TY - JOUR
T1 - Quantitation of Apurinic/Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts
AU - Chen, Haoqing
AU - Yao, Lihua
AU - Brown, Christina
AU - Rizzo, Carmelo J.
AU - Turesky, Robert J.
N1 - Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/6/4
Y1 - 2019/6/4
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.analchem.9b01351
DO - 10.1021/acs.analchem.9b01351
M3 - Article
C2 - 31055913
AN - SCOPUS:85066811668
SN - 0003-2700
VL - 91
SP - 7403
EP - 7410
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 11
ER -