Researchers at the University of Washington School of Public Health are using a newly developed panel of zebrafish genes and a rapid testing platform to identify chemicals that trigger oxidative stress. The method is cost-effective and can be performed more quickly and with less tissue than other methods, according to a research brief released May 3.
Oxidative stress is an imbalance between the production of free radicals, or oxygen-containing molecules with unpaired electrons, and antioxidant defenses. Overproduction of free radicals can cause oxidative damage to lipids, proteins and DNA, eventually leading to chronic diseases such as cancer, diabetes and heart disease. It is also a mechanism of toxicity for many chemicals.
“There are thousands of chemicals currently in use for which little toxicity data exists,” said Margaret Mills, a postdoctoral researcher at the School and lead study author. “Zebrafish are a powerful model organism for toxicology, allowing us to rapidly test many chemicals for their effects in a system that is relevant for both human and aquatic health.”
Cellular experience of oxidative stress may be illustrated by changes in expression for genes that encode antioxidant proteins to combat oxidative stress. For both zebrafish and humans, a protein known as Nrf2 regulates many of these antioxidant genes.
Researchers from the UW Superfund Research Program, housed within the School’s department of environmental and occupational health sciences, developed a targeted panel of 13 genes containing the following:
- Nrf2-dependent antioxidant genes
- Genes responsive to oxidative stress or DNA damage, but not dependent on Nrf2
- Three reference genes
Researchers from the center, led by Evan Gallagher, professor of environmental toxicology at the School, used the Affymetrix QuantiGene Plex (QGP) platform to examine the panel of genes in larval zebrafish exposed to model chemicals known to induce oxidative stress. The QGP is a fast and flexible way to measure expression from up to 80 genes at once.
“Adapting the QGP platform for use in zebrafish allows us to speed up gene expression analysis to match the rapid development of zebrafish,” Mills said.
For the QGP panel, Mills and Gallagher designed probes to bind transcripts from the specific antioxidant genes to uniquely labeled beads. When the beads were sorted, the identity of each bead correlated with the fluorescent signal from the bound transcript, allowing quantification of expression from multiple genes within a single sample.
The researchers compared the QGP results to those obtained through a conventional laboratory method called quantitative polymerase chain reaction.
Findings showed that both QGP and the conventional method revealed the same changes in gene expression in zebrafish exposed to the two chemicals and had comparable reagent costs. However, QGP required significantly less time and tissue to carry out the same analysis.
The UW Superfund Research Program is an interdisciplinary initiative that conducts research on the impacts of metal neurotoxicity on human and ecological health. Research focuses on metals that commonly occur at Superfund hazardous waste sites for which there are important data gaps impeding the full understanding of their neurotoxic effects on human and ecological health.
To learn more about the program, visit http://deohs.washington.edu/srp/.