Neely M Diana, Amarnath Venkataraman, Weitlauf Carl, Montine Thomas J
Department of Pathology and the Centers for Molecular Neuroscience and Molecular Toxicology, Vanderbilt University Medical School, MCN U4216, 21st Avenue South, Nashville, TN 37232-2561, USA.
Chem Res Toxicol. 2002 Jan;15(1):40-7. doi: 10.1021/tx010115w.
4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation and has been suggested to play a role in the pathogenesis in several common diseases as well as injuries caused by environmental toxicants. Although formed intracellularly in vivo, for practical reasons this molecule is applied extracellularly in order to analyze its biological effects. The focus of this study was to develop an approach that would enable intracellular HNE production in the absence of the many other products and processes that occur in cells experiencing generalized oxidative stress. To this end, we synthesized 1,1,4-tris(acetyloxy)-2(E)-nonene (HNE[Ac]3), a triester analogue of HNE that is itself unreactive but could be hydrolyzed intracellularly presumably by lipases and/or esterases into the highly reactive HNE. In vitro lipase rapidly converted HNE(Ac)(3) initially to 4-acetyloxy-2-nonenal (HNE[Ac]1) and then to HNE. Neuro 2A cell lysate also caused a rapid hydrolysis of HNE(Ac)3 into HNE(Ac)1 and HNE. Incubation of BSA with HNE(Ac)3 resulted in protein-adduct formation only in the presence of lipase. We demonstrated adduction of HNE to proteins in Neuro 2A cells exposed to HNE(Ac)3 by immunoblotting and immunocytochemistry using antibodies specific for HNE-Michael adducts on proteins. We have previously shown that microtubule organization is very sensitive to HNE. Analysis of Neuro 2A cell microtubules showed that this cytoplasmic organelle is similarly sensitive to HNE and HNE(Ac)3.
4-羟基-2-壬烯醛（HNE）已被公认为脂质过氧化的反应产物，并被认为在几种常见疾病的发病机制以及环境毒物所致损伤中起作用。尽管HNE在体内是在细胞内形成的，但出于实际原因，该分子在细胞外应用以分析其生物学效应。本研究的重点是开发一种方法，该方法能够在不存在经历全身性氧化应激的细胞中出现的许多其他产物和过程的情况下实现细胞内HNE的产生。为此，我们合成了1,1,4-三（乙酰氧基）-2（E）-壬烯（HNE[Ac]3），它是HNE的三酯类似物，其本身无反应性，但可能在细胞内被脂肪酶和/或酯酶水解成高反应性的HNE。体外脂肪酶能迅速将HNE(Ac)3首先转化为4-乙酰氧基-2-壬烯醛（HNE[Ac]1），然后再转化为HNE。Neuro 2A细胞裂解物也能使HNE(Ac)3迅速水解为HNE(Ac)1和HNE。仅在脂肪酶存在的情况下，牛血清白蛋白（BSA）与HNE(Ac)3孵育才会导致蛋白质加合物的形成。我们通过使用针对蛋白质上HNE-迈克尔加合物的特异性抗体进行免疫印迹和免疫细胞化学，证明了暴露于HNE(Ac)3的Neuro 2A细胞中HNE与蛋白质的加合。我们之前已经表明微管组织对HNE非常敏感。对Neuro 2A细胞微管的分析表明，这种细胞质细胞器对HNE和HNE(Ac)3同样敏感。
