In vitro vitellogenin production by carp (Cyprinus carpio) hepatocytes as a screening method for determining (anti)estrogenic activity of xenobiotics.
The yolk protein precursor vitellogenin (Vtg) is secreted by the liver of female as well as male fish, in response to estrogenic compounds. In this study, an in vitro assay was developed for measuring Vtg induction, using cultured primary hepatocytes from genetically uniform strains of carp (Cyprinus carpio). Vtg production was measured by indirect competitive ELISA, using a polyclonal antiserum against goldfish Vtg that cross-reacts with carp Vtg. Vtg was dose-dependently induced by 17beta-estradiol (E2) in hepatocytes of both sexes. E2 had a lowest observed effect concentration (LOEC) for Vtg induction of 2 nM, an EC50 between 50 and 150 nM, and a maximum response at 2 microM. The plasticizer and xenoestrogen bisphenol-A induced Vtg secretion by hepatocytes of both sexes at 50 and 100 microM. This carp hepatocyte (CARP-HEP) assay can also be used to detect antiestrogenic activity, which was measured as the reduction of E2-stimulated Vtg synthesis. Two well-known antiestrogenic compounds, tamoxifen and 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), were tested. TCDD caused a reduction in Vtg synthesis in female hepatocytes at concentrations <0.1 nM, making it approximately 10,000-fold more potent than tamoxifen. Carp hepatocytes were also sensitive to induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylase (EROD). Depending on the exposure time, 18 or 96 h, EROD EC50 values for TCDD were 27 or 6 pM, respectively. The CARP-HEP assay, using the 96-well plate format, offers good possibilities to screen large numbers of compounds for (anti)estrogenic properties. In addition, it can simultaneously determine aryl hydrocarbon receptor agonist properties, measured as CYP1A induction.
D000818:Animals; D001559:Benzhydryl Compounds; D002347:Carps / Q000378:metabolism*; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010636:Phenols / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014819:Vitellogenins / Q000096:biosynthesis*; D015262:Xenobiotics / Q000494:pharmacology*; D000098826:Bisphenol A Compounds