In silico identification of protein targets for chemical neurotoxins using ToxCast in vitro data and read-across within the QSAR toolbox.
ABSTRACT: There are many mechanisms of neurotoxicity that are initiated by the interaction of chemicals with different neurological targets. Under the U.S. Environmental Protection Agency's ToxCast program, the biological activity of thousands of chemicals was screened in biochemical and cell-based assays in a high-throughput manner. Two hundred sixteen assays in the ToxCast screening database were identified as targeting a total of 123 proteins having neurological functions according to the Gene Ontology database. Data from these assays were imported into the Organization for Economic Co-operation and Development QSAR Toolbox and used to predict neurological targets for chemical neurotoxins. Two sets of data were generated: one set was used to classify compounds as active or inactive and another set, composed of AC50s for only active compounds, was used to predict AC50 values for unknown chemicals. Chemical grouping and read-across within the QSAR Toolbox were used to identify neurologic targets and predict interactions for pyrethroids, a class of compounds known to elicit neurotoxic effects in humans. The classification prediction results showed 79% accuracy while AC50 predictions demonstrated mixed accuracy compared with the ToxCast screening data.
Project description:Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed.We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 ?M), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay.NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined.The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92-100%) and specificity (70-81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 ?M, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 ?M were active in the uterotrophic assay.We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had high sensitivity and specificity when compounds were in-domain of the models. Based on this research, we recommend a tiered screening approach wherein a) QSAR is used to identify compounds in-domain of the ER or AR binding models and predicted to bind; b) those compounds are screened in vitro to assess binding potency; and c) the stronger binders (AC50 < 1 ?M) are screened in vivo. This scheme prioritizes compounds for integrative testing and risk assessment. Importantly, compounds that are not in-domain, that are predicted either not to bind or to bind weakly, that are not active in in vitro, that require metabolism to manifest activity, or for which in vivo AR testing is in order, need to be assessed differently.Bhhatarai B, Wilson DM, Price PS, Marty S, Parks AK, Carney E. 2016. Evaluation of OASIS QSAR models using ToxCast™ in vitro estrogen and androgen receptor binding data and application in an integrated endocrine screening approach. Environ Health Perspect 124:1453-1461;?http://dx.doi.org/10.1289/EHP184.
Project description: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.
Project description:Modern toxicology is shifting from an observational to a mechanistic science. As part of this shift, high-throughput toxicity assays are being developed using alternative, nonmammalian species to prioritize chemicals and develop prediction models of human toxicity.The nematode Caenorhabditis elegans (C. elegans) was used to screen the U.S. Environmental Protection Agency's (EPA's) ToxCast™ Phase I and Phase II libraries, which contain 292 and 676 chemicals, respectively, for chemicals leading to decreased larval development and growth. Chemical toxicity was evaluated using three parameters: a biologically defined effect size threshold, half-maximal activity concentration (AC50), and lowest effective concentration (LEC).Across both the Phase I and Phase II libraries, 62% of the chemicals were classified as active ? 200 ?M in the C. elegans assay. Chemical activities and potencies in C. elegans were compared with those from two zebrafish embryonic development toxicity studies and developmental toxicity data for rats and rabbits. Concordance of chemical activity was higher between C. elegans and one zebrafish assay across Phase I chemicals (79%) than with a second zebrafish assay (59%). Using C. elegans or zebrafish to predict rat or rabbit developmental toxicity resulted in balanced accuracies (the average value of the sensitivity and specificity for an assay) ranging from 45% to 53%, slightly lower than the concordance between rat and rabbit (58%).Here, we present an assay that quantitatively and reliably describes the effects of chemical toxicants on C. elegans growth and development. We found significant overlap in the activity of chemicals in the ToxCast™ libraries between C. elegans and zebrafish developmental screens. Incorporating C. elegans toxicological assays as part of a battery of in vitro and in vivo assays provides additional information for the development of models to predict a chemical's potential toxicity to humans.Boyd WA, Smith MV, Co CA, Pirone JR, Rice JR, Shockley KR, Freedman JH. 2016. Developmental effects of the ToxCast™ Phase I and II chemicals in Caenorhabditis elegans and corresponding responses in zebrafish, rats, and rabbits. Environ Health Perspect 124:586-593; http://dx.doi.org/10.1289/ehp.1409645.
Project description:BACKGROUND: 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). OBJECTIVE: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.
Project description:BACKGROUND: Understanding health risks to embryonic development from exposure to environmental chemicals is a significant challenge given the diverse chemical landscape and paucity of data for most of these compounds. High-throughput screening (HTS) in the U.S. Environmental Protection Agency (EPA) ToxCast™ project provides vast data on an expanding chemical library currently consisting of > 1,000 unique compounds across > 500 in vitro assays in phase I (complete) and Phase II (under way). This public data set can be used to evaluate concentration-dependent effects on many diverse biological targets and build predictive models of prototypical toxicity pathways that can aid decision making for assessments of human developmental health and disease. OBJECTIVE: We mined the ToxCast phase I data set to identify signatures for potential chemical disruption of blood vessel formation and remodeling. METHODS: ToxCast phase I screened 309 chemicals using 467 HTS assays across nine assay technology platforms. The assays measured direct interactions between chemicals and molecular targets (receptors, enzymes), as well as downstream effects on reporter gene activity or cellular consequences. We ranked the chemicals according to individual vascular bioactivity score and visualized the ranking using ToxPi (Toxicological Priority Index) profiles. RESULTS: Targets in inflammatory chemokine signaling, the vascular endothelial growth factor pathway, and the plasminogen-activating system were strongly perturbed by some chemicals, and we found positive correlations with developmental effects from the U.S. EPA ToxRefDB (Toxicological Reference Database) in vivo database containing prenatal rat and rabbit guideline studies. We observed distinctly different correlative patterns for chemicals with effects in rabbits versus rats, despite derivation of in vitro signatures based on human cells and cell-free biochemical targets, implying conservation but potentially differential contributions of developmental pathways among species. Follow-up analysis with antiangiogenic thalidomide analogs and additional in vitro vascular targets showed in vitro activity consistent with the most active environmental chemicals tested here. CONCLUSIONS: We predicted that blood vessel development is a target for environmental chemicals acting as putative vascular disruptor compounds (pVDCs) and identified potential species differences in sensitive vascular developmental pathways.
Project description:In ToxCast™ Phase I, the U.S. EPA commissioned screening of 320 pesticides, herbicides, fungicides, and other chemicals in a series of high-throughput assays. The agency also developed a toxicological prioritization tool, ToxPi, to facilitate using ToxCast™ assays to predict biological function.We asked whether top-scoring PPAR? activators identified in ToxCast™ Phase I were genuine PPAR? activators and inducers of adipogenesis. Next, we identified ToxCast™ assays that should predict adipogenesis, developed an adipogenesis ToxPi, and asked how well the ToxPi predicted adipogenic activity.We used transient transfection to test the ability of ToxCast™ chemicals to modulate PPAR? and RXR?, and differentiation assays employing 3T3-L1 preadipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to evaluate the adipogenic capacity of ToxCast™ chemicals.Only 5/21 of the top scoring ToxCast™ PPAR? activators were activators in our assays, 3 were PPAR? antagonists, the remainder were inactive. The bona fide PPAR? activators we identified induced adipogenesis in 3T3-L1 cells and mBMSCs. Only 7 of the 17 chemicals predicted to be active by the ToxPi promoted adipogenesis, 1 inhibited adipogenesis, and 2 of the 7 predicted negatives were also adipogenic. Of these 9 adipogenic chemicals, 3 activated PPAR?, and 1 activated RXR?.ToxCast™ PPAR? and RXR? assays do not correlate well with laboratory measurements of PPAR? and RXR? activity. The adipogenesis ToxPi performed poorly, perhaps due to the performance of ToxCast™ assays. We observed a modest predictive value of ToxCast™ for PPAR? and RXR? activation and adipogenesis and it is likely that many obesogenic chemicals remain to be identified.Janesick AS, Dimastrogiovanni G, Vanek L, Boulos C, Chamorro-García R, Tang W, Blumberg B. 2016. On the utility of ToxCast™ and ToxPi as methods for identifying new obesogens. Environ Health Perspect 124:1214-1226;?http://dx.doi.org/10.1289/ehp.1510352.
Project description:The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.
Project description:The pregnane X receptor (PXR) is a key transcriptional regulator of many genes [e.g., cytochrome P450s (CYP2C9, CYP3A4, CYP2B6), MDR1] involved in xenobiotic metabolism and excretion.As part of an evaluation of different approaches to predict compound affinity for nuclear hormone receptors, we used the molecular docking program GOLD and a hybrid scoring scheme based on similarity weighted GoldScores to predict potential PXR agonists in the ToxCast database of pesticides and other industrial chemicals. We present some of the limitations of different in vitro systems, as well as docking and ligand-based computational models.Each ToxCast compound was docked into the five published crystallographic structures of human PXR (hPXR), and 15 compounds were selected based on their consensus docking scores for testing. In addition, we used a Bayesian model to classify the ToxCast compounds into PXR agonists and nonagonists. hPXR activation was determined by luciferase-based reporter assays in the HepG2 and DPX-2 human liver cell lines.We tested 11 compounds, of which 6 were strong agonists and 2 had weak agonist activity. Docking results of additional compounds were compared with data reported in the literature. The prediction sensitivity of PXR agonists in our sample ToxCast data set (n = 28) using docking and the GoldScore was higher than with the hybrid score at 66.7%. The prediction sensitivity for PXR agonists using GoldScore for the entire ToxCast data set (n = 308) compared with data from the NIH (National Institutes of Health) Chemical Genomics Center data was 73.8%.Docking and the GoldScore may be useful for prioritizing large data sets prior to in vitro testing with good sensitivity across the sample and entire ToxCast data set for hPXR agonists.
Project description:The U.S. Environmental Protection Agency's ToxCast program has screened thousands of chemicals for biological activity, primarily using high-throughput in vitro bioassays. Adverse outcome pathways (AOPs) offer a means to link pathway-specific biological activities with potential apical effects relevant to risk assessors. Thus, efforts are underway to develop AOPs relevant to pathway-specific perturbations detected in ToxCast assays. Previous work identified a "cytotoxic burst" (CTB) phenomenon wherein large numbers of the ToxCast assays begin to respond at or near test chemical concentrations that elicit cytotoxicity, and a statistical approach to defining the bounds of the CTB was developed. To focus AOP development on the molecular targets corresponding to ToxCast assays indicating pathway-specific effects, we conducted a meta-analysis to identify which assays most frequently respond at concentrations below the CTB. A preliminary list of potentially important, target-specific assays was determined by ranking assays by the fraction of chemical hits below the CTB compared with the number of chemicals tested. Additional priority assays were identified using a diagnostic-odds-ratio approach which gives greater ranking to assays with high specificity but low responsivity. Combined, the two prioritization methods identified several novel targets (e.g., peripheral benzodiazepine and progesterone receptors) to prioritize for AOP development, and affirmed the importance of a number of existing AOPs aligned with ToxCast targets (e.g., thyroperoxidase, estrogen receptor, aromatase). The prioritization approaches did not appear to be influenced by inter-assay differences in chemical bioavailability. Furthermore, the outcomes were robust based on a variety of different parameters used to define the CTB.
Project description:Identifying chemicals, beyond those already implicated, to test for potential endocrine disruption is a challenge and high throughput approaches have emerged as a potential tool for this type of screening. This review focused the Environmental Protection Agency's (EPA) ToxCast(TM) high throughput in vitro screening (HTS) program. Utility for identifying compounds was assessed and reviewed by using it to run the recently expanded chemical library (from 309 compounds to 1858) through the ToxPi(TM) prioritization scheme for endocrine disruption. The analysis included metabolic and neuroendocrine targets. This investigative approach simultaneously assessed the utility of ToxCast, and helped identify novel chemicals which may have endocrine activity. Results from this exercise suggest the spectrum of environmental chemicals with potential endocrine activity is much broader than indicated, and that some aspects of endocrine disruption are not fully covered in ToxCast.