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Yann Le Page,¹ Martin Scholze,² Olivier Kah,¹ and Farzad Pakdel¹

¹Endocrinologie Moléculaire de la Reproduction, Université de Rennes, Rennes, France; ²Centre for Toxicology, School of Pharmacy, University of London, London, United Kingdom

Abstract
The brain cytochrome P450 aromatase (Aro-B) in zebrafish is expressed in radial glial cells and is strongly stimulated by estrogens (E₂) ; thus, it can be used in vivo as a biomarker of xenoestrogen effects on the central nervous system. By quantitative real-time polymerase chain reaction, we first confirmed that the expression of Aro-B gene is robustly stimulated in juvenile zebrafish exposed to several xenoestrogens. To investigate the impact of environmental estrogenic chemicals on distinct estrogen receptor (ER) activity, we developed a glial cell-based assay using Aro-B as the target gene. To this end, the ER-negative glial cell line U251-MG was transfected with the three zebrafish ER subtypes and the Aro-B promoter linked to a luciferase reporter gene. E₂ treatment of U251-MG glial cells cotransfected with zebrafish ER- and the Aro-B promoter-luciferase reporter resulted in a 60- to 80-fold stimulation of luciferase activity. The detection limit was < 0.05 nM, and the EC₅₀ (median effective concentration) was 1.4 nM. Interestingly, in this glial cell context, maximal induction achieved with the Aro-B reporter was three times greater than that observed with a classical estrogen-response-element reporter gene (ERE-tk-Luc) . Dose-response analyses with ethynylestradiol (EE₂) , estrone (E₁) , -zeralenol, and genistein showed that estrogenic potency of these agents markedly differed depending on the ER subtype in the assay. Moreover, the combination of these agents showed an additive effect according to the concept of concentration addition. This confirmed that the combined additive effect of the xenoestrogens leads to an enhancement of the estrogenic potency, even when each single agent might be present at low effect concentrations. In conclusion, we demonstrate that our bioassay provides a fast, reliable, sensitive, and efficient test for evaluating estrogenic potency of endocrine disruptors on ER subtypes in a glial context. Key words: aromatase gene, brain, endocrine disruptors, estrogen receptors, glial cells, xenoestrogens, zebrafish. Environ Health Perspect 114:752-758 (2006) . doi:10.1289/ehp.8141 available via http://dx.doi.org/ [Online 8 December 2005]

Address correspondence to F. Pakdel, Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes cedex, France. Telephone: 33-2-23-23-51-32. Fax: 33-2-23-23-67-94. E-mail: farzad.pakdel@univ-rennes1.fr

We thank B. Jegou for positive input.

This work was supported by the European Community (project QLRT-2001-00603-EDEN) , the Centre National de la Recherche Scientifique, and the Ministry of Research and Technology.

The authors declare they have no competing financial interests.

Received 23 March 2005 ; accepted 7 December 2005.