Lloyd D R, Phillips D H
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey, SM2 5NG, UK.
Mutat Res. 1999 Mar 8;424(1-2):23-36. doi: 10.1016/s0027-5107(99)00005-6.
The role of metal ion-DNA interactions in the Fenton reaction-mediated formation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA and pBluescript K+ plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity for DNA and in the spectrum of oxidative DNA damage they induce in Fenton reactions. EDTA was included in these incubations according to two different strategies; the first (strategy 1) in which DNA and metal ions were mixed prior to the addition of EDTA, the second (strategy 2) in which EDTA and metal ions were mixed prior to the addition of DNA. The formation of the putative intrastrand cross-links, monitored by 32P-postlabelling, was not affected by the addition of between 10 microM and 5 mM EDTA to the copper(II) Fenton reaction according to strategy 1. In contrast, the level of cross-links declined significantly upon inclusion of 20 microM EDTA and above when added according to strategy 2. Similarly, formation of these lesions declined in the iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA when added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(II) Fenton reaction declined equally with both strategies with up to 25 mM EDTA. The formation of single- and double-strand breaks was investigated in plasmid DNA by agarose gel electrophoresis and subsequent densitometry. The formation of linear DNA in the iron(II) Fenton reaction decreased dramatically upon inclusion of EDTA according to strategy 2, while no such decline was observed using strategy 1. In contrast, the formation of linear DNA in the copper(II) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the formation of open-circular DNA was also observed upon inclusion of EDTA according to both strategies; however this decrease occurred at a lower EDTA concentration in strategy 2 (100 microM) compared to strategy 1 (200 microM), and the level of open-circular DNA reached a lower level (8. 5% compared to 24.2%). The nickel(II) Fenton reaction generated only open-circular DNA, and this was completely inhibited upon addition of 25 microM EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(II) Fenton reactions when EDTA was added according to strategy 2 than according to strategy 1. These results suggest that a site-specific mechanism is involved in the formation of double-strand breaks and, to a lesser extent, 8-OHdG and the putative intrastrand cross-links, while the formation of single-strand breaks is more likely to involve generation of hydroxyl radicals in solution.
研究了金属离子与DNA的相互作用在芬顿反应介导的假定链内交联、8-羟基脱氧鸟苷（8-OHdG）以及单链和双链断裂形成过程中的作用。将鲑鱼精DNA和pBluescript K+质粒与过氧化氢以及铜（II）、铁（II）或镍（II）一起孵育，这几种金属离子对DNA的亲和力以及它们在芬顿反应中诱导的氧化性DNA损伤谱均有所不同。根据两种不同策略在这些孵育体系中加入乙二胺四乙酸（EDTA）；第一种策略（策略1）是在加入EDTA之前将DNA和金属离子混合，第二种策略（策略2）是在加入DNA之前将EDTA和金属离子混合。通过32P后标记监测假定链内交联的形成，按照策略1向铜（II）芬顿反应中加入10微摩尔/升至5毫摩尔/升的EDTA，对交联的形成没有影响。相比之下，按照策略2加入20微摩尔/升及以上的EDTA时，交联水平显著下降。同样，按照策略2加入5毫摩尔/升的EDTA时，铁（II）芬顿反应中这些损伤的形成比策略1更显著地下降，而在镍（II）芬顿反应中形成的交联产率在两种策略下加入高达25毫摩尔/升的EDTA时均下降。通过琼脂糖凝胶电泳及后续的光密度测定研究了质粒DNA中单链和双链断裂的形成。按照策略2在铁（II）芬顿反应中加入EDTA后，线性DNA的形成显著减少，而使用策略1时未观察到这种下降。相比之下，按照两种策略在铜（II）芬顿反应中加入EDTA后，线性DNA的形成均减少。按照两种策略加入EDTA后，开环DNA的形成也减少；然而，与策略1（200微摩尔/升）相比，策略2（100微摩尔/升）在较低的EDTA浓度下就出现这种减少，并且开环DNA的水平达到更低水平（8.5%，而策略1为24.2%）。镍（II）芬顿反应仅产生开环DNA，按照两种策略加入25微摩尔/升的EDTA后，这种反应完全被抑制。按照策略2加入EDTA时，铜（II）和铁（II）芬顿反应中8-OHdG的形成比策略1少。这些结果表明，双链断裂以及在较小程度上8-OHdG和假定链内交联的形成涉及位点特异性机制，而单链断裂的形成更可能涉及溶液中羟基自由基的产生。
