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Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay
Citation:
HUNTER, E. S., H. P. NICHOLS, S. C. JEFFAY, M. L. HOOPES, AND M. BARRIER. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay. Presented at Society of Toxicology 49th Annual meeting, Salt Lake City, UT, March 07 - 11, 2010.
Impact/Purpose:
The U.S. Environmental Protection Agency (EPA) established the ToxCast" program in order to develop a high-throughput screening (HTS) approach for predicting the toxicity of large numbers of chemicals
Description:
An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ToxCast" program in order to develop a high-throughput screening (HTS) approach for predicting the toxicity of large numbers of chemicals. The library contains 309 well-characterized chemicals which are mostly pesticides. Initial evaluation used a Myosin Heavy Chain (MHC) antibody to monitor cardiomyocyte differentiation, which requires signaling from endodermal, ectodermal, and mesodermal cell types. For assessment of cytotoxicity, DNA staining (DRAQ5/Sapphire 700) was used to determine relative cell number for each assay well. Each ToxCast chemical was tested at 4 concentrations (0.0125 to 12.5 uM) in addition to DMSO vehicle control for calculation of AC50 values for cytotoxicity and MHC-positive differentiation. The results from this screen are being entered into the Toxlvliner" database for use in predictive modeling with other ToxCast HTS assays and ToxRefDB in vivo data, and for systems modeling with EPA's Virtual Embryo for evaluating prenatal developmental toxicity. Preliminary analysis ofthe data revealed patterns of activity for subsets of chemicals on differentiation at a lower concentration than on cell number. Interestingly, some chemicals showed increasing cell number or MHC levels with increasing dose. [This work is approved by EPA but does not necessarily reflect official Agency policy].