Evaluation of a Human Neural Stem Cell Culture Method for Prediction of the Neurotoxicity of Anti-epileptics

Abdal-jabbar Al-Rubai, Peter Wigmore and Margaret K. Pratten

Human neural stem cells have been proposed as an in vitro model to predict neurotoxicity. In this study, the potential of in vitro cultures of human-derived neurospheres to predict the effects of various anti-epileptic drugs (sodium valproate, phenytoin, carbamazepine and phenobarbitone) was evaluated. In general, these drugs had no significant effects on cell viability, total cellular protein, and  neuronal process length at low doses, but at high doses these parameters were reduced significantly. Therapeutic doses of sodium valproate and phenytoin had a clear effect on neurosphere size and cell migration, with a significant reduction in both parameters when compared with the control group. The other drugs (carbamazepine and phenobarbitone) reduced neurosphere size and cell migration only at higher doses. The expression levels of glial fibrillary protein and tubulin III, which were used to identify astrocytes and neuronal cells, respectively, were reduced in a dose-dependent manner that became significant at high doses. The levels of glial fibrillary protein did not indicate any occurrence of reactive astrocytosis.

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ESNATS Conference — The Use of Human Embryonic Stem Cells for Novel Toxicity Testing Approaches

Costanza Rovida, Manon Vivier, Bernward Garthoff and Jürgen Hescheler

The main achievements and results of the ESNATS project (Embryonic Stem Cell-based Novel Alternative Testing Strategies) were presented at the final project conference that was held on 15 September 2013, the day before the traditional EUSAAT (European Society for Alternatives to Animal Testing) Congress in Linz, Austria. The ESNATS project was an FP7 European Integrated Project, running from 2008 to 2013, the aim of which was to develop a novel toxicity testing platform based on embryonic stem cells (ESCs), and in particular, human ESC (hESCs), to accelerate drug development, reduce related R&D costs, and propose a powerful alternative to animal tests in the spirit of the Three Rs principles. Altogether, ESNATS offered the first proof of concept that hESCs can be used to create robust, reproducible and ready-to use test assays for predicting human toxicity. In the end, essentially five test systems were developed to an adequate level for entering possible pre-validation procedures. These methods are based on hESCs, and can be combined to study the possible effects, on the human embryo, of exposure to a chemical during the early stages of development. In addition to the presentations by the main project partners, external speakers were invited to give lectures on relevant topics, both in the field of neurotoxicity and, more generally, on the applicability of hESCs in the development of advanced in vitro tests.

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Neurotoxicity of Seven MEIC Chemicals Evaluated in Organotypic Cultures of Chick Embryonic Dorsal Root Ganglia

Václav Mandys, Katerina Jirsová and Jirí Vrana

The neurotoxic effects of seven selected Multicenter Evaluation of In Vitro Cytotoxicity programme chemicals (methanol, ethanol, isopropanol, sodium chloride, potassium chloride, iron [II] sulphate and chloroform) were evaluated in organotypic cultures of chick embryonic dorsal root ganglia (DRG), maintained in a soft agar culture medium. Two growth parameters of neurite outgrowth from the ganglia — the mean radial length of neurites and the area of neurite outgrowth — were used to evaluate the toxicities of the chemicals. Dose-dependent decreases of both parameters were observed in all experiments. IC50 values (the concentration causing 50% inhibition of growth) were calculated from the dose-response curves established at three time-points during culture, i.e. 24, 48 and 72 hours. The lowest toxic effect was observed in cultures exposed to methanol (the IC50 ranging from 580mM to 1020mM). The highest toxic effect was observed in cultures exposed to iron (II) sulphate (the IC50 ranging from 1.2mM to 1.7mM). The results of other recent experiments suggest that organotypic cultures of DRG can be used during in vitro studies on target organ toxicity within the peripheral nervous system. Moreover, these cultures preserve the internal organisation of the tissue, maintain intercellular contacts, and thus reflect the in vitro situation, more precisely than other cell cultures.
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Development of an In Vitro Test Battery for the Estimation of Acute Human Systemic Toxicity: An Outline of the EDIT Project

Cecilia Clemedson, Marika Nordin-Andersson, Henning F. Bjerregaard, Jørgen Clausen, Anna Forsby, Helena Gustafsson, Ulrika Hansson, Boris Isomaa, Carsten Jørgensen, Ada Kolman, Natalia Kotova, Gunter Krause, Udo Kristen, Kalle Kurppa, Lennart Romert and Ellen Scheers

The aim of the Evaluation-guided Development of New In Vitro Test Batteries (EDIT) multicentre programme is to establish and validate in vitro tests relevant to toxicokinetics and for organ-specific toxicity, to be incorporated into optimal test batteries for the estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity obtained with three human cell line tests (R2 = 0.77), in the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme. However, the results from the MEIC study indicated that at least two other types of in vitro test ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity - to determine key kinetic events (such as biotransformation and passage through biological barriers), and to predict crucial organ-specific mechanisms not covered by the tests in the MEIC battery. The EDIT programme will be a case-by-case project, but the establishment and validation of new tests will be carried through by a common, step-wise procedure. The Scientific Committee of the EDIT programme defines the need for a specific set of toxicity or toxicokinetic data. Laboratories are then invited to perform the defined tests in order to provide the "missing" data for the EDIT reference chemicals. The results obtained will be evaluated against the MEMO (the MEIC Monograph programme) database, i.e. against human acute systemic lethal and toxicity data. The aim of the round-table discussions at the 19th Scandinavian Society for Cell Toxicology (SSCT) workshop, held in Ringsted, Denmark on 6-9 September 2001, was to identify which tests are the most important for inclusion in the MEIC battery, i.e. which types of tests the EDIT programme should focus on. It was proposed that it is important to include in vitro methods for various kinetic events, such as biotransformation, absorption in the gut, passage across the blood-brain barrier, distribution volumes, protein binding, and renal clearance/accumulation. Models for target organ toxicity were also discussed. Because several of the outlier chemicals (paracetamol, digoxin, malathion, nicotine, paraquat, atropine and
potassium cyanide) in the MEIC in vivo-in vitro evaluation have a neurotoxic potential, it was proposed that
the development within the EDIT target organ programme should initially be focused on the nervous system.
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Metabolism and Neurotoxicity: The Significance of Genetically Engineered Cell Lines and New Three-Dimensional Cell Cultures

Sandra Coecke, Chantra Eskes, Joanne Gartion, Erwin van Vliet, Agnieszka Kinsner, Alessia Bogni, Laura Raimondo, Nicholaos Parissis and Ingrid Langezaal

Until now, no methods have been validated for the determination of neurotoxic effects or for the evaluation of metabolism-mediated hazards of chemical substances. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 30,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process, with an emphasis on metabolism-mediated toxicity and neurotoxicity.
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