In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.
Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons. Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.
Huang
D
D
Department of Neurosurgery, Baylor College of Medicine, Houston, Texas 77030, USA.
Shenoy
A
A
Cui
J
J
Huang
W
W
Liu
P K
PK
D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000737:chemistry; D001253:Astrocytes / Q000737:chemistry; D002545:Brain Ischemia / Q000378:metabolism*; D016923:Cell Death; D002540:Cerebral Cortex / Q000737:chemistry; D004249:DNA Damage; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006651:Histocytochemistry / Q000379:methods*; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009249:NADP / Q000302:isolation & purification; D009474:Neurons / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D018384:Oxidative Stress / Q000502:physiology*; D016548:Prosencephalon / Q000737:chemistry; D015427:Reperfusion Injury / Q000378:metabolism*
D009249:NADP; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III