Hu Chiung-Wen, Chang Yuan-Jhe, Hsu Yu-Wen, Chen Jian-Lian, Wang Tsu-Shing, Chao Mu-Rong
Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan; Department of Family and Community Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan.
Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan.
Free Radic Biol Med. 2016 Dec;101:348-355. doi: 10.1016/j.freeradbiomed.2016.10.505. Epub 2016 Oct 30.
Peroxynitrite is a major oxidizing and nitrating biological agent formed at sites of inflammation. Peroxynitrite can cause DNA damage and is thought to contribute to inflammation-related carcinogenesis. This study describes a sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct determination of peroxynitrite-derived 8-nitroguanine (8-nitroGua) in DNA hydrolysates. This method exhibited a sensitive detection limit of 3 fmol and inter- and intraday imprecision of <10% and was applied to systemically examine the formation and stability of peroxynitrite-derived 8-nitroGua in different DNA substrates under various conditions. The 8-nitroGua formation was maximal at pH 8. The formation rate of 8-nitroGua in different DNA substrates decreased in the order of monodeoxynucleoside>single-stranded DNA>double-stranded DNA. A stability test revealed that the half-life for the depurination of 8-nitroGua from DNA was short and affected by both the temperature and DNA structure. When present in monodeoxynucleoside, the half-life of 8-nitroGua was estimated to be ~6min at 25°C and 2.3h at ~0°C. In single-stranded DNA, the half-life varied from 1.6h at 37°C to 533h at -20°C, whereas the half-life increased from 2.4h at 37°C to 1115h at -20°C in double-stranded DNA. We demonstrated that the measurement of 8-nitroGua in isolated DNA is not practicable because 8-nitroGua is unstable and lost during DNA extraction from cell. Therefore, we suggest that directly detecting cellular 8-nitroGua following nuclear membrane lysis is an alternative measure of the nitrative damage of nucleic acids, accounting for both DNA and RNA lesions within cells.
过氧亚硝酸盐是在炎症部位形成的一种主要的氧化和硝化生物因子。过氧亚硝酸盐可导致DNA损伤，并被认为与炎症相关的致癌作用有关。本研究描述了一种灵敏且可靠的液相色谱-串联质谱（LC-MS/MS）方法，用于直接测定DNA水解产物中过氧亚硝酸盐衍生的8-硝基鸟嘌呤（8-nitroGua）。该方法的检测限灵敏，为3 fmol，日内和日间不精密度均<10%，并被用于系统研究不同DNA底物在各种条件下过氧亚硝酸盐衍生的8-硝基鸟嘌呤的形成和稳定性。8-硝基鸟嘌呤的形成在pH 8时最大。不同DNA底物中8-硝基鸟嘌呤的形成速率按单脱氧核苷>单链DNA>双链DNA的顺序降低。稳定性测试表明，8-硝基鸟嘌呤从DNA上脱嘌呤的半衰期较短，且受温度和DNA结构的影响。当存在于单脱氧核苷中时，8-硝基鸟嘌呤在25°C时的半衰期估计约为6分钟，在约0°C时为2.3小时。在单链DNA中，半衰期从37°C时的1.6小时到-20°C时的533小时不等，而在双链DNA中，半衰期从37°C时的2.4小时增加到-20°C时的1115小时。我们证明，在分离的DNA中测量8-硝基鸟嘌呤是不可行的，因为8-硝基鸟嘌呤不稳定，在从细胞中提取DNA的过程中会丢失。因此，我们建议在核膜裂解后直接检测细胞内的8-硝基鸟嘌呤是核酸硝化损伤的一种替代测量方法，它同时考虑了细胞内的DNA和RNA损伤。
