Quantitation of Apurinic/Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts.
Anal Chem
; 91(11): 7403-7410, 2019 06 04.
Article
en En
| MEDLINE
| ID: mdl-31055913
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Núcleo Celular
/
ADN-(Sitio Apurínico o Apirimidínico) Liasa
/
Riñón
/
Hígado
Límite:
Animals
Idioma:
En
Revista:
Anal Chem
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos