Centre for Advances Research in Environmental Genomics, Department of Biology, University of Ottawa, Ontario, Canada.
Aquat Toxicol. 2010 Nov 15;100(4):354-64. doi: 10.1016/j.aquatox.2010.08.016. Epub 2010 Sep 22.
Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54μg/L and 54μg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20β-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F₂(α) (PGF₂(α)). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54μg/L FLX while pheromone-stimulated milt volume decreased at 0.54μg/L and 54μg/L FLX. Fluoxetine (54μg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF₂(α)-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.
氟西汀（FLX）是一种作为选择性 5-羟色胺再摄取抑制剂的药物，用于治疗人类抑郁症。氟西汀和主要的活性代谢物去甲氟西汀（NFLX）通过污水处理厂排放到水生系统中。已经发现它们在野生鱼类中生物浓缩，引起了对潜在内分泌干扰影响的关注。本研究的目的是确定包括环境浓度在内的水氟西汀对性成熟雄性金鱼生殖轴的影响。我们最初克隆了金鱼 5-羟色胺转运体，以研究 5-羟色胺转运体的组织和时间表达，即 FLX 的靶标，以确定靶组织和敏感的暴露窗口。在接受车辆或性信息素刺激之前，显示神经内分泌脑中 5-羟色胺转运体表达最高的性成熟雄性金鱼在 14 天暴露于 0.54μg/L 和 54μg/L 的 FLX 下，该刺激由 4.3nM 17,20β-二羟基-4-孕烯-3-酮（17,20P）或 3nM 前列腺素 F₂(α)（PGF₂(α)）组成。评估的生殖终点包括性腺指数、精液量和血液中睾酮和雌二醇的性类固醇水平。通过测量促黄体激素、生长激素、促黄体激素、生长激素和促卵泡激素的垂体基因表达以及催产素和血管加压素的神经内分泌脑表达来研究神经内分泌功能。为了研究生殖轴在性腺水平上的变化，还测量了促性腺激素受体、黄体生成素受体和促卵泡激素受体的睾丸基因表达以及生长激素受体的表达。为了研究对精子发生的潜在影响，测量了精子发生标记物 vasa 的睾丸基因表达，并对睾丸组织学样本进行了定性分析。通过性腺芳香化酶的表达和活性分析以及雌激素标记物 esr1 的肝表达分析来测量雌激素指数。14 天后，54μg/L FLX 时基础精液量显著减少，而 0.54μg/L 和 54μg/L FLX 时信息素刺激的精液量减少。氟西汀（54μg/L）抑制了基础和信息素刺激的睾酮水平。在 17,20P 和 PGF₂(α)刺激组中，分别观察到 FLX 导致促卵泡激素和催产素表达的浓度依赖性降低。雌二醇水平和 esr1 的浓度依赖性增加随着 FLX 的增加而增加。本研究表明，FLX 以环境相关浓度破坏雄性鱼类的生殖生理学，讨论了潜在机制。
