title: Radioligand binding assay for accurate determination of nuclear retinoid X receptors: A case of triorganotin endocrine disrupting ligands.
abstract: Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.
authors.affiliation: Institute of Experimental Endocrinology, BMC, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic.
authors.forename: Lucia
authors.initials: L
authors.lastname: Toporova
authors.affiliation: Institute of Experimental Endocrinology, BMC, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Macejova
authors.affiliation: Institute of Experimental Endocrinology, BMC, Slovak Academy of Sciences, Dubravska cesta 9, 845 05 Bratislava, Slovak Republic. Electronic address: julius.brtko@savba.sk.
authors.forename: Julius
authors.initials: J
authors.lastname: Brtko
mesh_terms: D000077556:Alitretinoin; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D009947:Organotin Compounds / Q000378:metabolism* / Q000633:toxicity; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D047488:Retinoid X Receptors / Q000187:drug effects / Q000378:metabolism*; D018570:Risk Assessment; D014212:Tretinoin / Q000378:metabolism*; D014220:Trialkyltin Compounds / Q000378:metabolism* / Q000633:toxicity
chemical_list: D008024:Ligands; D009947:Organotin Compounds; D047488:Retinoid X Receptors; D014220:Trialkyltin Compounds; D000077556:Alitretinoin; C011559:tributyltin; D014212:Tretinoin; C012594:triphenyltin chloride
keywords: 9-cis Retinoic acid; Affinity; Endocrine disruptors; Maximal binding capacity; Radioligand binding assay; Retinoid X receptor; Tributylin; Triphenyltin