Background: Chemical toxicity testing is being transformed by advances in biology and computer modeling, concerns over animal use, and the thousands of environmental chemicals lacking toxicity data. ...The U.S. Environmental Protection Agency's ToxCast program aims to address these concerns by screening and prioritizing chemicals for potential human toxicity using in vitro assays and in silico approaches. Objectives: This project aims to evaluate the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by environmental chemicals and to build initial prioritization models of in vivo toxicity. Methods: We tested 309 mostly pesticide active chemicals in 467 assays across nine technologies, including high-throughput cell-free assays and cell-based assays, in multiple human primary cells and cell lines plus rat primary hepatocytes. Both individual and composite scores for effects on genes and pathways were analyzed. Results: Chemicals displayed a broad spectrum of activity at the molecular and pathway levels. We saw many expected interactions, including endocrine and xenobiotic metabolism enzyme activity. Chemicals ranged in promiscuity across pathways, from no activity to affecting dozens of pathways. We found a statistically significant inverse association between the number of pathways perturbed by a chemical at low in vitro concentrations and the lowest in vivo dose at which a chemical causes toxicity. We also found associations between a small set of in vitro assays and rodent liver lesion formation. Conclusions: This approach promises to provide meaningful data on the thousands of untested environmental chemicals and to guide targeted testing of environmental contaminants.
Background: The large and increasing number of chemicals released into the environment demands more efficient and cost-effective approaches for assessing environmental chemical toxicity. The U.S. ...Tox21 program has responded to this challenge by proposing alternative strategies for toxicity testing, among which the quantitative high-throughput screening (qHTS) paradigm has been adopted as the primary tool for generating data from screening large chemical libraries using a wide spectrum of assays. Objectives: The goal of this study was to develop methods to evaluate the data generated from these assays to guide future assay selection and prioritization for the Tox21 program. Methods: We examined the data from the Tox21 pilot-phase collection of approximately 3,000 environmental chemicals profiled in qHTS format against a panel of 10 human nuclear receptors (AR, ERα, FXR, GR, LXRβ, PPARγ, PPARδ, RXRα, TRβ, and VDR) for reproducibility, concordance of biological activity profiles with sequence homology of the receptor ligand binding domains, and structure-activity relationships. Results: We determined the assays to be appropriate in terms of biological relevance. We found better concordance for replicate compounds for the agonist-mode than for the antagonist-mode assays, likely due to interference of cytotoxicity in the latter assays. This exercise also enabled us to formulate data-driven strategies for discriminating true signals from artifacts, and to prioritize assays based on data quality. Conclusions: The results demonstrate the feasibility of qHTS to identify the potential for environmentally relevant chemicals to interact with key toxicity pathways related to human disease induction.
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.
Addressing the safety aspects of drugs and environmental chemicals has historically been undertaken through animal testing. However, the quantity of chemicals in need of assessment and the challenges ...of species extrapolation require the development of alternative approaches. Our approach, the US Environmental Protection Agency's ToxCast program, utilizes a large suite of in vitro and model organism assays to interrogate important chemical libraries and computationally analyze bioactivity profiles. Here we evaluated one component of the ToxCast program, the use of primary human cell systems, by screening for chemicals that disrupt physiologically important pathways. Chemical-response signatures for 87 endpoints covering molecular functions relevant to toxic and therapeutic pathways were generated in eight cell systems for 641 environmental chemicals and 135 reference pharmaceuticals and failed drugs. Computational clustering of the profiling data provided insights into the polypharmacology and potential off-target effects for many chemicals that have limited or no toxicity information. The endpoints measured can be closely linked to in vivo outcomes, such as the upregulation of tissue factor in endothelial cell systems by compounds linked to the risk of thrombosis in vivo. Our results demonstrate that assaying complex biological pathways in primary human cells can identify potential chemical targets, toxicological liabilities and mechanisms useful for elucidating adverse outcome pathways.
Exposure to environmental chemicals adds to the burden of disease in humans and wildlife to a degree that is difficult to estimate and, thus, mitigate. The ability to assess the impact of existing ...chemicals for which little to no toxicity data are available or to foresee such effects during early stages of chemical development and use, and before potential exposure occurs, is a pressing need. However, the capacity of the current toxicity evaluation approaches to meet this demand is limited by low throughput and high costs. In the context of EPA’s ToxCast project, we have evaluated a novel cellular biosensor system (Factorial) that enables rapid, high-content assessment of a compound’s impact on gene regulatory networks. The Factorial biosensors combined libraries of cis- and trans-regulated transcription factor reporter constructs with a highly homogeneous method of detection enabling simultaneous evaluation of multiplexed transcription factor activities. Here, we demonstrate the application of the technology toward determining bioactivity profiles by quantitatively evaluating the effects of 309 environmental chemicals on 25 nuclear receptors and 48 transcription factor response elements. We demonstrate coherent transcription factor activity across nuclear receptors and their response elements and that Nrf2 activity, a marker of oxidative stress, is highly correlated to the overall promiscuity of a chemical. Additionally, as part of the ToxCast program, we identify molecular targets that associate with in vivo end points and represent modes of action that can serve as potential toxicity pathway biomarkers and inputs for predictive modeling of in vivo toxicity.
The Deepwater Horizon oil spill has led to the use of >1 M gallons of oil spill dispersants, which are mixtures of surfactants and solvents. Because of this large scale use there is a critical need ...to understand the potential for toxicity of the currently used dispersant and potential alternatives, especially given the limited toxicity testing information that is available. In particular, some dispersants contain nonylphenol ethoxylates (NPEs), which can degrade to nonylphenol (NP), a known endocrine disruptor. Given the urgent need to generate toxicity data, we carried out a series of in vitro high-throughput assays on eight commercial dispersants. These assays focused on the estrogen and androgen receptors (ER and AR), but also included a larger battery of assays probing other biological pathways. Cytotoxicity in mammalian cells was also quantified. No activity was seen in any AR assay. Two dispersants showed a weak ER signal in one assay (EC50 of 16 ppm for Nokomis 3-F4 and 25 ppm for ZI-400). NPs and NPEs also had a weak signal in this same ER assay. Note that Corexit 9500, the currently used product, does not contain NPEs and did not show any ER activity. Cytotoxicity values for six of the dispersants were statistically indistinguishable, with median LC50 values ∼100 ppm. Two dispersants, JD 2000 and SAF-RON GOLD, were significantly less cytotoxic than the others with LC50 values approaching or exceeding 1000 ppm.
Understanding potential health risks is a significant challenge due to the large numbers of diverse chemicals with poorly characterized exposures and mechanisms of toxicities. The present study ...analyzes 976 chemicals (including failed pharmaceuticals, alternative plasticizers, food additives, and pesticides) in Phases I and II of the U.S. EPA’s ToxCast project across 331 cell-free enzymatic and ligand-binding high-throughput screening (HTS) assays. Half-maximal activity concentrations (AC50) were identified for 729 chemicals in 256 assays (7,135 chemical–assay pairs). Some of the most commonly affected assays were CYPs (CYP2C9 and CYP2C19), transporters (mitochondrial TSPO, norepinephrine, and dopaminergic), and GPCRs (aminergic). Heavy metals, surfactants, and dithiocarbamate fungicides showed promiscuous but distinctly different patterns of activity, whereas many of the pharmaceutical compounds showed promiscuous activity across GPCRs. Literature analysis confirmed >50% of the activities for the most potent chemical–assay pairs (54) but also revealed 10 missed interactions. Twenty-two chemicals with known estrogenic activity were correctly identified for the majority (77%), missing only the weaker interactions. In many cases, novel findings for previously unreported chemical–target combinations clustered with known chemical–target interactions. Results from this large inventory of chemical–biological interactions can inform read-across methods as well as link potential targets to molecular initiating events in adverse outcome pathways for diverse toxicities.
We describe a framework for estimating the human dose at which a chemical significantly alters a biological pathway in vivo, making use of in vitro assay data and an in vitro-derived pharmacokinetic ...model, coupled with estimates of population variability and uncertainty. The quantity we calculate, the biological pathway altering dose (BPAD), is analogous to current risk assessment metrics in that it combines dose−response data with analysis of uncertainty and population variability to arrive at conservative exposure limits. The analogy is closest when perturbation of a pathway is a key event in the mode of action (MOA) leading to a specified adverse outcome. Because BPADs are derived from relatively inexpensive, high-throughput screening (HTS) in vitro data, this approach can be applied to high-throughput risk assessments (HTRA) for thousands of data-poor environmental chemicals. We envisage the first step of HTRA to be an assessment of in vitro concentration−response relationships across biologically important pathways to derive biological pathway altering concentrations (BPAC). Pharmacokinetic (PK) modeling is then used to estimate the in vivo doses required to achieve the BPACs in the blood at steady state. Uncertainty and variability are incorporated in both the BPAC and the PK parameters and then combined to yield a probability distribution for the dose required to perturb the critical pathway. We finally define the BPADL as the lower confidence bound of this pathway-altering dose. This perspective outlines a framework for using HTRA to estimate BPAD values; provides examples of the use of this approach, including a comparison of BPAD values with published dose−response data from in vivo studies; and discusses challenges and alternative formulations.
Over the past 20 years, an increased focus on detecting environmental chemicals that pose a risk of adverse effects due to endocrine disruption has driven the creation of the U.S. Environmental ...Protection Agency (EPA) Endocrine Disruptor Screening Program (EDSP). Thousands of chemicals are subject to the EDSP; thus, processing these chemicals using current test batteries could require millions of dollars and decades. A need for increased throughput and efficiency motivated the development of methods using in vitro high throughput screening (HTS) assays to prioritize chemicals for EDSP Tier 1 screening (T1S).
In this study we used U.S. EPA ToxCast HTS assays for estrogen, androgen, steroidogenic, and thyroid-disrupting mechanisms to classify compounds and compare ToxCast results to in vitro and in vivo data from EDSP T1S assays.
We implemented an iterative model that optimized the ability of endocrine-related HTS assays to predict components of EDSP T1S and related results. Balanced accuracy was used as a measure of model performance.
ToxCast estrogen receptor and androgen receptor assays predicted the results of relevant EDSP T1S assays with balanced accuracies of 0.91 (p < 0.001) and 0.92 (p < 0.001), respectively. Uterotrophic and Hershberger assay results were predicted with balanced accuracies of 0.89 (p < 0.001) and 1 (p < 0.001), respectively. Models for steroidogenic and thyroid-related effects could not be developed with the currently published ToxCast data.
Overall, results suggest that current ToxCast assays can accurately identify chemicals with potential to interact with the estrogenic and androgenic pathways, and could help prioritize chemicals for EDSP T1S assays.
The presence of industrial chemicals, consumer product chemicals, and pharmaceuticals is well documented in waters in the U.S. and globally. Most of these chemicals lack health-protective guidelines ...and many have been shown to have endocrine bioactivity. There is currently no systematic or national prioritization for monitoring waters for chemicals with endocrine disrupting activity. We propose ambient water bioactivity concentrations (AWBCs) generated from high throughput data as a health-based screen for endocrine bioactivity of chemicals in water. The U.S. EPA ToxCast program has screened over 1800 chemicals for estrogen receptor (ER) and androgen receptor (AR) pathway bioactivity. AWBCs are calculated for 110 ER and 212 AR bioactive chemicals using high throughput ToxCast data from in vitro screening assays and predictive pathway models, high-throughput toxicokinetic data, and data-driven assumptions about consumption of water. Chemical-specific AWBCs are compared with measured water concentrations in data sets from the greater Denver area, Minnesota lakes, and Oregon waters, demonstrating a framework for identifying endocrine bioactive chemicals. This approach can be used to screen potential cumulative endocrine activity in drinking water and to inform prioritization of future monitoring, chemical testing and pollution prevention efforts.
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