Background: High-content imaging (HCI) allows simultaneous measurement of multiple cellular phenotypic changes and is an important tool for evaluating the biological activity of chemicals. Objectives: Our goal was to analyze dynamic cellular changes using HCI to identify the “tipping point” at which the cells did not show recovery towards a normal phenotypic state. Methods: HCI was used to evaluate the effects of 967 chemicals (in concentrations ranging from 0.4 to 200 μM) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A.x, α-tubulin, histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size, and cell number. A computational model was developed to interpret HCI responses as cell-state trajectories. Results: Analysis of cell-state trajectories showed that 336 chemicals produced tipping points and that HepG2 cells were resilient to the effects of 334 chemicals up to the highest concentration (200 μM) and duration (72 hr) tested. Tipping points were identified as concentration-dependent transitions in system recovery, and the corresponding critical concentrations were generally between 5 and 15 times (25th and 75th percentiles, respectively) lower than the concentration that produced any significant effect on HepG2 cells. The remaining 297 chemicals require more data before they can be placed in either of these categories. Conclusions: These findings show the utility of HCI data for reconstructing cell state trajectories and provide insight into the adaptation and resilience of in vitro cellular systems based on tipping points. Cellular tipping points could be used to define a point of departure for risk-based prioritization of environmental chemicals. This dataset is associated with the following publication: Shah , I., W. Setzer , J. Jack, K. Houck , R. Judson , T. Knudsen , J. Liu, M. Martin , D. Reif, A.M. Richard , R.S. Thomas , K. Crofton , D.J. Dix , and R.J. Kavlock. (Envir. Health Perspect.) Using ToxCast data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. ENVIRONMENTAL HEALTH PERSPECTIVES. National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC, USA, 1-33, (2015).

The data presented here support the application of the Stemina devTOXqP platform for predictive toxicology and further demonstrate its value in ToxCast as a novel resource that can generate testable hypotheses aimed at characterizing potential pathways for teratogenicity and HTS prioritization of environmental chemicals for an exposure-based assessment of developmental hazard. The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Rush, P. Kothiya, R. Judson, K. Houck, E. Hunter, N. Baker, J. Palmer, R. Thomas, and T. Knudsen. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 174(2): 189-209, (2020).

The data presented here support the application of the Stemina devTOXqP platform for predictive toxicology and further demonstrate its value in ToxCast as a novel resource that can generate testable hypotheses aimed at characterizing potential pathways for teratogenicity and HTS prioritization of environmental chemicals for an exposure-based assessment of developmental hazard. The dataset from the Stemina (STM) assay is annotated in the ToxCast portfolio as STM. Major findings from the analysis of ToxCast_STM dataset include (1) 19% of 1065 chemicals yielded a prediction of developmental toxicity, (2) assay performance reached 79%-82% accuracy with high specificity (> 84%) but modest sensitivity (< 67%) when compared with in vivo animal models of human prenatal developmental toxicity, (3) sensitivity improved as more stringent weights of evidence requirements were applied to the animal studies, and (4) statistical analysis of the most potent chemical hits on specific biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain. The results of this study will be useful to improving our ability to predict in vivo developmental toxicants based on in vitro data and in silico models. This dataset is associated with the following publication: Zurlinden, T., K. Saili, N. Rush, P. Kothiya, R. Judson, K. Houck, E. Hunter, N. Baker, J. Palmer, R. Thomas, and T. Knudsen. Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 174(2): 189-209, (2020).

The AOP concept was used to translate ToxCast high-throughput screening data into a ToxPi matrix of 1058 chemicals x 24 features ordered by predicted potential to disrupt angiogenesis. This dataset is associated with the following publication: Saili, K., J. Franzosa, N. Baker, R. Ellis-Hutchings, R. Settivari, E. Carney, R. Spencer, T. Zurlinden, N. Kleinstreuer, S. Li, M. Xia, and T. Knudsen. Systems Modeling of Developmental Vascular Toxicity. Current Opinion in Toxicology. Elsevier BV, AMSTERDAM, NETHERLANDS, 15(1): 55-63, (2019).

The AOP concept was used to translate ToxCast high-throughput screening data into a ToxPi matrix of 1058 chemicals x 24 features ordered by predicted potential to disrupt angiogenesis. This dataset is associated with the following publication: Saili, K., J. Franzosa, N. Baker, R. Ellis-Hutchings, R. Settivari, E. Carney, R. Spencer, T. Zurlinden, N. Kleinstreuer, S. Li, M. Xia, and T. Knudsen. Systems Modeling of Developmental Vascular Toxicity. Current Opinion in Toxicology. Elsevier BV, AMSTERDAM, NETHERLANDS, 15(1): 55-63, (2019).

Bioactivity data for p,p'-DDD and analogues from ToxCast assays conducted in liver cells were sourced from the EPA’s CompTox Chemistry Dashboard. The links also provide access to the ToxCast assay information and annotation data user guide. This dataset is associated with the following publication: Lizarraga, L., J. Dean, J. Kaiser, S. Wesselkamper, J. Lambert, and J. Zhao. A Case Study on the Application of An Expert-driven Read-Across Approach in Support of Quantitative Risk Assessment of p,p’-Dichlorodiphenyldichloroethane. REGULATORY TOXICOLOGY AND PHARMACOLOGY. Elsevier Science Ltd, New York, NY, USA, 103: 301-313, (2019).

Bioactivity data for p,p'-DDD and analogues from ToxCast assays conducted in liver cells were sourced from the EPA’s CompTox Chemistry Dashboard. The links also provide access to the ToxCast assay information and annotation data user guide. This dataset is associated with the following publication: Lizarraga, L., J. Dean, J. Kaiser, S. Wesselkamper, J. Lambert, and J. Zhao. A Case Study on the Application of An Expert-driven Read-Across Approach in Support of Quantitative Risk Assessment of p,p’-Dichlorodiphenyldichloroethane. REGULATORY TOXICOLOGY AND PHARMACOLOGY. Elsevier Science Ltd, New York, NY, USA, 103: 301-313, (2019).

Supplemental datafiles for journal article 'High-Throughput Transcriptomics Screen of ToxCast Chemicals in U-2 OS Cells'.

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to false positive (chemical is detoxified in vivo) as well as false negative results (chemical is bioactivated in vivo) and thus potential mischaracterization of chemical hazard. We have addressed this challenge by introducing the ten most prevalent human liver cytochrome P450 (CYP) enzymes into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as singlets or as a mixture in relative proportions expressed in the liver. Initial experiments using luminogenic CYP450 substrates demonstrate that cell models express metabolic enzymes from the transfected mRNAs and activities are significantly increased when co-transfected with a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates, with metabolite formation occurring through 18 hours post-treatment. As a demonstration of how this method can be used to retrofit existing HTS assays, a proof-of-concept screen for cytotoxicity in HEK293T cells was conducted using 56 test compounds. The results demonstrate that the xenobiotic metabolism conferred by transfection of CYP-encoding mRNAs shifts the dose-response relationship for certain test chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting metabolic competence to existing cell-based HTS assays. This dataset is associated with the following publication: DeGroot, D., A. Swank, R. Thomas, M. Strynar, M. Lee, P. Carmichael, and S. Simmons. mRNA transfection retrofits cell-based assays with xenobiotic metabolism. JOURNAL OF PHARMACOLOGICAL & TOXICOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 92: 77-94, (2018).

The US EPA’s ToxCast program is designed to assess chemical perturbations of molecular and cellular endpoints using a variety of high-throughput screening (HTS) assays. However, existing HTS assays have limited or no xenobiotic metabolism which could lead to false positive (chemical is detoxified in vivo) as well as false negative results (chemical is bioactivated in vivo) and thus potential mischaracterization of chemical hazard. We have addressed this challenge by introducing the ten most prevalent human liver cytochrome P450 (CYP) enzymes into a human cell line (HEK293T) with low endogenous metabolic capacity. The CYP enzymes were introduced via transfection of modified mRNAs as singlets or as a mixture in relative proportions expressed in the liver. Initial experiments using luminogenic CYP450 substrates demonstrate that cell models express metabolic enzymes from the transfected mRNAs and activities are significantly increased when co-transfected with a CYP accessory protein, P450 oxidoreductase (POR). Transfected HEK293T cells demonstrate the ability to produce predicted metabolites following treatment with well-studied CYP substrates, with metabolite formation occurring through 18 hours post-treatment. As a demonstration of how this method can be used to retrofit existing HTS assays, a proof-of-concept screen for cytotoxicity in HEK293T cells was conducted using 56 test compounds. The results demonstrate that the xenobiotic metabolism conferred by transfection of CYP-encoding mRNAs shifts the dose-response relationship for certain test chemicals such as aflatoxin B1 (bioactivation) and fenazaquin (detoxification). Overall, transfection of CYP-encoding mRNAs is an effective and portable solution for retrofitting metabolic competence to existing cell-based HTS assays. This dataset is associated with the following publication: DeGroot, D., A. Swank, R. Thomas, M. Strynar, M. Lee, P. Carmichael, and S. Simmons. mRNA transfection retrofits cell-based assays with xenobiotic metabolism. JOURNAL OF PHARMACOLOGICAL & TOXICOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 92: 77-94, (2018).