Pharmaceuticals as neuroendocrine disruptors: lessons learned from fish on Prozac.

Pharmaceuticals are increasingly detected in a variety of aquatic systems. One of the most prevalent environmental pharmaceuticals in North America and Europe is the antidepressant fluoxetine, a selective serotonin reuptake inhibitor (SSRI) and the active ingredient of Prozac. Usually detected in the range below 1 µg/L, fluoxetine and its active metabolite norfluoxetine are found to bioaccumulate in wild-caught fish, particularly in the brain. This has raised concerns over potential disruptive effects of neuroendocrine function in teleost fish, because of the known role of serotonin (5-HT) in the modulation of diverse physiological processes such as reproduction, food intake and growth, stress and multiple behaviors. This review describes the evolutionary conservation of the 5-HT transporter (the therapeutic target of SSRIs) and reviews the disruptive effects of fluoxetine on several physiological endpoints, including involvement of neuroendocrine mechanisms. Studies on the goldfish, Carassius auratus, whose neuroendocrine regulation of reproduction and food intake are well characterized, are described and represent a reliable model to study neuroendocrine disruption. In addition, fish studies investigating the effects of fluoxetine, not only on reproduction and food intake, but also on stress and behavior, are discussed to complement the emerging picture of neuroendocrine disruption of physiological systems in fish exposed to fluoxetine. Environmental relevance and key lessons learned from the effects of the antidepressant fluoxetine on fish are highlighted and may be helpful in designing targeted approaches for future risk assessments of pharmaceuticals disrupting the neuroendocrine system in general.

The fibrate drug gemfibrozil disrupts lipoprotein metabolism in rainbow trout.

Gemfibrozil (GEM) is a fibrate drug consistently found in effluents from sewage treatment plants. This study characterizes the pharmacological effects of GEM on the plasma lipoproteins of rainbow trout (Oncorhynchus mykiss). Our goals were to quantify the impact of the drug on: 1) lipid constituents of lipoproteins (phospholipids (PL), triacylglycerol (TAG), and cholesterol), 2) lipoprotein classes (high, low and very low density lipoproteins), and 3) fatty acid composition of lipoproteins. Potential mechanisms of GEM action were investigated by measuring lipoprotein lipase activity (LPL) and the hepatic gene expression of LPL and of the peroxisome proliferator-activated receptor (PPAR) α, ß, and γ isoforms. GEM treatment resulted in decreased plasma lipoprotein levels (-29%) and a reduced size of all lipoprotein classes (lower PL:TAG ratios). However, the increase in HDL-cholesterol elicited by GEM in humans failed to be observed in trout. Therefore, HDL-cholesterol cannot be used to assess the impact of the drug on fish. GEM also modified lipoprotein composition by reducing the abundance of long-chain n-3 fatty acids, thereby potentially reducing the nutritional quality of exposed fish. The relative gene expression of LPL was increased, but the activity of the enzyme was not, and we found no evidence for the activation of PPAR pathways. The depressing effects of GEM on fish lipoproteins demonstrated here may be a concern in view of the widespread presence of fibrates in aquatic environments. Work is needed to test whether exposure to environmental concentrations of these drugs jeopardizes the capacity of fish for reproduction, temperature acclimation or migratory behaviors.

Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus.

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 17,20ß-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.