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2J - TIE (TUP/128) Development of a Toxicity Identification and Evaluation method for estrogenic chemicals in fish bile. Houtman, Corine1, van Oostveen, Annemiek1, Brouwer, Abraham1, Lamoree, Marja1, Legler, Juliette1, 1 Institute for Environmental Studies, Vrije Universiteit, Amsterdam, The Netherlands, The Netherlands ABSTRACT- In fish, conjugates of many estrogenic chemicals, endogenous as well as xenobiotic, are excreted via bile into the intestine. Therefore, measurement of estrogenic activity in bile could yield useful information about the animal's internal exposure to (xeno-)estrogens. Estrogenic activity in male fish bile has been reported (Legler et al., Environ. Toxicol. Chem. 21: 473-479, 2002), but the contribution of natural hormones to this activity is unknown. Here we report our progress on the development of a Toxicity Identification and Evaluation method for the identification of estrogenic chemicals in fish bile. In this approach the Estrogen Responsive Chemical Activated Luciferase Gene Expression (ER-CALUX) assay is used to assess estrogenic activity in bile samples and to direct fractionation and chemical analysis of estrogenic compounds. First, bile samples were enzymatically deconjugated to convert estrogen glucuronides and sulphates present into their biologically active forms. Subsequently, estrogenic compounds were extracted from the deglucuronidation mixture. To select the optimal method of extraction, the recovery of estrogenic activity with three different methods of extraction was compared. Bile extracts were then fractioned according to polarity using RP-HPLC. In this way, fractions containing natural hormones can be separated from fractions containing compounds with different polarity. GC-MS/MS analysis will be performed to assess the contribution of natural estrogenic hormones to the total estrogenic activity observed. This TIE approach is a promising tool to assess the internal exposure of fish to endogenous and xenobiotic estrogenic compounds. Key words: estrogen, fish bile, Toxicity Identification and Evaluation (TIE), bioassay |
