No abstract

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis.

No abstract

The human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and II drug-

To characterize the specificity of synthetic compounds for nuclear receptors, we established stable cell lines expressing the luciferase gene and different wild-type or chimaeric receptors. MCF-7 cells, which express the oestrogen receptor alpha (ER alpha), and HeLa cells, which do not express the oestrogen receptor, were transfected with a plasmid containing the luciferase gene downstream from a minimum promoter (beta-globin) and an oestrogen-responsive element, generating the MELN and the HELN cell lines, respectively. MELN cells enabled the detection of compounds that bind to the ER alpha or interfere with its pathway. HELN cells were used to establish stable transfectants expressing different nuclear receptors containing the DNA-binding domain of the oestrogen receptors. We thus established ER alpha or ER beta reporter cell lines by transfecting ER alpha or ER beta expression plasmids, and also retinoic acid receptor alpha, beta or gamma reporter cell lines by transfecting the chimaeric RAR gene, in which the DNA-binding domain was replaced by the ER alpha DNA-binding domain.