Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells.
Evidence of covalent binding of the dietary flavonoid quercetin to DNA and protein in human intestinal and hepatic cells.
复制标题
膳食类黄酮槲皮素与人类肠道和肝细胞中的 DNA 和蛋白质共价结合的证据。
DOI：
10.1016/s0006-2952(03)00151-5
复制
发表时间：
2003
影响因子：
5.8
通讯作者：
Walle,UKristina
中科院分区：
文献类型：
--
作者：
Walle,Thomas;Vincent,TimothyS;Walle,UKristina
富含栎素的食物具有预防人类疾病的潜力。然而，对其生物学命运和作用机制的了解有限。这项研究扩展了以前观察到的过氧化物酶将槲皮素氧化成苯醌/甲基醌中间体的研究结果，并首次证明了[14C]槲皮素与大分子的共价结合。这是首次使用辣根过氧化物酶和过氧化氢与人肝微粒体蛋白捕获中间体来证明的。为了将这一观察扩展到细胞水平，将人肠道Caco-2细胞和肝脏Hep G2细胞与[14C]栎素孵育长达2小时，并提取细胞DNA和蛋白质。细胞对[14C]槲皮素的摄取速度很快，经大量纯化后，用液体闪烁光谱仪测定了[14C]槲皮素与DNA和蛋白质的共价结合。两种细胞均表现出DNA结合，最大结合水平为5-15pmol/mg DNA。在两种细胞类型中，与蛋白质的共价结合水平都相当高，为75-125pmol/mg蛋白质。为了确定蛋白结合的潜在特异性，用[14C]槲皮素处理Hep G2细胞，细胞裂解产物经SDS-PAGE染色和放射自显影。几条不同的放射性标记蛋白条带与主要的考马斯蓝染色的细胞蛋白不对应。我们认为，这种特异性结合可能介导了部分栎皮素的抗增殖和其他细胞作用。
Quercetin-rich foods have the potential to prevent human disease. However, knowledge of its biological fate and mechanism of action is limited. This study extends previous observations of the oxidation of quercetin by peroxidases to quinone/quinone methide intermediates and, for the first time, demonstrates covalent binding of [14C ]quercetin to macromolecules. This was first demonstrated using horseradish peroxidase and hydrogen peroxide with human liver microsomal protein to trap the intermediates. To extend this observation to the cellular level, human intestinal Caco-2 cells and hepatic Hep G2 cells were incubated for up to 2hr with [14C ]quercetin, and cellular DNA and protein were isolated. The cellular uptake of [14C ]quercetin was rapid, and the covalent binding of [14C ]quercetin to DNA and protein was determined by liquid scintillation spectrometry after extensive purification. Both cell types demonstrated DNA binding with a maximum level of 5–15pmol/mg DNA. The level of covalent binding to protein was considerably higher in both cell types, 75–125pmol/mg protein. To determine potential specificity in the protein binding, Hep G2 cells were treated with [14C ]quercetin, and the cell lysate was subjected to SDS–PAGE followed by staining and autoradiography. Several distinct radiolabeled protein bands did not correspond to the major Coomassie blue stained cellular proteins. We propose that this specific binding may mediate part of the antiproliferative and other cellular actions of quercetin.