Abstract : To date, studies on endocrine disruptors mixtures have mainly focused on the potential additive effects of estrogenic compounds. Until now, most of in vitro and in vivo assays that have been used to address mixture effect in fish are based on ERa and liver vitellogenin expression, while mixture effect of estrogens on other ER subtypes and other ER-regulated genes is still poorly explored. In this context, the aim of our project is to determine the effects of binary mixtures of estrogenic compounds on the three zebrafish ER subtypes and on the brain specific ER-regulated cyp19a1b gene. For that purpose, newly established zebrafish-derived tools are used: (1) in vitro stable reporter gene assay (ERE-luciferase/ zfER-a, -b1 or -b2) in a zebrafish liver cell line (ZFL) (2) in vitro transient reporter gene assay (ZfCyp19a1b-luciferase/ zfER-a, -b1 or -b2) in a human glial cell line (U251-MG) and (3) in vivo cyp19a1b-GFP transgenic zebrafish. Concentration-response relationships for all single chemicals were measured, modeled, and used to design the binary mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition (CA) and independent action (IA) models. Two different statistical approaches (Jonker’s dose-response surface models and Hewlett and Streibig’s isobole-based models implemented by Sorensen) were used to assess deviations from the CA model and to characterize interactions between the components of the mixtures (synergism/antagonism). Our study showed that binary mixtures of model estrogenic compounds generally lead to additive effects, even if some deviations could be observed. As regards binary mixtures of estrogenic compounds with different estrogenic potency, our study highlights an antagonism between E2 and genistein in mixture both in vitro (U251-MG) and in vivo in a glial cell context. To our knowledge this is the first report of an antagonism between these two compounds in brain context. Conversely, in another cell context, i.e. the liver (ZFL cells), preliminary results on the effect of mixtures of E2 and genistein showed a slight synergistic effect between the two compounds. The future development of our work will concern the effects of mixtures of estrogenic compounds with indirect estrogenic ones(needing metabolization prior to elicit estrogenic activity).
