Profiling Cytosine Oxidation in DNA by LC-MS/MS

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• 作者：Francois Samson-Thibault ; Guru S. Madugundu ; Shanshan Gao ; Jean Cadet ; J. Richard Wagner
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：September 17, 2012
• 年：2012
• 卷：25
• 期：9
• 页码：1902-1911
• 全文大小：406K
• 年卷期：v.25,no.9(September 17, 2012)
• ISSN：1520-5010

文摘

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 (路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鈥?00 Gy) and to well-known Fenton-like reactions (Fe²⁺ or Cu⁺ with H₂O₂ and ascorbate). The following cytosine modifications were quantified as modified 2鈥?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鈥?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.