Date Published: February 17, 2017
Publisher: Public Library of Science
Author(s): Margaret G. Mills, Evan P. Gallagher, Jonathan H Freedman.
Chemical-induced oxidative stress and the biochemical pathways that protect against oxidative damage are of particular interest in the field of toxicology. To rapidly identify oxidative stress-responsive gene expression changes in zebrafish, we developed a targeted panel of antioxidant genes using the Affymetrix QuantiGene Plex (QGP) platform. The genes contained in our panel include eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1a, gstp1, gclc, nqo1, prdx1, gpx1a, sod1, sod2), a stress response gene (hsp70), an inducible DNA damage repair gene (gadd45bb), and three reference genes (actb1, gapdh, hprt1). We tested this platform on larval zebrafish exposed to tert-butyl hydroperoxide (tBHP) and cadmium (Cd), two model oxidative stressors with different modes of action, and compared our results with those obtained using the more common quantitative PCR (qPCR) method. Both methods showed that exposure to tBHP and Cd induced expression of prdx1, gstp1, and hmox1a (2- to 12-fold increase via QGP), indicative of an activated Nrf2 response in larval zebrafish. Both compounds also elicited a general stress response as reflected by elevation of hsp70 and gadd45bb, with Cd being the more potent inducer. Transient changes were observed in sod2 and gpx1a expression, whereas nqo1, an Nrf2-responsive gene in mammalian cells, was minimally affected by either tBHP or Cd chemical exposures. Developmental expression analysis of the target genes by QGP revealed marked upregulation of sod2 between 0-96hpf, and to a lesser extent, of sod1 and gstp1. Once optimized, QGP analysis of these experiments was accomplished more rapidly, using far less tissue, and at lower total costs than qPCR analysis. In summary, the QGP platform as applied to higher-throughput zebrafish studies provides a reasonable cost-effective alternative to qPCR or more comprehensive transcriptomics approaches to rapidly assess the potential for chemicals to elicit oxidative stress as a mechanism of chemical toxicity.
Oxidative stress (OS) contributes to a wide range of human diseases, such as neurodegeneration [1–4], cancer [5–7], cardiac conditions such as hypertension and atherosclerosis [8–11], and cell death associated with ischemia/reperfusion injuries [12,13]. It is also a common mechanism leading to cellular damage caused by chemical exposure in humans [14–16] and wildlife species [17–21].
In the current study we show that analysis of antioxidant gene expression by QuantiGene Plex (QGP) is comparable to that of qPCR. QGP probes have been validated for use in other species [42,43,59], but ours is the first published use of the assay for quantifying expression of zebrafish genes. Despite comparable reagent costs, the QGP assay required less (and often vastly less) time and tissue to complete, and allowed us to avoid the lengthy and contamination-prone steps involved in qPCR. We found that while the two methods did not agree on the precise–fold changes in expression induced by exposure of larval zebrafish to tBHP or Cd, they did agree on the particular genes whose expressions were significantly modulated by two prototypical oxidative stressors, and the extent of -fold change in gene expression. The relative effects of the two chemicals, whether tBHP or Cd induced a greater change in expression of a particular gene, was also consistent between the two platforms, with a few exceptions for which the biological significance is probably low, as these cases all involve either increases in gene expression of less than 2-fold (in the cases of nqo1 and gadd45bb) or a decrease in expression to 0.9x (in the case of sod2). We also conclude that the optional step of purifying RNA from zebrafish tissue before use in the QGP assay is not necessary and does not provide any advantage in comparing QGP results to qPCR results.