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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.

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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.

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Affinity labelling of the estradiol-178 dehydrogenase of human placenta has been performed using derivatives of estradiol-17P carrying alkylating groups in nine different positions on the steroid nucleus.The active-site-directed character of the inhibitions is confirmed by the following observations : the affinity labels are substrates or competitive inhibitors, the enzyme is protected against inactivation and alkylation by the substrate and by the coenzyme, the stoichiometry of the alkylation is two moles of inhibitor per 68000 g of enzyme (dimer).The alkylation of a histidine residue which is fast and extensive when the alkylating side chain is on the C-3 carbon atom, is dramatically decreased when the alkylating side chain is shifted towards rings B and D. These results allow the location of this histidine in the vicinity of ring A and probably on the P face of the steroid nucleus.The reactivity of a cysteine located on the active site was quite different, showing increasing alkylation when the alkylating substituent of the affinity labels was shifted from C-3 to C-16 of the steroid nucleus.The correlation of this result and that obtained using an alkylating analog of NAD (3-chloroacetylpyridine-adenine dinucleotide) indicates that this cysteine is located in the catalytic region of the active site, at the junction of the ring D of the steroid nucleus with the nicotinamide moiety of the coenzyme.Affinity labelling of steroid dehydrogenases was performed by Warren [I-31 on the bacterial 20p-hydroxysteroid dehydrogenase, by Engel [4,5] and by ourselves [6-81 on the estradiol-17P dehydrogenase of human placenta.This estradiol dehydrogenase (B enzyme) transfers the 4 -p r o 3 hydrogen atom of nicotinamide to 17-0x0-steroids which are converted into 17~-hydroxysteroids (S alcohols) [9,10]. This stereospecificity is strict and implicates, according to Prelog [ll], that during the transition state, the CI face of the substrate is always presented to the B face of nicotinamide. This mechanistic feature and the relative rigidity of the substrate molecule make estradiol-178 dehydrogenase an enzyme very suitable for affinity labelling : the substrate binding site can be studied using several substrate analogs, each carrying an alkylating substituent in a ~ Enzyme. EstradiolLl7fi dehydrogenase (EC 1.1.1.62). Trivial names. 16a-Broinoacetoxy-estrone 3-acetate, 17-0x0-1,3,5( lO)-estratrien-3,16cc-dio~ 3-acetate 16-bromoacetate; I6c-bromoacetoxy-estradiol3-methyl ether, 3-methoxy-I ,3,5( 10)-estratrien16a,l7p-diol 16-broinoacetate; 3-iodoacetoxy-estrone, 17-oxo-1,3,5-(1 O)-estratrien-3-yl iodoacetate ; 3-iodoacetoxy-estradiol, 17/M~y-droxy-l,3,5(10)-estratrien-3-yI iodoacetate. different position : under such conditions the region to be explored can be selected by the position and the orientation of the alkylating group on the steroid nucleus (Fig. 1). Thus, the localization of labelled amino acid in the active site can be performed with some accuracy, provided the orientation of the substrate analog during alkylation is i...