Keyhani Ezzatollah, Abdi-Oskouei Fatemeh, Attar Farnoosh, Keyhani Jacqueline
Laboratory for Life Sciences, and Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, 13145 Tehran, Iran.
Ann N Y Acad Sci. 2006 Dec;1091:52-64. doi: 10.1196/annals.1378.054.
Purified Salmonella typhimurium DNA was incubated for 1h at 37 degrees C with various concentrations (10-100 muM) of transition metal ions (Fe(2+), Fe(3+), Cu(2+), Ni(2+), Cd(2+)), with various concentrations (0.1-100 mM) of H(2)O(2), and with various concentrations of each transition metal ion in the presence of various concentrations of H(2)O(2). Damage to DNA was assessed by electrophoresis of the reaction mixtures in 1% agarose gel. Breakage of the DNA strands would produce a series of DNA fragments resulting in a smear in the gel, while intact DNA produced a single band. Results showed that no damage to the DNA was detectable after incubation with either H(2)O(2) alone or either of the metal ions alone. However, all of the metal ions investigated triggered DNA breakage in the presence of H(2)O(2). The extent of breakage depended on the metal ion and on its concentration, as well as on the H(2)O(2) concentration. Addition of either EDTA or catalase to the reaction mixture completely inhibited the DNA degradation, confirming the involvement of both the metal ion and the H(2)O(2) in the breakage of DNA strands. Production of the hydroxyl radical when H(2)O(2) and a metal ion were both present in the reaction mixture was evidenced by the thiobarbituric acid method. The most extensive damage was caused by Cu(2+) followed, in decreasing order, by Fe(2+), Fe(3+), Ni(2+), and Cd(2+).
将纯化的鼠伤寒沙门氏菌DNA与各种浓度（10 - 100μM）的过渡金属离子（Fe(2+)、Fe(3+)、Cu(2+)、Ni(2+)、Cd(2+)）、各种浓度（0.1 - 100 mM）的H₂O₂以及在各种浓度H₂O₂存在下的各种浓度的每种过渡金属离子在37℃孵育1小时。通过在1%琼脂糖凝胶中对反应混合物进行电泳来评估DNA损伤。DNA链的断裂会产生一系列DNA片段，导致凝胶中出现拖尾现象，而完整的DNA则产生一条带。结果表明，单独与H₂O₂或单独与任何一种金属离子孵育后，均未检测到对DNA的损伤。然而，所有研究的金属离子在H₂O₂存在下均引发了DNA断裂。断裂程度取决于金属离子及其浓度，以及H₂O₂浓度。向反应混合物中添加EDTA或过氧化氢酶可完全抑制DNA降解，证实金属离子和H₂O₂均参与了DNA链的断裂。通过硫代巴比妥酸法证明，当反应混合物中同时存在H₂O₂和金属离子时会产生羟基自由基。造成最广泛损伤的是Cu(2+)，其次是Fe(2+)、Fe(3+)、Ni(2+)和Cd(2+)，损伤程度依次递减。
