High-throughput screening tools facilitate calculation of a combined exposure-bioactivity index for chemicals with endocrine activity.

High-throughput and computational tools provide a new opportunity to calculate combined bioactivity of exposure to diverse chemicals acting through a common mechanism. We used high throughput in vitro bioactivity data and exposure predictions from the U.S. EPA's Toxicity and Exposure Forecaster (ToxCast and ExpoCast) to estimate combined estrogen receptor (ER) agonist activity of non-pharmaceutical chemical exposures for the general U.S. population. High-throughput toxicokinetic (HTTK) data provide conversion factors that relate bioactive concentrations measured in vitro (µM), to predicted population geometric mean exposure rates (mg/kg/day). These data were available for 22 chemicals with ER agonist activity and were estimated for other ER bioactive chemicals based on the geometric mean of HTTK values across chemicals. For each chemical, ER bioactivity across ToxCast assays was compared to predicted population geometric mean exposure at different levels of in vitro potency and model certainty. Dose additivity was assumed in calculating a Combined Exposure-Bioactivity Index (CEBI), the sum of exposure/bioactivity ratios. Combined estrogen bioactivity was also calculated in terms of the percent maximum bioactivity of chemical mixtures in human plasma using a concentration-addition model. Estimated CEBIs vary greatly depending on assumptions used for exposure and bioactivity. In general, CEBI values were <1 when using median of the estimated general population chemical intake rates, while CEBI were ≥1 when using the upper 95th confidence bound for those same intake rates for all chemicals. Concentration-addition model predictions of mixture bioactivity yield comparable results. Based on current in vitro bioactivity data, HTTK methods, and exposure models, combined exposure scenarios sufficient to influence estrogen bioactivity in the general population cannot be ruled out. Future improvements in screening methods and computational models could reduce uncertainty and better inform the potential combined effects of estrogenic chemicals.

Anchoring a dynamic in vitro model of human neuronal differentiation to key processes of early brain development in vivo.

We characterize temporal pathway dynamics of differentiation in an in vitro neurotoxicity model with the aim of informing design and interpretation of toxicological assays. Human neural progenitor cells (hNPCs) were cultured in differentiation conditions up to 21 days. Genes significantly changed through time were identified and grouped according to temporal dynamics. Quantitative pathway analysis identified gene ontology (GO) terms enriched among significantly changed genes and provided a temporal roadmap of pathway trends in vitro. Gene expression in hNPCs was compared with publicly available gene expression data from developing human brain tissue in vivo. Quantitative pathway analysis of significantly changed genes and targeted analysis of specific pathways of interest identified concordance between in vivo and in vitro expression associated with proliferation, migration, differentiation, synapse formation, and neurotransmission. Our analysis anchors gene expression patterns in vitro to sensitive windows of in vivo development, helping to define appropriate applications of the model.

Differential epigenetic effects of chlorpyrifos and arsenic in proliferating and differentiating human neural progenitor cells.

Understanding the underlying temporal and mechanistic responses to neurotoxicant exposures during sensitive periods of neuronal development are critical for assessing the impact of these exposures on developmental processes. To investigate the importance of timing of neurotoxicant exposure for perturbation of epigenetic regulation, we exposed human neuronal progenitor cells (hNPCs) to chlorpyrifos (CP) and sodium arsenite (As; positive control) during proliferation and differentiation. CP or As treatment effects on hNPCs morphology, cell viability, and changes in protein expression levels of neural differentiation and cell stress markers, and histone H3 modifications were examined. Cell viability, proliferation/differentiation status, and epigenetic results suggest that hNPCs cultures respond to CP and As treatment with different degrees of sensitivity. Histone modifications, as measured by changes in histone H3 phosphorylation, acetylation and methylation, varied for each toxicant and growth condition, suggesting that differentiation status can influence the epigenetic effects of CP and As exposures.

Stage-specific signaling pathways during murine testis development and spermatogenesis: A pathway-based analysis to quantify developmental dynamics.

Shifting the field of developmental toxicology toward evaluation of pathway perturbation requires a quantitative definition of normal developmental dynamics. This project examined a publicly available dataset to quantify pathway dynamics during testicular development and spermatogenesis and anchor toxicant-perturbed pathways within the context of normal development. Genes significantly changed throughout testis development in mice were clustered by their direction of change using K-means clustering. Gene Ontology terms enriched among each cluster were identified using MAPPfinder. Temporal pathway dynamics of enriched terms were quantified based on average expression intensity for all genes associated with a given term. This analysis captured processes that drive development, including the peak in steroidogenesis known to occur around gestational day 16.5 and the increase in meiosis and spermatogenesis-related pathways during the first wave of spermatogenesis. Our analysis quantifies dynamics of pathways vulnerable to toxicants and provides a framework for quantifying perturbation of these pathways.