Alpha-naphthoflavone acts as an antagonist of 2,3,7, 8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor.

alpha-Naphthoflavone (ANF) has previously been shown to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the Ah receptor under conditions in vitro. However, ANF also prevents TCDD-elicited cytochrome P450lA1 induction, immunosuppression, and down-regulation of the estrogen receptor in vivo and within intact isolated cells. These data suggest that ANF is a TCDD antagonist. This study investigated the ability of ANF to transform the Ah receptor contained in rat hepatic cytosol or mouse hepatoma cells to a form that recognizes the dioxin-responsive enhancer element (DRE) upstream of the cytochrome P450lA1 gene. Gel retardation analysis indicated that TCDD- or beta-naphthoflavone (BNF)-bound receptor was able to bind to the DRE, whereas essentially no receptor-DRE complexes were observed using cytosol incubated with ANF concentrations as high as 1000 nM. Furthermore, an excess of ANF, when added to cytosol just before TCDD, blocked, in a concentration-dependent manner, the ability of TCDD to transform the receptor to a form that bound to the DRE. These studies indicated that ANF binds to the receptor and confers on it a conformation that cannot recognize the DNA recognition sequence contained in the DRE. Although an excess of the agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) readily reversed the inhibitory actions of ANF, ANF was unable to reverse the effects of TCDD, TCDF, or BNF on the receptor. These studies suggested that TCDD binding, unlike that of ANF, results in a receptor conformation that has higher affinity for the ligand. Treatment of mouse hepatoma Hepa 1c1c7 cells with TCDD or BNF resulted in receptor contained in nuclear extracts that bound to the DRE. Only a very minor ligand-dependent protein-DNA complex was detected when cells were treated with ANF. These data indicated that ANF acts as an antagonist of TCDD by directly binding to the Ah receptor and eliciting a protein conformation that has very low affinity for DNA.