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ASSESSMENT OF CHEMICAL EFFECTS ON NEURONAL DIFFERENTIATION USING THE ARRAYSCAN HIGH CONTENT SCREENING SYSTEM
Citation:
RADIO, N., T. M. FREUDENRICH, K. M. CROFTON, T. J. SHAFER, AND W. R. MUNDY. ASSESSMENT OF CHEMICAL EFFECTS ON NEURONAL DIFFERENTIATION USING THE ARRAYSCAN HIGH CONTENT SCREENING SYSTEM. Presented at Society of Toxicology, Charlotte, NC, March 25 - 29, 2007.
Description:
The development of alternative methods for toxicity testing is driven by the need for scientifically valid data that can be obtained in a rapid and cost-efficient manner. In vitro systems provide a model in which chemical effects on cellular events can be examined using techniques that are amenable to high-throughput analysis. We used the new technology of high content screening (HCS) to examine the effect of chemicals on the processes of differentiation and neurite outgrowth in Neuroscreen-1 cells (NS-1, a PC12 cell clone). HCS was performed using the Cellomics ArrayScan VTiTM, an automated epifluorescence microscope and image analysis system using cells in a microtiter plate format. To establish the parameters for differentiation, NS-1 cells were grown on a 96-well plate in the absence (negative control) or presence (positive control) of increasing concentrations of nerve growth factor (NGF). Morphological measures indicative of NS-1 cell differentiation and growth (cell size, neurite elaboration, neurite length) were assessed after fixation and staining for Hoechst (nucleus) and beta-tubulin (cell body and neurites). Treatment with NGF caused a concentration-related increase in cell body area, average number of neurites per cell, and neurite length. Maximal effects of NGF occurred at 30-100 ng/ml. Differentiation of NS-1 cells induced by NGF was observable at 48 hr and reached maximum at 72 hr. All subsequent studies used 100 ng/ml NGF to induce differentiation and included non-NGF treated wells as negative controls. NGF-treated cells were exposed to chemicals 2 hr after plating, and differentiation was assessed 72 or 96 hr later. DMSO (vehicle) did not effect differentiation at a concentration of 0.1%. A MAP kinase inhibitor (U0126) and a PKC inhibitor (Bis I) both inhibited differentiation at concentrations that were not cytotoxic. These studies demonstrate the utility of HCS to assess chemical effects on neuronal differentiation and growth in an in vitro model. (This is an abstract of a proposed presentation and does not necessarily reflect EPA policy).