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.
Bibliographical noteFunding Information:
This work is funded by the National Cancer Institute (P01 CA160032) and National Center for Advancing Translational Sciences of the National Institutes of Health Grant UL1TR000114. Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota, funded in part by Cancer Center Support Grant CA 077598.