Mills Margaret G, Gallagher Evan P
Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Seattle, Washington, United States of America.
PLoS One. 2017 Feb 17;12(2):e0171025. doi: 10.1371/journal.pone.0171025. eCollection 2017.
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.
化学诱导的氧化应激以及抵御氧化损伤的生化途径在毒理学领域备受关注。为了快速识别斑马鱼中氧化应激反应性基因表达的变化，我们利用Affymetrix QuantiGene Plex（QGP）平台开发了一组靶向抗氧化基因。我们的基因面板包含八个假定的Nrf2（Nfe2l2a）依赖性抗氧化基因（hmox1a、gstp1、gclc、nqo1、prdx1、gpx1a、sod1、sod2）、一个应激反应基因（hsp70）、一个可诱导的DNA损伤修复基因（gadd45bb）以及三个参考基因（actb1、gapdh、hprt1）。我们在暴露于叔丁基过氧化氢（tBHP）和镉（Cd）的斑马鱼幼体上测试了这个平台，tBHP和Cd是两种具有不同作用模式的氧化应激模型，并且将我们的结果与使用更常用的定量PCR（qPCR）方法得到的结果进行了比较。两种方法均显示，暴露于tBHP和Cd会诱导prdx1、gstp1和hmox1a的表达（通过QGP增加2至至12倍），这表明斑马鱼幼体中的Nrf2反应被激活。两种化合物还引发了一般应激反应，表现为hsp70和gadd45bb升高，其中Cd是更强效的诱导剂。观察到sod2和gpx1a表达有短暂变化，而nqo1（哺乳动物细胞中的Nrf2反应性基因）在tBHP或Cd化学暴露下受影响最小。通过QGP对靶基因进行的发育表达分析显示，sod2在0至96小时胚胎期（hpf）之间有明显上调，sod1和gstp1上调程度较小。一旦优化，这些实验的QGP分析比qPCR分析完成得更快，使用的组织更少，总成本更低。总之，应用于高通量斑马鱼研究的QGP平台为qPCR或更全面的转录组学方法提供了一种合理的性价比高的替代方案，以快速评估化学物质引发氧化应激作为化学毒性机制的可能性。
