A Short-Term Study Investigating the Estrogenic Potency of Diethylstilbesterol in the Fathead Minnow (Pimephales promelas): Exposure Phase

Diethylstilbestrol (DES) is a synthetic estrogen that has been banned for use in humans, but still is employed in livestock and aquaculture operations in some parts of the world. Detectable concentrations of DES in effluent and surface waters have been reported to range from slightly below 1 to greater than 10 ng/L. Little is known, however, concerning the toxicological potency of DES in fish. In this study, sexually-mature fathead minnows (Pimephales promelas) of both sexes were exposed to 1, 10 or 100 ng DES/L water in a flow-through system. Tissue concentrations of DES and changes in a number of estrogen-responsive endpoints, including alterations to the hepatic transcriptome in females, were measured in the fish at the end of a 4-d exposure, and after a 4-d depuration/recovery period in clean water. The objectives of the study were to measure accumulation of DES in fish tissues, characterize effects of the chemical on physiological endpoints related to reproductive performance, and to evaluate DES's potency relative to concentrations reported in aquatic environments. The current series includes n=12 microarrays associated with exposure-phase samples (collected from fish exposed continuously for 4 d). Fish were exposed to 0, 1, 10, or 100 ng DES/L delivered in a continuous flow (45 ml/min) of sand filtered, UV treated, Lake Superior Water, without the use of carrier solvents. Flow of DES into the test system was initiated without fish present and continued for 1 d. Exposures then were started by placing three male and three female sexually-mature fathead minnows into each tank. There were eight replicate tanks for the control and 100 ng/L treatments, and six replicate tanks for the 1 and 10 ng/L groups. The fish were held at 25+1ºC under a 16:8 L:D photoperiod and were fed frozen adult brine shrimp twice daily (San Francisco Bay Brand, Newark, CA, USA). Fish used for the experiment were from an on-site culture, and all procedures involving animals conformed to guidelines approved by the local Animal Care and Use Committee. After the 4-d exposure, fish from three tanks from each treatment (n=9 per sex) were sampled for determination of various biological endpoints. At this time fish from two additional tanks from the control and 100 ng/L treatments also were sampled for determination of DES tissue residues (n=6 per sex). Delivery of DES to the test system was subsequently stopped, and fish from the remaining three tanks per treatment group were sampled 4 d later (n= 9 per sex). The fathead minnows were euthanized with buffered MS-222 (Argent, Redmond, VA, USA) and weighed. Fish were scored for occurrence and relative expression of nuptial tubercles, which in males can be reduced by ER agonists. Blood was collected from the caudal vein/artery with a microhematocrit tube, and plasma was separated by centrifugation and stored at -80ºC. Gonads were removed from males and a subsample (8.5±3.0 mg; mean±SD) was immediately immersed in tissue culture media for determination of ex vivo steroid production. Livers were removed from both sexes and flash-frozen for gene expression analyses using microarray (females) or real-time QPCR (males). Hepatic transcripts from three females per treatment group, in each phase of the experiment (except n=2 from recovery phase 10 ng/L) were analyzed using a custom 15,000 feature microarray (GEO Platform Accession GPL9248). Data sets for the exposure phase (n=12 microarrays) and recovery phase (n=11) were normalized independently using Fastlo (Ballman et al., 2004) implemented in R (http://www.r-project.org/), but analyzed using parallel approaches.

己烯雌酚（Diethylstilbestrol, DES）是一种合成雌激素，已被禁止用于人类，但在全球部分地区的畜牧与水产养殖活动中仍有使用。据报道，废水及地表水中可检出的DES浓度范围为略低于1 ng/L至高于10 ng/L不等。然而，目前关于DES对鱼类的毒理学效力尚不清楚。本研究将性成熟的两性黑头呆鱼（Pimephales promelas）置于流水暴露系统中，以1、10或100 ng/L的DES水溶液进行暴露处理。在4天暴露结束后，以及将鱼转移至清洁水中进行4天净化/恢复培养后，分别测定鱼体组织中的DES浓度，以及多项雌激素响应终点的变化，包括雌性个体肝脏转录组的改变。

本研究的目的为：测定DES在鱼体组织中的蓄积情况，表征该化学物对与繁殖性能相关的生理终点的影响，并基于水生环境中已报道的浓度，评估DES的毒效强度。

本数据集包含与暴露阶段样本相关的n=12个微阵列（microarrays）样本，这些样本采集自连续暴露4天的鱼体。实验中，鱼体暴露于以砂滤、紫外消毒的苏必利尔湖湖水（流速45 ml/min）为介质的0、1、10或100 ng/L DES溶液，未使用助溶剂。试验系统的DES投加先在无鱼状态下启动并持续1天，随后向每个养殖槽中放入3条雄性和3条性成熟的黑头呆鱼。对照组与100 ng/L处理组各设8个重复养殖槽，1 ng/L和10 ng/L组各设6个重复养殖槽。

实验鱼饲养于25±1℃环境，光周期为16:8（光照:黑暗），每日投喂两次冷冻成年卤虫（San Francisco Bay Brand, Newark, CA, USA）。实验用鱼取自本单位养殖种群，所有涉及动物的实验操作均符合当地动物护理与使用委员会批准的指南。

4天暴露结束后，每个处理组的3个养殖槽中的鱼（每个性别n=9）被采样以测定各类生物学终点。此时，对照组与100 ng/L处理组额外的2个养殖槽中的鱼也被采样，用于测定鱼体组织中的DES残留量（每个性别n=6）。随后停止向试验系统投加DES，每个处理组剩余的3个养殖槽中的鱼在4天后被采样（每个性别n=9）。

黑头呆鱼经缓冲MS-222（Argent, Redmond, VA, USA）安乐死后称重。对雄鱼的追星（nuptial tubercles）的出现情况与相对表达量进行评分，因为雌激素受体激动剂可降低雄鱼的追星发育。通过微量血细胞比容管从尾静脉/尾动脉采集血液，离心分离血浆后于-80℃保存。采集雄鱼的性腺，取亚样本（8.5±3.0 mg；平均值±标准差）立即浸入组织培养基中，用于离体类固醇生成量测定。采集雌雄个体的肝脏，速冻后分别通过微阵列（雌性）或实时荧光定量PCR（real-time QPCR）进行基因表达分析。

本实验各阶段中，每个处理组的3条雌性个体的肝脏转录本（恢复阶段10 ng/L组n=2除外）均采用定制的15,000探针基因芯片（GEO平台登录号GPL9248）进行分析。暴露阶段（n=12个微阵列）与恢复阶段（n=11个微阵列）的数据集分别使用R语言（http://www.r-project.org/）中实现的Fastlo算法（Ballman等，2004）进行归一化，但采用平行分析方法。