Partial characterization of specific cantharidin binding sites in mouse tissues

The mode of action of cantharidin, the natural vesicant of blister beetles, is examined by radioligand binding studies with mouse tissues. [³H]Cantharidin undergoes specific and saturable binding with the liver cytosol, which is characterized as follows: K(d) and B(max) values of 30 nM and 1.8 pmol/mg of protein, respectively; linearity with respect to protein concentration; pH optimum of 6.5 to 7.5; association and dissociation half-times of 20 min and 12 hr, respectively; and 50% inhibition by Mg²⁺ at 70 μM, Ca²⁺ at 224 μM, pyrophosphate at 27 μM, and nucleotide triphosphates at 52-81 μM. The binding site undergoes a loss of activity at 45° or higher. The toxicological relevance of this specific [³H]cantharidin binding site of mouse liver cytosol is established in three ways. First, the potency of 15 active cantharidin analogs for inhibiting [³H]cantharidin binding is correlated with their acute toxicity to mice (r = 0.8299). Second, 26 related compounds that are inactive in inhibiting [³H]cantharidin binding are also of little or no toxicity to mice. Finally, the binding of [³H]cantharidin to liver cytosol from mice poisoned with increasing amounts of unlabeled cantharidin is inhibited in a dose-dependent manner. [³H]Cantharidin also specifically binds to cytosol fractions of blood, brain, heart, kidney, lung, pancreas, skin, spleen, and stomach. The characteristics of the specific binding site in brain are very similar to those determined in liver with respect to K(d), B(max), association/dissociation kinetics, and sensitivity to inhibitors. It therefore appears that the toxicity of cantharidin and related oxabicycloheptanes, including the herbicide endothal, is attributable to binding at a specific site in liver and possibly other tissues.