Bodil Korsgaard

Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio)

The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg x ml(-1) and the practical detection limit was 40 ng Vtg x g(-1) fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng x l(-1) of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 x l(-1) induced a 200-fold increase in Vtg levels.

Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

The impact of 17β-estradiol (E2) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17β-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E2 and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E2-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E2- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na+,K+-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E2 and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na+,K+-ATPase activity, relative α-subunit Na+,K+-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E2- and 4-NP-treated fish was strongly correlated with a decreased gill Na+,K+-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17β-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E2 activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.

The Preservatives Ethyl‐, Propyl‐ and Butylparaben are Oestrogenic in an in vivo Fish Assay

The widely used phenolic preservatives ethylparaben, propylparaben, butylparaben and their common metabolite p-hydroxybenzoic acid were tested for their ability to evoke an oestrogenic response in vivo. Yolk protein induction in sexually immature rainbow trout was used as an oestrogen-specific endpoint after repeated injections of the compounds. All tested parabens were oestrogenic in doses between 100 and 300 mg/kg, while the metabolite showed no activity. Ethylparaben was found to be approximately sixty times weaker than propyl- and butylparaben which had oestrogenic potencies comparable to those previously found for bisphenol A.

In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncorhynchus mykiss)

The in vivo estrogenic activity of the two branched alkylphenols, tert-octylphenol and technical nonylphenol, and the two linear isoforms, n-octylphenol and n-nonylphenol, was compared. The compounds were administered to juvenile rainbow trout (Oncorhynchus mykiss) by either intraperitoneal injection or water exposure and their estrogenic potential was evaluated by ELISA measurements of induced plasma vitellogenin. Intraperitoneal injections (50 mg/kg) of the two branched alkylphenols resulted in a significant vitellogenic response after 12 days whereas no significant induction was seen with the two linear isomers. Water exposure for 9 days to a nominal concentration of 150 micrograms/l of the alkylphenols elicited the same response pattern as seen for the injection experiment. Furthermore, in the present vitellogenin assay tert-octylphenol was giving a higher estrogenic response compared to technical nonylphenol using either of the two exposure routes.

Isolated and combined exposure to ammonia and nitrite in rainbow trout (Oncorhynchus mykiss) : effects on electrolyte status, blood respiratory properties and brain glutamine/glutamate concentrations

Abstract Rainbow trout ( Oncorhynchus mykiss ) were exposed for up to 4 days to 100, 300 and 500 μM ammonia and 0, 300 and 600 μM nitrite. Each ammonia concentration was combined with each nitrite concentration, giving a total of nine exposure groups. High mortality was observed in trout exposed to 500 μM ammonia in combination with 600 μM nitrite. Other exposure groups only showed sporadic mortality. Interactive effects (i.e. synergism or antagonism) of ammonia and nitrite exposure were not observed on the physiological parameters measured. Ammonia and nitrite, however, both caused a significant and additive decrease in muscle potassium concentrations after 4 days of exposure. The decrease was approximately 50 μmol g −1 dry weight for both nitrite and ammonia at the highest exposure concentrations. Nitrite was accumulated in plasma to approximately twice the ambient concentration, which was associated with a small significant increase in blood [lactate]. Plasma ammonia concentration only increased significantly upon ammonia exposure. The extracellular osmolality and Cl − , Na + and amino acid concentrations remained constant. A transient increase in haematocrit and blood haemoglobin concentration was observed in trout exposed to the highest ammonia concentration. Accumulation of nitrite induced methaemoglobin formation and thereby functional anaemia, while no changes in red cell organic phosphates occurred. Plasma glutamate oxaloacetate transaminase (GOT) activity increased after 4 days exposure to the highest ammonia concentration. No changes occurred in plasma glutamate pyruvate transaminase (GPT) activity. Brain glutamate concentrations decreased while the brain glutamine concentrations increased in trout exposed to ammonia, suggesting detoxification of ammonia by its reaction with glutamate to form glutamine. Brain [glutamate] did not decrease below a threshold of approximately 2.37 μmol g −1 wet weight.

The effect of 4-nonylphenol on the synthesis of vitellogenin in the flounder Platichthys flesus

Dose–response effects of the putative estrogenic compound 4-nonylphenol (4-NP) on the de novo synthesis of vitellogenin were investigated in the flounder Platichthys flesus. Male flounders received intraperitoneal injections during a period of 2 weeks with 10, 50, 100, 150 and 200 μg g−1 week−1 of 4-NP. Controls received the peanut oil vehicle only. The dose–response effects of the treatment were investigated on different plasma indicators of vitellogenin, alkali-labile protein phosphorous, total calcium and protein. Increased concentrations of these indicated a dose–response induced synthesis of vitellogenin in the liver of the 4-NP-treated male flounders. Vitellogenin from plasma of estradiol-treated male flounders was purified by gel filtration followed by ion-exchange chromatography and subsequently subjected to native gel electrophoresis. The purified vitellogenin fraction appeared as a high molecular protein band of 540 kDa in the electropherograms. The presence of vitellogenin in the plasma of the 4-NP-treated fish could then be directly detected by the native gel electrophoresis method. A high molecular weight vitellogenin band from plasma of the 4-NP-treated males appeared in the electropherograms in exactly the same position as the purified vitellogenin and vitellogenin in plasma of estradiol-treated male flounders. No vitellogenin band could be detected in the plasma of control males. The dose–response effect of the 4-NP-treatment was also reflected by an increased thickness of the vitellogenin band of the electropherograms by increasing doses of the 4-NP. De novo induction of vitellogenin synthesis in the liver was also indicated by increases of the hepatosomatic indices and of hepatic total RNA in the 4-NP-treated fish. No changes could be observed in the hepatic DNA concentrations. A significant increase of plasma GPT-concentrations indicated that 4-NP also elicited toxic effects on the fish. This was further indicated by a high mortality in the group treated with the highest dose of 4-NP (200 μg g−1). No effect could be observed on the gonadosomatic indices of the treated fish when compared to the peanut oil-injected controls. The present experiments were carried out towards the end of the reproductive season with testicular size observed to be near maximal in the control group. This is the most likely reason why no effect of the 4-NP injections could be observed on the testicular morphology or growth in the present experiments.

Estrogenicity of xenobiotics in rainbow trout (Oncorhynchus mykiss) using in vivo synthesis of vitellogenin as a biomarker

Abstract The estrogenicity of several xenobiotics was evaluated using in vivo vitellogenin (Vtg) synthesis in immature rainbow trout as a biomarker. 17β-estradiol, DES and ethinyl estradiol were tested as positive controls. The xenobiotic compounds tested were technical nonylphenol, bisphenol A, butylbenzylphthalate (BBP) and dibutylphthalate (DBF). Measurements of the Vtg concentration was performed with a direct sandwich ELISA. 17β-estradiol, DES and ethinyl estradiol caused up to 100 000-fold increases in the Vtg-levels. Nonylphenol and bisphenol A had the highest estrogenic potency of the xenobiotics increasing the vitellogenin level approximately 300-fold while BBP was only weakly estrogenic, increasing the concentration about 3 times. DPB did not raise the vitellogenin contration above the detection limit.

Vitellogenin in Zoarces viviparus: Purification, quantification by ELISA and induction by estradiol-17β and 4-nonylphenol

Vitellogenin was isolated by gel filtration and ion-exchange chromatography from plasma of estradiol-treated male Zoarces viviparus. The purification of vitellogenin was followed through each step by native polyacrylamide gelelectrophoresis (PAGE). The purified vitellogenin was used to raise anti-vitellogenin antibodies in rabbits. The specificity of the affinity purified antibody raised against vitellogenin was assessed by Western blot analysis. No crossreactivity was observed with plasma from non-induced control males. A direct enzyme-linked immunosorbent assay (ELISA) was developed by use of the raised anti-vitellogenin. The detection limit for the purified standard vitellogenin by the ELISA method was 5 ng ml-1 Vitellogenin levels were quantified in plasma of pregnant female Zoarces viviparus, which were held in aquaria with different concentrations of the estrogenic compound 4-nonylphenol dissolved in the ambient seawater. A marked effect of exposure to ambient 4-nonylphenol was observed by significant dose-dependent increases in the level of plasma vitellogenin of the pregnant fish as well as of embryos exposed in vitro.

Vitellogenin, lipid and carbohydrate metabolism during vitellogenesis and pregnancy, and after hormonal induction in the blenny Zoarces viviparus (L.).

1. Ovarian vitellogenic growth in Zoarces viviparus lasts about 2 months. Vitellogenesis is immediately followed by ovulation, fertilization and a pregnancy period of 4 months. Vitellogenin is observed in the blood during vitellogenesis, but declines during the first month of pregnancy. 2. The largest amount of liver lipid is found before vitellogenesis is initiated. During pregnancy the liver is depleted of lipid and glycogen, and total lipid and phospholipid is accumulating in the blood. 3. Estradiol treatment during pregnancy results in a dose-dependent increase in vitellogenin and lipids of the blood. 4. In late pregnancy, birth can be provoked with progesterone alone, or with combined progesterone and estradiol treatment.

Molecular cloning, characterisation, and tissue distribution of oestrogen receptor alpha in eelpout (Zoarces viviparus)

A cDNA encoding the eelpout (Zoarces viviparus) oestrogen receptor alpha (eERalpha) has been isolated from eelpout liver, cloned and sequenced. The cDNA contains a complete open reading frame encoding 570 amino acid residues (mw: 63.0 kDa). The amino acid sequence of eERalpha showed a high degree of identity to ERalpha of other teleost species. The tissue distribution of eERalpha mRNA was examined using Northern blotting, RT-PCR and in situ hybridisation (ISH). All three methods identified a pronounced expression of eERalpha in liver, pituitary, testis and ovary. In the brain ISH experiments showed that ERalpha mRNA was highly expressed in distinct regions of the preoptic area and the mediobasal hypothalamus. We have provided evidence that the receptor is auto-regulated by 17beta-oestradiol (E(2)) not only in liver but also in the testis, indicating an important role for E(2) during spermatogenesis in male eelpout. RT-PCR analysis showed a broader expression pattern including significant expression in the brain, kidney, heart, and gut of adult eelpout. In eelpout embryos eERalpha expression has also been identified, indicating a possible role for the receptor in early development. This study contributes to the accumulating evidence that in fish E(2) is not only involved in the regulation of liver specific proteins, but has a much broader range of targets.