Fluoxetine exposure disrupts food intake and energy storage in the cichlid fish Cichlasoma dimerus (Teleostei, Cichliformes).

Human pharmaceuticals are pollutants of special concern due to their widespread consumption over the last decades, their high persistence in the environment, and the reported alterations produced on non-target organism. The antidepressant fluoxetine (FLX) exerts its effect by inhibiting serotonin (5-HT) reuptake at the presynaptic membrane, thus increasing brain serotonergic activity. In vertebrates, there is a clear inverse relationship between hypothalamic 5-HT levels and food intake, therefore we hypothesized that FLX would inhibit food intake, and in consequence alter energy metabolism in freshwater fish. The aim of this study was to analyze the effect of FLX on feeding behavior and energy storage of the cichlid fish Cichlasoma dimerus. Adult fish were intraperitoneally injected daily with 2 or 20 µg.g-1 FLX or saline for a 5-day period, during which the 20 µg.g-1 FLX-injected fish exhibited a marked reduction in food intake, consistent with a decrease in total body weight and total hepatocyte area observed at the end of the experiment. Although not statistically significant, a marked 50% decrease in glycogen and lipid content and an increase in protein levels in liver was observed for the 20 µg.g-1 FLX dose. This was evidenced histochemically by a weak PAS positive reaction and an intense Coomasie Blue stain. Taken together, these results suggest that the SSRI antidepressant FLX produces an anorectic effect in adults of C. dimerus, which could alter normal physiological function and, in consequence, have a negative impact on fish growth, reproduction, and population success.

Effects of waterborne exposure to the antidepressant fluoxetine on swimming, shoaling and anxiety behaviours of the mosquitofish Gambusia holbrooki.

Chemical pollution from pharmaceuticals is increasingly recognized as a major hazard to the aquatic biota. Among the wide variety of pharmaceuticals, fluoxetine (FLX) is one of the most widely prescribed antidepressants, and therefore, it is frequently identified in the aquatic environment. As FLX is designed to alter human behaviour and many physiological pathways are conserved across vertebrates, this drug may affect the behaviour of fish living in FLX-polluted environments. Here, we exposed groups of female mosquitofish Gambusia holbrooki to waterborne FLX for 14 days, under semi-static conditions with daily renewal of test solutions. Following exposure, we conducted a set of behavioural assays in individual fish, aimed at assessing the effects of FLX on their locomotor activity and behavioural responses. We found that FLX impaired swimming behaviour at high concentrations (25 µg/L and 50 µg/L) but not at low concentrations close to environmental levels (1 µg/L and 5 µg/L). When swimming activity was assessed 5 min after transfer of the focal fish to the testing tank, 50 µg/L FLX was the only concentration showing significant effects. However, when the same trials were performed 24 h later, 25 µg/L FLX turned out to be an effect concentration in addition to 50 µg/L. Interestingly, these concentrations would elicit fish plasma concentrations comprised within the range of human therapeutic doses. When subjected to a light/dark preference test, fish showed tendency to remain less time in the dark area at high FLX concentrations, thus suggesting an anti-anxiety response. Shoaling behaviour was not affected by FLX exposure. Our study contributes to the growing body of literature evaluating the effects of FLX on animal behaviour. Regarding the experimental design used in behavioural testing, our findings suggest that focal fish should be subjected to long habituation periods, namely of at least a few hours, in order to better assess the effects of drug exposure.

Endocrine disruption of phenanthrene in the protogynous dusky grouper Epinephelus marginatus (Serranidae: Perciformes).

The dusky grouper Epinephelus marginatus is a protogynous hermaphrodite fish, that maintains high levels of plasma steroids as juveniles, as substrates for sex inversion. These fish are exposed to marine pollution from oil spills during cargo handling. Polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene (Phe), are the main crude oil components and are toxic to fish, acting as endocrine disruptors (ED). This is the first study that investigated impacts of Phe as an ED in E. marginatus juveniles. An in vivo sublethal exposure (96h) to Phe was carried out at two concentrations (0.1mg/L and 1mg/L); exposure to the vehicle (ethanol; ETOH) was also performed. Plasma levels of 17ß-estradiol (E2), testosterone (T) and 11-ketotestosterone (11-KT) were measured by ELISA. Gonads, liver and spleen were processed for histological analysis. In an in vitro bioassay, gonad fragments were incubated with Phe (8.91mg/L) or ETOH. Steroid levels in the culture media were measured by ELISA. The in vivo exposure to Phe triggered an increase of the area of the hepatocytes, increased number of melanomacrophagic centers and hemosiderosis in the spleen; ETOH induced similar effects on spleen. E2 and T levels did not change in plasma or in vitro media. In plasma, ETOH decreased 11-KT levels. Phenanthrene sharply reduced 11-KT levels in vitro. Although in vivo bioassay results were not unequivocal owing to ethanol effects, Phe might disrupt steroidogenesis in juvenile grouper, possibly causing dysfunctions during sex change and gonadal maturity, considering the importance of 11-KT in developing ovaries.

The SSRI fluoxetine exhibits mild effects on the reproductive axis in the cichlid fish Cichlasoma dimerus (Teleostei, Cichliformes).

Among the wide variety of pharmaceuticals released into the environment, Fluoxetine (FLX), a selective serotonin reuptake inhibitor, is one of the most prescribed for the treatment of major depression. It inhibits serotonin (5-HT) reuptake at the presinaptic membrane, increasing serotonergic activity. In vertebrates, including fish, the serotonergic system is closely related to the Hypothalamic Pituitary Gonadal (HPG) axis which regulates reproduction. As FLX can act as an endocrine disrupting compound (EDC) by affecting several reproductive parameters in fish, the aim of this study was to provide an integral assessment of the potential effect of FLX on the reproductive axis of the Neotropical freshwater fish Cichlasoma dimerus. Adult fish were intraperitoneally injected with 2 µg g-1 FLX or saline every third day for 15 days. No significant differences were found on serotonergic turnover (5-HIAA/5-HT ratio). Pituitary ßLH content in FLX injected females was significantly higher than control females; no significant differences were seen for ßFSH content. Sex steroids remained unaltered, both in males and females fish, after FLX treatment. No plasma vitellogenin was induced in treated males. Some alterations were seen in testes of FLX injected males, such as the presence of foam cells and an acidophilic PAS positive, Alcian-Blue negative secretion in the lobular lumen. Although there is no clear consensus about the effect of this drug on reproductive physiology, these results indicate that FLX is acting as a mild EDC in adults of C. dimerus.

Mechanism of action of endosulfan as disruptor of gonadal steroidogenesis in the cichlid fish Cichlasoma dimerus.

The organochlorine pesticide endosulfan (ES) is used in several countries as a wide spectrum insecticide on crops with high commercial value. Due to its high toxicity to non-target animals, its persistence in the environment and its ability to act as an endocrine disrupting compound in fish, ES use is currently banned or restricted in many other countries. Previous studies on the cichlid fish Cichlasoma dimerus have shown that waterborne exposure to ES can lead to both decreased pituitary FSH content and histological alterations of testes. As gonadotropin-stimulated sex steroids release from gonads was inhibited by ES in vitro, the aim of the present study was to elucidate possible mechanisms of disruption of ES on gonadal steroidogenesis in C. dimerus, as well as compare the action of the active ingredient (AI) with that of currently used commercial formulations (CF). Testis and ovary fragments were incubated with ES (AI or CF) and/or steroidogenesis activators or precursors. Testosterone and estradiol levels were measured in the incubation media. By itself, ES did not affect hormone levels. Co-incubation with LH and the adenylate cyclase activator forskolin caused a decrease of the stimulated sex steroids release. When co-incubated with precursors dehydroandrostenedione and 17αhydroxyprogesterone, ES did not affect the increase caused by their addition alone. No differences were observed between the AI and CFs, suggesting that the effect on steroidogenesis disruption is mainly caused by the AI. Results indicate that action of ES takes place downstream of LH-receptor activation and upstream of the studied steroidogenic enzymes.

Endocrine disruptive potential of endosulfan on the reproductive axis of Cichlasoma dimerus (Perciformes, Cichlidae).

Endosulfan (ES), a persistent organochlorine pesticide, is widely used despite its toxicity to non-target animals. Upon reaching water bodies, ES can cause negative effects on aquatic animals, including disruption of hormonal systems. However, the action of ES on fish reproductive axis has been hardly studied thus far. The aim of the present work was to assess the endocrine disruptive potential of endosulfan on the pituitary gonadotropins levels and on the testes function due to ES in the South American freshwater fish Cichlasoma dimerus, using in vitro and in vivo approaches. In vitro experiments showed that ES inhibited the LH-stimulated steroidogenesis in gonads; no change was observed in gonadotropins release from pituitaries in culture. Laboratory waterborne ES (0.1, 0.3 and 1 µg/L) exposure for two months caused decrease in ßFSH pituitary content and γGT activity in the testes (Sertoli cell function marker). Testicular histology revealed pathologies such as scarce intermediate stages of spermatogenesis, release of immature germ cells into the lobular lumen, presence of foam cells and interstitial fibrosis. As FSH and FSH-mediated steroidogenesis regulate spermatogenesis and Sertoli cell function, the effect of ES on FSH could be responsible for the morphological alterations observed in testes. In vitro, ES disrupted steroidogenesis in gonads, therefore similar effects in vivo cannot be ruled out. Based on this evidence, ES exhibits an endocrine disruptive action on the reproductive axis of C. dimerus, causing disruption at the pituitary and/or at the gonad level. These effects could acquire ecological significance under prolonged exposure to the pesticide in nature.