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




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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.



Author: Margaret G. Mills, Evan P. Gallagher

Source: http://plos.srce.hr/



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