Grantee Research Project Results
2022 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
Current 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, 2021 through July 31,2022
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. Forty four of the approximately 1800 Phase 2 compounds exhibited high NADPH consumption, representing good substrate-enzyme coupling in our heterologous expression system. In addition, we have identified a number of Phase 2 compounds that exhibit high reactive oxygen production (CYP enzyme uncoupling). These compounds may be toxic to fish through a different mechanism, and will be evaluated alongside metabolically active compounds.
NADPH consumption as a measure of Phase 2 compound activity with expressed CYP1A, plotted against DCF formation as a measure of P450 uncoupling. High coupling and low DCF production indicate good substrates.
Figure 1 NADPH consumption as a measure of Phase 2 compound activity with expressed CUP1A, plotted against DCF formation as a measure of P450 uncoupling. High coupling and low DCF production indicate good substrates.
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 three of the four knockout CYP model zebrafish lines proposed in the original grant. These include CYP1A, CYP3A65, and CYP3C1. 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.
Future Activities:
For Specific Aim 1, all of the proposed enzymes are in hand (CYP1A, CYP3A65, and CYP3C1). CYP2Y3 has been synthesized, codon optimized, and cloned into the expression plasmid. In vitro high throughput screening for the three remaining CYP enzymes will be completed in Y3, including detailed confirmation of reactive oxygen production and enzyme kinetics.
For Specific Aim 2, in silico ligand screening of the Phase 2 libraries will be performed using additional software, to proceed through the middle of year 3.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, in vivo models for CYP2Y3 will be generated in year 3, and stable knockout lines for CYP3A65 and CYP3C1 fully established. High throughput chemical analysis of hits arising from the screening of the first two enzymes will be performed, using novel GCxGC methods. Toxcast library materials will be used as the standards for GCxGC analyses.
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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Eide M, Zhang X, Karlsen OA, Goldstone JV, Stegeman J, Jonassen I, Goksøyr A. The chemical defensome of five model teleost fish. Scientific Reports 2021;11(1):1-3, doi:10.1038/s41598-021-89948-0. |
R840029 (2022) |
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Supplemental Keywords:
Cytochrome P450, CRISPR-CasRelevant Websites:
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.