Danio rerio larvae exposed to SSRIs Fluoxetine and Sertraline

Pharmaceutical chemicals used in human medicine are released into surface waters via municipal effluents and pose a risk for aquatic organisms. Among these substances are selective serotonin reuptake inhibitors (SSRIs) which can affect aquatic organisms at sub ppb concentrations. To better understand biochemical pathways influenced by SSRIs, evaluate changes in the transcriptome, and identify gene transcripts with potential for biomarkers of exposure to SSRIs; larval zebrafish Danio rerio were exposed (96 h) to two concentrations (25 and 250 µg/L) of the SSRIs, fluoxetine and sertraline, and changes in global gene expression were evaluated (Affymetrix GeneChip® Zebrafish Array). Significant changes in gene expression (>=1.7 fold change, p<0.05) were determined with Partek® Genomics Suite Gene Expression Data Analysis System and ontology analysis was conducted using Molecular Annotation System 3. The number of genes differentially expressed after fluoxetine exposure was 288 at 25 µg/L and 131 at 250 µg/L; and after sertraline exposure was 33 at 25 µg/L and 52 at 250 µg/L. Five genes were differentially regulated in all treatments relative to control, suggesting that both SSRIs share some similar molecular pathways. Among them, expression of the gene coding for FK506 binding protein 5 (FKBP5), which is annotated to stress response regulation, was highly down-regulated in all treatments (results confirmed by qRT-PCR). Gene ontology analysis indicated that regulation of stress response and cholinesterase activity were critical functions influenced by these SSRIs, and suggested that changes in the transcription of FKBP5 or acetylcholinesterase could be useful biomarkers of SSRIs exposure in wild fish. Zebrafish (Danio rerio) were obtained from the Zebrafish Research Facility maintained at the Center for Environmental Biotechnology at the University of Tennessee. Fish husbandry, spawning, and experimental procedures were conducted with approval from the UT Insititutional Animal Care and Use Committee (Protocol #1690-1007). Water for holding fish and conducting experiments (hereafter referred to as fish water) consisted of MilliQ water (Millipore, Bedford, MA) with ions added: 19 mg/L NaHCO3, 1 mg/L sea salt (Instant Ocean Synthetic Sea Salt, Mentor, OH), 10 mg/L CaSO4, 10 mg/L MgSO4, 2 mg/L KCl. Embroyos were obtained by spawning adult fish with no history of contaminant exposure. Fertilization of embryos took place at the same time (<15 minutes), such that larvae used in experiments were of similar age at the time of exposure. All activities (maintenance of adult fish, spawning, and experiments) were conducted in an environmental chamber with a temperature of 27 +/- 1 C and 14:10h light:dark photoperiod. Larval zebrafish (72 hpf) were exposed for 96 h in 200ml fish water containing appropirate amount of SSRI stock (i.e. fluoxetine or sertraline). There were four SSRIs treatments (25 and 250 ug/L fluoxetine and 25 and 250 ug/L sertraline) and one control (no SSRIs) with triplicate beakers and each beaker contained about 100 larval fish. During exposure for 96 hours, beakers were kept covered to prevent water evaporation and fish were not fed (i.e., fish consumed their yolk sac).

用于人类医学的药用化学品通过市政污水排放进入地表水体，对水生生物构成潜在风险。其中一类物质为选择性5-羟色胺再摄取抑制剂（selective serotonin reuptake inhibitors，SSRIs），其在亚ppb浓度下即可对水生生物产生影响。
为更清晰地解析SSRIs所调控的生化通路、评估转录组变化，并筛选可作为SSRIs暴露生物标志物的潜在基因转录本，研究人员将斑马鱼（Danio rerio）幼鱼暴露于两种浓度（25 µg/L与250 µg/L）的氟西汀（fluoxetine）与舍曲林（sertraline）这两种SSRIs中，暴露时长为96小时。随后通过Affymetrix GeneChip® 斑马鱼基因芯片（Affymetrix GeneChip® Zebrafish Array）对全局基因表达变化进行检测。
采用Partek® 基因组套件基因表达数据分析系统（Partek® Genomics Suite Gene Expression Data Analysis System）筛选出表达差异显著的基因（差异倍数≥1.7倍，p<0.05），并通过分子注释系统3.0（Molecular Annotation System 3）完成基因本体论分析。氟西汀暴露组中，25 µg/L浓度下共有288个差异表达基因，250 µg/L浓度下则为131个；舍曲林暴露组中，25 µg/L浓度下有33个差异表达基因，250 µg/L浓度下为52个。在所有处理组中，共有5个基因的表达量均出现显著变化，提示这两种SSRIs存在部分共通的分子通路。
其中，编码FK506结合蛋白5（FK506 binding protein 5，FKBP5）的基因，其功能注释为应激反应调控，在所有处理组中均呈现显著下调，该结果经实时荧光定量PCR（qRT-PCR）验证。基因本体论分析结果显示，应激反应调控与胆碱酯酶活性是这两种SSRIs影响的关键功能通路，提示FKBP5或乙酰胆碱酯酶的转录变化可作为野生鱼类暴露于SSRIs的潜在生物标志物。
本研究所用的斑马鱼均取自田纳西大学环境生物技术中心维护的斑马鱼研究设施。鱼类饲养、产卵及实验流程均已获得田纳西大学机构动物护理与使用委员会（UT Institutional Animal Care and Use Committee）批准（协议编号：1690-1007）。用于饲养鱼类与实验的水体（以下简称养殖用水）由MilliQ水（Millipore, 马萨诸塞州贝德福德）配制而成，添加的离子成分包括：19 mg/L碳酸氢钠、1 mg/L海盐（Instant Ocean合成海盐，俄亥俄州门托）、10 mg/L硫酸钙、10 mg/L硫酸镁、2 mg/L氯化钾。
实验所用胚胎通过无污染物暴露史的成年斑马鱼产卵获得。胚胎受精时间均控制在15分钟以内，确保实验所用幼鱼在暴露时的年龄基本一致。所有操作（成鱼饲养、产卵及实验）均在环境舱内完成，环境参数设置为：温度27±1℃，光周期14:10小时（光照:黑暗）。
72 hpf（受精后72小时）的斑马鱼幼鱼被置于200 ml养殖用水中，加入适量的SSRIs储备液（即氟西汀或舍曲林），暴露时长为96小时。实验共设置4个SSRIs处理组（25 µg/L与250 µg/L氟西汀、25 µg/L与250 µg/L舍曲林）及1个对照组（无SSRIs添加），每组设置3个重复烧杯，每个烧杯中约含100尾斑马鱼幼鱼。在96小时的暴露期间，烧杯均加盖以防止水分蒸发，且期间不投喂饵料（幼鱼可通过自身卵黄囊获取营养）。