Flavone antagonists bind competitively with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation

Previous analyses suggested that potent aryl hydrocarbon receptor (AhR) ant agonists were planar, with a lateral electron-rich center. To further defin e structural requirements and mechanism for antagonism, ten additional flav one 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) inhi bit TCDD-elicited binding of AhR to dioxin-responsive elements (DRE) in vit ro; and 3) inhibit TCDD-induced transcription of DRE;dependent luciferase i n stably transfected hepatoma cells, the most potent flavones contained a 3 '-methoxy group and a 4'-substituent having one or more terminal atoms of h igh electron density (-N-3, - NO2, or -NCS). Furthermore, these had low ago nist activity as assessed by their inability to elicit AhR DRE binding or t o 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'-nitroflavone s 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 contai ned 90 kDa heat shock protein. In contrast, AhR in TCDD-treated cells was p rimarily immunoprecipitated from nuclear extracts and was associated with A rnt but not 90 kDa heat shock protein. Immunocytofluorescence analysis in i ntact 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 localizat ion.