Abstract
The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC₅₀) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation.