Diverse neurotoxicants target the differentiation of embryonic neural stem cells into neuronal and glial phenotypes

Abstract

The large number of compounds that need to be tested for developmental neurotoxicity drives the need to establish in vitro models to evaluate specific neurotoxic endpoints. We used neural stem cells derived from rat neuroepithelium on embryonic day 14 to evaluate the impact of diverse toxicants on their ability to differentiate into glia and neurons: a glucocorticoid (dexamethasone), organophosphate insecticides (chlorpyrifos, diazinon, parathion), insecticides targeting the GABAA receptor (dieldrin, fipronil), heavy metals (Ni2+, Ag+), nicotine and tobacco smoke extract. We found three broad groupings of effects. One diverse set of compounds, dexamethasone, the organophosphate pesticides, Ni2+ and nicotine, suppressed expression of the glial phenotype while having little or no effect on the neuronal phenotype. The second pattern was restricted to the pesticides acting on GABAA receptors. These compounds promoted the glial phenotype and suppressed the neuronal phenotype. Notably, the actions of compounds eliciting either of these differentiation patterns were clearly unrelated to deficits in cell numbers: dexamethasone, dieldrin and fipronil all reduced cell numbers, whereas organophosphates and Ni2+ had no effect. The third pattern, shared by Ag+ and tobacco smoke extract, clearly delineated cytotoxicity, characterized major cell loss with suppression of differentiation into both glial and neuronal phenotypes; but here again, there was some selectivity in that glia were suppressed more than neurons. Our results, from this survey with diverse compounds, point to convergence of neurotoxicant effects on a specific “decision node” that controls the emergence of neurons and glia from neural stem cells.

In vitro models reveal differences in the developmental neurotoxicity of an environmental polycylic aromatic hydrocarbon mixture compared to benzo[a]pyrene: Neuronotypic PC12 Cells and embryonic neural stem cells

“…In every experiment, we verified the ability of dexamethasone to evoke significant reductions in cell numbers and specifically to impair emergence of the glial phenotype. The dexamethasone results are not shown here because they are not relevant to PAH effects other than to validate each NSC experiment, and because they replicated the previously published effects (Slotkin et al 2016). …”

“…The techniques for NSC preparation, culturing and assays have all appeared previously (Slotkin et al 2016). Primary neural stem cells (passage zero; MTI-GlobalStem, Gaithersburg, MD) were isolated from rat cortical neuroepithelium on embryonic day 14 and were frozen in DMEM/F-12 medium with N2 supplement (MTIGlobalStem) and 10% DMSO.…”

“…As with the PC12 cell studies, the NSC studies included a positive control, 10 μM dexamethasone (Slotkin et al 2016). In every experiment, we verified the ability of dexamethasone to evoke significant reductions in cell numbers and specifically to impair emergence of the glial phenotype.…”

“…The PC12 model has been used in thousands of studies to evaluate many developmental neurotoxicants, including a full characterization of BaP (Slotkin et al 2013, 2014; Slotkin and Seidler 2009). In contrast, the NSC model considers an earlier decision node, the point at which neural stem cells choose to become neurons or glia; for that purpose, we used NSCs derived from rat neuroepithelium on embryonic day 14, when phenotypic separation into neurons and glia is determined (Slotkin et al 2016; Rodier 1988). We recently showed how diverse developmental neurotoxicants have the ability to divert neurodifferentiation toward or away from these two phenotypes (Slotkin et al 2016).…”

Brominated and organophosphate flame retardants target different neurodevelopmental stages, characterized with embryonic neural stem cells and neuronotypic PC12 cells

“…Studies of the emergence of neural phenotypes in embryonic stem cells readily demonstrate adverse effects, but the point-of-departure concentrations are not separable from those of outright cytotoxicity (Behl et al 2015), and thus do not provide a convincing case for specific effects on neurodevelopment. It is important to note, though, that embryonic stem cells are not the same as neural stem cells, which have already made a commitment to a neural phenotype; indeed, toxicant effects on embryonic stem cells can differ substantially from those of neural stem cells, as shown with a standard developmental neurotoxicant, such as nicotine (Berger et al 1998; Culbreth et al 2012; Ishizuka et al 2012; Liszewski et al 2012; Slotkin et al 2016). Furthermore, the choice of endpoints for neurodifferentiation is critically important.…”