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High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway
Citation:
CHANDLER, K. J., E. S. HUNTER, S. C. JEFFAY, H. P. NICHOLS, M. Hoopes, M. BARRIER, J. Habig, AND T. B. KNUDSEN. High-throughput Screening of ToxCast" Phase I Chemicals in an Embryonic Stem Cell Assay Reveals Potential Disruption of a Critical Developmental Signaling Pathway. Presented at Teratology, Louisville, KY, June 26 - 30, 2010.
Impact/Purpose:
Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (HCS) approaches. Several in vitro models, such as the embryonic stem cell test (EST), are used to evaluate the effects of xenobiotics and as predictive models of developmental toxicity.
Description:
Little is known about the developmental toxicity of the expansive chemical landscape in existence today. Significant efforts are being made to apply novel methods to predict developmental activity of chemicals utilizing high-throughput screening (HTS) and high-content screening (HCS) approaches. Several in vitro models, such as the embryonic stem cell test (EST), are used to evaluate the effects of xenobiotics and as predictive models of developmental toxicity. An Adherent Cell Differentiation and Cytotoxicity (ACDC) 11 mouse embryonic stem cell (mESC) assay was used to interrogate the well-characterized ToxCast™ Phase I chemical library for potential developmental activity. Differentiation and cytotoxicity were measured by In-Cell Western" analysis of Myosin Heavy Chain (MHC), a cardiomyocyte marker, and DRAQ5™/Sapphire700TM, respectively. Chromosome and MHC analysis of 11 mESCs through passage 27 showed the line remained euploid and maintained consistent levels of cardiomyocyte differentiation in control conditions. A dose-response curve was generated between 0.0125-12.5 JlM for each of 309 ToxCast" chemicals and half-maximal activity (AC50) was computed by curve-fitting methods. This analysis revealed an AC50 for MHC expression in 45 of 309 chemicals. The majority of these actives (41/45) inhibited cardiomyocyte differentiation. Comparison with 467 ToxCast™ assays revealed significant correlations between the inhibition of cardiomyocyte differentiation and perturbation of transcription factor signaling such as POU2Fl/0CTl (p=0.01), JUN (p=O.OI), and EGRI (p=0.03) as well as BMPR2 transcription (p=0.002). The BMP signaling pathway, which includes EGR1, JUN, and BMPR2, has been shown to play an important role in mESC self-renewal, embryonic development, cardiomyocyte differentiation, and cardiopulmonary function. These findings suggest this subset of ToxCast™ Phase I chemicals perturbs developmental signaling in pluripotent mESCs. [This work is approved by EPA but does not necessarily reflect official Agency policy].