In situdetection of AP sites and DNA strand breaks bearing 3'‐phosphate termini in ischemic mouse brain

Abstract

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.

Increased Cytochrome c–Mediated DNA Fragmentation and Cell Death in Manganese–Superoxide Dismutase–Deficient Mice After Exposure to Subarachnoid Hemolysate

“…32 Reactive oxygen species have been implicated in both pathways. 12,17,18,25,37 As described above, it was possible that superoxide in the presence of the iron carried by heme may have formed the reactive hydroxyl radical as a mediator of cell death. 36 Another possible route to cell death and DNA damage may have been through formation of peroxynitrite from nitric oxide and superoxide.…”

“…36 The hydroxyl radical and oxidative stress have been shown to mediate DNA damage. 8,17,18 Decreased levels of Mn-SOD may have permitted greater superoxide production and, in the presence of iron, greater hydroxyl radical formation.…”

“…36 Another possible route to cell death and DNA damage may have been through formation of peroxynitrite from nitric oxide and superoxide. 12,17,38 Peroxynitrite may damage cells through the formation of the hydroxyl radical and nitrogen dioxide, which may attack endothelial membranes. 38 Diminished levels of Mn-SOD may have permitted increased formation of peroxynitrite, which would ultimately have led to DNA damage, DNA fragmentation, and cell death.…”

“…[5][6][7][8][9][10] Oxidative stress in brain has been associated with superoxide production, mutagenesis of nuclear genes, irreversible and reversible DNA damage, and mitochondrial injury. [11][12][13][14][15][16][17][18] Cytochrome c, a mitochondrial membrane protein, functions in the respiratory chain to transport electrons from coenzyme QH 2 -cytochrome c reductase to the cytochrome oxidase. 19 Recent attention has focused on its relationship to DNA fragmentation.…”

Noninvasive delivery of gene targeting probes to live brains for transcription MRI

“…Although BBB leakage after CSD may be short-lived (approximately 1 wk), such minor and transient disruption is sufficient to allow SPION retention and MR probe uptake in brain cells. On the other hand, the GCI model of BCAO is an acute neurological disorder thought to simulate the neurological conditions associated with cardiac arrest; it seldom induces necrosis but elicits oxidative DNA damage and apoptotic DNA fragmentation (27)(28)(29). Several reports have described how GCI induces BBB leakage in rat and mouse brains (30; Savitz et al, unpublished data); we report here that BBB leakage in a C57BL6 mouse GCI model can be detected as early as 10 h and as late as 9 wk after BCAO when assessing Gd retention.…”