1. Development of a High-Throughput Assay to Identify 5-α Reductase Inhibitors for Orthogonal Evaluation in an Androgen-Dependent Human 3D Prostate Microtissue
Leads: Chad Deisenroth, Josh Harrill, Menghang Xia
Goal: Develop additional human-based, cellular tests to more completely model androgen pathway activity.
- Issue: Environmental factors that alter the metabolism or bioactivity of androgen, a male sex hormone, can harm human reproductive and sexual development. Current guidelines rely on animal testing to evaluate the potential for certain chemicals to disrupt normal androgenic functions. Efforts to evaluate androgen-active chemicals using predictive computational models with in vitro data are limited; unlike animal models, computational models have insufficient coverage of key metabolic outcomes and observable characteristics resulting from gene-environment interactions.
- Project Focus: Develop a high-throughput assay to evaluate the effects of blocking the activity of enzyme 5α-reductase and develop a 3-D human prostate microtissue assay that may enable better assessments of androgen-active chemicals. Together, the assays can provide greater depth for predicting the negative effects of androgen-disrupting compounds.
2. Cell Line Selection for High-Throughput Transcriptomics (HTT)
Leads: Nisha Sipes, Josh Harrill, Woody Setzer
Goal: Develop a strategy for selecting maximally diverse cell types/lines to maximally cover biological targets and pathways for high-throughput chemical screening using gene expression (i.e. transcriptomics).
- Issue: HTT is an efficient means of screening chemicals for bioactivity across a broad range of potential molecular targets. However, no single laboratory (in vitro) model will express all molecular targets or accurately model the diversity of chemical disturbances observed in different cell types of the human body. As in vitro models derived from diverse tissues and sources have different gene/protein expression patterns, they may respond differently to chemical exposures. A HTT screening panel using diverse cell lines would provide a more comprehensive understanding of chemical bioactivity than studies in any single cell type.
- Project Focus: Use computational modeling of existing transcriptomics data to select a set of “diverse” cell lines, measure transcriptomic responses of “diverse” cells following exposure to a panel of chemicals, determine the degree to which “diverse” cell lines respond differently to chemicals, and analyze the responses among pooled cell lysates.
3. Profiling Environmental, Drug, and Food-Related Chemicals that Inhibit Acetylcholinesterase Activity
Leads: Menghang Xia, Michael Santillo
Goal: Develop a high-throughput in vitro test system to identify and characterize new compounds that block the activity of acetylcholinesterase.
- Issue: Acetylcholinesterase (AchE) inhibitors are compounds related to foods, drugs, and the environment that can be toxic to humans.
- Project Focus: To advance existing methods, metabolism will be incorporated into the in vitro screening system, which, in addition to in-depth mechanistic studies, may improve the ability to detect emerging chemical hazards.
- “Use of high-throughput enzyme-based assay with xenobiotic metabolic capability to evaluate the inhibition of acetylcholinesterase activity by organophosphorous pesticides.” Li S, Zhao J, Huang R, Santillo MF, Houck KA, Xia M; Toxicology in Vitro, 2019, 56:93-100. http://www.sciencedirect.com/science/article/pii/S088723331830540X
Profiling the Tox21 Chemical Collection for Acetylcholinesterase Inhibition. Li S, Zhao J, Huang R, Travers J, Klumpp-Thomas C, Yu W, MacKerell AD, Sakamuru S, Ooka M, Xue F, Sipes NS, Hsieh J-H, Ryan K, Simeonov A, Santillo MF, Xia M; Environmental Health Perspectives, 2021, 129(4):047008. https://ehp.niehs.nih.gov/doi/pdf/10.1289/EHP6993
6. Predictive Modeling of Developmental Toxicity with Human Pluripotent Stem Cells
Leads: Thomas Knudsen, Nicole Kleinstreuer, Annie Lumen
Goal: Evaluate a human-based, induced pluripotent stem cell (iPSC) test system to predict developmental toxicity.
- Issue: Traditionally, information from studies using laboratory animals has been used to predict the impact of chemical exposure on the human fetus. However, these studies are slow, costly and unrealistic for assessing tens of thousands of environmental chemicals in commerce, and this approach may not provide adequate coverage of human biology.
- Project Focus: Evaluate an assay (test) based on reducing cellular ornithine’s release relative to cystine uptake by iPSCs. Coupled with computational predictive modeling, the in vitro test system findings can be extrapolated to living systems (in vivo) and dose-activity measures translated to the whole-body level. This approach enables exposure-based risk characterization for chemicals considered high priority for developmental toxicity. This cross-program project will work towards the characterization, validation, and implementation of this platform by modeling the predictive and health-protective potential of the assay with regards to fundamental principles of abnormal development in the womb.
11. Development of High-Throughput Assays to Detect Chemicals with the Potential to Induce Skin Sensitization, Eye Irritation, or Corrosion
Leads: Menghang Xia, Nicole Kleinstreuer
Goal: To develop high-throughput screening (HTS) assays that can evaluate chemicals in the Tox21 10K library for the potential to cause skin sensitization, eye irritation, or serious eye damage.
- Issue: Assessing the sensitization or irritation potential is a key element in the safety evaluation of topically applied or exposed chemicals and drugs. European legislation now mandates the use of alternative test methods for these compounds, instead of testing them on animals. Developing HTS assays for these compounds would provide a faster and cheaper alternative testing method than current in vitro assays.
- Project Focus: 1) Develop a quantitative HTS assay to determine which chemicals in the Tox21 10K library are potential skin sensitizers. The identified sensitizers will be tested using the hCLAT assay, cytokine secretion assay, and bio-printed skin model, and the results compared to available in vivo data. 2) Use immortalized human corneal epithelial cells, instead of Statens Seruminstitut Rabbit Cornea (SIRC) cells, to develop a quantitative HTS assay to screen the Tox21 10K library for chemicals that can cause eye irritation or serious eye damage.
12. Evaluating Thiol Reactivity of Tox21 Chemicals Using the MSTI Assay
Leads: Grace Patlewicz, Keith Houck, Menghang Xia, Steve Simmons
Goal: To generate chemical reactivity information for the Tox21 10K library and determine which chemicals are electrophiles.
- Issue: Existing Tox21 high-throughput screening assays do not characterize non-specific reactive chemicals. The ability to characterize these chemicals and determine which are electrophiles would enhance the prediction of adverse events, such as skin sensitization and liver and kidney toxicities, which may be caused by chemical electrophiles binding to cellular nucleophiles.
- Project Focus: Adapt the (E)-2-(4-mercaptostyryl)-1,3,3-trimethyl-3H-indol-1-ium thiol (MSTI), a biochemical reactivity assay, to provide semi-quantitative kinetic information, that can be used to predict toxicities mediated by electrophiles binding to cellular nucleophiles.
- “Evaluating the utility of a high throughput thiol-containing fluorescent probe to screen for reactivity: A case study with the Tox21 library.” Grace Patlewicz, Katie Paul-Friedman, Keith Houck, Li Zhang, Ruili Huang, Menghang Xia, Jason Brown, Steven O. Simmons; Computational Toxicology, 2023, 26:100271. https://www.sciencedirect.com/science/article/pii/S2468111323000129