Grantee Research Project Results
2024 Progress Report: Toxicokinetic screening of zebrafish cytochrome P450 enzymes for in vitro-in vivo extrapolation
EPA Grant Number: R840029Title: Toxicokinetic screening of zebrafish cytochrome P450 enzymes for in vitro-in vivo extrapolation
Investigators: Goldstone, Jared , Wilson, Joanna
Institution: Woods Hole Oceanographic Institution , McMaster University
EPA Project Officer: Spatz, Kyle
Project Period: August 1, 2020 through May 10, 2025
Project Period Covered by this Report: August 1, 2023 through July 31,2024
Project Amount: $799,999
RFA: Advancing Toxicokinetics for Efficient and Robust Chemical Evaluations (2019) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
We propose to determine the metabolic activities of the key xenobiotic cytochrome P450 Phase I enzymes in zebrafish, using novel high-throughput screening methodologies in vitro and in silico, and transgenic knockout animals for in vivo assessments of toxicokinetic parameters in sensitive lifestages. Zebrafish are important test organisms for mechanistic toxicological research and for the safety assessment of manufactured and environmental chemicals, yet aspects of metabolism critical to the use of this model are not fully understood. Furthermore, zebrafish embryos and early ovolarvae provide access to early life stages that are differently sensitive to pollutants, and serve as models for both human and wildlife exposures. Our goal is to develop HTS methods for and determine functionality of cytochrome P450 enzymes that may be most important in pollutant metabolism in zebrafish.
Progress Summary:
High throughput screening of zebrafish cytochrome P450 (CYP) enzymes with Toxcast Phase 2 compounds has been successfully performed for zebrafish CYP1A, and CYP2Y3, and CYP3A65 and CYP3C1 have been screened with similar libraries. In addition to substrates identified for these enzymes, we have identified a number of Phase 2 compounds that exhibit high reactive oxygen production (CYP or CYP oxidoreductase [POR] enzyme uncoupling). These compounds may be toxic to fish through a different mechanism, and will be evaluated alongside metabolically active compounds.
In silico studies performed to complement the in vivo results have pointed to the current limitations in open-source protein-ligand docking software. We have performed and validated homology modeling zebrafish CYP enzymes, and previously done detailed docking analyses of individual compounds. Despite the methodological advances in proprietary, close-source docking tools, we have not found the high throughput docking methods to be predictive of in vitro experimental results.
We have successfully generated and maintain the four knockout CYP model zebrafish lines proposed in the original grant. These include CYP1A, CYP3A65, CYP3C1, and CYP2Y3. In vivo metabolic work using GCxGC separation is ongoing, following the establishment of methods with excellent resolution (peak separation). These substrates (hydroxypyrene, aminoanthracene, phenanthrol) are among the top 10 hits for CYP1A, and interestingly also CYP2Y3.
Future Activities:
For Specific Aim 2, in silico ligand screening of the Phase 2 libraries will be completed using commercial software. An additional scoring metric will be developed. The obtained docking scores will be compared to the in vitro results generated in Aim 1, with the eventual goal of predicting the affinity of any chemical with CYPs of toxicological relevance.
For Specific Aim 3, high throughput chemical analysis of hits arising from the screening of all of the enzymes will be completed, using novel GCxGC methods.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
| Other project views: | All 3 publications | 2 publications in selected types | All 2 journal articles |
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Perone DM, Annalora AJ, Goldstone JV, Dickey AN, Salanga MC, Francolini RD, Wright FA, Marcus CB, Tanguay RL, Garcia-Jaramillo M. Zebrafish Cyp1b1 knockout alters eye and brain metabolomic profiles, affecting ocular and neurobehavioral function.Toxicology and Applied Pharmacology 2025:117246. |
R840029 (2024) |
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Supplemental Keywords:
Cytochrome P450, CRISPR-CasRelevant Websites:
WHOI Stegeman Lab - Environmental Toxicology Exit
McMaster Wilson Lab - Aquatic Toxicology Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.