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Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) antagonists were planar, with a lateral electronrich center. To further define structural requirements and mechanism for antagonism, ten additional flavone derivatives were synthesized. Based on their ability to 1) compete with 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) for binding to the AhR; 2) inhibit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vitro; and 3) inhibit TCDD-induced transcription of DRE-dependent luciferase in stably transfected hepatoma cells, the most potent flavones contained a 3´-methoxy group and a 4´-substituent having one or more terminal atoms of high electron density (–N3, –NO2, or –NCS). Furthermore, these had low agonist activity as assessed by their inability to elicit AhR z DRE binding or to induce luciferase. Compounds containing bulkier 3´or 4´-substituents, or a 3´-OH group were less potent antagonists, and some were partial agonists. In rat liver cytosol, 3´-methoxy-4´-azido- and 3´-methoxy-4´-nitroflavones bound competitively (with TCDD) to the AhR, indicating that they bind to the TCDD-binding site. When hepatoma cells were exposed to these flavones, AhR complexes were primarily immunoprecipitable from the cytosol and contained 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was primarily immunoprecipitated from nuclear extracts and was associated with Arnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in intact cells further indicated that the potent antagonist inhibited nuclear uptake of AhR and blocked TCDD-dependent down-regulation of AhR. Together, these data indicate that the most potent antagonists bind the AhR with high affinity but cannot initiate receptor transformation and nuclear localization.
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