An assay for the detection of xenoestrogens based on a promoter containingoverlapping EREs

Xenoestrogens could be implicated in the decrease of male fertility and in the increased incidence of testicular and breast cancers in humans. To pred ict their deleterious effects, various in vivo or in vitro tests have been proposed to assay the xenoestrogenic activity. We have designed an assay fo r the detection of xenoestrogens based on a novel estrogen responsive unit formed by two overlapping estrogen response elements (overEREs). This const ruct is able to mediate a synergistic activation of transcription by 17 bet a-estradiol. We have used the overERE unit to assay the estrogenic activity of synthetic compounds, mostly organochlorine compounds. By using the over ERE construct, we were able to detect the estrogenic activity of compounds at concentrations 10- to 100-fold lower than a single ERE (i.e., we detecte d the estrogenic effect of endosulfan at a concentration of 10(-5) M with E RE, whereas the overERE unit allowed us to detect a significant estrogenic activity of endosulfan at a lower concentration (10(-6) M). Some compounds did not exhibit any estrogenic activity when tested with a classical ERE, w hereas they were potent xenoestrogens when the overERE was used (i.e., Beta nal). The assays we have developed are very sensitive and can be performed quickly. Moreover, because the promoter that we used contains only an overl apping ERE as a regulatory unit, the interference of the tested molecules w ith other regulatory pathways can be avoided.