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 degrees 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 Ni(2+)-induced carcinogenesis. The inhibitory effect of chelators suggests a possible prevention strategy against Ni(2+)-induced toxicity and carcinogenesis.
Shi
X
X
Laboratory of Experimental Pathology, National Cancer Institute, Bethesda, Maryland 20892.
Mao
Y
Y
Ahmed
N
N
Jiang
H
H
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002374:Catalase / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D003676:Deferoxamine / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009532:Nickel / Q000494:pharmacology*; D010545:Peroxides / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000494:pharmacology; D020122:tert-Butylhydroperoxide
D010545:Peroxides; D017382:Reactive Oxygen Species; D000431:Ethanol; D009532:Nickel; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D020122:tert-Butylhydroperoxide; D004492:Edetic Acid; D002374:Catalase; D013482:Superoxide Dismutase; D003849:Deoxyguanosine; D005978:Glutathione; D003676:Deferoxamine