Identification of a transcriptional fingerprint of estrogen exposure in rainbow trout liver. Oncorhynchus mykiss

The goal of this study was to identify a set of hepatic genes regulated by ligand-dependent activation of the estrogen receptor in juvenile rainbow trout (Oncorhynchus mykiss) that can serve as a biomarker of estrogen exposure. A custom rainbow trout oligo DNA microarray, which contains probes targeting approximately 1450 genes relevant to carcinogenesis, toxicology, endocrinology and stress physiology was utilized to identify transcriptional “fingerprints” of in vivo dietary exposure to 17ß-estradiol (E2), tamoxifen (TAM), estradiol + tamoxifen (E2+TAM), diethylstilbestrol (DES), dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and cortisol (CORT). Estrogen exposure altered the expression of up to 49 genes involved in reproduction, immune response, cell growth, transcriptional regulation, protein synthesis and modification, drug metabolism, redox regulation and signal transduction. E2, DES and DHEA regulated 18 genes in common, mostly those associated with vitellogenesis, cell proliferation and signal transduction. Interestingly, DHEA uniquely regulated several complement component genes of importance to immune response. While the effect of TAM on E2-induced changes in gene expression was mostly antagonistic, TAM alone increased expression of VTG1 and other genes associated with egg development and immune response. Few genes responded to CORT treatment, and DHT significantly altered expression of only one gene targeted by the OSUrbt array. Hierarchical cluster and principal components analyses revealed distinct patterns of gene expression corresponding to estrogens and non-estrogens, though unique patterns could also be detected for individual chemicals. A set of estrogen-responsive genes has been identified that can serve as a biomarker of environmental exposure to xenoestrogens. Keywords: estrogen transcriptional profile, chemical transcriptional fingerprint Overall design: Eight experimental treatments were selected to examine hepatic gene expression in response to estrogens, a selective estrogen receptor modulator (SERM) and non-estrogens. Treatments and concentrations (calculated with respect to diet wet weight) are as follows: control (CON), 5 ppm 17ß-estradiol (E2), 50 ppm tamoxifen (TAM), 5 ppm E2 + 50 ppm TAM (E2+TAM), 2 ppm diethylstilbestrol (DES), 500 ppm dehydroepiandrosterone (DHEA), 5 ppm dihydrotestosterone (DHT) and 5 ppm cortisol (CORT). Test chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were added directly to the oil portion of the OTD. Experimental treatments were administered for 14 days, and feeding occurred five days per week. Total hepatic RNA was extracted in TRIzol reagent according to supplier’s instructions. Equal amounts of RNA from each individual liver sample were sub-pooled according to tank (four males pooled per tank) resulting in three biological replicates for each treatment. A reference RNA pool was made by combining equal amounts of RNA from all individual male CON liver samples (12 liver samples total). For detection of gene expression on the OSUrbt array, the Genisphere 3DNA Array 350 kit (Hatfield, PA) was used in a dye-swap, reference sample design following the supplier’s protocol. During reverse transcription (SuperScript II, Invitrogen) of total RNA (7 µg) using the supplied Genisphere oligo d(T) primers, each biological sample was tagged separately with a capture sequence for one of two fluorescent dendromer reagents, Cy3 or Cy5. Each reverse transcription reaction also included spiked in mRNA corresponding to the SpotReport Alien Oligo controls. Corresponding reference RNA was reverse transcribed with the capture sequence for the opposite reagent (Cy3 or Cy5). To account for any differences in dye labeling efficiency, technical replicates were performed for two of the three biological replicates in which the capture sequence tags were swapped between the sample and reference. Thus, five arrays per experimental treatment were processed, including the CON treatment.

本研究旨在鉴定幼年虹鳟（Oncorhynchus mykiss）中依赖配体的雌激素受体激活所调控的一组肝脏基因，此类基因可作为雌激素暴露的生物标志物（biomarker）。本研究使用一款定制化虹鳟寡核苷酸DNA微阵列，该芯片包含针对约1450个与致癌作用、毒理学、内分泌学及应激生理学相关的基因的探针，用于鉴定经口暴露于17β-雌二醇（17ß-estradiol, E2）、他莫昔芬（tamoxifen, TAM）、雌二醇+他莫昔芬（E2+TAM）、己烯雌酚（diethylstilbestrol, DES）、脱氢表雄酮（dehydroepiandrosterone, DHEA）、二氢睾酮（dihydrotestosterone, DHT）及皮质醇（cortisol, CORT）的体内转录“指纹”特征。

雌激素暴露可改变多达49个基因的表达，这些基因涉及生殖、免疫应答、细胞生长、转录调控、蛋白质合成与修饰、药物代谢、氧化还原调控及信号转导通路。E2、DES与DHEA共同调控18个基因，其中多数与卵黄生成、细胞增殖及信号转导相关。值得注意的是，DHEA可特异性调控数个与免疫应答密切相关的补体成分基因。尽管他莫昔芬对E2诱导的基因表达变化主要表现为拮抗作用，但单独使用他莫昔芬可上调VTG1及其他与卵子发育、免疫应答相关的基因的表达。皮质醇处理仅引发少量基因的应答，而DHT仅显著改变OSUrbt微阵列靶向的1个基因的表达。

层级聚类分析（hierarchical cluster）与主成分分析（principal components analysis, PCA）结果显示，雌激素与非雌激素类物质对应截然不同的基因表达模式，同时各受试化合物也呈现出独特的表达特征。本研究已鉴定出一组雌激素应答基因，可作为环境中外源雌激素（xenoestrogens）暴露的生物标志物。

关键词：雌激素转录谱，化合物转录指纹

总体实验设计：设置8组实验处理，以探究雌激素、选择性雌激素受体调节剂（selective estrogen receptor modulator, SERM）及非雌激素类物质诱导的肝脏基因表达变化。各处理组及对应浓度（以日粮湿重计算）如下：对照组（CON）、5 ppm 17β-雌二醇（E2）、50 ppm他莫昔芬（TAM）、5 ppm E2 + 50 ppm TAM（E2+TAM）、2 ppm己烯雌酚（DES）、500 ppm脱氢表雄酮（DHEA）、5 ppm二氢睾酮（DHT）及5 ppm皮质醇（CORT）。受试化合物均购自Sigma-Aldrich（美国密苏里州圣路易斯市），直接添加至OTD的油相组分中。实验处理持续14天，每周投喂5次。

按照供应商说明书，使用TRIzol试剂提取总肝脏RNA。将每个肝脏样本的RNA按养殖缸等量混合（每缸4尾雄鱼的RNA混合），每组处理获得3个生物学重复。参照RNA混合池由所有对照组雄鱼肝脏样本的RNA等量混合制成（共计12个肝脏样本）。

为在OSUrbt微阵列上检测基因表达，采用Genisphere 3DNA Array 350试剂盒（美国宾夕法尼亚州哈特菲尔德市），按照供应商方案实施染料互换的参照样本设计实验。在使用SuperScript II反转录酶（Invitrogen公司）及配套的Genisphere寡聚d(T)引物对总RNA（7 µg）进行反转录时，每个生物学样本分别用两种荧光树状聚合物试剂（Cy3或Cy5）的捕获序列进行标记。每个反转录反应体系均加入SpotReport Alien寡核苷酸对照对应的mRNA作为内参。参照RNA则使用与样本相反的捕获序列试剂（Cy3或Cy5）进行反转录。为校正染料标记效率的差异，对3个生物学重复中的2个实施技术重复，交换样本与参照的捕获序列标签。因此，每组实验处理（包括对照组）均需处理5张微阵列芯片。