Quantitation of Apurinic/Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts

• Published in: Analytical chemistry (Washington) 2019-06, Vol.91 (11), p.7403-7410
• Main Authors: Chen, Haoqing, Yao, Lihua, Brown, Christina, Rizzo, Carmelo J, Turesky, Robert J
• Format: Article
• Language: English
• Subjects: Apyrimidinic sites; Chemistry; Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Hydroxylamine; In vivo methods and tests; Kidneys; Lesions; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Mustard; Nucleotides; Oxidative stress; Quantitation
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b01351

The apurinic/apyrimidinic (AP) site is a common lesion of DNA damage. The levels of AP sites reported in the literature cover a wide range, which is primarily due to the artifactual generation or loss of AP sites during processing of the DNA. Herein, we have developed a method for quantitating AP sites with a largely reduced level of artifacts by derivatizing AP sites before DNA isolation. A rapid digestion of nuclear protein was performed to minimize enzymatic DNA repair, followed by direct derivatization of AP sites in the nuclear lysate with O-(pyridin-3-yl-methyl)­hydroxylamine, yielding an oxime derivative that is stable through the subsequent DNA processing steps. Quantitation was done using highly selective and sensitive liquid chromatography–tandem mass spectrometry, with a limit of quantitation at 2.2 lesions per 108 nucleotides (nts, 0.9 fmol on column). The method was applied in vivo to measure AP sites in rats undergoing oxidative stress [liver, 3.31 ± 0.47/107 nts (dosed) vs 0.91 ± 0.06/107 nts (control); kidney, 1.60 ± 0.07/107 nts (dosed) vs 1.13 ± 0.12/107 nts (control)]. The basal AP level was significantly lower than literature values. The method was also used to measure AP sites induced by the chemotherapeutic nitrogen mustard in vitro.