Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT).
Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di-tert-butyl-4-methylphenol (BHT).
复制标题
DOI：
10.1021/tx970124y
复制
发表时间：
1997-10
影响因子：
4.1
通讯作者：
M. Reed;D. Thompson
中科院分区：
文献类型：
--
作者：
M. Reed;D. Thompson
几种烷基酚（例如，2，6-二叔丁基-4-甲基苯酚，BHT）形成反应性醌甲基化物中间体（例如，2，6-二叔丁基-4-亚甲基-2，5-环己二烯酮，BHT-QM）。为了研究蛋白质烷基化在烷基酚毒性中的作用，我们使用免疫化学方法来鉴定BHT烷基化的蛋白质靶点。将合成的BHT-N-乙酰半胱氨酸（BHT-NAC）与匙孔血蓝蛋白偶联，并用作抗原，在新西兰白色兔中产生多克隆抗体。兔血清中含有一种对BHT-NAC具有高度特异性的抗体，通过竞争性ELISA测定。将BHT抗体用作探针，以在与大鼠肝微粒体或组织切片的体外孵育中以及在暴露于BHT的雄性Sprague-Dawley大鼠的肝组织中寻找BHT-蛋白加合物的存在。蛋白质凝胶的Western印迹显示BHT依赖的蛋白质烷基化在很宽的分子量范围内。在约34.5、52、64.5、74和97 kDa处观察到显著的重复条带。在微粒体孵育中，加合物的检测受到细胞色素P450抑制剂、氘代BHT和NADPH缺失的抑制。在暴露于合成制备的BHT-QM的大鼠肝微粒体中观察到与酶介导孵育中相似的蛋白质烷基化模式。在灌胃剂量高达1000 mg/kg BHT的大鼠中，在给药后24 h观察到的蛋白质烷基化量最大，且呈剂量依赖性。通过N-末端测序分离并鉴定了两种烷基化蛋白：线粒体β-氧化酶、烯酰辅酶A水合酶和来自埃兹蛋白/膜突蛋白/根蛋白家族的质膜/细胞骨架连接蛋白。
Several alkylphenols (e.g., 2,6-di-tert-butyl-4-methylphenol, BHT) form reactive quinone methide intermediates (e.g., 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, BHT-QM) upon oxidation by cellular enzymes. In order to pursue the role of protein alkylation in alkylphenol toxicity, we used an immunochemical approach to identify protein targets alkylated by BHT. Synthetic BHT-N-acetylcysteine (BHT-NAC) was coupled to keyhole limpet hemocyanin and used as an antigen from which polyclonal antibodies were raised in New Zealand white rabbits. Rabbit serum contained an antibody which was highly specific for BHT-NAC, as determined by competitive ELISA. The BHT antibody was used as a probe to look for the presence of BHT-protein adducts in in vitro incubations with rat liver microsomes or tissue slices and also in vivo in liver tissue from male Sprague-Dawley rats exposed to BHT. Western blotting of protein gels revealed BHT-dependent protein alkylation over a wide molecular weight range. Prominent recurrent bands were observed at approximately 34.5, 52, 64.5, 74, and 97 kDa. Detection of adducts was inhibited in microsomal incubations by cytochrome P450 inhibitors, deuterated BHT, and the omission of NADPH. Similar protein alkylation patterns were observed in rat liver microsomes exposed to synthetically prepared BHT-QM as in the enzyme-mediated incubations. In rats gavaged with up to 1000 mg/kg BHT, the amount of protein alkylation observed was maximal at 24 h postdosing and was dose-dependent. Two alkylated proteins were isolated and identified by N-terminal sequencing: a mitochondrial beta-oxidation enzyme, enoyl-CoA hydratase, and a plasma membrane/cytoskeletal linker protein from the ezrin/moesin/radixin family.