Oxidative DNA damage mediated by copper(II), iron(II) and nickel(II) Fenton reactions: evidence for site-specific mechanisms in the formation of double-strand breaks, 8-hydroxydeoxyguanosine and putative intrastrand cross-links

The role of metal ion-DNA interactions in the Fenton reaction-mediated form ation of putative intrastrand cross-links, 8-hydroxydeoxyguanosine (8-OHdG) and single- and double-strand breaks was investigated. Salmon sperm DNA an d pBluescript K + plasmid were incubated with hydrogen peroxide and either copper(II), iron(II), or nickel(II), which differ in both their affinity fo r DNA and in the spectrum of oxidative DNA damage they induce in Fenton rea ctions. EDTA was included in these incubations according to two different s trategies; the first (strategy 1) in which DNA and metal ions were mixed pr ior to the addition of EDTA, the second (strategy 2) in which EDTA and meta l ions were mixed prior to the addition of DNA, The formation of the putati ve intrastrand cross-links, monitored by P-32-postlabelling, was not affect ed by the addition of between 10 mu M and 5 mM EDTA to the copper(II) Fento n reaction according to strategy 1. In contrast, the level of cross-links d eclined significantly upon inclusion of 20 mu M EDTA and above when added a ccording to strategy 2. Similarly, formation of these lesions declined in t he iron(II) Fenton reaction more dramatically upon addition of 5 mM EDTA wh en added according to strategy 2 compared to strategy 1, while the yield of cross-links formed in the nickel(ll) 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 electrophores is 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 contra st, the formation of linear DNA in the copper(LT) Fenton reaction decreased upon inclusion of EDTA according to both strategies. A decrease in the for mation of open-circular DNA was also observed upon inclusion of EDTA accord ing to both strategies; however this decrease occurred at a lower EDTA conc entration in strategy 2 (100 mu M) compared to strategy 1 (200 mu M), and t he 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 thi s was completely inhibited upon addition of 25 mu M EDTA according to both strategies. There was less formation of 8-OHdG in the copper(II) and iron(I I) Fenton reactions when EDTA was added according to strategy 2 than accord ing 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 sin gle-strand breaks is more likely to involve generation of hydroxyl radicals in solution. (C) 1999 Elsevier Science B.V. All rights reserved.