Proteome studies of carp and frog exposed to endocrine disrupting compounds

Vitellogenin (Vtg) is a well established plasma biomarker for endocrine disrupting compounds with estrogenic properties in fish. In the EU-project EASYRING carp were exposed to estrogens (17-ethynyl-estradiol, EE2), anti-estrogens (tamoxifen), androgens (17-methyldihydrotestosterone), and anti-androgens (flutamide), as well as to caging in polluted stretches of the Rivers Po and Lambro in Northern Italy. Monoclonal antibodies to carp Vtg were used for specific detection of Vtg in 1D- and 2D-western blotting, and in a quantitative sandwich ELISA and a qualitative lateral flow immunoassay (LFIA) dipstick, for measuring Vtg levels in plasma and mucus. Proteomic-based analysis using 2D-gel electrophoresis and mass spectrometry (MALDI-TOF, LC-MS/MS) identified Vtg as a prominent protein in liver, plasma, and mucus after EE2 exposure. At 64 ng EE2/l induction of multiple Vtg forms was observed in all tissues analyzed. In plasma a total of 30 EE2 inducible Vtg spots was identified, a response accompanied by alterations in the levels of other proteins identified as novel biomarker candidates. The induction and presence of numerous Vtg forms was confirmed by 1-D and 2-D western blot analysis. Quantitative Vtg ELISA showed a dose-dependent increase in plasma and mucus-Vtg, significantly different from control after 4 ng EE2/l in mucus, and 16 ng EE2/l in plasma. Regression analysis of plasma vs. mucus Vtg levels gave strong positive correlations for all groups, except for the fish from tamoxifen and field exposure. Analysis of river sediments using YES revealed a mixture of estrogens, and anti-androgens, suggesting that plasma vs. mucus Vtg profiles may be influenced by the nature of endocrine disrupting chemicals present in the environment.