COMPARISON OF THE FORMATION OF 8-HYDROXY-2'-DEOXYGUANOSINE AND SINGLE-STRAND AND DOUBLE-STRAND BREAKS IN DNA-MEDIATED BY FENTON REACTIONS

The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single-and double-strand breaks in DNA by Fenton-type reactions has been investig ated. Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and on e of nine different transition-metal ions (25 mu M-1 mM). Modified DNA was isolated and subjected to analysis by liquid chromatography coupl ed to an electrochemical detection system (LC-ECD), to evaluate the fo rmation of 8-OHdG. The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III ) (5800), and copper(II) (5200). The chromium(VI) Fenton reaction gene rated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the y ield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in contro l incubations of DNA with hydrogen peroxide alone. Similar treatment o f the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transitionmetal ion (1-100 mu M) followed by quantitativ e agarose gel electrophoresis demonstrated that open-circle DNA, resul ting from single-strand breaks, was generated in Fenton reactions invo lving all nine metal ions. In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and va nadium(III) ions. Formation of linear DNA, under conditions that gener ated relatively few single-strand breaks, suggests that these four tra nsition-metal ions partake in Fenton reactions to generate true double -strand breaks. Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting tha t they arise by a similar mechanism.