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 f emale as well as male fish, in response to estrogenic compounds. In this st udy, an in vitro assay was developed for measuring Vtg induction, using cul tured primary hepatocytes from genetically uniform strains of carp (Cyprinu s carpio). Vtg production was measured by indirect competitive ELISA, using a polyclonal antiserum against goldfish Vtg that cross-reacts with carp Vt g. Vtg was dose-dependently induced by 17 beta-estradiol (E2) in hepatocyte s of both sexes; E2 had a lowest observed effect concentration (LOEC) for V tg induction of 2 nM, an EC50 between 50 and 150 nM, and a maximum response at 2 mu M. The plasticizer and xenoestrogen bisphenol-A induced Vtg secret ion by hepatocytes of both sexes at 50 and 100 mu M. This carp hepatocyte ( CARP-HEP) assay can also be used to detect antiestrogenic activity,which wa s measured as the reduction of E2- stimulated Vtg synthesis. Two well-known antiestrogenic compounds, tamoxifen and 2,3,7,8-tetrachlorodibenzo-p-dioxi n (TCDD), were tested. TCDD caused a reduction in Vtg synthesis in female h epatocytes at concentrations <0.1 nM,making it approximately 10,000-fold mo re potent than tamoxifen. Carp hepatocytes were also sensitive to induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deeth ylase (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-we ll plate format, offers good possibilities to screen large numbers of compo unds for (anti)estrogenic properties. In addition, it can simultaneously de termine aryl hydrocarbon receptor agonist properties, measured as CYP1A ind uction. (C) 1999 Academic Press.