Thymidine decomposition induced by low-energy electrons and soft X rays under N2 and O2 atmospheres

12 pags.; 3 figs.; 2 tabs.; 1 schemeA novel technique has been employed to investigate the simultaneous damage to DNA components induced by soft X rays (1.5 keV) and low-energy electrons (0-30 eV) in thin films of thymidine deposited on glass and tantalum substrates and irradiated under atmospheric pressure and temperature. The films were surrounded by either an N2 or O2 environment. The formation of four radiation-induced products is reported in this article: base release, 5-hydroxymethyl-2′-deoxyuridine (5-HMdUrd), 5-formyl-2′- deoxyuridine (5-FordUrd) and 5,6-dihydrothymidine (5,6-DHThd). Analysis with LC-MS/MS shows larger damage yields in the samples deposited on tantalum than in those deposited on glass, which is attributed to the interaction of the additional low-energy electrons that are photoemitted from the metal surface. From a comparison of the results obtained from N2 and O2 environment, we report a dramatic effect from 6 O2: an approximately threefold increase in the yield of products, attributed to the reaction of O2 with initial carbon-centered thymidine radicals generated in the film during irradiation. © 2014 by Radiation Research Society.A. G. S. and G. G. acknowledge partial support from the Spanish Ministerio de Economiay Competitividad (Project FIS2009-10245) and the EU COST program (Action MP1002 Nano-IBCT).Peer Reviewe

Radiation-Induced Formation of 2′,3′-Dideoxyribonucleosides in DNA: A Potential Signature of Low-Energy Electrons

We have identified a series of modifications of the 2′-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. The modifications consist of 2′,3′-dideoxyribonucleoside derivatives of T, C, A, and G, as identified by enzymatic digestion and LC-MS/MS. Under dry conditions, the yield of these products was 6- to 44-fold lower than the yield of 8-oxo-7,8-dihydroguanine. We propose that 2′,3′-dideoxyribonucleosides are generated from the reaction of low-energy electrons with DNA, leading to cleavage of the C3′–O bond and formation of the corresponding C3′-deoxyribose radical

Profiling Cytosine Oxidation in DNA by LC-MS/MS

Spontaneous and oxidant-induced damage to cytosine is probably the main cause of CG to TA transition mutations in mammalian genomes. The reaction of hydroxyl radical (·OH) and one-electron oxidants with cytosine derivatives produces numerous oxidation products, which have been identified in large part by model studies with monomers and short oligonucleotides. Here, we developed an analytical method based on LC-MS/MS to detect 10 oxidized bases in DNA, including 5 oxidation products of cytosine. The utility of this method is demonstrated by the measurement of base damage in isolated calf thymus DNA exposed to ionizing radiation in aerated aqueous solutions (0–200 Gy) and to well-known Fenton-like reactions (Fe²⁺ or Cu⁺ with H₂O₂ and ascorbate). The following cytosine modifications were quantified as modified 2′-deoxyribonucleosides upon exposure of DNA to ionizing radiation in aqueous aerated solution: 5-hydroxyhydantoin (Hyd-Ura) > 5-hydroxyuracil (5-OHUra) > 5-hydroxycytosine (5-OHCyt) > 5,6-dihydroxy-5,6-dihydrouracil (Ura-Gly) > 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (Imid-Cyt). The total yield of cytosine oxidation products was comparable to that of thymine oxidation products (5,6-dihydroxy-5,6-dihydrothymine (Thy-Gly), 5-hydroxy-5-methylhydantotin (Hyd-Thy), 5-(hydroxymethyl)­uracil (5-HmUra), and 5-formyluracil (5-ForUra)) as well as the yield of 8-oxo-7,8-dihydroguanine (8-oxoGua). The major oxidation product of cytosine in DNA was Hyd-Ura. In contrast, the formation of Imid-Cyt was a minor pathway of DNA damage, although it is the major product arising from irradiation of the monomers, cytosine, and 2′-deoxycytidine. The reaction of Fenton-like reagents with DNA gave a different distribution of cytosine derived products compared to ionizing radiation, which likely reflects the reaction of metal ions with intermediate peroxyl radicals or hydroperoxides. The analysis of the main cytosine oxidation products will help elucidate the complex mechanism of oxidative degradation of cytosine in DNA and probe the consequences of these reactions in biology and medicine

Generation of Guanine–Thymine Cross-Links in Human Cells by One-Electron Oxidation Mechanisms

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [¹⁵N, ¹³C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ∼0.21 and 1.19 d­(G*-T*) lesions per 10⁶ DNA bases at laser intensities of 50 and 280 mJ/cm²/pulse, respectively