Binding of 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin to a common Ah receptor site in mouse and rat hepatic cytosols.

Abstract

The Ah receptor, which mediates induction of aryl hydrocarbon hydroxylase (cytochrome P1-450), previously was identified and characterized using 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin as the radioligand. Polycyclic aromatic hydrocarbons such as 3-methylcholanthrene induce the hydroxylase and compete with 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin for Ah receptor sites. 3-[3H]Methylcholanthrene is known to bind to several classes of cytosolic protein. Our main goal was to determine whether 3-[3H]methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin bind to an identical receptor site, or whether each inducer might function by binding to its own unique receptor. Binding of the two radioligands in rodent hepatic cytosols was characterized using 2-h sucrose-density-gradient separations in a vertical-tube ultracentrifuge rotor. These experiments show the following results. 1 The Ah receptor from hepatic cytosols of genetically ‘responsive’ C57BL/6J mice and inducible Sprague-Dawley rats sediments in the 8–10-S region of sucrose gradients when labeled either with 3-[3H]methylcholanthrene or 2,3,7,8-tetrahloro[3H]dibenzo-p-dioxin. 2 No specific 8–10-S binding peak is detectable in cytosol from genetically ‘nonresponsive’ DBA/2J mice using either radioligand. 3 A high capacity binder for 3-[3H]methylcholanthrene sediments in the 4–5-S region of the gradient and is present in hepatic cytosols from all mice studied regardless of whether they are genetically ‘responsive’ or ‘nonresponsive’ for aryl hydrocarbon hydroxylase induction. 4 In genetic crosses, inheritance of the specific 8–10-S component (which binds both 3-[3H]methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin) segregates with the Ah locus, whereas the 4–5-S component does not. 5 The concentration of specific 8–10-S receptor sites detected in a variety of cytosols is the same when 3-[3H]-methylcholanthrene is used as the radioligand as when 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin is used. 6 The rank-order potency with which ten different chemicals inhibit binding of 3-[3H]methylcholanthrene to the 8–10-S peak is the same as the rank-order of those chemicals in competing with 2,3,7,8-tetra-hloro[3H]-dibenzo-p-dioxin in this region; this constitutes the strongest evidence that the two radioligands share a common binding site. 7 The rank-order in which these competitors inhibit 3-[3H]methylcholanthrene binding to the Ah receptor (8–10-S component) is not well correlated with their ability to inhibit 3-[3H]methylcholanthrene binding in the 4–5-S region. 8 Scatchard plot analyses indicate that 3-[3H]methylcholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin bind to the Ah receptor with very similar affinities; the Kd for each radioligand is ∼ 3 nM in hepatic cytosol from C57BL/6J mice and ∼ 1 nM in hepatic cytosol from Sprague-Dawley rats. Thus the far greater potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer of aryl hydrocarbon hydroxylase in vivo is not due simply to any exceptionally high affinity of binding to the cytosolic Ah receptor. Overall evidence indicates that 3-[3H]methyl-cholanthrene and 2,3,7,8-tetrachloro[3H]dibenzo-p-dioxin both induce aryl hydrocarbon hydroxylase by acting through the same receptor site. Although the two ligands have similar affinity for cytosolic Ah receptor sites, the relatively long biological half-life of the dioxin may account for its greater potency in vivo.