Grantee Research Project Results
2021 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, 2020 through July 31,2021
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:
Due to the significant delays in receiving the ToxCast Compound libraries work has been delayed by approximately 12 months, and we do not have the anticipated HTS screening results nor the intermediate screening in vitro or in vivo screening results we proposed to have prior to the Covid emergency.
Standard HTS assay measurements for the previously expressed CYP1A enzyme include the analysis of NADPH consumption by the metabolic activation of the enzyme with methoxyresorufin. This produces a linear response in the loss of NADPH absorption. Deviations from linearity in these positive controls serve as a quality control measure. Negative controls include no-enzyme, no-NADPH, and no-substrate controls.
Future Activities:
Aim 1: Protein expression for HTS screening was optimized prior to the proposal submission. Three of the four proposed enzymes were in hand in the freezer prior to 2019 (CYP1A, CYP3A65, and CYP3C1), as previously published. During the second quarter of Y2, CYP2Y3 will be synthesized, codon optimized, and cloned into the expression plasmid. Protein expression will be optimized, and P450 content will be confirmed using standard methods as noted in the proposal.
High throughput screening (HTS) of the Phase 2 compound library will be performed for all four enzymes during Y2. Work on CYP1A will be started immediately upon disentanglement of the three ToxCast libraries. Additional enzymes will be screened following quality checks on the CYP1A data, and cross validation with previously obtained data. The ToxCast libraries were supplied with varying concentrations, rather than the requested 20 uM concentrations. Well selection and dilution to a standard concentration will be performed using robots to eliminate the variability induced by human pipetting.
Aim 2: In silico ligand screening of the Phase 2 libraries will begin in Q2 of Y2, to proceed throughout the remainder of Y2. In vitro confirmation of hits from the first enzyme screens will be performed in Q3 and Q4.
Aim 3:In vivo models for CYP2Y3 and CYP3A65 will be generated and genotyped in Y2. A technician should be in place by Q2, while reagents have been designed and purchased in previous years. In vivo screening of metabolite generation from the first two enzyme screens will be performed in Q3 and Q4. GCxGC analysis of hits arising from the screening of the first two enzymes will be performed in Q4. Toxcast library materials will be used as the standards for GCxGC analyses.
Journal Articles:
No journal articles submitted with this report: View all 1 publications for this projectProgress 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.