Science Inventory

SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity

Citation:

Word, L., C. Willis, S. Padilla, C. Weitekamp, K. Carstens, L. Everett, B. Knapp, K. Britton, R. Judson, AND J. Harrill. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity. Society of Toxicology Conference 2024, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25565328

Impact/Purpose:

This research for presentation at the society of toxicology conference helps to provide needed insight into cumulative impacts from chemical and non-chemical stressors (specifically, cortisol hormone levels), which will advance the understanding of susceptibility, risk determination, and application of adverse outcome pathways in environmental justice communities.

Description:

Environmental justice seeks to affirm individual’s rights to a healthy environment within communities that are subjected to a disproportionate burden of environmental hazard. These communities often experience higher levels of chronic stress and reduced quality of life, which may impact their response to environmental chemical exposures. However, current new approach methodologies (NAMs) do not account for the modifying effect of stress on health outcomes from exposure to harmful chemicals. The interaction between chemical exposure and stress levels will likely be chemical specific, therefore, there is a need for methods that can rapidly screen chemicals for their interaction with endogenous stress hormones. To fill this critical limitation in existing methods, we propose a chemical screening approach that explores how differences in physiological stress levels shift dose-response relationships following chemical exposure. We are evaluating chemical effects at multiple levels of the stress hormone, cortisol, using an in vitro high-throughput cell painting model and a zebrafish developmental medium-throughput model. For the cell-based assay, we are phenotypically profiling human osteosarcoma cells (U-2 OS) that express fluorescent protein markers for the nucleus and the cytoskeleton, specifically, microtubules and actin. We are screening 147 chemicals and extracting 787 distinct features for the phenotypic analysis. These 147 chemicals were selected based on their relevance to environmental justice, interaction with the glucocorticoid receptor pathway, and/or known toxicity pathways. We used a glucocorticoid receptor translocation assay to select cortisol levels, where the low concentration was set equal to the AC50 (0.03 µM) and the high concentration was set to the level of maximal response of the translocation assay in U-2 OS cells (10 µM). Cells were pre-treated with no cortisol, low cortisol, or high cortisol for either 6 hours or 24 hours prior to exposure to the test chemicals, and imaging was captured after 24 hours of co-exposure to the test chemical and cortisol. Developing zebrafish embryos will also be exposed to these chemicals to evaluate phenotypic and behavioral effects of chemical exposure in the presence of no, low, or high cortisol added during development. Our pilot experiment with cortisol co-exposure included nine different chemicals that are known to impact different parts of the cytoskeleton. Our results showed a chemical-dependent effect from differences in cortisol levels: high cortisol levels caused a detectable shift in the benchmark concentrations (BMCs) for docetaxel, latrunculin A, latranculin B, paclitaxel, and staurosporine; and an effect was not detected in this initial pilot experiment for etoposide, fenbendazole, jasplakinolide, or oxibendazole. These results are based on Mahalanobis Distance calculations and were consistent across the 6-hour and 24-hour pre-treatment experiments, showing good replicability thus far. Additionally, developmental cortisol exposure in zebrafish embryos only elicited minor developmental changes at very high concentrations with ≤ 30 µM cortisol showing no lethality or dysmorphology. The shift in chemical potency at different levels of cortisol co-exposure (0 µM, 0.03 µM, or 10 µM) was mechanism- and chemical-dependent in this initial pilot experiment. The upcoming full screen on 147 chemicals covers a more diverse array of mechanisms of action, which will allow for additional insight into the range of effect sizes. This multi-pronged approach provides both in vitro and in vivo data for mechanistic insight into the quantitative impact of stress on adverse outcome pathways (AOPs). Thus, this research will help provide needed insight into cumulative impacts from chemical and non-chemical stressors, which will advance the understanding of susceptibility, risk determination, and application of AOPs in environmental justice communities.