Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice. Relationship to cytotoxicity.
Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice. Relationship to cytotoxicity.
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对乙酰氨基酚和 2,6-二甲基对乙酰氨基酚在苯巴比妥诱导和未诱导小鼠的培养肝细胞中进行选择性蛋白质芳基化。
DOI：
10.1016/0006-2952(89)90653-9
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发表时间：
1989
影响因子：
5.8
通讯作者：
Khairallah,EA
中科院分区：
文献类型：
--
作者：
Birge,RB;Bartolone,JB;McCann,DJ;Mangold,JB;Cohen,SD;Khairallah,EA
为了评价共价结合在对乙酰氨基酚(APAP)肝毒性机制中的重要性，我们在原代培养的小鼠肝细胞中比较了2，6-二甲基对乙酰氨基酚(2，6-DMA)和APAP的作用。对电泳分离蛋白的免疫化学分析表明，细胞毒性剂量的APAP后，大多数共价结合发生在44和58kD的两条主要条带上(Bartoloneet al.，Biochem Pharmacol36：1193-1196,1987)。在等摩尔浓度下，2，6-DMA结合蛋白的广泛性仅为APAP的15%，并且在未诱导的小鼠肝细胞中没有细胞毒性。然而，当从苯巴比妥诱导的小鼠中获得肝细胞时，APAP给药导致蛋白质芳基化增加，并更快地开始细胞毒性。此外，在苯巴比妥诱导的小鼠细胞中，2，6-DMA不仅导致结合增加，而且还具有明显的细胞毒性。由于亲和纯化的抗APAP抗体不与2，6-DMA发生交叉反应，因此我们制备了一种新的针对2，6-DMA的抗体，经亲和纯化后，用Western blotting检测了2，6-DMA的蛋白加合物。结果表明，在苯巴比妥诱导和未诱导的小鼠肝细胞中，2，6-DMA的结合也是高度选择性的，最显著的靶点是58kD的胞浆蛋白。然而，与APAP相反，经2，6-DMA处理后，仅检测到与44kD蛋白的极少量结合。虽然在苯巴比妥诱导的小鼠的处理细胞中增加了几个额外的蛋白质加合物，但58kD的蛋白质显然是与APAP和2，6-DMA细胞毒性有关的最显著的芳基化靶标。这些数据表明，共价结合的特异性以及与主要靶点的结合程度可能在随后的毒性中发挥重要作用。
To evaluate the mechanistic importance of covalent binding in acetaminophen (APAP)-induced hepatotoxicity, we compared the effects of 2,6-dimethylacetaminophen (2,6-DMA) to those of APAP in primary cultures of mouse hepatocytes. Immunochemical analysis of electrophoretically separated proteins has shown that the majority of covalent binding after a cytotoxic dose of APAP occurs on two major bands of 44 and 58 kD (Bartoloneet al.,Biochem Pharmacol36: 1193–1196, 1987). At equimolar concentrations, 2,6-DMA bound proteins only 15% as extensively as did APAP and was not cytotoxic in hepatocytes from uninduced mice. However, when the hepatocytes were obtained from phenobarbital-induced mice, APAP administration resulted in increased protein arylation and a more rapid onset of cytotoxicity. Furthermore, in the cells from phenobarbital-induced mice, 2,6-DMA not only resulted in increased binding but also in overt cytotoxicity. Since our affinity-purified anti-APAP antibody did not cross-react with 2,6-DMA, a new antibody specific for 2,6-DMA was prepared and, after affinity purification, was used to detect 2,6-DMA protein adducts by Western blotting. Results indicated that, in hepatocytes from both phenobarbital-induced and non-induced mice, the binding of 2,6-DMA was also highly selective with the most prominent target being the 58 kD cytosolic protein. However, by contrast to APAP, only minimal binding to the 44 kD protein was detected after 2,6-DMA treatment. Although several additional protein adducts were increased in treated cells from phenobarbital-induced mice, the 58 kD protein was clearly the most prominently arylated target associated with both APAP and 2,6-DMA cytotoxicity. These data suggest that both the specificity of covalent binding as well as the extent of binding to the major targets may play an important role in the ensuing toxicity.