An in vitro developmental neurotoxicity screening assay for retinoic acid-induced neuronal differentiation using the human NT2/D1 cell line

Abstract

Traditional approaches (e.g., neurobehavior, neuropathology) can detect alterations in apical endpoints indicative of developmental neurotoxicity (DNT). However, there is an increasing desire to understand mode-of-action (MOA) for DNT effects; thus, this short communication describes initial work on a neuronal differentiation assay. Basically, our laboratory used the human NT2/D1 cell line to develop an assay to evaluate toxicants for effects on all-trans retinoic acid (RA)-induced neuronal differentiation. Based on literature reports, we selected a neuronal protein, neuronal class III β-tubulin (β3-tubulin), as a marker of differentiation. For this assay, cultured RA-treated NT2 cells were trypsinized to individual cells, methanol fixed, and labeled with a β3-tubulin specific monoclonal antibody (TUJ1). Characterization studies using 100,000 cells/sample showed that NT2 cells had appreciable expression of β3-tubulin starting around day 7 of the differentiation process with a peak expression noted around day 12. Methylmercury, 22(R)-hydroxycholesterol, N-(4-hydroxyphenol)retinamide (4HPR), and 9-cis retinoic acid were selected as initial test compounds. Of these, only 9-cis RA, which is known to affect the RA pathway, was positive for specific impacts on differentiation. These results demonstrate the feasibility of using a flow cytometry method targeting specific cellular biomarkers for evaluating effects on neuronal differentiation. Additional assays are needed to detect compounds targeting other (non-RA) neuronal differentiation pathways. Ultimately, a battery of in vitro assays would be needed to evaluate the potential MOAs involved in altered neuronal differentiation.