Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays.

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.

Development and validation of a direct homologous quantitative sandwich ELISA for fathead minnow (Pimephales promelas) vitellogenin.

Vitellogenin (Vtg) is an established and sensitive endpoint for analysis of exposure to (anti-)oestrogens and their mimics in fish [Sumpter, J.P., 1995. Feminized responses in fish to environmental estrogens. Toxicol. Lett. 82, 737-742; Arukwe, A., Goksøyr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. ]. In some instances, links have been drawn between high level induction of Vtg and adverse health effects in fish [Herman, R.L., Kincaide, H.L., 1988. Pathological effects of orally administered estradiol to rainbow trout. Aquaculture 72, 165-172; Schwaiger, J., Spieser, O.H., Bauer, C., Ferling, H., Mallow, U., Kalbfus, W., Negele, R.D., 2000. Chronic toxicity of nonylphenol and ethinyloestraiol: haematological and histopathological effects in juvenile common carp (Cyprinus carpio). Aquat. Toxicol. 51, 69-78]. The widespread use of Vtg as a biomarker has led to the development of a variety of assays to quantitatively measure Vtg concentrations in tissue samples from fish, and hence a need for a standardization of the performance criteria and validation of such assays [Goksøyr, A., Eidem, J.K., Kristiansen, S.I., Nilsen, B.M., 2003. On the need for a standardized set-up for validation studies of fish vitellogenin assays as an endpoint in endocrine disruptor testing and screening-a proposal. ]. One of the most popular test fish species for assessing chemical effects is the fathead minnow (Pimephales promelas), which is now used widely for studies into endocrine disruption [Panter, G.H., Hutchinson, T.H., Lange, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti)estrogenic substances. Environ. Toxicol. Chem. 21, 319-326; Hutchinson, T.H., Yokota, H., Hagino, S., Ozato, K., 2003. Development of fish tests for endocrine disruptors. Pure Appl. Chem. 75, 2343-2353]. This paper describes the development and validation of a new, homologous enzyme-linked immunosorbent assay (ELISA) for quantification of Vtg in this fish species.

Development of quantitative vitellogenin-ELISAs for fish test species used in endocrine disruptor screening.

The yolk protein precursor vitellogenin (Vtg) in plasma has proved to be a simple and sensitive biomarker for assessing exposure of fish to environmental estrogens. Within international bodies such as the Organization for Economic Cooperation and Development (OECD) work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp (Cyprinus carpio), fathead minnow (Pimephales promelas), zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). In this study we have developed quantitative enzyme linked immunosorbent assays (ELISAs) for Vtg in common carp/fathead minnow, zebrafish and Japanese medaka. The assays were developed using a combination of monoclonal and polyclonal fish Vtg antibodies in a sandwich format, using stabilized Vtg from the test species as a standard. The carp Vtg ELISA has a working range of 1-63 ng/mL, a minimal detection limit of 0.6 ng/mL, and may also be used for quantification of Vtg in fathead minnow. In fathead minnow whole-body homogenate samples, the practical detection limit is 400 ng/mL due to the matrix effect. The zebrafish Vtg ELISA has a working range of 0.5-63 ng/mL, a minimal detection limit of 0.4 ng/mL, and a practical detection limit of 200 ng/mL in whole-body homogenate samples. The medaka Vtg ELISA has a working range of 0.25-16 ng/mL, a minimal detection limit of 0.1 ng/mL, and a practical detection limit of 125 ng/mL in whole-body homogenate samples. The intra- and inter-assay variations were below 20% for all assays. The assays were evaluated with sets of representative samples spanning the wide dynamic range of Vtg-levels found in fish exposed to environmental estrogens, and all three assays are currently undergoing international inter-laboratory validation.

Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio).

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.