Abstract
In living tissues under inflammatory conditions, superoxide radicals (O(2)*)) are generated and are known to cause oxidative DNA damage. However, the mechanisms of action are poorly understood. It is shown here that the combination of O(2)* with guanine neutral radicals, G(-H)* in single- or double-stranded oligodeoxyribonucleotides (rate constant of 4.7 +/- 1.0 x 10(8) m(-1) s(-1) in both cases), culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo-7,8-dihydroguanine). The G(-H)* and O(2)* radicals were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of guanine in the 5'-d(CC[2AP]-TCGCTACC) strands and the trapping of the ejected electrons by molecular oxygen (Shafirovich, V., Dourandin, A., Huang, W., Luneva, N. P., and Geacintov, N. E. (2000) Phys. Chem. Chem. Phys. 2, 4399-4408). The addition of Cu,Zn-superoxide dismutase, known to react rapidly with superoxide, dramatically enhances the life-times of guanine radicals from 4 to 7 ms to 0.2-0.6 s in the presence of 5 microm superoxide dismutase. Oxygen-18 isotope labeling experiments reveal two pathways of 8-oxo-7,8-dihydroguanine formation including either addition of O(2)* to the C-8 position of G(-H)* (in the presence of oxygen), or the hydration of G(-H)* (in the absence of oxygen). The formation of the guanine lesions via combination of guanine and superoxide radicals is greatly reduced in the presence of typical antioxidants such as trolox and catechol that rapidly regenerate guanine by the reductive "repair" of G(-H)* radicals. The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed.
Highlights
IntroductionThe mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed
Radicals; Electron Transfer Versus Radical Addition—The direct spectroscopic time-resolved measurements described here demonstrate that in single- and double-stranded oligonucleotides, the combination of G(ϪH)1⁄7 and superoxide radicals is relatively rapid and is characterized by rate constants that are smaller by 1 order of magnitude less than those for the free nucleoside dG(ϪH)1⁄7 and free nucleotide dGMP(ϪH)1⁄7 radicals (Table I)
O2 molecules from the These observations can be combination explained in terms of a competition of two basic processes: (i) radical addition followed by the formation of end products, and (ii) oxidation of O2. by G(ϪH)1⁄7 with the regeneration of G and O2 molecules
Summary
The mechanistic aspects of the radical reactions that either regenerate undamaged guanine in DNA or lead to oxidatively modified guanine bases are discussed. The one-electron oxidation of guanine residues first produces the guanine radical cation The superoxide radical is an important biological intermediate that is formed in living cells [7,8,9] and in ionization reactions that generate electrons that are rapidly captured by dissolved oxygen in aqueous solutions (e.g. Ref. 10). Whenever G(ϪH)1⁄7 radicals in DNA are formed by oxidative one-electron transfer mechanisms followed by deprotonation [3, 4], the reactive combination of G(ϪH)1⁄7 with O2. Neither the rates of such reactions nor the nature of the oxidation products formed have been investigated. Yguanosine 5Ј-monophosphate; 8-oxoGua, 8-oxo-7,8-dihydroguanine; 8-oxodGuo, 8-oxo-7,8-dihydro-2Ј-deoxyguanosine, Iz, 2,5-diamino-4H-imidazolone; dZ, 2,2-diamino-4-[(2-deoxy--D-erythro-pentofuranosyl)amino]2,5-dihydrooxazol-5-one; O2., superoxide radical anion; SOD, superoxide dismutase; MALDI-TOF, matrix-assisted laser desorption/ionization with time-of-flight detection; MS, mass-spectrometry; HPLC, high pressure liquid chromatography; BQ, 1,4-benzoquinone
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