title: Comprehensive analysis of the formation and stability of peroxynitrite-derived 8-nitroguanine by LC-MS/MS: Strategy for the quantitative analysis of cellular 8-nitroguanine.
abstract: 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.
authors.affiliation: 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.
authors.forename: Chiung-Wen
authors.initials: CW
authors.lastname: Hu
authors.affiliation: Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan.
authors.forename: Yuan-Jhe
authors.initials: YJ
authors.lastname: Chang
authors.affiliation: Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan; Department of Optometry, Da-Yeh University, Changhua 515, Taiwan.
authors.forename: Yu-Wen
authors.initials: YW
authors.lastname: Hsu
authors.affiliation: School of Pharmacy, China Medical University, Taichung 404, Taiwan.
authors.forename: Jian-Lian
authors.initials: JL
authors.lastname: Chen
authors.affiliation: Department of Biomedical Sciences, Chung Shan Medical University, Taichung 402, Taiwan.
authors.forename: Tsu-Shing
authors.initials: TS
authors.lastname: Wang
authors.affiliation: Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan. Electronic address: chaomurong@gmail.com.
authors.forename: Mu-Rong
authors.initials: MR
authors.lastname: Chao
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003412:Cricetulus; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D042783:Endothelial Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D057230:Limit of Detection; D015588:Observer Variation; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: C095838:8-nitroguanine; D030421:Peroxynitrous Acid; D006147:Guanine; D004247:DNA; C427203:calf thymus DNA
keywords: Calf thymus DNA; Half-life; LC-MS/MS; Peroxynitrite; Stability