Look-alikes may not act alike: Gene expression regulation and cell-type-specific responses of three valproic acid analogues in the neural embryonic stem cell test (ESTn).

In vitro assays to assess developmental neurotoxicity of chemicals are highly desirable. The murine neural embryonic stem cell test (ESTn) can mimic parts of early differentiation of embryonic brain and may therefore be useful for this purpose. The aim of this study was to investigate whether this test is able to rank the toxic potencies of three valproic acid analogues and to study their mode of action by investigating their individual effects on four cell types: stem cells, neurons, astrocytes and neural crest cells. Using immunocytochemical read-outs and qPCR for cell type-specific genes, the effects of valproic acid (VPA), 2-ethylhexanoic acid (EHA) and 2-ethyl-4-methylpentanoic (EMPA) were assessed. VPA and EHA but not EMPA downregulated cell type-specific differentiation makers and upregulated stem cell related markers (Fut4, Cdh1) at different time points during differentiation. Expression of Gfap, a marker for astrocytes, was dramatically downregulated by VPA and EHA, but not by EMPA. This finding was verified using immunostainings. Based on the number and extent of genes regulated by the three compounds, relative potencies were determined as VPA > EHA > EMPA, which is consistent with in vivo developmental toxicity potency ranking of these compounds. Thus, ESTn using a combination of morphology, gene and protein expression readouts, may provide a medium-throughput system for monitoring the effects of compounds on differentiation of cell types in early brain development.