Knud Ladegaard Pedersen

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.

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.

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.

Sex-specific accumulation of Cd-metallothionein in the abdominal muscle of the coral prawn Metapenaeopsis crassissima from a natural population

Abstract Female coral prawns Metapenaeopsis crassissima from natural populations in Shark Bay, Western Australia contain high concentrations of Cd in both their midgut gland and abdominal muscle. The biochemical form of this Cd was examined by gel filtration chromatography, HPLC, MALDI mass spectrometry and N-terminal sequence analysis. The majority of the Cd was soluble (mean values: midgut 91%, muscle 57%; n = 4), and eluted (gel filtration) in a single 10 kDa-peak which could be further resolved into five peaks by reversed-phase HPLC. Mass spectral data on the peaks showed molecular masses of 5778, 5876 and 6008. N-terminal sequencing of two of the peaks gave typical decapod crustacean MT-sequences, one of which contained an N-terminal residue of methionine. The methionine variant of MT has previously only been reported in laboratory studies of Cd-stressed crustaceans. The presence of the methionine variant of MT in a natural population of crustaceans containing high Cd levels suggests its possible use as a biomarker of toxic response to Cd exposure.

Monitoring and risk assessment for endocrine disruptors in the aquatic environment: a biomarker approach.

Evidence that a number of chemicals affect wildlife populations or individuals via interaction with endocrine systems has been increasing in recent years. Worldwide effects of tributyltin from antifouling paints on mollusc populations (Langston 1996; Oehlmann et al. 1996), effects of polychlorinated biphenyls on Baltic and Wadden Sea seals (Reijnders 1986; Brouwer et al. 1989), masculinisation of North American fish affected by pulp and paper mill effluents (Howell 1980; Munkittrick et al. 1991, 1992), feminisation of male fish in British rivers receiving effluents from waste water plants (Jobling and Sumpter 1993; Purdom et al. 1994; Harries et al. 1997), demasculinisation of alligators in Lake Apopka after a chemical spill (Guillette et al. 1994, 1995a,b, 1996) and effects on North American birds (Fry and Toone 1981; Fry 1995) are some of the most prominent and best documented examples, all attributed to chemicals exerting endocrine disrupting effects.