Hook Sharon E, Skillman Ann D, Small Jack A, Schultz Irvin R
Battelle, Marine Research Operations, Sequim, WA, USA.
Aquat Toxicol. 2006 May 25;77(4):372-85. doi: 10.1016/j.aquatox.2006.01.007. Epub 2006 Feb 20.
The increased availability and use of DNA microarrays has allowed the characterization of gene expression patterns associated with exposure to different toxicants. An important question is whether toxicant induced changes in gene expression in fish are sufficiently diverse to allow for identification of specific modes of action and/or specific contaminants. In theory, each class of toxicant may generate a gene expression profile unique to its mode of toxic action. In this study, isogenic (cloned) rainbow trout Oncorhynchus mykiss were exposed to sublethal levels of a series of model toxicants with varying modes of action, including ethynylestradiol (xeno-estrogen), 2,2,4,4'-tetrabromodiphenyl ether (BDE-47, thyroid active), diquat (oxidant stressor), chromium VI, and benzo[a]pyrene (BaP) for a period of 1-3 weeks. An additional experiment measured trenbolone (anabolic steroid; model androgen) induced gene expression changes in sexually mature female trout. Following exposure, fish were euthanized, livers removed and RNA extracted. Fluorescently labeled cDNA were generated and hybridized against a commercially available Atlantic Salmon/Trout array (GRASP project, University of Victoria) spotted with 16,000 cDNA's. The slides were scanned to measure abundance of a given transcript in each sample relative to controls. Data were analyzed via Genespring (Silicon Genetics) to identify a list of up- and downregulated genes, as well as to determine gene clustering patterns that can be used as "expression signatures". The results indicate each toxicant exposure caused between 64 and 222 genes to be significantly altered in expression. Most genes exhibiting altered expression responded to only one of the toxicants and relatively few were co-expressed in multiple treatments. For example, BaP and Diquat, both of which exert toxicity via oxidative stress, upregulated 28 of the same genes, of over 100 genes altered by either treatment. Other genes associated with steroidogenesis, p450 and estrogen responsive genes appear to be useful for selectively identifying toxicant mode of action in fish, suggesting a link between gene expression profile and mode of toxicity. Our array results showed good agreement with quantitative real time polymerase chain reaction (qRT PCR), which demonstrates that the arrays are an accurate measure of gene expression. The specificity of the gene expression profile in response to a model toxicant, the link between genes with altered expression and mode of toxic action, and the consistency between array and qRT PCR results all suggest that cDNA microarrays have the potential to screen environmental contaminants for biomarkers and mode of toxic action.
DNA微阵列可用性和使用的增加，使得与接触不同毒物相关的基因表达模式得以表征。一个重要的问题是，毒物诱导的鱼类基因表达变化是否足够多样，以便识别特定的作用模式和/或特定的污染物。理论上，每一类毒物可能会产生与其毒性作用模式独特相关的基因表达谱。在本研究中，将同基因（克隆）虹鳟鱼（Oncorhynchus mykiss）暴露于一系列具有不同作用模式的亚致死水平模型毒物中，包括乙炔雌二醇（外源雌激素）、2,2,4,4'-四溴二苯醚（BDE - 47，甲状腺活性物质）、敌草快（氧化应激源）、六价铬和苯并[a]芘（BaP），暴露时间为1 - 3周。另一个实验测量了群勃龙（合成代谢类固醇；模型雄激素）诱导的性成熟雌性虹鳟鱼基因表达变化。暴露后，将鱼安乐死，取出肝脏并提取RNA。生成荧光标记的cDNA，并与用16,000个cDNA点样的市售大西洋鲑鱼/虹鳟鱼微阵列（维多利亚大学GRASP项目）杂交。扫描玻片以测量每个样品中给定转录本相对于对照的丰度。通过Genespring（Silicon Genetics）分析数据，以识别上调和下调基因列表，以及确定可作为“表达特征”的基因聚类模式。结果表明，每种毒物暴露导致64至222个基因的表达发生显著变化。大多数表达改变的基因仅对一种毒物有反应，并且在多种处理中共表达的相对较少。例如，BaP和敌草快都通过氧化应激发挥毒性作用，在超过100个因任何一种处理而改变的基因中，它们上调了28个相同的基因。其他与类固醇生成、p450和雌激素反应基因相关的基因似乎有助于选择性地识别鱼类中毒物的作用模式，这表明基因表达谱与毒性作用模式之间存在联系。我们的微阵列结果与定量实时聚合酶链反应（qRT PCR）显示出良好的一致性，这表明微阵列是基因表达的准确测量方法。对模型毒物反应的基因表达谱的特异性、表达改变的基因与毒性作用模式之间的联系以及微阵列和qRT PCR结果之间的一致性，都表明cDNA微阵列有潜力筛选环境污染物的生物标志物和毒性作用模式。
