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 (Fe(2+) or Cu(+) with H(2)O(2) 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.
Département de Médecine nucléaire et Radiobiologie, Faculté de Médecine, 3001 12e Avenue Nord, Université de Sherbrooke, Québec, Canada J1H 5N4.
Francois
F
Samson-Thibault
Guru S
GS
Madugundu
Shanshan
S
Gao
Jean
J
Cadet
J Richard
JR
Wagner
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D010084:Oxidation-Reduction; D011839:Radiation, Ionizing; D053719:Tandem Mass Spectrometry
C045076:Fenton's reagent; D009587:Nitrogen Isotopes; D003596:Cytosine; D004247:DNA; C427203:calf thymus DNA; D006861:Hydrogen Peroxide; D007501:Iron