Sandra Coecke

Workgroup Report: Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity into International Hazard and Risk Assessment Strategies

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19–21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

A Human Stem Cell-Based Model for Identifying Adverse Effects of Organic and Inorganic Chemicals on the Developing Nervous System

The aim of our study was to investigate whether a human neural stem cell line derived from umbilical cord blood (HUCB‐NSC) can serve as a reliable test model for developmental neurotoxicity (DNT). We assessed the sensitivity of HUCB‐NSCs at different developmental stages to a panel of neurotoxic (sodium tellurite, methylmercury chloride, cadmium chloride, chlorpyrifos, and L‐glutamate) and non‐neurotoxic (acetaminophen, theophylline, and D‐glutamate) compounds. In addition, we investigated the effect of some compounds on key neurodevelopmental processes like cell proliferation, apoptotic cell death, and neuronal and glial differentiation. Less differentiated HUCB‐NSCs were generally more sensitive to neurotoxicants, with the notable exception of L‐glutamate, which showed a higher toxicity to later stages. The relative potencies of the compounds were: cadmium chloride > methylmercury chloride ≫ chlorpyrifos ≫ L‐glutamate. Fifty nanomolar methylmercury chloride (MeHgCl) inhibited proliferation and induced apoptosis in early‐stage cells. At the differentiated stage, 1 μM MeHgCl induced selective loss of S100β‐expressing astrocytic cells. One millimolar L‐glutamate did not influence the early stages of HUCB‐NSC development, but it affected late stages of neuronal differentiation. A valuable system for in vitro DNT assessment should be able to discriminate between neurotoxic and non‐neurotoxic compounds and show different susceptibilities to chemicals according to developmental stage and cell lineage. Although not exhaustive, this work shows that the HUCB‐NSC model fulfils these criteria and may serve as a human in vitro model for DNT priority setting. STEM CELLS 2009;27:2591–2601

Micro-stamped surfaces for the patterned growth of neural stem cells

We present a method for patterning neural stem cells based on pre-patterning polypeptides on a cell-repellent surface (poly(ethylene) oxide-like, PEO-like, plasma-deposited films). The method ensures cell attachment and stability for several weeks, as well as it allows cell migration and differentiation. Various patterns of approximately 1 nm thick cell adhesive poly-L-lysine (PLL) have been created on a cell-repellent PEO-like matrix by microcontact printing using different array configurations and printing conditions. The cell-repellent property of PEO-like film determined the confinement of the cells on the printed patterns. Optimization of the printing method showed that the most homogeneous patterns over large areas were obtained using PLL diluted in carbonate buffer (100mM) at pH 8.4. Neural stem cells cultured on the PLL patterns in low serum and in differentiating medium over 20 days exhibited a good confinement to the polypeptide domains. The number of cells attached increased linearly with the micro-stamped PLL area. The cells were able to extend random axon-like projections to the outside of the patterns and presented high amount of ramifications when cultured in differentiating medium. Migration and axon-like outgrowth have been successfully guided by means of an interconnected squares configuration. The surfaces are suitable for controlling the patterning of stem cells and provide a platform for the assessment of the way how different cell arrangements and culture conditions influence cell interactions and cell developmental processes.

Inflammatory neurodegeneration induced by lipoteichoic acid from Staphylococcus aureus is mediated by glia activation, nitrosative and oxidative stress, and caspase activation.

In this study we investigated the mechanisms of neuronal cell death induced by lipoteichoic acid (LTA) and muramyl dipeptide (MDP) from Gram‐positive bacterial cell walls using primary cultures of rat cerebellum granule cells (CGCs) and rat cortical glial cells (astrocytes and microglia). LTA (± MDP) from Staphylococcus aureus induced a strong inflammatory response of both types of glial cells (release of interleukin‐1β, tumour necrosis factor‐α and nitric oxide). The death of CGCs was caused by activated glia because in the absence of glia (treatment with 7.5 µm cytosine‐d‐arabinoside to inhibit non‐neuronal cell proliferation) LTA + MDP did not cause significant cell death (less than 20%). In addition, staining with rhodamine‐labelled LTA confirmed that LTA was bound only to microglia and astrocytes (not neurones). Neuronal cell death induced by LTA (± MDP)‐activated glia was partially blocked by an inducible nitric oxide synthase inhibitor (1400 W; 100 µm), and completely blocked by a superoxide dismutase mimetic [manganese (III) tetrakis (4‐benzoic acid)porphyrin chloride; 50 µm] and a peroxynitrite scavenger [5,10,15,20‐tetrakis (4‐sulfonatophenyl) porphyrinato iron (III); 100 µm] suggesting that nitric oxide and peroxynitrite contributed to LTA‐induced cell death. Moreover, neuronal cell death was inhibited by selective inhibitors of caspase‐3 (z‐DEVD‐fmk; 50 µm) and caspase‐8 (z‐Ile‐Glu(O‐Me)‐Thr‐Asp(O‐Me) fluoromethyl ketone; 50 µm) indicating that they were involved in LTA‐induced neuronal cell death.

mRNA Expression is a Relevant Tool to Identify Developmental Neurotoxicants Using an In Vitro Approach

So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Co-operation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF-68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100beta) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation.

Advancing the science of developmental neurotoxicity (DNT): Testing for better safety evaluation

test Guidelines OPPtS 8706300 on DNt (US ePA, 1998) and in 2007 the Organization for economic Cooperation and Development (OeCD) endorsed a new OeCD DNt test Guideline 426 (OeCD, 2007). these guidelines are largely based on animal studies and are used as higher tiered, triggered tests based on structure activity relationships or evidence of neurotoxicity in standard adult, developmental, or reproduction studies (Makris et al., 2009). experts at the conference stated that these in vivo tests are unsuitable for screening large numbers of chemicals for many reasons including low throughput, high cost, and questions regarding reliability. there was also consensus that new, reliable, and efficient screening and assessment tools are needed for better identification, prioritization, and evaluation of chemicals with the potential to induce developmental neurotoxicity. the information obtained from these screening studies will likely also help to refine animal tests and to inform epidemiological studies.

Meeting report: Validation of toxicogenomics-based test systems: ECVAM-ICCVAM/NICEATM considerations for regulatory use

This is the report of the first workshop “Validation of Toxicogenomics-Based Test Systems” held 11–12 December 2003 in Ispra, Italy. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and organized jointly by ECVAM, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). The primary aim of the workshop was for participants to discuss and define principles applicable to the validation of toxicogenomics platforms as well as validation of specific toxicologic test methods that incorporate toxicogenomics technologies. The workshop was viewed as an opportunity for initiating a dialogue between technologic experts, regulators, and the principal validation bodies and for identifying those factors to which the validation process would be applicable. It was felt that to do so now, as the technology is evolving and associated challenges are identified, would be a basis for the future validation of the technology when it reaches the appropriate stage. Because of the complexity of the issue, different aspects of the validation of toxicogenomics-based test methods were covered. The three focus areas include a) biologic validation of toxicogenomics-based test methods for regulatory decision making, b) technical and bioinformatics aspects related to validation, and c) validation issues as they relate to regulatory acceptance and use of toxicogenomics-based test methods. In this report we summarize the discussions and describe in detail the recommendations for future direction and priorities.

Relevance of in vitro neurotoxicity testing for regulatory requirements: Challenges to be considered

The current testing requirements for both adult and developmental neurotoxicity evaluation are based on in vivo animal models and the neurotoxic potency of compounds is mainly determined by neurobehavioural and neuropathological effects. In vitro studies are considered complementary to animal tests because they provide an understanding of the molecular/cellular mechanisms involved in neurotoxicity. However, the selection of relevant in vitro neuronal/glial specific endpoints applied to various neuronal cellular models should be done in a careful way to build reliable and feasible testing strategies since usually these endpoints have to be tested in various complementary in vitro systems. The requirements for applying a more complex test strategy where toxicokinetic aspects are included together with different tools to compensate for the lack of in vitro metabolic competence are discussed. Taking into consideration the recent European Commission chemical legislation concerning registration, evaluation and authorisation of chemicals (REACH) it has become a priority to develop new intelligent testing strategies integrating computational models and in vitro assays based on cell culture models and endpoints that are amenable for adaptation to high throughput screening to be able to test a large number of chemicals.

The value of alternative testing for neurotoxicity in the context of regulatory needs

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.

Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA(A) receptors), proliferation and differentiation of astrocytes (GFAP and S100beta) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA(A) receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA(A) receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100beta) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.