ToxSci Advance Access published online on June 6, 2008
Toxicological Sciences, doi:10.1093/toxsci/kfn114
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Published by Oxford University Press 2008.
Assessment of chemical effects on neurite outgrowth in PC12 cells using high content screening
1 Neurotoxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protections Agency, B105-06, Research Triangle Park, NC 27711 USA 2 The Curriculum in Toxicology, UNC School of Medicine, CB #7270, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA
Correspondence: William R. Mundy, Ph.D., USEPA, Neurotoxicology Division, B105-06, Research Triangle Park, NC 27711, Phone: 919-541-7729. FAX: 919-541-0700. E-mail: mundy.william{at}epa.gov.
Received March 14, 2008; revision received May 30, 2008; accepted June 2, 2008
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Abstract |
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Identification of chemicals that pose a hazard to the developing nervous system is the first step in reducing human exposure and preventing health risks to infants and children. In response to the need for more efficient methods to identify potential developmental neurotoxicants, the present study evaluated the utility of an automated high content screening system to detect chemical effects on neurite outgrowth in Neuroscreen-1 cells (NS-1), a subclone of PC12 cells. Plating 2,000 NS-1 cells/well with 100 ng/ml nerve growth factor (NGF) for 96 hours produced optimal neurite growth in a 96-well format. Using this protocol, five chemicals that had been previously shown to inhibit neurite outgrowth in PC12 cells were examined. Inhibition of neurite outgrowth (assessed as total neurite length per cell) was observed for all five chemicals. For three of the chemicals, inhibition was associated with decreased cell viability. To demonstrate the utility of this approach for screening, a further set of chemicals (eight known in vivo developmental neurotoxicants and eight chemicals with little evidence of in vivo neurotoxicity) were tested over a wide concentration range (1 nM – 100 µM). Trans-retinoic acid, dexamethasone, cadmium, and methylmercury inhibited neurite outgrowth, although dexamethasone and cadmium only affected neurite outgrowth at concentrations that decreased viability. Amphetamine facilitated neurite outgrowth, while valproic acid, diphenylhydantoin, and lead had no effect. Of the chemicals that were not neurotoxic, there were no effects on cell viability, but two (dimethyl phthalate and omeprazole) increased neurite outgrowth at the highest concentration tested. These results demonstrate that a high content screening system can rapidly quantify chemical effects on neurite outgrowth in vitro. Concentration-response data for both neurite outgrowth and cell viability allowed for the determination of the specificity of chemical effects on a neurodevelopmental endpoint. Further studies will examine the utility of other in vitro preparations for cell-based assays of neurite outgrowth.