Grantee Research Project Results
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 Amount: $799,999
RFA: Advancing Toxicokinetics for Efficient and Robust Chemical Evaluations (2019) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Description:
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.
Objective:
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.
Approach:
InVivo screening of heterologously expressed zebrafish CYPs will be performed by analyzing the oxidation of the essential cofactor NADPH to identify putative substrates and uncouplers in the ToxCast Phase 2 library of more than 1800 compounds. We will focus on CYP1A, CYP3A65, CYP3C1, and CYP2Y3, which we believe represent many of the key xenobiotic-metabolizing CYPs in zebrafish. We will determine detailed parent and primary metabolite profiles for top hit compounds, performing more detailed kinetic assays. A novel multiplexed multidimensional GCxGC-MS identification will be used to characterize the metabolite profiles for individual hit compounds. Iterative ligand docking studies will be used to support ligand interactions, to examine how substrates dock into active sites in relation to product profiles, and to predict additional substrates. In vitro-in vivo extrapolation is a key goal in chemical screening and hazard identification. Using transgenic knock-out strains of zebrafish, we will determine the kinetics of identified substrates in whole embryos.
Expected Results:
This proposal will provide detailed metabolite screening of the ToxCast Phase 2 chemicals for a key test species, zebrafish, and provide foundational in vitro-in vivo extrapolation data in a sensitive lifestage. This data will inform EPA and EPA stakeholders, and provide additional routes to in vitro and in silico screening methods to refine and reduce animal testing methods.
Publications and Presentations:
Publications have been submitted on this project: View all 3 publications for this projectJournal Articles:
Journal Articles have been submitted on this project: View all 2 journal articles for this projectSupplemental Keywords:
Tox21, toxics, pesticides, PAH, metabolism, sensitive populations, enzymesProgress and Final Reports:
The 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.