Absence of 2′-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations
Absence of 2′-deoxyoxanosine and presence of abasic sites in DNA exposed to nitric oxide at controlled physiological concentrations
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DOI：
10.1021/tx034046s
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发表时间：
2003-09-01
影响因子：
4.1
通讯作者：
Dedon, PC
中科院分区：
文献类型：
--
作者：
Dong, M;Wang, C;Dedon, PC
一氧化氮（NO.）在低浓度下是一种重要的生理分子，而高浓度则与慢性炎症（如癌症）相关疾病的病理生理学有关。然而，一项广泛的体外研究表明，氧化和亚硝化反应主导了NO的复杂化学反应。DNA损伤的频谱及其在体内的后果都没有严格的定义。我们已经接近了这个问题，主要努力来定义由NO产生的亚硝化DNA损伤的光谱。生物条件下衍生的活性氮种。质粒pUC19 DNA暴露于稳态浓度为1.3 muM NO的环境中。190 mma O-2（计算稳态浓度为40 mmn2o3和3 pM NO2）。（在散装溶液中）在最近开发的反应器中，避免了不希望的NO气相化学反应。近似于组织炎症部位的情况。通过质粒拓扑异构体分析和新型LC/MS分析，分别确定了亚硝化诱导的碱基和核碱基脱胺产物的光谱。LC/MS分析显示，在50杯DNA中，2'-脱氧黄嘌呤（dX）、2'-脱氧肌苷（dI）和2'-脱氧尿苷（dU）以几乎相同的速率形成(k = 1.2 x 10(5) M-1 s(-1))，暴露于NO 12小时后，每106 nt形成80个类似的病变。在反应堆里。虽然与HNO2的反应会产生高水平的2'-脱氧氧苷（dO），但在500杯暴露于NO的DNA中没有检测到dG脱氨产生的两种产物之一dO。在反应器中放置24小时(
Nitric oxide (NO.) is a physiologically important molecule at low concentrations, while high levels have been implicated in the pathophysiology of diseases associated with chronic inflammation, such as cancer. While an extensive study in vitro suggests that oxidative and nitrosative reactions dominate the complicated chemistry of NO.-mediated genotoxicity, neither the spectrum of DNA lesions nor their consequences in vivo have been rigorously defined. We have approached this problem with a major effort to define the spectrum of nitrosative DNA lesions produced by NO.-derived reactive nitrogen species under biological conditions. Plasmid pUC19 DNA was exposed to steady state concentrations of 1.3 muM NO. and 190 muM O-2 (calculated steady state concentrations of 40 fM N2O3 and 3 pM NO2. in the bulk solution) in a recently developed reactor that avoids the undesired gas phase chemistry of NO. and approximates the conditions at sites of inflammation in tissues. The resulting spectrum of nitrosatively induced abasic sites and nucleobase deamination products was defined using plasmid topoisomer analysis and a novel LC/MS assay, respectively. With a limit of detection of 100 fmol and a sensitivity of 6 lesions per 10(7) nt in 50 mug of DNA, the LC/MS analysis revealed that 2'-deoxyxanthosine (dX), 2'-deoxyinosine (dI), and 2'-deoxyuridine (dU) were formed at nearly identical rates (k = 1.2 x 10(5) M-1 s(-1)) to the extent of similar to80 lesions per 106 nt after 12 h exposure to NO. in the reactor. While reactions with HNO2 resulted in the formation of high levels of 2'-deoxyoxanosine (dO), one of two products arising from deamination of dG, dO, was not detected in 500 mug of DNA exposed to NO. in the reactor for up to 24 h (