Generation of Guanine鈥揟hymidine Cross-Links in DNA by Peroxynitrite/Carbon Dioxide

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• 作者：Byeong Hwa Yun ; Nicholas E. Geacintov ; Vladimir Shafirovich
• 刊名：Chemical Research in Toxicology
• 出版年：2011
• 出版时间：July 18, 2011
• 年：2011
• 卷：24
• 期：7
• 页码：1144-1152
• 全文大小：956K
• 年卷期：v.24,no.7(July 18, 2011)
• ISSN：1520-5010

文摘

Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO₃^{鈥⑩€?/sup> and 鈥?/sup>NO₂ radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2鈥?deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine鈥搕hymine product G*-T* with a covalent bond between the C8 (G*) and the thymine N3 (T*) atoms (CreanNucleic Acids Res. 2008, 36, 742鈭?55). In this work, we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5鈥?.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well-known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitro-G), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxo-G), and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2鈥?deoxy oligoribonucleotides 5鈥?dGpT and 5鈥?dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and trioligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction monitoring mode. The NIm and 8-nitro-G are the major products formed (0.05% each), and lesser amounts of 8-oxo-G (0.02%) and d(G*pT*) and d(G*-T*) enzymatic digestion products (0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand and intrastrand cross-linked products.}