GENERATION OF PUTATIVE INTRASTRAND CROSS-LINKS AND STRAND BREAKS IN DNA BY TRANSITION-METAL ION-MEDIATED OXYGEN RADICAL ATTACK

Generation of putative intrastrand cross-links and strand breaks was i nvestigated in salmon sperm DNA exposed to Fenton-type oxygen radical- generating systems. P-32-Postlabeling analysis of DNA treated with hyd rogen peroxide and either copper(II), chromium(VI), cobalt(II), iron(I I), nickel(II), or vanadium(III) resulted in the detection of between four and eight radioactive TLC spots that are probably hydroxyl radica l-mediated oxidative DNA lesions. The copper Fenton system generated t he highest total yield of these DNA lesions (75.6 per 10(8) nucleotide s), followed by cobalt (47.5), nickel (26.2), chromium (25.1), iron (2 1.7), and vanadium (17.1). Two spots, common to all these Fenton syste ms, were the major oxidation products in each case. Similar Fenton-typ e treatment of the purine dinucleotides dApdG and dApdA resulted in pr oducts that were chromatographically identical on anion-exchange TLC a nd on reverse-phase HPLC to the two major products generated in DNA. T hese 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. Ge neration of the putative intrastrand cross-links increased in a concen tration-dependent manner up to 1 mM cobalt, nickel, or chromium(VI) io ns. However, in experiments with copper, iron, or vanadium ions, maxim um levels were obtained at 250, 150, and 150 mu M, respectively, and t he yield declined with higher concentrations of these three metal ions . Agarose gel electrophoresis demonstrated extensive DNA strand breaka ge with copper, iron, chromium(III), or vanadium, but not with nickel, chromate(VI), cobalt, cadmium, or zinc Fenton systems. The results de monstrate that generation of the putative intrastrand cross-links and strand breaks in DNA, mediated by Fenton reactions, occurs by independ ent mechanisms.