title: Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus.
abstract: 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.
authors.affiliation: Centre for Advances Research in Environmental Genomics, Department of Biology, University of Ottawa, Ontario, Canada.
authors.forename: Jan A
authors.initials: JA
authors.lastname: Mennigen
authors.forename: Wudu E
authors.initials: WE
authors.lastname: Lado
authors.forename: Jake M
authors.initials: JM
authors.lastname: Zamora
authors.forename: Paula
authors.initials: P
authors.lastname: Duarte-Guterman
authors.forename: Valérie S
authors.initials: VS
authors.lastname: Langlois
authors.forename: Chris D
authors.initials: CD
authors.lastname: Metcalfe
authors.forename: John P
authors.initials: JP
authors.lastname: Chang
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Moon
authors.forename: Vance L
authors.initials: VL
authors.lastname: Trudeau
mesh_terms: D000818:Animals; D001141:Aromatase / Q000378:metabolism; D001483:Base Sequence; D047628:Estrogen Receptor alpha / Q000378:metabolism; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism; D005473:Fluoxetine / Q000633:toxicity*; D015870:Gene Expression / Q000187:drug effects; D006054:Goldfish / Q000235:genetics / Q000502:physiology*; D008099:Liver / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010121:Oxytocin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D011460:Prostaglandins F / Q000235:genetics / Q000378:metabolism; D012098:Reproduction / Q000187:drug effects*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000187:drug effects; D017367:Selective Serotonin Reuptake Inhibitors / Q000633:toxicity*; D013737:Testis / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D001141:Aromatase; D047628:Estrogen Receptor alpha; D029941:Fish Proteins; D005473:Fluoxetine; D010121:Oxytocin; D011460:Prostaglandins F; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D013739:Testosterone; D014874:Water Pollutants, Chemical; C008215:isotocin; C034238:prostaglandin F3alpha