HPLC INVESTIGATION ON NI(II)-MEDIATED DNA-DAMAGE IN THE PRESENCE OF T-BUTYL HYDROPEROXIDE AND GLUTATHIONE

By use of HPLC with UV and electrochemical detection, the present stud y demonstrates that reaction of Ni2+ with t-butyl hydroperoxide in the presence of glutathioine (GSH) generates 8-hydroxy-2'-deoxyguanosine (8-OH-dG) from 2'-deoxyguanosine (dG) and from dG residues in calf thy mus DNA at physiological pH. No significant amount of 8-OH-dG was gene rated in the absence of GSH, indicating an important role of GSH in en hancing the reactivity of Ni2+ toward lipid hydroperoxide to oxidize d G or dG residues in DNA. The rate of dG conversion to 8-OH-dG depends on the concentration of the reagents. During a two hour incubation of 0.75 mM dG, 10 mM t-butyl hydroperoxide, 1 mM Ni2+, and 2 mM GSH at ro om temperature under ambient air, dG was converted to 8-OH-dG with a y ield of about 0.2%. For dG residues in DNA, 24 hour incubation at 37 d egrees C yielded 0.1% 8-OH-dG. The 8-OH-dG generation from both dG and dG residues in DNA was inhibited by superoxide dismutase, catalase, a nd ethanol (hydroxyl radical scavenger), implying the involvement of o xygen free radicals in the 8-OH-dG generation process. The metal ion c helators, deferoxamine and EDTA, efficiently inhibited the 8-OH-dG for mation. Similar results were obtained for the conversion of dG residue s in calf thymus DNA to 8-OH-dG. Electrophoretic assays of DNA strand breaks showed that Ni2+ caused DNA double-strand breaks in the presenc e of t-butyl hydroperoxide and GSH. Because GSH is ubiquitously presen t in cellular systems at relatively high concentration, and the exposu re of cells to Ni2+ results in the generation of lipid hydroperoxides, the 8-OH-dG generation and DNA double-strand breaks caused by the rea ction of Ni2+ with lipid hydroperoxides in the presence of GSH may be an important mechanism in Ni2+-induced carcinogenesis. The inhibitory effect of chelators suggests a possible prevention strategy against Ni 2+-induced toxicity and carcinogenesis.