title: Evaluation of 309 environmental chemicals using a mouse embryonic stem cell adherent cell differentiation and cytotoxicity assay.
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.
authors.affiliation: National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, United States of America. chandler.kelly@epa.gov
authors.forename: Kelly J
authors.initials: KJ
authors.lastname: Chandler
authors.forename: Marianne
authors.initials: M
authors.lastname: Barrier
authors.forename: Susan
authors.initials: S
authors.lastname: Jeffay
authors.forename: Harriette P
authors.initials: HP
authors.lastname: Nichols
authors.forename: Nicole C
authors.initials: NC
authors.lastname: Kleinstreuer
authors.forename: Amar V
authors.initials: AV
authors.lastname: Singh
authors.forename: David M
authors.initials: DM
authors.lastname: Reif
authors.forename: Nisha S
authors.initials: NS
authors.lastname: Sipes
authors.forename: Richard S
authors.initials: RS
authors.lastname: Judson
authors.forename: David J
authors.initials: DJ
authors.lastname: Dix
authors.forename: Robert
authors.initials: R
authors.lastname: Kavlock
authors.forename: Edward S
authors.initials: ES
authors.lastname: Hunter
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Knudsen
mesh_terms: D000818:Animals; D001681:Biological Assay; D002448:Cell Adhesion / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects*; D023381:Endpoint Determination; D004785:Environmental Pollutants / Q000633:toxicity*; D008297:Male; D051379:Mice; D008954:Models, Biological; D015999:Multivariate Analysis; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D004785:Environmental Pollutants