New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools

Characterization of disturbed neural crest migration as mechanism of developmental toxicity of prescription drugs

In the last years, different individual human embryonic stem cell-based developmental toxicity test systems have been established and have been proven to offer new possibilities to explore toxicological hazard directly on relevant and non-transformed human cells. A further achievement has been the combination of these assays to comprehensive batteries able to predict human developmental toxicity. In the framework of the European project ESNATS (Embryonic Stem cell-based Novel Alternative Testing Strategies), we developed a test battery which allows the inclusion of any developmental toxicity assay, and that explores the responses of such test systems to a wide range of compounds. As a first step, we selected and characterized a heterogeneous group of compounds with a wide applicability domain, which ranged from environmental pollutants to several prescription drugs. To evaluate the feasibility of the suggested test framework, we performed the initial screen in a well-characterized assay that evaluates ‘migration inhibition of neural crest cells’ (MINC assay), which finally resulted in the identification of 11 hits (e.g. geldanamycin, arsenite, PBDE-99). Next, transcriptome analysis for some selected MINC hits was performed. The transcriptome changes triggered by these substances in human neural crest cells (NCC) were recorded and analyzed. Transcript profiling allowed a clear separation of different toxicants. Furthermore, a diagrammatic system was developed to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. KEGG pathways or overrepresented gene ontology terms). Finally, the transcript data were mined for potential markers of toxicity. We found that the inclusion of transcriptome data largely increased the information from the MINC phenotypic test. As a final step, one of the MINC-positive compounds, the prescription drug interferon-β (IFNβ), was chosen to be further characterized as potential developmental toxicity hazard. We could confirm the adverse effects of IFNβ on NCC migration in different functional assays. The analysis of transcriptome changes suggested a role of altered JAK-STAT signaling in toxicity, which was confirmed by detailed measurements of interferon effects on signaling in the presence of specific kinase inhibitors.publishe

On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains

In many countries, animal experiments can only be performed when their necessity has been demonstrated in a legal document. As the usefulness of animals in research is also a significant societal and political issue, criteria to structure debates and evaluations are needed. Here, background information is given on laboratory animal studies. Moreover, parameters that may be considered in judging their usefulness are suggested. The discussion is strictly focused on animals used as tools/test systems/models to provide information on humans. In this context, general features and performance characteristics of models are discussed. Examples are given for well-recognized criteria (e.g., robustness, relevance, predictivity) to judge the usefulness of predictive models. The main hypothesis put forward here is that a benefits evaluation (usefulness metrics) is only possible within sharply circumscribed "use domains". Examples are given for the research fields of drug and vaccine research, toxicology, disease pathogenesis, and basic biological research. Efficacy, safety, and quality studies are highlighted as "use domains" within the field of drug discovery and production. A further separation into individual diseases, drug targets or symptoms is suggested for, e.g., efficacy studies or pathophysiology. Finally, an outlook is given on the evaluation of model advantages and disadvantages to arrive at their "net benefit". Moreover, the need to compare the net benefits of animal models versus that of their alternatives is highlighted.publishe

Corners: EUToxRisk

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On the usefulness of animals as a model system (part I) : Overview of criteria and focus on robustness

Banning or reduction of the use of animals for laboratory experiments is a frequently-discussed societal and scientific issue. Moreover, the usefulness of animals needs to be considered in any decision process on the permission of specific animal studies. This complex issue is often simplified and generalized in the media around the question, "Are animals useful as a model?" To render an often emotional discussion about animal experimentation more rational, it is important to define "usefulness" in a structured and transparent way. To achieve such a goal, many sub-questions need to be asked, and the following aspects require clarification: (i) consistency of animal-derived data (robustness of the model system); (ii) scientific domain investigated (e.g., toxicology vs disease modelling vs therapy); (iii) measurement unit for "benefit" (inte-grating positive and negative aspects); (iv) benchmarking to alternatives; (v) definition of success criteria (how good is good enough); (vi) the procedure to assess benefit and necessity. This series of articles discusses the overall benchmarking process by specifying the six issues. The goal is to provide guidance on what needs to be clarified in scientific and political discussions. This framework should help in the future to structure available information, to identify and fill information gaps, and to arrive at rational decisions in various sub-fields of animal use. In part I of the series, we focus on the robustness of animal models. This describes the capacity of models to produce the same output/response when faced with the "same" input. Follow-up articles will cover the remaining usefulness aspects.publishe

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

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

A human pluripotent carcinoma stem cell-based model for in vitrodevelopmental neurotoxicity testing: Effects of methylmercury, leadand aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After the characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity then was evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial) in the control culture and in the cells exposed to the compounds. The obtained results suggest that lead chloride and aluminum nitrate at low concentrations were toxic primary to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The obtained results suggest that neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of the potential neurotoxic compounds.JRC.I.2-Public Health Policy Suppor

miRNA expression profiling in a human stem cell-based model as a tool for developmental neurotoxicity testing

The main aim of this study was to evaluate whether microRNA (miRNA) profiling could be a useful tool for in vitro developmental neurotoxicity (DNT) testing. Therefore, to identify the possible DNT biomarkers among miRNAs, we have studied the changes in miRNA expressions in a mixed neuronal/glial culture derived from carcinoma pluripotent stem cells (NT2 cell line) after exposure to MetHgCl during the process of neuronal differentiation (2-36 DIV). The obtained results identified the presence of a miRNA signature which was specific for neural differentiation in the control culture and another for the response to MetHgCl-induced toxicity. In differentiated neuronal control cultures we observed the down-regulation of the stemness phenotype-linked miR-302 cluster and the over-expression of several miRNAs specific for neuronal differentiation (e.g. let-7, miR-125b and miR-132). In the cultures exposed to MetHgCl (400nM) we observed an over-expression of a signature composed of 5 miRNAs (miR-302b, miR-367, miR-372, miR-196b and miR-141) that are known to be involved in the regulation of developmental processes and cellular stress response mechanisms. Using gene ontology terms and pathway enrichment analysis of the validated targets of the miRNAs deregulated by the toxic treatment, the possible effect of MetHgCl exposure on signaling pathways involved in axon guidance and learning and memory processes was revealed. The obtained data suggest that miRNA profiling could provide simplified functional evaluation of the toxicity pathways involved in developmental neurotoxicity in comparison with the transcriptomics studies.JRC.I.5-Systems Toxicolog

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

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.publishe