Battelle PNWD, Marine Sciences Laboratory, Sequim, WA, USA.
Aquat Toxicol. 2010 Sep 1;99(3):320-9. doi: 10.1016/j.aquatox.2010.05.011. Epub 2010 May 20.
Time is often not characterized as a variable in ecotoxicogenomic studies. In this study, temporal changes in gene expression were determined during exposure to crude oil and a subsequent recovery period. Juvenile rainbow trout, Oncorhynchus mykiss, were exposed for 96 h to the water accommodated fractions of 0.4, 2 or 10 mgl(-1) crude oil loadings. Following 96 h of exposure, fish were transferred to recovery tanks. Gill and liver samples were collected after 24 and 96 h of exposure, and after 96 h of recovery for RNA extraction and microarray analysis. Fluorescently labeled cDNA was hybridized against matched controls, using salmonid cDNA arrays. Each exposure scenario generated unique patterns of altered gene expression. More genes responded to crude oil in the gill than in the liver. In the gill, 1137 genes had altered expression at 24 h, 2003 genes had altered expression levels at 96 h of exposure, yet by 96 h of recovery, no genes were significantly altered in expression. In the liver at 10 mgl(-1), only five genes were changed at 24 h, yet 192 genes had altered expression after 96 h recovery. At 2 mgl(-1) in the liver, many genes had altered regulation at all three time points. The 0.4 mgl(-1) loading also showed 289 genes upregulated at 24 h after exposure. The Gene Ontology terms associated with altered expression in the liver suggested that the processes of protein synthesis, xenobiotic metabolism, and oxidoreductase activity were altered. The concentration-responsive expression profile of cytochrome P450 1A, a biomarker for oil exposure, did not predict the majority of gene expression profiles in any tissue or dose, since direct relationships with dose were not observed for most genes. While the genes and their associated functions agree with known modes of toxic action for crude oil, the gene lists obtained do not match our previously published work, presumably due to array analysis procedures. These results demonstrate that changes in gene expression with time and dose may be complicated, and should be characterized in controlled laboratory settings before attempts are made to interpret responses in field-collected organisms. Further, processes for analyzing microarray data need to be developed such that standardized gene lists are developed, or that analysis does not rely on lists of significantly altered genes before arrays can be further evaluated as a monitoring tool.
在生态毒基因学研究中，时间通常不作为一个变量。本研究旨在确定暴露于原油及其后续恢复期时基因表达的时间变化。将幼年虹鳟（Oncorhynchus mykiss）暴露于 0.4、2 或 10mg/L 原油负荷的水可容纳分数中 96 小时。暴露 96 小时后，将鱼转移到恢复池中。在暴露 24 和 96 小时以及恢复 96 小时后，采集鳃和肝组织样本以提取 RNA 并进行微阵列分析。用鲑鱼 cDNA 阵列对荧光标记的 cDNA 与匹配对照进行杂交。每种暴露情况都会产生独特的改变基因表达的模式。与肝脏相比，原油在鳃中引起更多基因的表达改变。在鳃中，有 1137 个基因在 24 小时时表达改变，有 2003 个基因在 96 小时时表达改变，但在 96 小时恢复时，没有基因的表达显著改变。在肝脏中，10mg/L 时，仅有 5 个基因在 24 小时时改变，而 192 个基因在 96 小时恢复时表达改变。在肝脏中，2mg/L 时，许多基因在所有三个时间点都有改变的调控。0.4mg/L 负荷也显示出 289 个基因在暴露后 24 小时上调。与肝脏中改变表达相关的基因本体论术语表明，蛋白质合成、外来生物代谢和氧化还原酶活性等过程发生了改变。细胞色素 P450 1A 的浓度反应表达谱是一种用于评估石油暴露的生物标志物，但它不能预测任何组织或剂量的大多数基因表达谱，因为大多数基因与剂量之间没有直接关系。虽然这些基因及其相关功能与原油的已知毒性作用模式一致，但获得的基因列表与我们之前的发表工作不匹配，这可能是由于微阵列分析程序不同。这些结果表明，随着时间和剂量的变化，基因表达的变化可能会很复杂，并且应该在尝试解释野外采集的生物的反应之前，在受控的实验室环境中对其进行特征描述。此外，需要开发用于分析微阵列数据的过程，以便开发标准化的基因列表，或者在进一步评估微阵列作为监测工具之前，不要依赖于显著改变的基因列表。
