In vitro activation of the dioxin receptor to a DNA-binding form by food-borne heterocyclic amines

The carcinogenic heterocyclic amines, which are formed upon cooking of protein-rich food, are activated in the body mainly by cytochrome P450 IA1 and IA2. Several of these so-called food mutagens have, by enzymatic and immunoblotting techniques, been shown to be weak inducers of cytochrome P450 IA in the rat. To elucidate whether this induction occurs via the dioxin receptor, the capacity of the heterocyclic amines to activate in vitro the dioxin receptor to a DNA-binding form was determined. The activation of the receptor in Hepa cell cytosol was analyzed with a gel-retardation assay using a [³²P]3′-end-labeled synthetic oligonucleotide, corresponding to nucleotides -1026 to -999 of the rat cytochrome P450 IA1 gene (XRE1), as probe. Five out of 14 heterocyclic amines had the ability to induce specific DNA-binding activity in the Hepa cell cytosol in a dose-dependent manner. The concentrations eliciting 50% of the maximal effect (EC₅₀) were found to be between 30 and 135 μM. Thus, in comparison to high-affinity ligands such as benzo[a]pyrene (B[a]P; EC₅₀ 11.2 nM), and 2,3,7,8-tetrachloro-1,6-dibenzo-p-dioxin (TCDD; EC₅₀ 1.9 nM), the activating capacity of the heterocyclic amines is low. Binding to the dioxin receptor has been shown to be dependent on the three-dimensional size of a molecule in relation to a 6.8 × 13.7 Å rectangle. The relatively low EC₅₀ values found for the heterocyclic amines might be explained by the smaller size of these molecules when compared to that of B[a]P and TCDD.