Human Ntera2 cells as a predictive in vitro test system for developmental neurotoxicity

Abstract

Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3Rs" (reduction, replacement, and refinement) of animal testing, alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. The goal of this study was to establish components for a human cell-based test system to assess DNT potential of chemicals at an early stage of brain development. A human neural precursor cell line should be tested for suitability for semi-automated high-throughput DNT screening. We established assays suitable for detecting disturbances in two basic processes of brain development in 96-well scale: neuronal differentiation and migration using the human Ntera2 (NT2) cell line. We assessed the effects of four test compounds with well-established DNT potential in comparison with three compounds without specific DNT potential. We found that human NT2 cell cultures treated with the morphogen, retinoic acid, imitate neuronal differentiation, and migration in vitro. The developmental neurotoxicants methylmercury chloride, sodium arsenite, sodium valproate, and methylazoxymethanol significantly reduced the expression of the neuronal marker β-tubulin type III and decreased the migration distance in developing NT2 cells. Both endpoints, differentiation and migration, can be read out directly in a standard fluorescence plate reader, enabling high-throughput screening. We conclude that NT2 cell tests are likely to become valuable components of a human cell-based modular in vitro DNT test systems.

An evaluation of a human stem cell line to identify risk of developmental neurotoxicity with antiepileptic drugs

“…In other recent studies, valproic acid inhibited mouse neurosphere formation in vitro (Zhou et al, 2011) and increased neuronal apoptosis in cells differentiating from human progenitor cells in vitro (Wang et al, 2011). Moreover, a study that used the NT2 teratocarcinoma stem cell line reported that valproic acid (in the high millimolar range) reduced differentiating cell viability and (in the micromolar range) reduced neural differentiation and migration (Stern et al, 2014). The molecular mechanisms underlying the impact of valproic acid on neural development are not well understood and may involve the regulation of several hundred genes and multiple pathways (see Hill et al, 2013) but were not the purpose of this investigation.…”

“…Importantly, we used human EC stem cells because the neurons derived from them display key morphological, molecular and functional properties of mature neurons including voltage-and ligand-gated ion channels (Coyne et al, 2011;Stewart et al, 2004). Moreover, teratocarcinoma stem cells have been shown to have value in screening for developmental neurotoxicity with heavy metals such as lead, mercury and aluminum (Laurenza et al, 2013;Stern et al, 2014).…”

“…In the present report therefore, we have begun to explore the value of TERA2.cl.SP12 cells in developmental neurotoxicity studies, since EC stem cell lines have already proven useful addressing the impact of metals such as mercury, lead and aluminum (Laurenza et al, 2013;Stern et al, 2014). The specific objectives of our study were to begin to assess the predictive validity of hECSCs in neurotoxicology by determining the impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine and two control drugs, ciprofloxacin and perfluorooctanoic acid on cell viability, cell cycle and their differentiation into neurons.…”

Rotenone exerts developmental neurotoxicity in a human brain spheroid model

“…Diverse workshops and meetings during the past 10 years have summarized the problems, the available assays and roadmaps for DNT in vitro assays and methods (Coecke et al, 2007; Lein et al, 2007; Crofton et al, 2011; Crofton et al, 2014; Bal-Price et al, 2015a; Bal-Price et al, 2015b). Several in vitro studies have been able to reproduce some of the relevant processes that occur in the brain such as neuronal differentiation (Hartley et al, 1999), migration (Stern et al, 2014), cell-cell interaction, neuritic development, synaptogenesis, myelinogenesis (Liu et al, 2012) and neurotransmission (Hogberg et al, 2011). In addition, they showed to be predictive of DNT (Hogberg et al, 2010; Hogberg et al, 2011; Laurenza et al, 2013; Pallocca et al, 2013).…”

Highlight report: critical evaluation of key evidence on health hazards of the general European population by exposure to arsenic

“…A particular research focus is how arsenic influences signaling pathways relevant in cell stress response and carcinogenesis (Chen et al 2014;Wang et al 2014;Weng et al 2014;Liu et al 2013;Jiang et al 2013;Pastoret et al 2013). Recently, also epigenetic alterations (Gribble et al 2014), developmental toxicity (Zimmer et al 2014;Stern et al 2014) and metabolism as well as transport of arsenic Yu et al 2013;Xu et al 2013;Watanabe and Hirano 2013) have been intensively studied. The present review of GundertRemy and colleagues which comprehensively compares arsenic exposure likely to cause adverse effects in humans to current exposure levels of the general European population demonstrates that research of arsenic is of high practical relevance and should lead to regulatory action to reduce the risk of the general population.…”