques will be used to study which AFRs affect intracellular calcium homeostasis and basal or evoked neurotransmitter release in PC12 cells. The effects of those AFRs that are considered to have a low neurotoxic potential are used to measure effects on the functioning of nicotinic acetylcholine receptors expressed in Xenopus oocytes using the two-electrode voltage-clamp technique. Supposedly safe AFRs with the potential of large scale use will then be selected for assessing effects on synaptic plasticity in mouse hippocampal slices.

Alfons Kroese – Neuro-immune modulation of the gastrointestinal tract

Mast cells are often found in close proximity of sensory nerve endings in the intestine and there is a direct bi-directional communication between mast cells and the neurons. Intestinal inflammation has consequences for the functioning of the enteric nervous system, especially resulting from the activation of neurons by inflam­ma­tory media­tors such as prostaglandi­nes and interleukins. Alterations in this regulatory process, so as for instance occur during acute pancreatitis, food allergy and intoxication, can induce changes in permeability and integrity of the intestinal barrier.

The aim of the project is the morphological, electrophysiological and neuropharmacological identification of the role, the mechanisms and the effects of the bi-directional interactions between the enteric nervous system and the enteric immune system on the functioning of the gastrointestinal tract under normal and inflammatory conditions.

Cellular electrophysiology and neuropharmacology using sharp and patch clamp microelectrode techniques for studying voltage-gated and ligand-gated ion channels and the excitability of neurons. Confocal laser scanning microscopy using fluorescent dyes for measurement of intracellular Ca²⁺ concentration. Immunohistochemistry with antibodies for identification of membrane receptors and cellular neuropeptides. Enteric neurons in wholemount preparations, cultured neurons, cell lines and primary cultured mast cells. Quantification of the integrity and permeability of the intestinal barrier in vitro in Ussing chambers.

Harm Heusinkveld – 3R strategy for neurotoxicity hazard, risk and safety

Humans are exposed to numerous chemicals via the environment. To prevent adverse health effects, risk and safety assessment are obligatory before chemicals are allowed on the market. Regulation regarding chemical legislation and exposure has been based on animal research for many years. Currently, there is societal and scientific incentive to (partly) replace animal research by human cellular assays. An integrated, system biology approach will reveal which combination of experiments and methods is best suited to predict human neurotoxicity, which is a clear societal and regulatory need. As such the N3rvousSystem project not only improves risk and safety assessment but also makes a strong contribution to the reduction and replacement of animal experiments.

Effects of selected reference chemicals on neuronal function and calcium signaling will be investigated in human and mouse embryonic stem cell derived neurons using state of the art multi-electrode array recordings and single cell fluoresce microscopy, respectively, and combined with information on phosphoproteomics and pathway analysis. This in vitro data will be combined with information from the intact animal (specific neurobehavioral readouts and kinetic information collected at collaborating institutes) in a testing strategy for neurotoxicity hazard.

Elsa Antunes – ATHON: toxicity of non-dioxin-like PCBs in food

PCBs are prevalent contaminants in fatty food of animal origin, such as meat, certain fish and dairy products. The present PCB exposure of general population in several countries is in the same range as the exposure level at which subtle neurotoxic effects have been observed in infants following perinatal exposure. Unlike dioxin-like PCBs (DL-PCBs) congeners, there is little insufficient information on the mechanisms of action of NDL-PCBs congeners and their relevance and presence in food to perform a health risk assessment. Because NDL-PCBs congeners constitute a large part of PCB congeners found in food and human tissues, it is of major importance to understand the toxicology of NDL-PCBs.

The aim of this project is to study the neurotoxic and endocrine disruptive effect of non-dioxin-like PCBs (NDL-PCBs). Therefore, the effects NDL-PCBs on pre- and postsynaptic aspects of neurotransmission, steroidogenesis, steroid metabolism, androgen receptor expression, and enzymes activities in vitro will be investigated.

The effects of NDL-PCBs on the function of the human GABAA expressed in Xenopus oocytes will be assed by measuring the effects on GABA-evoked Cl^– currents. Amperometry and calcium-imaging techniques will be used to study which congeners (or metabolites) affect intracellular calcium homeostasis and basal or evoked neurotransmitter release in neuroendocrine PC12 cells. Several cell lines will be used to study effects of several PCBs congeners and their hydroxyl- and methylsulfonyl-metabolites on steroidogenesis and steroid metabolism. Special attention will be given to androgen receptor expression, aromatase and COMT activity.

Laura Hondebrink – Neuroreceptor interactions of drug of abuse

A large part of the population (~25%) in the Western world has once or more frequently been exposed to cannabis or other drugs of abuse. The use of these drugs of abuse is associated with health problems, addiction and social problems and has major financial consequences for both society and addicted users. Surprisingly, for many designer drugs the working mechanism and the (secondary?) mechanism (s?) causing the adverse health effects are not known. This holds in particular for chronic exposure, developmental exposure and for exposure to mixtures of drugs of abuse (e.g., alcohol and cocaine). Another issue is that many new designer drugs with unknown working mechanism and health effects are put onto the market every year. It should also be noted that some people are more sensitive to these drug effects than other people. It is therefore of importance to study the basis of these inter-individual differences in vulnerability. Contrasting to these adverse effects, some of these drugs have been ascribed a medicinal and possible neuroprotective effects. Therefore, it has become an urgent matter to advance the understanding of the working mechanisms underlying both the neurotoxic and neuroprotective potential of new and already existing designer drugs.

The aim of this project is to gain insight in the neurophysiological mechanisms underlying the effects of designer drugs during acute, chronic and developmental exposure. Interindividual differences as well as combinations of drugs will also be examined.

Effects on activation/inhibition of postsynaptic neurotransmitter receptors and presynaptic release mechanisms will be investigated. Electrophysiological techniques (voltage clamp, amperometry) will be combined with calcium imaging techniques to determine the mechanisms in which drugs exert their effects. These techniques will first be applied in in vitro models, and validated using ex vivo brain slices and/or models for neurodevelopment. Interindividual differences in receptor subunit compositions will be examined with a heterologues expression system; Xenopus oocytes. Experiments in a parallel PhD project will be performed in human volunteers; these data will be used to relate mechanistic findings to clinically relevant exposure data.

Jakub Rychter – Role of mast cells in the impairment of the intestinal barrier

The main objective of the project is the determination of the role of mucosal mast cells in the impairment of the intestinal mucosal barrier during pathological conditions such as acute pancreatitis (AP). Functional and morphological properties of the intestinal barrier of mice with induced AP will be characterized in vitro with the aim to quantify its impairment. With the aim to obtain direct evidence for a role of the mast cells in the impairment of the intestinal barrier, the barrier properties will be measured also on tissues from (KO) mice that lack mucosal mast cells or mast cell protease-1. The applicability of this approach on existing models of food allergies will also be tested. The possibility to suppress the barrier impairment will be investigated by application of probiotics to these models. Further, the pathways involved in mast cell activation by physiological signalling molecules (neurotransmitters and neuropeptides) will be characterized in a cultured mucosal mast cell model. This will, apart from providing detailed insight in the mechanisms associated with the activation of mast cells, allow for the development of verifiable hypotheses for the (presumably neural) origin of the activating substances and for the neuro-immune interactions involved in impairment of the barrier.

To characterize the mucosal barrier condition, electrical resistance and permeability of isolated mouse ileum and colon will be measured in vitro in a Ussing chamber. The Ussing chamber experiments will be performed on an AP mice model (using cerulein). The degree of intactness of the mucosal barrier will be determined morphologically by immunohistochemical labelling of functionally relevant proteins in the tight junctions between the epithelial cells.

Using electrophysiological techniques for characterization of membrane ion channels and optical recordings of the intracellular calcium concentration, in vitro mast cell responses to activating substances will be obtained to characterize membrane receptors, cellular responses and intracellular signalling pathways. Also, the vesicular release of serotonin during degranulation will be quantified using amperometry with single vesicle resolution.

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