Developmental Neurotoxicity Evaluation Research Articles

Developmental neurotoxicity evaluation of three Chinese herbal medicines in zebrafish larvae by means of two behavioral assays: Touch-evoked response and light/dark transition

Chinese herbal medicine (CHM) is used among pregnant women. However, the question of its safety during pregnancy remains unclear. The use of these products relies on history of use data but there are specific toxicities like developmental neurotoxicity that are clearly understudied. Here we use the zebfrafish embryo developmental toxicity assay (ZEDTA) in combination with two behavioral assays: touch-evoked response and Light/Dark (L/D) transition assay to evaluate the neuro/developmental toxicity of three herbal products commonly used in CHM [Chinese name (abbreviation; part of the plant and Scientific name]: tian ma (TM; tuber form Gastrodia elata Blume), lei gong teng (LGT; root and rhizome of Tripterygium wilfordii Hook.f) and cha ye (green tea, leaves from Camellia sinensis (L.) Kuntze). In case significant alterations were detected, single components with potential exposure during pregnancy were identified in the literature and further tested. TM had no neurodevelopmental toxic potential in zebrafish embryos, while LGT and its main compounds triptolide and celastrol induced significant alterations in behavior. Developmental exposure to EGCG, the main catechin of green tea, also produced significant alterations in zebrafish embryos behavior after developmental exposure. A combination of ZEDTA with L/D Transition assay is proposed as a useful combination of alternative methods for DNT assessment of CHM products together with other New Approach Methodologies (NAMs).

S03-03: New approach methodologies (NAMs) for developmental neurotoxicity evaluation (DNT) and their use for regulatory purposes

S03-03: New approach methodologies (NAMs) for developmental neurotoxicity evaluation (DNT) and their use for regulatory purposes

Developmental neuro-toxicity evaluation of endocrine-disrupting chemical butylparaben

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation.

There is a call for a paradigm shift in developmental neurotoxicity (DNT) evaluation, which demands the implementation of faster, more cost-efficient, and human-relevant test systems than current in vivo guideline studies. Under the umbrella of the Organisation for Economic Co-operation and Development (OECD), a guidance document is currently being prepared that instructs on the regulatory use of a DNT in vitro battery (DNT IVB) for fit-for-purpose applications. One crucial issue for OECD application of methods is validation, which for new approach methods (NAMs) requires novel approaches. Here, mechanistic information previously identified in vivo, as well as reported neurodevelopmental adversities in response to disturbances on the cellular and tissue level, are of central importance. In this study, we scientifically validate the Neurosphere Assay, which is based on human primary neural progenitor cells (hNPCs) and an integral part of the DNT IVB. It assesses neurodevelopmental key events (KEs) like NPC proliferation (NPC1ab), radial glia cell migration (NPC2a), neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5), and thyroid hormone-dependent oligodendrocyte maturation (NPC6). In addition, we extend our work from the hNPCs to human induced pluripotent stem cell-derived NPCs (hiNPCs) for the NPC proliferation (iNPC1ab) and radial glia assays (iNPC2a). The validation process we report for the endpoints studied with the Neurosphere Assays is based on 1) describing the relevance of the respective endpoints for brain development, 2) the confirmation of the cell type-specific morphologies observed in vitro, 3) expressions of cell type-specific markers consistent with those morphologies, 4) appropriate anticipated responses to physiological pertinent signaling stimuli and 5) alterations in specific in vitro endpoints upon challenges with confirmed DNT compounds. With these strong mechanistic underpinnings, we posit that the Neurosphere Assay as an integral part of the DNT in vitro screening battery is well poised for DNT evaluation for regulatory purposes.

Environmental exposures impact the nervous system in a life stage-specific manner

Abstract Exposure to environmental pollutants like chemicals or air pollution is major health concern for the human population. Especially the nervous system is a sensitive target for environmental toxins with exposures leading to life stage-dependent neurotoxicity. Developmental and adult neurotoxicity are characterized by specific adverse outcomes ranging from neurodevelopmental disorders to neurodegenerative diseases like Alzheimer’s and Parkinson’s disease. The risk assessment process for human health protection is currently undergoing a paradigm change toward new approach methods that allow mechanism-based toxicity assessment. As a flagship project, an in vitro battery of test methods for developmental neurotoxicity evaluation is currently supported by the Organization for Economic Co-operation and Development (OECD). A plethora of stem cell-based methods including brain spheres and organoids are currently further developed to achieve time- and cost-saving tools for linking MoA-based hazards to adverse health effects observed in humans.

Human Oligodendrocytes and Myelin In Vitro to Evaluate Developmental Neurotoxicity.

Neurodevelopment is uniquely sensitive to toxic insults and there are concerns that environmental chemicals are contributing to widespread subclinical developmental neurotoxicity (DNT). Increased DNT evaluation is needed due to the lack of such information for most chemicals in common use, but in vivo studies recommended in regulatory guidelines are not practical for the large-scale screening of potential DNT chemicals. It is widely acknowledged that developmental neurotoxicity is a consequence of disruptions to basic processes in neurodevelopment and that testing strategies using human cell-based in vitro systems that mimic these processes could aid in prioritizing chemicals with DNT potential. Myelination is a fundamental process in neurodevelopment that should be included in a DNT testing strategy, but there are very few in vitro models of myelination. Thus, there is a need to establish an in vitro myelination assay for DNT. Here, we summarize the routes of myelin toxicity and the known models to study this particular endpoint.

Integrating biokinetics and in vitro studies to evaluate developmental neurotoxicity induced by chlorpyrifos in human iPSC-derived neural stem cells undergoing differentiation towards neuronal and glial cells

For some complex toxicological endpoints, chemical safety assessment has conventionally relied on animal testing. Apart from the ethical issues, also scientific considerations have been raised concerning the traditional approach, highlighting the importance for considering real life exposure scenario. Implementation of flexible testing strategies, integrating multiple sources of information, including in vitro reliable test methods and in vitro biokinetics, would enhance the relevance of the obtained results. Such an approach could be pivotal in the evaluation of developmental neurotoxicity (DNT), especially when applied to human cell-based models, mimicking key neurodevelopmental processes, relevant to human brain development.Here, we integrated the kinetic behaviour with the toxicodynamic alterations of chlorpyrifos (CPF), such as in vitro endpoints specific for DNT evaluation, after repeated exposure during differentiation of human neural stem cells into a mixed culture of neurons and astrocytes. The upregulation of some cytochrome P450 and glutathione S-transferase genes during neuronal differentiation and the formation of the two major CPF metabolites (due to bioactivation and detoxification) supported the metabolic competence of the used in vitro model. The alterations in the number of synapses, neurite outgrowth, brain derived neurotrophic factor, the proportion of neurons and astrocytes, as well as spontaneous electrical activity correlated well with the CPF ability to enter the cells and be bioactivated to CPF-oxon. Overall, our results confirm that combining in vitro biokinetics and assays to evaluate effects on neurodevelopmental endpoints in human cells should be regarded as a key strategy for a quantitative characterization of DNT effects.

Assessment of developmental neurotoxicity induced by chemical mixtures using an adverse outcome pathway concept

BackgroundIn light of the vulnerability of the developing brain, mixture risk assessment (MRA) for the evaluation of developmental neurotoxicity (DNT) should be implemented, since infants and children are co-exposed to more than one chemical at a time. One possible approach to tackle MRA could be to cluster DNT chemicals in a mixture on the basis of their mode of action (MoA) into ‘similar’ and ‘dissimilar’, but still contributing to the same adverse outcome, and anchor DNT assays to common key events (CKEs) identified in DNT-specific adverse outcome pathways (AOPs). Moreover, the use of human in vitro models, such as induced pluripotent stem cell (hiPSC)-derived neuronal and glial cultures would enable mechanistic understanding of chemically-induced adverse effects, avoiding species extrapolation.MethodsHiPSC-derived neural progenitors differentiated into mixed cultures of neurons and astrocytes were used to assess the effects of acute (3 days) and repeated dose (14 days) treatments with single chemicals and in mixtures belonging to different classes (i.e., lead(II) chloride and methylmercury chloride (heavy metals), chlorpyrifos (pesticide), bisphenol A (organic compound and endocrine disrupter), valproic acid (drug), and PCB138 (persistent organic pollutant and endocrine disrupter), which are associated with cognitive deficits, including learning and memory impairment in children. Selected chemicals were grouped based on their mode of action (MoA) into ‘similar’ and ‘dissimilar’ MoA compounds and their effects on synaptogenesis, neurite outgrowth, and brain derived neurotrophic factor (BDNF) protein levels, identified as CKEs in currently available AOPs relevant to DNT, were evaluated by immunocytochemistry and high content imaging analysis.ResultsChemicals working through similar MoA (i.e., alterations of BDNF levels), at non-cytotoxic (IC20/100), very low toxic (IC5), or moderately toxic (IC20) concentrations, induce DNT effects in mixtures, as shown by increased number of neurons, impairment of neurite outgrowth and synaptogenesis (the most sensitive endpoint as confirmed by mathematical modelling) and increase of BDNF levels, to a certain extent reproducing autism-like cellular changes observed in the brain of autistic children.ConclusionsOur findings suggest that the use of human iPSC-derived mixed neuronal/glial cultures applied to a battery of assays anchored to key events of an AOP network represents a valuable approach to identify mixtures of chemicals with potential to cause learning and memory impairment in children.

ヒトiPS細胞の神経分化能を指標にした発達神経毒性評価

ヒトiPS細胞の神経分化能を指標にした発達神経毒性評価

Electrophysiological evaluation of developmental neurotoxicity induced by the prenatal exposure to 1-bromopropane

Electrophysiological evaluation of developmental neurotoxicity induced by the prenatal exposure to 1-bromopropane

Current Availability of Stem Cell-Based In Vitro Methods for Developmental Neurotoxicity (DNT) Testing.

There is evidence that chemical exposure during development can cause irreversible impairments of the human developing nervous system. Therefore, testing compounds for their developmentally neurotoxic potential has high priority for different stakeholders: academia, industry, and regulatory bodies. Due to the resource-intensity of current developmental neurotoxicity (DNT) in vivo guidelines, alternative methods that are scientifically valid and have a high predictivity for humans are especially desired by regulators. Here, we review availability of stem-/progenitor cell-based in vitro methods for DNT evaluation that is based on the concept of neurodevelopmental process assessment. These test methods are assembled into a DNT in vitro testing battery. Gaps in this testing battery addressing research needs are also pointed out.

Development of the Concept for Stem Cell-Based Developmental Neurotoxicity Evaluation.

Human brain development consists of a series of complex spatiotemporal processes that if disturbed by chemical exposure causes irreversible impairments of the nervous system. To evaluate a chemical disturbance in an alternative assay, the concept evolved that the complex procedure of brain development can be disassembled into several neurodevelopmental endpoints which can be represented by a combination of different alternative assays. In this review article, we provide a scientific rationale for the neurodevelopmental endpoints that are currently chosen to establish assays with human stem/and progenitor cells. Assays covering these major neurodevelopmental endpoints are thought to assemble as building blocks of a DNT testing battery.

Development of the Adverse Outcome Pathway (AOP): Chronic binding of antagonist to N-methyl-d-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities of children

The Adverse Outcome Pathways (AOPs) are designed to provide mechanistic understanding of complex biological systems and pathways of toxicity that result in adverse outcomes (AOs) relevant to regulatory endpoints. AOP concept captures in a structured way the causal relationships resulting from initial chemical interaction with biological target(s) (molecular initiating event) to an AO manifested in individual organisms and/or populations through a sequential series of key events (KEs), which are cellular, anatomical and/or functional changes in biological processes. An AOP provides the mechanistic detail required to support chemical safety assessment, the development of alternative methods and the implementation of an integrated testing strategy.An example of the AOP relevant to developmental neurotoxicity (DNT) is described here following the requirements of information defined by the OECD Users' Handbook Supplement to the Guidance Document for developing and assessing AOPs. In this AOP, the binding of an antagonist to glutamate receptor N-methyl-d-aspartate (NMDAR) receptor is defined as MIE. This MIE triggers a cascade of cellular KEs including reduction of intracellular calcium levels, reduction of brain derived neurotrophic factor release, neuronal cell death, decreased glutamate presynaptic release and aberrant dendritic morphology. At organ level, the above mentioned KEs lead to decreased synaptogenesis and decreased neuronal network formation and function causing learning and memory deficit at organism level, which is defined as the AO. There are in vitro, in vivo and epidemiological data that support the described KEs and their causative relationships rendering this AOP relevant to DNT evaluation in the context of regulatory purposes.

Prenatal exposure to valproic acid alters the development of excitability in the postnatal rat hippocampus

Prenatal valproic acid (VPA) exposure is a well-known animal model of autism spectrum disorder (ASD) that produces alterations in embryonic and adult neurogenesis as well as adolescent/adulthood neurobehavioral phenotypes. However, the effects of prenatal VPA exposure on neural network excitability, especially during the synaptogenic period around eye opening, are not fully understood. In this study, we orally administered VPA (300 mg/kg) to pregnant Wistar rats on gestation day 15 and subsequently performed field potential recording in the CA1 area of hippocampal slices obtained from control (saline-exposed) and VPA-exposed rat pups between postnatal day (PND) 13 and PND18. In control slices, we observed an abrupt enhancement of stimulation-dependent responses including population spike (PS) amplitudes and field excitatory postsynaptic potential (fEPSP) slopes at PND16, which coincided with the average day of eye opening. In contrast, VPA-exposed pups exhibited delayed eye opening (PND17) and gradual rather than abrupt increases in PS amplitudes and fEPSP slopes over the duration of the synaptogenic period. We next investigated the involvement of ambient GABA (γ-aminobutyric acid) in PS generation using bicuculline methiodide (BMI), a GABA type A (GABAA) receptor antagonist. In control slices, BMI enhanced PS amplitudes during PND14–15 (before eye opening) and had little effect thereafter during PND16–17; a subsequent regression model analysis of BMI ratios (the ratio of PS amplitudes in the presence and absence of BMI) indicated a possible developmental change between these periods. In contrast, almost identical regression models were obtained for BMI ratios during PND14–15 and PND16–17 in the VPA-exposed group, indicating the absence of a developmental change. Our results suggest that prenatal VPA exposure accelerates the development of hippocampal excitability before eye opening. Moreover, our experimental model can be used as a novel approach for the evaluation of developmental neurotoxicity.

Comparative performance analysis of human iPSC-derived and primary neural progenitor cells (NPC) grown as neurospheres in vitro

Developmental neurotoxicity (DNT) testing performed in rats is resource-intensive (costs, time, animals) and bears the issue of species extrapolation. Thus, reliable alternative human-based approaches are needed for predicting neurodevelopmental toxicity. Human induced pluripotent stem cells (hiPSCs) represent a basis for an alternative method possibly being part of an alternative DNT testing strategy.Here, we compared two hiPSC neural induction protocols resulting in 3D neurospheres: one using noggin and one cultivating cells in neural induction medium (NIM protocol). Performance of Nestin+/SOX2+ hiPSC-derived neural progenitor cells (NPCs) was compared to primary human NPCs. Generally, primary hNPCs first differentiate into Nestin+ and/or GFAP+ radial glia-like cells, while the hiPSC-derived NPCs (hiPSC-NPC) first differentiate into βIII-Tubulin+ neurons suggesting an earlier developmental stage of hiPSC-NPC. In the ‘Neurosphere Assay’, NIM generated hiPSC-NPC produced neurons with higher performance than with the noggin protocol. After long-term differentiation, hiPSC-NPC form neuronal networks, which become electrically active on microelectrode arrays after 85days. Finally, methylmercury chloride inhibits hiPSC-NPC and hNPC migration with similar potencies.hiPSC-NPCs-derived neurospheres seem to be useful for DNT evaluation representing early neural development in vitro. More system characterization by compound testing is needed to gain higher confidence in this method.

Using mechanistic information in application of an Adverse Outcome Pathway (AOP) concept for developmental neurotoxicity evaluation

Using mechanistic information in application of an Adverse Outcome Pathway (AOP) concept for developmental neurotoxicity evaluation

Systematic review on methods for developmental neurotoxicity evaluation based on an EFSA Report

Systematic review on methods for developmental neurotoxicity evaluation based on an EFSA Report

Application of the Adverse Outcome Pathway (AOP) concept for evaluation of developmental neurotoxicity triggered by N-methyl-d-aspartate receptor (NMDAR) antagonists

Application of the Adverse Outcome Pathway (AOP) concept for evaluation of developmental neurotoxicity triggered by N-methyl-d-aspartate receptor (NMDAR) antagonists

205 ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY ON NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

Human embryonic stem cells (hESCs) have a potential for differentiation into neuronal cells. Neural differentiation of hESCs is currently used in many fields of neurological study. Therefore, evaluation of developmental neurotoxicity in hESCs is possible during the embryonic period. In the current study, we investigated the neuronal toxicity of hydroxyurea, cytosine arabinoside, and penicillin G at multiple doses (low, medium, high) for 28 days for neural differentiation. For assessment of neural toxicity, we examined the expression of marker genes that represent neural cell development. The mRNA levels of the marker genes were evaluated by real-time PCR in hESCs. Morphological changes of hESCs during neuronal differentiation were also estimated. The results for low and medium doses of hydroxylurea showed a significant increase of dopaminergic neuron marker gene (NR4A2) and GABA neuron marker gene (GAD2). However, glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) showed a significant decrease. Results for another drug, cytosine arabinoside, showed a significant decrease of glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) at a high dose. In addition, cytosine arabinoside caused a significant decrease of dopaminergic neuron marker gene (NR4A2) without significant change in GAD2. For the control, penicillin G, no significant difference in expression of neural specific-genes was observed at all tested doses. These findings suggest that neural-specific genes are perturbed by hydroxyurea and cytosine arabinoside, which may be involved in abnormal neural development during the embryonic neurogenesis period in hESCs.

The classification of motor neuron defects in the zebrafish embryo toxicity test (ZFET) as an animal alternative approach to assess developmental neurotoxicity

Rodents are widely used to test the developmental neurotoxicity potential of chemical substances. The regulatory test procedures are elaborate and the requirement of numerous animals is ethically disputable. Therefore, non-animal alternatives are highly desirable, but appropriate test systems that meet regulatory demands are not yet available. Hence, we have developed a new developmental neurotoxicity assay based on specific whole-mount immunostainings of primary and secondary motor neurons (using the monoclonal antibodies znp1 and zn8) in zebrafish embryos. By classifying the motor neuron defects, we evaluated the severity of the neurotoxic damage to individual primary and secondary motor neurons caused by chemical exposure and determined the corresponding effect concentration values (EC50). In a proof-of-principle study, we investigated the effects of three model compounds thiocyclam, cartap and disulfiram, which show some neurotoxicity-indicating effects in vertebrates, and the positive controls ethanol and nicotine and the negative controls 3,4-dichloroaniline (3,4-DCA) and triclosan. As a quantitative measure of the neurotoxic potential of the test compounds, we calculated the ratios of the EC50 values for motor neuron defects and the cumulative malformations, as determined in a zebrafish embryo toxicity test (zFET). Based on this index, disulfiram was classified as the most potent and thiocyclam as the least potent developmental neurotoxin. The index also confirmed the control compounds as positive and negative neurotoxicants. Our findings demonstrate that this index can be used to reliably distinguish between neurotoxic and non-neurotoxic chemicals and provide a sound estimate for the neurodevelopmental hazard potential of a chemical. The demonstrated method can be a feasible approach to reduce the number of animals used in developmental neurotoxicity evaluation procedures.