PARENT SESSION

2A - Mixture toxicity
Poster Hall
8:30 AM - Monday, 28 April 2003
Chair: Hermens, J.¹, ¹
Co-chair: Toy, R.², Backhaus, T.³, ^{2 3}

(MOP/72) Additive effects of multi-component mixtures of similarly acting (xeno-)estrogens in the in vitro ER-CALUX reporter gene assay.

Legler, Juliette¹, Hopman, Gerda¹, Scholze, Martin², Backhaus, Thomas², ¹ Institute for Environmental Studies, Vrije Universiteit, Amsterdam, Netherlands² Department of Biology and Chemistry, University of Bremen, Bremen, Germany

ABSTRACT- The E.U. project "Analysing Combination Effects of mixtures of estrogenic chemicals in marine and freshwater organisms" (ACE) aims to analyse and assess the effects of multi-component mixtures of (xeno-)estrogens on biological systems ranging from the subcellular level to populations of fish. In this study, up to 6 (xeno-)estrogenic compounds, including estradiol, estrone, bisphenol A, nonylphenol, octylphenol, and genistein, were tested in the in vitro Estrogen Responsive Chemical Activated Luciferase Gene eXpression (ER-CALUX) reporter gene assay to investigate combination effects. The ER-CALUX assay sensitively measures transactivation of the estrogen receptor following 24 hour exposure to (xeno-)estrogens in T47D human breast cancer cells stably transfected with an estrogen-regulated luciferase reporter gene construct. Compounds were first tested individually to examine dose-response relationships and determine EC50s. Interestingly, genistein exposure resulted in a bi-phasic curve with maximal luciferase activity exceeding that of E2 by a factor of about three. When tested together at equipotent ratios based on the EC50 level, the 5-component mixture (excluding genistein) showed additive effects, which fit the prediction of mixture toxicity using the principle of concentration addition. However, inclusion of genistein in the mixture resulted in a bi-phasic curve with super-maximal luciferase activity. Future studies will focus on investigating the mechanisms underlying the bi-phasic curve, as well as increasing the number of (xeno-)estrogens to be tested in mixtures to include up to 20 components. This study has been financed by the EU, contract no. EVK1-2001-00091.

Key words: mixture, estrogen, addition, genistein