Douki T, Delatour T, Bianchini F, Cadet J
CEA/Département de Recherche Fondamentale sur la Matière Condensé, SESAM/LAN, Grenoble, France.
Carcinogenesis. 1996 Feb;17(2):347-53. doi: 10.1093/carcin/17.2.347.
Gas chromatography coupled to electron impact mass spectrometry (GC-EIMS) analysis following hydrolysis of DNA is a widely used assay for the detection of oxidized nucleobases and nucleosides. However, evidence was recently provided for an oxidation of guanine residues of hydrolysed DNA during the silylation prior to GC-EIMS analysis. This reaction accounts for the overestimation of the yield of 8-oxo-7,8-dihydroguanine by GC-EIMS. In the present work, we showed that adenine, cytosine, thymine and thymidine also give rise to oxidized derivatives during the derivatization. This was inferred from the measurement of the amount of 5-formyluracil, 5-hydroxymethyluracil, 5-hydroxycytosine (5-OHCyt), 8-oxo-7,8-dihydroadenine (8-OxoAde) and 5-hydroxymethyl-2'-deoxyuridine (5-HMdUrd) in a series of experiments based on the use of purified bases and nucleosides. Isotopically labelled oxidized bases and 5-HMdUrd were used as internal standards to control the quantitative aspect of the silylation reaction. Support for an artefactual oxidation of hydrolysed DNA was provided by the comparison of the amount of 8-OxoAde and 5-OHCyt detected within native and gamma-irradiated DNA by HPLC-EC and GC-EIMS. To prevent the artefactual formation of oxidized bases during the silylation, an approach based on an HPLC prepurification was developed to remove the precursors of the oxidized bases measured in the DNA sample. The HPLC/GC-EIMS assay was successfully applied to the quantitation of 8-OxoAde and 5-OHCyt in calf thymus DNA. In addition, the detection of the dose-dependent formation of 5-HMdUrd in isolated DNA exposed to ionizing radiation was achieved using the same approach.
DNA水解后通过气相色谱-电子轰击质谱联用（GC-EIMS）分析是检测氧化核苷酸碱基和核苷的一种广泛应用的方法。然而，最近有证据表明，在GC-EIMS分析之前的硅烷化过程中，水解DNA的鸟嘌呤残基会发生氧化。该反应导致GC-EIMS对8-氧代-7,8-二氢鸟嘌呤产率的高估。在本研究中，我们表明腺嘌呤、胞嘧啶、胸腺嘧啶和胸腺嘧啶核苷在衍生化过程中也会产生氧化衍生物。这是通过在一系列基于使用纯化碱基和核苷的实验中测量5-甲酰基尿嘧啶、5-羟甲基尿嘧啶、5-羟基胞嘧啶（5-OHCyt）、8-氧代-7,8-二氢腺嘌呤（8-OxoAde）和5-羟甲基-2'-脱氧尿苷（5-HMdUrd）的量推断出来的。同位素标记的氧化碱基和5-HMdUrd用作内标以控制硅烷化反应的定量方面。通过比较高效液相色谱-电化学检测（HPLC-EC）和GC-EIMS在天然DNA和γ射线辐照DNA中检测到的8-OxoAde和5-OHCyt的量，为水解DNA的人为氧化提供了支持。为了防止硅烷化过程中氧化碱基的人为形成，开发了一种基于HPLC预纯化的方法，以去除DNA样品中测量的氧化碱基的前体。HPLC/GC-EIMS测定法成功应用于小牛胸腺DNA中8-OxoAde和5-OHCyt的定量。此外，使用相同方法实现了在暴露于电离辐射的分离DNA中5-HMdUrd剂量依赖性形成的检测。
