Olinski R, Nackerdien Z, Dizdaroglu M
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899.
Arch Biochem Biophys. 1992 Aug 15;297(1):139-43. doi: 10.1016/0003-9861(92)90651-c.
Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.
据报道，在经γ射线照射或过氧化氢处理的培养人细胞的染色质中，胸腺嘧啶与酪氨酸之间形成了DNA - 蛋白质交联。从细胞中分离出染色质，随后进行水解和衍生化。通过气相色谱/质谱联用的选择离子监测对衍生化水解产物进行分析，结果表明形成了3 - [(1,3 - 二氢 - 2,4 - 二氧代嘧啶 - 5 - 基) - 甲基] - L - 酪氨酸（胸腺嘧啶 - 酪氨酸交联物）。在对照细胞中也观察到这种DNA - 蛋白质交联物的存在，其水平约为每10(6)个DNA核苷酸中有7个分子。细胞暴露于8.7至82 Gy（J·kg-1）剂量的电离辐射下，胸腺嘧啶 - 酪氨酸交联物的量线性增加，比背景水平高出约四倍。在高于82 Gy的剂量下，产量接近平稳状态。用0.5至10 mM的过氧化氢处理细胞也以浓度依赖的方式增加了胸腺嘧啶 - 酪氨酸交联物的量。在培养基中添加二甲基亚砜和邻菲罗啉可部分抑制交联物的形成。在过氧化氢处理之前添加过氧化氢酶抑制剂氰化钾，使交联物的产量高于单独用过氧化氢处理时观察到的水平。在无过氧化氢的情况下，用抗坏血酸对细胞进行24小时预处理会导致胸腺嘧啶 - 酪氨酸交联物的形成。细胞染色质中的这种DNA - 蛋白质交联物被认为是通过涉及自由基加成反应和/或涉及胸腺嘧啶和酪氨酸自由基的自由基 - 自由基结合的机制形成的。有人提出，染色质结合的金属离子介导的羟基自由基会导致在过氧化氢处理的细胞中形成胸腺嘧啶 - 酪氨酸交联物。
