An in vitro Developmental Neurotoxicity Screening Assay for Retinoic Acid-Induced Neuronal Differentiation Using the Human NT2/D1 Cell Line.

An in vitro Developmental Neurotoxicity Screening Assay for Retinoic Acid-Induced Neuronal Differentiation Using the Human NT2/D1 Cell Line. Neurotoxicology. 2019 Apr 10;: Authors: Taylor MA, Kan HL, Gollapudi BB, Marty MS 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 (RA) were selected as initial test compounds. Of these, only 9-cis RA, which is known to a...
Source: Neurotoxicology - Category: Neurology Authors: Tags: Neurotoxicology Source Type: research