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 study 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 thymus DNA at physiological pH. No significant amount of 8-OH-dG was generated in the absence of GSH, indicating an important role of GSH in enhancing the reactivity of Ni²⁺ toward lipid hydroperoxide to oxidize dG 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 room temperature under ambient air, dG was converted to 8-OH-dG with a yield of about 0.2%. For dG residues in DNA, 24 hour incubation at 37°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, and ethanol (hydroxyl radical scavenger), implying the involvement of oxygen free radicals in the 8-OH-dG generation process. The metal ion chelators, deferoxamine and EDTA, efficiently inhibited the 8-OH-dG formation. Similar results were obtained for the conversion of dG residues in calf thymus DNA to 8-OH-dG. Electrophoretic assays of DNA strand breaks showed that Ni²⁺ caused DNA double-strand breaks in the presence of t-butyl hydroperoxide and GSH. Because GSH is ubiquitously present in cellular systems at relatively high concentration, and the exposure of cells to Ni²⁺ results in the generation of lipid hydroperoxides, the 8-OH-dG generation and DNA double-strand breaks caused by the reaction 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 Ni²⁺-induced toxicity and carcinogenesis.