Nanna Brande-Lavridsen

Effects of cortisol, growth hormone and prolactin on gill claudin expression in Atlantic salmon.

We recently showed that a series of tight junction proteins of the claudin family are regulated in the gill of salmon during salinity acclimation. The aim of the present study was to investigate the role of cortisol, growth hormone (GH) and prolactin (PRL) on regulation of expression of these isoforms. Experiments on primary cultures of gill tissue showed that cortisol stimulates claudin 10e, 27a and 30 mRNA levels while no significant effects were observed on claudin 28a and 28b. The associated receptor signalling pathway was examined using glucocorticoid and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The observed in vitro responses were blocked by RU486, suggesting the involvement of a glucocorticoid type receptor. Injections of FW salmon with cortisol increased the expression of claudin 10e, 27a, and 30 but did not affect claudin 28a and 28b significantly. While GH had no effect on its own, the combination of GH and cortisol reduced claudin 28b levels. Injection of SW salmon with PRL selectively increased the expression of claudin 28a but had no effect on the other examined isoforms. The data shows that FW- (27a and 30) and SW-induced (10e) claudins are all stimulated by cortisol while the major osmoregulatory hormones GH and PRL had no effect on these salinity sensitive isoforms. This suggests that other hormones and/or osmotic conditions interact with cortisol to determine claudin composition in the gill.

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT).

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 μg/L and 101/293 μg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 μg/L and ~30/68 μg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.

Effects of prochloraz and ethinylestradiol on sexual development in Rana temporaria.

A wide range of environmental xenobiotics that mimic hormones (endocrine-disrupting chemicals) may cause alterations in sexual development or reproductive function in aquatic organisms such as amphibians when exposed during early sensitive stages. We exposed tadpoles of the Common frog, Rana temporaria, from hatch to metamorphosis, to two different endocrine disruptors, the synthetic estrogen 17 alpha-ethinylestradiol and the fungicide prochloraz. The object of the study was to assess the effects of these two compounds on the sexual development of the tadpoles by investigating sex ratio, gonadal development, sex steroid concentrations and vitellogenin induction. Histology revealed that a large percentage of all groups were juvenile hermaphrodites at metamorphosis. Tadpoles exposed to 115 and 251 microg/L prochloraz showed a significant increased proportion of males. However, the testosterone concentrations were depressed in those groups. Ethinylestradiol in concentrations of 77 and 159 ng/L EE(2) increased whole-body calcium levels in a dose-dependent manner indicating induction of the egg yolk protein vitellogenin, verified also by gel electrophoresis. The study shows that ethinylestradiol may induce vitellogenesis and prochloraz may affect the sexual development in Common frogs.

Effects of Ethinylestradiol and the fungicide Prochloraz on metamorphosis and thyroid gland morphology in Rana temporaria

Several environmental xenobiotics have been found to affect the metamorphosis of amphibians. In this study we exposed tadpoles of the Common frog Rana temporaria from hatching to metamorphosis to two known endocrine disruptors, the estrogenic pharmaceutical 17� -ethinylestradiol and the antiandrogenic/antiestrogenic fungicide prochloraz to determine their effect on 1) days to metamorphosis and size at metamorphosis, 2) body concentrations of triiodothyronine (T3) and corticosterone, and 3) thyroid morphology. We found effects of both compounds on each of these response variables. A low dose of prochloraz (115 μg/l) and all doses of ethinylestradiol also caused a delay in metamorphosis. T3 levels were elevated in metamorphs exposed to high concentration of prochloraz (252 μg/l) but the group showed a delay in metamorphosis. A low dose of prochloraz (115 μg/l) and all doses of ethinylestradiol also caused a delay in metamorphosis but no changes in T3 levels. The delayed metamorphs weighed more than controls. Thyroid histology revealed significant differences in the high prochloraz exposure group only. Ethinylestradiol and prochloraz, however not in environmentally relevant doses, may therefore impact the thyroid axis, and may cause other sublethal effects especially in combination with other stressors likely encountered.

Estrogenic effect of the phytoestrogen biochanin A in zebrafish, Danio rerio, and brown trout, Salmo trutta

UNLABELLED Isoflavones with estrogenic activity produced in Fabaceae plants are known to leach from agricultural areas to freshwater systems, but the effect of waterborne isoflavones in fish has not been thoroughly characterized. Therefore, the estrogenic effect of waterborne biochanin A was investigated in zebrafish (Danio rerio) and juvenile brown trout (Salmo trutta). Exposure of juvenile brown trout to 10 μg biochanin AL(-1) or higher caused marked vitellogenin induction after 9-10 days of exposure and so did exposure to 186 μg biochanin AL(-1) for 6h. Following 8d of exposure, a NOEC for induction of vitellogenin production in male zebrafish was 70 and LOEC 114 μg biochanin AL(-1). Exposure to 209 μg biochanin AL(-1) from hatch to 60 days post hatch (dph) caused a skewing of the sex ratio toward more phenotypic female zebrafish, but did not cause induction of vitellogenin in male and undifferentiated fish. IN CONCLUSION (1) biochanin A elicits estrogenic effects in trout at environmentally realistic concentrations, (2) brown trout plasma vitellogenin concentrations respond to lower biochanin A exposure concentrations than vitellogenin concentrations in zebrafish homogenates and (3) concerning vitellogenin induction, the hypothesis should be tested if short term tests with zebrafish may show a higher sensitivity than partial life cycle tests.

17β-estradiol causes abnormal development in embryos of the viviparous eelpout

Elevated frequencies of malformations among the offspring of Baltic eelpout (Zoarces viviparus) have been observed in aquatic environments receiving high anthropogenic input suggesting that manmade chemicals could be the causative agent. However, causal links between exposure to chemicals and abnormal development have never been confirmed in laboratory experiments. The purpose of this study was to investigate if exposure to 17β-estradiol (E2) causes abnormal development in larvae of the viviparous eelpout. Wild female eelpout were collected immediately after fertilization and exposed to E2 concentrations ranging from 5.7 to 133 ng L(-1) for 6 weeks in a flow through test system. The experiment shows that E2 concentrations of 53.6 and 133 ng L(-1) cause severe abnormal development among eelpout embryos. Reduced amount of ovarian fluid and increased weight of the ovarian sac indicate disturbance of ovarian function. Female plasma concentrations of E2 and vitellogenin increase in a monotonic concentration-response relationship with significant induction in the low concentration range. Our findings support the plausibility that the abnormal development among eelpout embryos encountered in monitoring programs may actually be caused by exposure to chemicals in the environment.