CHARACTERIZING TOXICITY PATHWAYS OF FLUOXETINE TO PREDICT ADVERSE OUTCOMES IN ADULT PIMEPHALES PROMELAS. CHARACTERIZING TOXICITY PATHWAYS OF FLUOXETINE TO PREDICT ADVERSE OUTCOMES IN ADULT PIMEPHALES PROMELAS

Fluoxetine (FLX) is the most common selective serotonin reuptake inhibitor pharmaceutical used for the treatment of psychological disorders such as depression and anxiety. Increased prescription use of FLX by human populations has led to its pseudo-persistence in effluent-receiving waterways. Despite emerging concerns of FLX becoming a potential threat to the aquatic environment, there is currently limited information on effects of FLX on aquatic wildlife, including its specific mechanism of action and associated toxicity pathways in fish. The main goal of this study was to identify and validate key molecular toxicity pathways that are predictive of apical endpoints induced by exposure to FLX using the fathead minnow (Pimephales promelas), a model species common to North American freshwater systems. Adult fathead minnows were exposed to three concentrations of FLX (2.42, 10.7, and 56.7 µg/L) and a control for 21 days. After 96 hours, a subset of fish was sampled, and liver and brain tissue were collected to characterize molecular toxicity pathways using whole transcriptome and proteome analyses. In addition, at the end of the 21-day exposure, individuals were assessed for apical outcomes of regulatory relevance including histopathology and fecundity. Differential gene expression observed in the liver of fish exposed to the highest FLX treatment and revealed dysregulation of pathways associated with biosynthesis and metabolism of fatty acids, which may be an upstream molecular response that led to lipid-type vacuolation of hepatocytes, as observed in the histology analysis. Whole proteome analysis of the same fish revealed dysregulation of PPAR signalling, which may be associated with the enrichment of lipid-related pathways observed in the transcriptome. In addition, there was an indication of pathway enrichment of transcription- and translation-related pathways in the proteome. Common dysregulated genes in the brain of the treated fish were related to cellular signalling processes that are influenced by serotonin levels and shown to be involved in reproductive behaviour and, in turn, reproductive success. This was confirmed through a significant decrease in fecundity following the 21-day exposure. Pathways to measurable adverse outcomes are complex, however, this research does provide some important clues to the mechanistic toxicity that FLX inflicts. Overall design: Fathead minnows (Pimephales promelas) were exposed to fluoxetine for 96h. Samples taken for transcriptomic analyses: Liver Samples: Negative Water Control (n=5, 2 individuals pooled per sample); 10.7µg FLX/L (n=5, 2 individuals pooled per sample); 56.7µg FLX/L (n=5, 2 individuals pooled per sample); Brain Samples: Negative Water Control (n=5, 2 individuals pooled per sample); 10.7µg FLX/L (n=5, 2 individuals pooled per sample); 56.7µg FLX/L (n=5, 2 individuals pooled per sample)

氟西汀（Fluoxetine, FLX）是目前临床应用最广泛的选择性5-羟色胺再摄取抑制剂（selective serotonin reuptake inhibitor）类药物，用于治疗抑郁、焦虑等精神疾病。人类对氟西汀的处方用量持续增加，导致其在接纳污水的水体中呈现假持久性。尽管氟西汀对水生环境的潜在威胁已日益受到关注，但目前关于其对水生野生生物的毒性效应、具体作用机制及鱼类相关毒性通路的研究仍较为匮乏。
本研究以北美淡水生态系统常见的模式生物黑头软口鲦（Pimephales promelas）为研究对象，旨在筛选并验证可预测氟西汀暴露诱导的顶端效应终点（apical endpoints）的关键分子毒性通路。将成年黑头软口鲦暴露于3种浓度的氟西汀（2.42、10.7和56.7 μg/L）及对照组中，暴露时长为21天。在暴露96小时后，采集部分受试鱼的肝脏与脑组织，通过全转录组和蛋白质组分析表征其分子毒性通路。此外，在21天暴露结束后，对受试鱼开展具有监管相关性的顶端效应评估，包括组织病理学检查与繁殖力测定。
在最高浓度氟西汀处理组的鱼肝脏中，观察到差异基因表达现象，且脂肪酸生物合成与代谢相关通路出现失调，这可能是导致肝细胞脂质空泡变性的上游分子响应，与组织病理学分析结果一致。对同一批受试鱼的全蛋白质组分析显示，过氧化物酶体增殖物激活受体（PPAR）信号通路出现失调，这可能与转录组中富集的脂质相关通路存在关联。此外，蛋白质组分析还提示转录与翻译相关通路存在富集现象。在处理组鱼的脑组织中，共同失调的基因与受5-羟色胺水平调控的细胞信号传导过程相关，这类过程已被证实参与生殖行为及繁殖成功率调控。21天暴露后受试鱼的繁殖力显著下降，进一步验证了这一关联。
尽管可引发可测量不良结局的通路极为复杂，但本研究为氟西汀所产生的机制性毒性提供了重要线索。
整体实验设计：将黑头软口鲦（Pimephales promelas）暴露于氟西汀中96小时。用于转录组分析的样本采集方案如下：肝脏样本：阴性水对照组（n=5，每份样本混合2尾个体）、10.7μg FLX/L组（n=5，每份样本混合2尾个体）、56.7μg FLX/L组（n=5，每份样本混合2尾个体）；脑组织样本：阴性水对照组（n=5，每份样本混合2尾个体）、10.7μg FLX/L组（n=5，每份样本混合2尾个体）、56.7μg FLX/L组（n=5，每份样本混合2尾个体）。