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

Chen, Haoqing, Yao, Lihua, Brown, Christina, Rizzo, Carmelo J., Turesky, Robert J.
Analytical chemistry 2019 v.91 no.11 pp. 7403-7410
DNA, DNA damage, DNA repair, derivatization, drug therapy, kidneys, liquid chromatography, liver, nucleotides, oxidative stress, rats, tandem mass spectrometry
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.