Lloyd D R, Phillips D H, Carmichael P L
Section of Molecular Carcinogenesis, Haddow Laboratories, Sutton, Surrey, U.K.
Chem Res Toxicol. 1997 Apr;10(4):393-400. doi: 10.1021/tx960158q.
Generation of putative intrastrand cross-links and strand breaks was investigated in salmon sperm DNA exposed to Fenton-type oxygen radical-generating systems. 32P-Postlabeling analysis of DNA treated with hydrogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(II), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radical-mediated oxidative DNA lesions. The copper Fenton system generated the highest total yield of these DNA lesions (75.6 per 10(8) nucleotides), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (21.7), and vanadium (17.1). Two spots, common to all these Fenton systems, were the major oxidation products in each case. Similar Fenton-type treatment of the purine dinucleotides dApdG and dApdA resulted in products that were chromatographically identical on anion-exchange TLC and on reverse-phase HPLC to the two major products generated in DNA. These results extend our earlier studies suggesting that these products were the result of a free radical-mediated intrastrand cross-linking reaction. Incubations involving cadmium(II), chromium(III), or zinc(II) ions with hydrogen peroxide did not generate DNA oxidation products at levels greater than in incubations with hydrogen peroxide alone. Generation of the putative intrastrand cross-links increased in a concentration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) ions. However, in experiments with copper, iron, or vanadium ions, maximum levels were obtained at 250, 150, and 150 microM, respectively, and the yield declined with higher concentrations of these three metal ions. Agarose gel electrophoresis demonstrated extensive DNA strand breakage with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results demonstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independent mechanisms.
在暴露于芬顿型氧自由基产生系统的鲑鱼精子DNA中，研究了推定的链内交联和链断裂的产生情况。用过氧化氢与铜（II）、铬（VI）、钴（II）、铁（II）、镍（II）或钒（III）处理DNA后进行32P后标记分析，结果检测到4至8个放射性薄层色谱斑点，这些斑点可能是羟基自由基介导的氧化性DNA损伤。铜芬顿系统产生的这些DNA损伤的总产率最高（每10^8个核苷酸75.6个），其次是钴（47.5）、镍（26.2）、铬（25.1）、铁（21.7）和钒（17.1）。所有这些芬顿系统共有的两个斑点是每种情况下的主要氧化产物。对嘌呤二核苷酸dApdG和dApdA进行类似的芬顿型处理，得到的产物在阴离子交换薄层色谱和反相高效液相色谱上的色谱行为与DNA中产生的两种主要产物相同。这些结果扩展了我们早期的研究，表明这些产物是自由基介导的链内交联反应的结果。涉及镉（II）、铬（III）或锌（II）离子与过氧化氢的孵育产生的DNA氧化产物水平不高于仅用过氧化氢孵育的水平。推定的链内交联的产生以浓度依赖的方式增加，直至1 mM钴、镍或铬（VI）离子。然而，在使用铜、铁或钒离子的实验中，分别在250、150和150 microM时获得最高水平，并且随着这三种金属离子浓度的增加，产率下降。琼脂糖凝胶电泳显示，铜、铁、铬（III）或钒会导致广泛的DNA链断裂，但镍、铬酸盐（VI）、钴、镉或锌芬顿系统不会。结果表明，由芬顿反应介导的DNA中推定的链内交联和链断裂的产生是通过独立机制发生的。
