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.

Figure 1. ES cell assay overview.

ES cells are seeded onto gelatin-coated 96-well plates at Day 0 in the absence of pluripotent factors. Eight chemicals (color outlined circles) are introduced on Day 1 at four concentrations (yellow→red; 0.0125, 0.125, 1.25, 12.5 uM) and are subsequently refreshed on Days 6–8. In-Cell Western™ analysis is initiated on Day 9 to assess differentiation (MYH6/MYH7) and cytotoxicity (DRAQ5/Sapphire700). Vehicle (grey outlined circles) and antibody controls (black outlined wells, Day 9–10) are included on each plate.

Figure 2. Dose-response curves generated for cytotoxicity and differentiation.

Images of experimental plate show cell number and differentiation signal for chemicals 17–24 across the concentration range tested (A). Dose-response curves for six chemicals from the above plate as well as AC₅₀ values are shown (B).

Figure 3. ToxCast™Phase I chemical activity across cytotoxicity and differentiation ES cell assay endpoints.

Heatmaps depict chemical effects on cytotoxicity and differentiation across dose. Unsupervised, two-dimensional hierarchical clustering of 320 ToxCast chemicals across four ES cell assay endpoints (cytotoxicity increase, cytotoxicity decrease, differentiation increase, differentiation decrease) and 4 doses each using Euclidean distance for measure and Ward's method for linkage analysis (A). Two-dimensional individual clusters for averaged dose normalized to controls for cytotoxicity and differentiation endpoints (B). Heatmap scales represent relative activity based on AC₅₀ = −log10(M), ranging from 0 (white) to 2 (yellow).

Figure 4. Associations between ToxCast™ assays and ES cell endpoints.

Univariate associations revealed multiple ToxCast assays correlate with ES cell cytotoxicity and differentiation. ES cell cytotoxicity and differentiation correlated with limited in vivo endpoints in ToxRefDB.

Figure 5. Multivariate models predict ES cell cytotoxicity and differentiation.

ROC curves generated using data from ToxCast assays and ES cells to drive machine learning algorithms that produced predictive models of ES cell cytotoxicity (A) and differentiation (B).

Figure 6. Cartoon depicting chemical targets across multiple components of ROS signaling in ToxCast platforms.

Chemicals that decreased ES cell differentiation by at least 50% also targeted multiple components of ROS signaling. The statistical associations between decreased ES cell differentiation and ToxCast assay platforms are highlighted (p≤0.1).