Graziano M J, Pessah I N, Matsuzawa M, Casida J E
Department of Entomological Sciences, University of California, Berkeley 94720.
Mol Pharmacol. 1988 Jun;33(6):706-12.
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: Kd and Bmax 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 microM, Ca2+ at 224 microM, pyrophosphate at 27 microM, and nucleotide triphosphates at 52-81 microM. The binding site undergoes a loss of activity at 45 degrees 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 = 0.829). 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 Kd, Bmax, 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.
通过对小鼠组织进行放射性配体结合研究，考察了斑蝥素（一种芫菁科昆虫的天然发泡剂）的作用方式。[3H]斑蝥素与肝脏胞质溶胶发生特异性和可饱和结合，其特征如下：解离常数（Kd）和最大结合量（Bmax）分别为30 nM和1.8 pmol/mg蛋白质；与蛋白质浓度呈线性关系；最适pH为6.5至7.5；结合半衰期和解离半衰期分别为20分钟和12小时；70 microM的Mg2+、224 microM的Ca2+、27 microM的焦磷酸盐和52 - 81 microM的三磷酸核苷酸可产生50%的抑制作用。该结合位点在45摄氏度或更高温度下活性丧失。小鼠肝脏胞质溶胶中这种特异性[3H]斑蝥素结合位点的毒理学相关性通过三种方式得以确立。首先，15种活性斑蝥素类似物抑制[3H]斑蝥素结合的效力与其对小鼠的急性毒性相关（r = 0.829）。其次，26种对抑制[3H]斑蝥素结合无活性的相关化合物对小鼠也几乎没有毒性。最后，[3H]斑蝥素与用越来越大量未标记斑蝥素中毒的小鼠肝脏胞质溶胶的结合呈剂量依赖性抑制。[3H]斑蝥素还特异性结合血液、脑、心脏、肾脏、肺、胰腺、皮肤、脾脏和胃的胞质溶胶部分。脑内特异性结合位点的特征在解离常数、最大结合量、结合/解离动力学以及对抑制剂的敏感性方面与在肝脏中测定的非常相似。因此，斑蝥素及相关的氧杂双环庚烷（包括除草剂安磺灵）的毒性似乎归因于在肝脏以及可能其他组织中的特异性位点结合。
