Grantee Research Project Results
2023 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
EPA Project Officer: Spatz, Kyle
Project Period: August 1, 2020 through July 30, 2023 (Extended to July 30, 2024)
Project Period Covered by this Report: August 1, 2022 through July 31,2023
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, CYP3A65, and CYP3C1. 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. High throughput docking scoring methods do not accurately reflect the observed in vitro experimental results. These observations warrant the evaluation of proprietary, close-source docking tools for in silico predictions.
We have successfully generated the four knockout CYP model zebrafish lines proposed in the original grant. These include CYP1A, CYP3A65, CYP3C1, and CYP2Y3. In vivo metabolic work is pending the identification of additional in vitro substrates in the expectation that we will observe overlapping substrate specificity between CYP isoforms, in order to take advantage of analytical efficiencies. GCxGC separation methods have been established for simultaneous injections of multiple substrates, with excellent resolution (peak separation; see Figure). These substrates (hydroxypyrene, aminoanthracene, phenanthrol) are among the top 10 hits for CYP1A, and interestingly also CYP2Y3. Product identification has been confirmed by mass spectrometry fragment analysis.
Future Activities:
For Specific Aim 1, all of the proposed in vitro high throughput screening for CYP1A, CYP3A65, and CYP3C1 has been completed in Y3. Detailed confirmation of reactive oxygen production and enzyme kinetics will be completed for CYP2Y3.
For Specific Aim 2, in silico ligand screening of the Phase 2 libraries will be performed using additional software. 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 performed, using novel GCxGC methods.
Journal Articles:
No journal articles submitted with this report: View all 1 publications for this projectSupplemental Keywords:
Cytochrome P450, CRISPR-CasRelevant Websites:
WHOI Stegeman Lab Environmental 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.