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. Zebrafish are capable of oxidative metabolism of many and perhaps all of the same xenobiotics that are metabolized by mammals. This metabolism is largely carried out by members of the CYP1, CYP2, and CYP3 families, as in humans. 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 determine functionality of cytochrome P450 enzymes that may be most important to pollutant metabolism in zebrafish.
We are screening the Tox21 compound library for interaction with recombinant CYP proteins in vitro, determine metabolism rates for key substrates and identify prominent metabolites using novel multidimensional chromatography, supported by in silico ligand docking. CRISPR-Cas knockout technologies will be used to determine roles of specific CYPs in metabolism of compounds in vivo, leading to direct toxicokinetic in vitro-in vivo extrapolation (IVIVE) in this key toxicological species. This research will address the ‘3R’ goal of reduction and refinement of animal use, and eventual replacement with non-animal systems.
Humans and wildlife are exposed to a wide range of contaminants in the environment and the role of these chemical pollutants in developmental neurotoxicity and neurodegenerative disorders has become a global environmental health concern. Legacy chemicals such as polychlorinated biphenyls (PCBs) persist to date in the environment where they have globally accumulated in wildlife tissue and human cord blood, amniotic fluid, and mother's milk. Pesticides potentially used as chemical threat agents, including organophosphate pesticides, are also neurotoxic.
During the early stages of life, the nervous system is generally more sensitive to chemical exposure compared to fully developed adults, and therefore it is essential to identify the neurotoxic mechanisms that underpin the behavioral effects of environmental exposure to different PCBs during gestation and early life stages if we want to successfully treat and remediate health effects in humans and wildlife.
We use methods including transcriptomics, behavioral studies, and advanced imaging techniques to determine the mechanisms, and devise potential therapies, for developmental neurotoxins.
Cytochrome P450 20A1 (CYP20A1) is the last human “orphan” P450 enzyme, with unknown physiological substrate(s) and functions. Orthologs of CYP20 occur as single genes in every vertebrate genome sequenced to date with a highly conserved sequence, and the CYP20 family is clearly identifiable even in early-diverging animal lineages. The conserved nature of CYP20A1 argues that it plays a vital role in vertebrate biology, yet we know very little about its function. Previous research, including ours, points to a connection between CYP20A1 and neurobehavior in humans. A significant clue is the high-level expression of CYP20A1 in the hippocampus and substantia nigra, regions of the brain associated with learning, memory and ubiquitous neuropathies including Alzheimers and Parkinson’s disease
We have generated knockout CYP20A1-/- zebrafish lines, and used them for metabolomics and behavioral studies. The zebrafish line exhibits phenotypes comparable to some human anxiety disorders, including hyperactivity or panic disorders, which collectively impact 10% of the world's population.
Deep sea P450s
The deep sea is the most expansive and one of the most species-rich habitats on the planet, yet is less well mapped than the moon. About 80% of the volume occupied by life is found at depths below 1000 meters. The hydrostatic pressure produced by the overlying water can reach 1100 bar (110 MPa), yet microbes, invertebrates and vertebrates (fishes) have evolved ability to live at these pressures. Adaptions to pressure may lead to changes in the number of genes present in a given species (gains and losses), modulate gene expression levels, and lead to subtle changes of protein sequence to allow optimal function at high pressure.
We are working on P450 gene expression in deep sea fishes, and are interested in the regulation and functioning of these proteins under high hydrostatic pressure. More broadly, we are interested in examining genomic signatures of hydrostatic pressure adaption across metazoans, and the evolution of barotolerance.