Use of [¹²⁵I]4′-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behavior

We have synthesized [₁₂₅I]4′-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [₁₂₅I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [₁₂₅I]4′-iodoflavone with the AhR was detected as a single radioactive peak (∼9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A ∼6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [₁₂₅I]4′-iodoflavone-AhR 6 S complex, compared with [₁₂₅I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4′-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4′-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [₁₂₅I]4′-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.