Kretz-Rommel A, Boelsterli U A
Institute of Toxicology, Swiss Federal Institute of Technology (ETH), Schwerzenbach.
Mol Pharmacol. 1994 Feb;45(2):237-44.
The nonsteroidal anti-inflammatory drug diclofenac can be bioactivated to the reactive acyl glucuronide, which covalently binds to hepatocellular proteins in rat hepatocytes. Short term cultured rat hepatocytes were used to further study the formation and nature of protein adducts after exposure to diclofenac. Incubation of cells with [14C]diclofenac (30 microM) for up to 24 hr was associated with a time-dependent increase in radioactivity bound to proteins. Upon subcellular fractionation of hepatocytes exposed to diclofenac for 2 hr, the majority of the radiolabel appeared in the microsomal fraction. By 24 hr, the specific binding had decreased by 50% in this cell compartment. In contrast, the hepatocellular plasma membrane fraction, which also was associated with high specific binding of diclofenac-derived radioactivity by 2 hr, exhibited a approximately 3-fold increase in adduct formation by 24 hr. Lesser amounts of radioactivity were associated with cytosolic proteins. After resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, the radioactivity was associated with a major protein band with an apparent molecular mass of 60 kDa that was present in both microsomes and plasma membranes. Further, we developed an antidiclofenac antibody against diclofenac-protein adducts by Protein A chromatography of a polyclonal antiserum raised in rabbits against a diclofenac-keyhole limpet hemocyanin adduct. The antidiclofenac antibody did recognize diclofenac-protein adducts on Western blots of homogenates of cultured rat hepatocytes exposed to diclofenac. The major detected adducts included the 60-kDa protein, which was present at all diclofenac concentrations used. In addition, the antibody recognized proteins with apparent molecular masses of 50, 80, and 126 kDa that were not evident in the radiochemical assay. There were no detectable cross-reactive epitopes of proteins recognized by the antibody on Western blots of cultured hepatocytes not treated with diclofenac. Moreover, immunoblots of liver homogenates from rats treated with diclofenac (30 mg/kg/day, intraperitoneally, for 4 days) also exhibited adducts with the 60- and 80-kDa proteins. Collectively, these results suggest that binding of diclofenac to rat hepatocyte proteins is selective and that a 60-kDa microsomal membrane protein (or protein subunit) that accumulates in the plasma membrane fraction appears to be the major target for alkylation both in cultured hepatocytes exposed to diclofenac and in vivo.
非甾体抗炎药双氯芬酸可被生物活化为具有反应活性的酰基葡萄糖醛酸，后者能与大鼠肝细胞中的肝细胞蛋白共价结合。使用短期培养的大鼠肝细胞进一步研究双氯芬酸暴露后蛋白加合物的形成及性质。将细胞与[14C]双氯芬酸（30微摩尔）孵育长达24小时，与蛋白结合的放射性呈时间依赖性增加。对暴露于双氯芬酸2小时的肝细胞进行亚细胞分级分离后，大部分放射性标记出现在微粒体部分。到24小时时，该细胞区室中的特异性结合下降了50%。相比之下，肝细胞质膜部分在2小时时也与双氯芬酸衍生的放射性高特异性结合有关，到24小时时加合物形成增加了约3倍。与胞质蛋白相关的放射性较少。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和荧光显影分离蛋白后，放射性与一条主要蛋白带相关，该蛋白带的表观分子量为60 kDa，存在于微粒体和质膜中。此外，我们通过对兔抗双氯芬酸-钥孔血蓝蛋白加合物产生的多克隆抗血清进行蛋白A层析，制备了抗双氯芬酸蛋白加合物的抗体。抗双氯芬酸抗体在暴露于双氯芬酸的培养大鼠肝细胞匀浆的蛋白质免疫印迹上确实能识别双氯芬酸-蛋白加合物。检测到的主要加合物包括60 kDa的蛋白，在所有使用的双氯芬酸浓度下均存在。此外，该抗体还识别表观分子量为50、80和126 kDa的蛋白，这些蛋白在放射化学分析中不明显。在未用双氯芬酸处理的培养肝细胞的蛋白质免疫印迹上，该抗体识别的蛋白没有可检测到的交叉反应表位。此外，用双氯芬酸（30毫克/千克/天，腹腔注射，共4天）处理的大鼠肝脏匀浆的免疫印迹也显示与60 kDa和80 kDa蛋白形成加合物。总的来说，这些结果表明双氯芬酸与大鼠肝细胞蛋白的结合具有选择性，并且一种在质膜部分积累的60 kDa微粒体膜蛋白（或蛋白亚基）似乎是暴露于双氯芬酸的培养肝细胞和体内烷基化的主要靶点。
