A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae

Abstract

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 and apoptotic effects of copper and cadmium in the zebrafish liver cell line ZFL

“…2 A , 3 A ). In general, cell response to Cu started earlier at 4 h and lasted till 24 h. In contrast, cell response to Cd started later at 12 h and lasted longer till 48 h. In one study, the expression of zsod1 , zsod2 and zgst showed no change, was suppressed and was induced, respectively, in zebrafish larvae after 3-h Cd exposure [ 61 ]. This result is similar to the 4-h Cd exposure result in our study.…”

Effect of contaminants of emerging concern on liver mitochondrial function in Chinook salmon

“…Expression of hepatic genes involved in the cellular antioxidant response and mitochondrial function were quantified in fish from the dietary exposure laboratory study using a customized Quantigene® plex 2.0 (QGP) panel (Affymetrix, Fremont, CA) (Mills and Gallagher, 2017). The antioxidant response genes included glutamate-cysteine ligase catalytic subunit ( GCLC ), phospholipid hydroperoxide glutathione peroxidase (GPx4), and mitochondrial superoxide dismutase (SOD2).…”

Comparative effects of cadmium, zinc, arsenic and chromium on olfactory-mediated neurobehavior and gene expression in larval zebrafish (Danio rerio)

“…To investigate if Cr, As, Cd and Zn share a similar mechanism of olfactory toxicity through whole larval oxidative stress, targeted gene expression was performed. Analysis of eight putative Nrf2 (Nfe2l2a)-dependent antioxidant genes (hmox1 a, 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) was completed after 24 hr metal exposures using the QuantiGene Plex (QGP) assay following procedures described in Mills & Gallagher (2017). In addition, a second QGP assay was developed that targeted two homologs of metallothionein (mt2 and mtbl) and contained two reference genes (actb1, and hprt1) and that was analyzed using the same protocol.…”