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(Stroke. 1999;30:2456-2463.)
© 1999 American Heart Association, Inc.
Original Contributions |
Early Decrease of XRCC1, a DNA Base Excision Repair Protein, May Contribute to DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice
From the Departments of Neurosurgery, Neurology and Neurological Sciences, and Program in Neurosciences, Stanford University School of Medicine, Palo Alto, Calif.
Correspondence to Pak H. Chan, PhD, Neurosurgical Laboratories, Stanford University, 701B Welch Rd, #148, Palo Alto, CA 94304. E-mail phchan{at}leland.stanford.edu
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Abstract |
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Background and Purpose—DNA damage and the DNA repair mechanism are known to be involved in ischemia/reperfusion injury in the brain. The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase ß. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after transient focal cerebral ischemia (FCI).
Methods—Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal blockade of the middle cerebral artery. XRCC1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase–mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). The spatial relationship between XRCC1 expression and DNA damage was examined by double staining with XRCC1 and TUNEL after FCI.
Results—Immunohistochemistry showed the nuclear expression of XRCC1 in all regions of the control brains and that it was predominant in the hippocampus. The XRCC1 level was markedly reduced in the caudate putamen at 10 minutes, further decreased in the entire middle cerebral artery territory at 1 hour, and remained reduced until 4 and 24 hours after FCI. Western blot analysis of the normal control brain showed a characteristic band of 70 kDa, which decreased after FCI. A significant amount of DNA fragmentation was detected by DNA gel electrophoresis 24 hours but not 4 hours after FCI. Double staining showed that the neurons that lost XRCC1 immunoreactivity became TUNEL positive.
Conclusions—These results suggest that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA fragmentation after FCI.
Key Words: apoptosis • cerebral ischemia, focal • DNA fragmentation • DNA repair
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The DNA protein x-ray repair cross-complementing group 1 (XRCC1) plays a central role in the DNA base excision repair (BER) pathway, which is responsible for repairing damage caused by various kinds of insults, including oxidative stress, by interacting with DNA ligase III and with DNA polymerase ß.1 2 3 4 5 6 The critical role of XRCC1 in the cellular response to DNA damage was found when the repair defects of the mutant Chinese hamster ovary cell lines were corrected by transfection with the human XRCC1 gene.1 Such a role was confirmed in 4 other mutant cell lines lacking the XRCC1 protein.6 Although no catalytic domain was found in XRCC1 itself, it was reported to interact with DNA ligase III by its N-terminal domain containing a BRCA1 C-terminus (BRCT) module, which is a widespread motif in DNA repair and DNA damage–responsive cell cycle checkpoint proteins.3 5 7 XRCC1 also interacts with DNA polymerase ß by its N-terminal half of the XRCC1 protein.3 In response to DNA-damaging agents, a preferential decrease in XRCC1 mRNA was found in vitro, suggesting its link to DNA damage.8 The predominant expression of XRCC1 mRNA and/or protein was found in rat brains8 and in baboon brains.9 However, detailed cellular/subcellular distribution of XRCC1 in the central nervous system and its role in pathophysiological conditions in vivo are totally unknown.
DNA damage and repair are drawing more attention in the field of acute central nervous system injury, including cerebral ischemia and brain trauma.10 Liu and colleagues11 have suggested that free radicals could attack the nuclear genes and cause genetic instability after mouse forebrain ischemia. As for the relationship of the DNA BER proteins to DNA damage after ischemia, we have shown that a decrease in apurinic/apyrimidinic endonuclease (APE/Ref-1) DNA BER protein preceded the occurrence of DNA fragmentation in the entire ischemic lesion after transient focal cerebral ischemia (FCI)12 and that loss of APE/Ref-1 was closely associated with the occurrence of DNA fragmentation in hippocampal CA1 neurons after transient global ischemia.13 These facts suggest that the reduction of DNA BER protein and a failure of the DNA repair mechanism may contribute to DNA damage after ischemia/reperfusion.
To further investigate the relationship between DNA BER protein expression and DNA damage after ischemia, we analyzed the expression of the XRCC1 protein, which is known to play a central role in DNA BER, before and after transient FCI. We further sought to clarify both temporal and anatomic relationships between XRCC1 alteration and the occurrence of DNA fragmentation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Focal Cerebral Ischemia
Adult male CD-1 mice (weight, 35 to 40 g) were subjected to transient focal ischemia by intraluminal middle cerebral artery (MCA) blockade with a nylon suture, as described.14 The mice were anesthetized with 2.0% isoflurane in 30% oxygen and 70% nitrous oxide with the use of a face mask. The rectal temperature was controlled at 37°C with a homeothermic blanket. Cannulation of a femoral artery allowed the monitoring of blood pressure and arterial blood gases, with samples for analysis taken immediately after cannulation, 10 minutes after occlusion, and 10 minutes after reperfusion. After the midline skin incision, the left external carotid artery was exposed, and its branches were electrocoagulated. An 11.0-mm 5-0 surgical monofilament nylon suture, blunted at the end, was introduced into the left internal carotid artery through the external carotid artery stump. After 60 minutes of MCA occlusion, blood flow was restored by the withdrawal of the nylon suture.
Immunohistochemistry of XRCC1
Anesthetized animals were perfused with 10 U/mL heparin
and subsequently with 4% formaldehyde in 0.1 mol/L PBS (pH 7.4) after
10 minutes, 1 hour, 4 hours, and 24 hours of reperfusion after
ischemia. Brains were removed, postfixed for 12 hours in 4%
formaldehyde, sectioned at 50 µm on a vibratome, and processed
for immunohistochemistry. As a negative control, sections were
incubated without primary antibodies. For histological
assessment, alternate slices from each brain section were stained with
cresyl violet. The sections were first reacted with mouse monoclonal
antibody against XRCC1 (Lab Vision) at a dilution of 1:100. To
avoid cross-reaction between the secondary antibody and mouse
immunoglobulin in the tissue, immunohistochemistry was performed with
the use of the DAKO ARK peroxidase kit. After development with
diaminobenzidine (DAB), the sections were mounted on slides and
counterstained with methyl green solution.
Western Blot Analysis
Whole cell protein extraction was performed. To confirm the
early reduction of XRCC1 expression in the ischemic brain, we
used the samples obtained from the ischemic core on the
ipsilateral side and homologous tissue from the contralateral side.
Approximately 30 mg of both ipsilateral striatum and homologous tissue
from the contralateral side was cut into pieces after 4 hours of
reperfusion and put into 10x volume of Tris-glycine SDS sample buffer
(Novex). Samples were then gently homogenized 20x in a
Teflon Dounce homogenizer (Wheaton). Equal amounts of
the samples (10 µL) were loaded per lane. The primary antibodies were
either 1:1000 dilution of monoclonal antibody against XRCC1 (Lab
Vision) or 1:10 000 dilution of anti–ß-actin monoclonal antibody
(Sigma). For XRCC1 detection, Western blots were performed with
horseradish peroxidase–conjugated anti-mouse immunoglobulin G with the
Boehringer Mannheim chemiluminescent system. As the internal
control, Western blot analysis of ß-actin was performed with
horseradish peroxidase–conjugated anti-mouse immunoglobulin G reagents
(Amersham International).
Gel Electrophoresis
Animals were killed 4 and 24 hours after 60 minutes of MCA
occlusion. Thirty to 50 mg wet weight of ischemic tissue was
taken from the third 2-mm section along with homologous tissue from the
contralateral side after the brain was cut coronally. Samples were
incubated overnight in 0.6 mL lysis buffer (0.5% SDS, 10 mmol/L
Tris-HCl, and 0.1 mol/L EDTA) with 0.6 mg proteinase K
(Boehringer Mannheim) at 55°C. The DNA was extracted with
equal volumes of phenol and phenol-chloroform-isoamyl alcohol (25:24:1)
and precipitated overnight in 0.2 mol/L sodium chloride in 100%
ethanol at -80°C. The DNA was washed with 75% ethanol 2 times, air
dried, and resuspended in DNase-free water (Sigma). The DNA
concentration was measured with To-Pro-1 dye (Molecular Probes). Gel
electrophoresis for detecting DNA laddering was performed according to
the manufacturer’s instructions (Trevigen). Before electrophoresis, 1
µg of DNA was incubated with 50 µg/mL of DNase-free RNase
(Boehringer Mannheim) for 30 minutes at 37°C. Then the
samples were reacted with Klenow enzyme (Trevigen) and dNTP (Trevigen)
in 1x Klenow buffer (Trevigen) for 10 minutes at room temperature.
Samples were mixed with loading buffer and subjected to electrophoresis
on 1.5% agarose gel. Then the gel was washed with 0.25 mol/L HCl, 0.4
mol/L NaOH/0.8 mol/L NaCl, and 0.5 mol/L Tris buffer (pH 7.5). DNA was
transferred to a nylon membrane overnight in 10x SSC. The membrane was
first blocked with 5% powdered milk (BioRad) in PBS for 30 minutes and
incubated with streptavidin–horseradish peroxidase conjugate
(Trevigen) for 30 minutes. Finally, the bands were visualized by the
chemiluminescence method with the use of PeroxyGlow (Trevigen), and the
films were exposed to x-ray film.
In Situ Labeling of DNA Fragmentation
Frozen brain sections at the level of the caudate putamen that
showed typical infarction were stained by an in situ technique
(terminal deoxynucleotidyl transferase–mediated
uridine 5'-triphosphate-biotin nick end-labeling [TUNEL] reaction) to
detect the DNA free 3'-OH ends, as described.15 Briefly,
frozen brain sections were fixed for 30 minutes in 3.7% formaldehyde
in 0.1 mol/L PBS, pH 7.4. The slides were placed in 1x terminal
deoxynucleotidyl transferase (TdT) buffer (Life
Technologies) for 15 minutes, followed by reaction with TdT enzyme
(Life Technologies) and biotinylated 16-dUTP (Boehringer
Mannheim) at 37°C for 60 minutes. The slides were then washed 2x in
SSC (150 mmol/L sodium chloride, 15 mmol/L sodium citrate, pH
7.4) for 15 minutes, followed by a washing in PBS 2x for 15 minutes.
Avidin-biotin horseradish peroxidase solution (ABC kit, Vector
Laboratories) was applied to the sections for 30 minutes, then the
slides were washed for 15 minutes with 0.175 mol/L sodium acetate.
Staining was visualized with the use of 0.025% DAB and 0.075%
H2O2 in PBS with 0.4 mg/mL
nickel sulfate. The slides were rinsed with water, stained with methyl
green for 10 minutes, dehydrated, and mounted.
Double Labeling With XRCC1 and DNA Fragmentation
To clarify the spatial relationship between XRCC1 expression and
DNA damage, we performed double staining of XRCC1 antibody and TUNEL,
as previously described.13 After transcardiac
perfusion, fixed sections were immunostained with XRCC1
antibody as described above, the sections were mounted on glass slides,
passed through ethanol (70%, 95%, and 100%), and then immersed in
chloroform for 5 minutes. The sections were rehydrated by passage
through a decreasing ethanol series, rinsed with water, and processed
to TUNEL staining as described above. Staining was visualized with the
use of 0.025% DAB and 0.075%
H2O2 with nickel sulfate.
The slides were rinsed with water, stained with methyl green for 10
minutes, dehydrated, and mounted.
Quantification and Statistical Analysis
The number of XRCC1-immunoreactive cells and methyl
green–positive cells was counted in a high-power field (x400) and
expressed by the percentage of XRCC1-positive cells. The number of
TUNEL-positive cells was also counted, as previously
described.15 The quantitative analysis of these
cells was evaluated by factorial ANOVA between each group. Significance
between groups was assigned at P<0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Physiological Data and Cerebral Infarction
Physiological parameters showed no significant differences in mean arterial blood pressure and arterial blood gas analysis between each time point (Table
). The
time-dependent increase of infarction in the mouse brains with the use
of intraluminal suture blockade is consistent with previous
reports that employed the same focal stroke model in
mice.14 15
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Immunohistochemistry Showed the Constitutive Expression of XRCC1 in
Normal Adult Mouse Brain, Which Decreases After Ischemia
The XRCC1 protein was constitutively expressed in the entire
region of the normal mouse brain (Figure 1A through 1D). It was mainly expressed
in the nucleus and had a regional predominance in the hippocampus,
including the CA1 (Figure 1C) and CA3 regions and the dentate
gyrus (Figure 1D), compared with the MCA territory cortex
(Figure 1A) and caudate putamen (Figure 1B). After 10
minutes of reperfusion that followed 1 hour of FCI, reduction of XRCC1
was observed in the lateral caudate putamen (Figure 1E) but not
in the cortex (data not shown). After 1 hour of reperfusion, XRCC1
expression was reduced in the entire MCA territory, including the
caudate putamen (Figure 1F) and cortex (data not shown). This
reduction was sustained at 4 hours (Figure 1G) and 24 hours
(Figure 1H) in the caudate putamen, as well as in the MCA
territory cortex. In contrast, the contralateral brain including the
caudate putamen (Figure 1I) showed no significant alteration of
XRCC1 expression until 24 hours after FCI. There was no
immunoreactivity in the control specimens, which were treated without a
primary antibody (data not shown).
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Western Blot Analysis of XRCC1 Protein Expression After
Transient MCA Occlusion
XRCC1 immunoreactivity was evident as a band of molecular mass 70
kDa of the whole cell fraction from the nonischemic hemisphere
(Figure 2, lane 1, top panel) and was
significantly decreased 4 hours after ischemia (Figure 2, lane 2). A consistent amount of ß-actin
immunoreactivity between each lane is shown in the bottom panel,
suggesting that the amount of the loaded protein was
consistent. These data not only confirm the specificity of the
antibody for XRCC1 used in this study but also suggest that XRCC1
decreased after transient focal ischemia.
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DNA Laddering Was Detected by Genomic DNA Gel
Electrophoresis
To detect the occurrence of apoptosis as
characterized by intranucleosomal DNA fragmentation, we
analyzed DNA from both the ischemic brain and the
homologous sample on the contralateral side. DNA laddering was absent
in both the control tissue and ischemic tissue 4 hours after
ischemia (Figure 3, lanes C and
4). A significant amount of DNA laddering was detected 24 hours after
ischemia (Figure 3, lane 24).
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Early Decrease of XRCC1 Preceded the Occurrence of DNA
Fragmentation Detected by TUNEL Staining After FCI
To elucidate the spatial and temporal profile of DNA
fragmentation, we examined in situ labeling of DNA breaks in the
infarcted brain sections (Figure 4), as
described.15 TUNEL staining did not label normal neuronal
cells in the noninfarcted area (Figure 4A) or the cells in the
infarcted area 4 hours after ischemia (Figure 4B). In
contrast, 2 different patterns of staining were observed in the
neuronal cells in the infarcted area 24 hours after ischemia
(Figure 4C). Some neuronal cells in the infarcted area were
densely labeled in their nuclei, accompanied by small particles around
the nuclei that resembled apoptotic bodies (Figure 4C, arrows). These cells are compatible with those in the apoptotic
cell death process, as previously described.15 16 17 Besides
these typical apoptotic neuronal cells, slightly TUNEL-stained
cells were also observed (Figure 4C, arrowheads). These cells
showed diffuse nuclear and cytoplasmic TUNEL staining, which is
consistent with necrotic cells.15 16 17 To quantify
the apoptotic neurons, the number of TUNEL-positive cells was
counted 4 and 24 hours after FCI. As illustrated in Figure 5, only a small number of TUNEL-positive
cells were detected 4 hours after ischemia. They were
significantly increased 24 hours after ischemia both in the
caudate putamen (Figure 5A; P<0.0001) and the cortex
(Figure 5B; P<0.01). As shown in Figure 5, the reduction of XRCC1 expression preceded the occurrence of DNA
fragmentation, which was detected by gel electrophoresis and TUNEL at
24 hours but not at 4 hours after FCI. In addition, there was no
significant difference in the number of XRCC1-positive cells between
nonischemic controls and the contralateral side after FCI (data
not shown).
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Double Staining With XRCC1 and TUNEL Showed the Spatial
Relationship Between XRCC1 Loss and DNA Damage
Double staining with XRCC1 and TUNEL 24 hours after
ischemia showed a significant amount of TUNEL-positive cells
with the characteristic features of apoptosis (densely labeled
in their nuclei, accompanied by apoptotic bodies) in the entire
MCA territory on the ipsilateral side, including the cortex (Figure 6A, arrowheads). None of these cells
showed XRCC1 immunoreactivity. On the other hand, some cells had a
faint expression of XRCC1 (Figure 6A, asterisks), but none were
TUNEL positive. No TUNEL-positive cells were seen in the contralateral
side at this time point (Figure 6B). There was no
immunoreactivity in the control specimens, which were treated without a
primary antibody (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The present study provides the first evidence that the early decrease of the DNA BER protein XRCC1, which is constitutively expressed in normal mouse brain, may play a role, at least in part, in the DNA-damaged cell death pathway in transient FCI. The immunohistochemistry of the normal mouse brain showed for the first time that the XRCC1 protein was constitutively expressed in the entire mouse brain and has a nuclear distribution and a regional predominance in the hippocampus. As early as 10 minutes after reperfusion, a significant reduction of the XRCC1 expression was seen in the lateral caudate putamen (Figure 1E
), extended to the entire MCA
territory after 1 hour of reperfusion (Figure 1F), and remained
reduced until 4 and 24 hours after transient FCI. This reduction was
confirmed by Western blot analysis of whole cell extract
(Figure 2). DNA fragmentation was not detected 4 hours after
FCI, while it markedly appeared 24 hours after FCI, as shown by DNA gel
electrophoresis (Figure 3) and TUNEL staining (Figure 4).
Finally, the spatial relationship between the loss of XRCC1 expression
and DNA fragmentation was shown by the double staining with XRCC1 and
TUNEL (Figure 6A). These results indicate that early reduction
of XRCC1 precedes the occurrence of DNA fragmentation in the
ischemic MCA territory that is destined to show necrosis and/or
apoptosis and that the reduction of XRCC1 may contribute to
DNA-damaged cell death after FCI. Although XRCC1 has no known catalytic
activity for undergoing DNA repair, it is believed to play a central
role in the DNA BER pathway by interacting with DNA polymerase ß and
DNA ligase III.1 2 3 4 5 6 Furthermore, using the same FCI model,
we previously reported that the DNA BER enzyme APE/Ref-1 was reduced in
the entire MCA territory on the ischemic side before the peak
of DNA fragmentation.12 Thus, it is suggested that the
tissue in the ischemic MCA is deficient in at least 3 major BER
enzymes, such as APE/Ref-1, DNA polymerase ß, and DNA ligase III,
which can be extremely detrimental for the cells in overcoming DNA
damage caused by ischemia/reperfusion injury. Programmed cell death and the DNA-repairing mechanism are both assumed to play important roles in cerebral ischemia.10 13 15 16 18 19 However, little is known about the interaction of these processes, except the data suggesting a link between apoptosis and the cleavage of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP).20 The interleukin-1ß–converting enzyme family caspases are the human homologues of the nematode Caenorhabditis elegans, Ced-3,21 and are considered to play a critical role in programmed cell death.20 One substrate for caspase is PARP, whose proteolytic cleavage results in a dysfunctional PARP that is unable to contribute to repair or genomic maintenance.20 Furthermore, the Ca2+/Mg2+-dependent endonuclease that generates internucleosomal DNA cleavage characteristic of apoptosis is negatively regulated by poly(ADP)-ribosylation. Therefore, inactivation of PARP could increase DNA cleavage and contribute to programmed cell death. On the other hand, excessive activation of PARP is believed to also be deleterious because it might cause energy depletion and ultimate cell death.22 In fact, most recent studies with PARP knockout mice implicate the deleterious role of PARP in FCI.23 24 Although these studies did not provide the data that suggested its role in DNA damage, they clearly show the marked reduction of the infarct volume in PARP knockout mice after transient cerebral ischemia,23 24 suggesting that PARP activation plays a detrimental role in neuronal damage after ischemia. Interestingly, a recent study showed that XRCC1 interacts with PARP by its central region containing a BRCT module.7 In this study, overexpression of XRCC1 in Cos-7 and HeLa cells resulted in the marked decrease of PARP activity in vitro, which suggests that XRCC1 may negatively regulate the activity of PARP. On the basis of these findings, the early reduction of XRCC1 during reperfusion in the present study could contribute to the early activation of PARP and subsequent energy depletion and then could be extremely detrimental for the cells in overcoming ischemia/reperfusion injury.
The selective reduction of XRCC1 after FCI was implied by our
following findings. Western blot analysis showed the
significant loss of XRCC1 4 hours after FCI (Figure 2, top
panel), while there was no alteration of ß-actin after FCI (Figure 2, bottom panel). Furthermore, immunohistochemistry showed the
marked reduction of XRCC1 in the entire ischemic area 1 hour
after FCI (Figure 1), when the immunoreactivity of the nuclear
neuronal marker NeuN showed much less alteration in the
ischemic area (data not shown). The exact mechanism by which
the selective reduction of XRCC1 occurs after FCI is unclear. It is
conceivable that XRCC1 reduction is caused by a selective decrease of
XRCC1 protein synthesis, an increase of protein degradation, a
difference in the half-life of the protein, or a difference in
posttranslational regulation. Further examination is warranted to
clarify this issue. Nevertheless, we have previously reported that
APE/Ref-1, another DNA BER protein, decreased rapidly after focal
cerebral ischemia/reperfusion in mice,12 and our
preliminary study using the same stroke model demonstrates that mice
overexpressing the endogenous antioxidant enzyme superoxide
dismutase-1 (SOD1) show less reduction of APE/Ref-1 after
ischemia/reperfusion compared with wild-type
mice.25 Therefore, it is conceivable that the
mechanism of the selective loss of XRCC1 could also be linked to
oxidative stress. In fact, our transient FCI model is known to be
associated with reactive oxygen species, as shown by our previous study
using SOD1 transgenic mice and knockout mice deficient in
SOD1.14 15 26 27 28
With regard to programmed cell death after ischemia, we have reported that mitochondrial cytochrome c was released to the cytosol after transient FCI in rats17 and in mice.29 Since translocated cytochrome c is known to be apoptogenic because of its role in caspase activation and subsequent DNA fragmentation,30 these results suggest that cytosolic release of cytochrome c may cause downstream caspase activation and subsequent DNA fragmentation after FCI. Consistent with this finding, the activation and cleavage of caspase 3 has been shown after FCI in mice,31 and inhibition of caspase family proteases resulted in significant protection against focal ischemia/reperfusion injury.32 From the point of oxidative stress and programmed cell death, our recent study showed that a 50% reduction of manganese SOD in knockout mice resulted in a marked increase of cytochrome c release and subsequent DNA fragmentation after FCI,29 suggesting that mitochondrial production of reactive oxygen species may contribute to the release of mitochondrial cytochrome c, thereby exacerbating DNA fragmentation after FCI. The correlation between the mitochondrial cascade and the postischemic modification of DNA repair proteins such as XRCC1 and APE/Ref-1 is totally unclear. Since it is well known that reperfusion increases mitochondrial production of reactive oxygen species,28 mitochondrial oxidative stress could contribute indirectly to the early decrease of DNA BER proteins. The future examination of DNA BER using mutant mice deficient in and/or overexpressing mitochondrial manganese SOD is warranted to address this important issue.
In conclusion, we have shown that XRCC1 rapidly decreased after transient FCI in mice and that this reduction preceded the occurrence of ischemic apoptosis and infarction. Furthermore, a spatial relationship between XRCC1 loss and DNA damage was shown by double staining with XRCC1 immunohistochemistry and TUNEL. These results indicate that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA-damaged cell death after transient FCI.
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Acknowledgments |
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This study was supported by National Institutes of Health grants NS14543, NS25372, NS36147, NS38653, and N01NS82386. Dr Chan is a recipient of the Jacob Javits Neuroscience Investigator Award. We thank Cheryl Christensen for editorial assistance and Liza Reola, Bernard Calagui, and Jane O. Kim for technical assistance.
Received May 25, 1999; revision received August 2, 1999; accepted August 5, 1999.
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24. Endres M, Wang ZQ, Namura S, Waeber C, Moskowitz MA. Ischemic brain injury is mediated by the activation of poly(ADP-ribose)polymerase. J Cereb Blood Flow Metab. 1997;17:1143–1151.[Medline] [Order article via Infotrieve]
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Fujimura M, Morita-Fujimura Y, Narasimhan P, Copin J-C,
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Stroke.. 1999;30:2408–2415.
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Editorial Comment
Guest Editors, Department of Neurology, Washington University School of Medicine, St Louis, Missouri
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Normal cellular metabolism, particularly mitochondrial respiration, produces reactive oxygen species (ROS) (eg, hydrogen peroxide and superoxide anion and hydroxyl radicals) as end products.1 Under physiological conditions, excessive ROS are neutralized by endogenous antioxidants (eg, ascorbate,
-tocopherol, ß-carotene, and glutathione) and antioxidant enzymes
(eg, superoxide dismutase, catalase, and glutathione peroxidase).
Severe oxidative stress may overwhelm the antioxidant mechanisms and
lead to oxidative DNA damage.2 Among the DNA lesions caused
by ROS are base modifications, single-strand breaks, double-strand
breaks, strand scissions, and the crosslinking of bases.3
The second line of defense against oxidative DNA damage is the DNA
repair machinery. Oxidative DNA damage can be rapidly and efficiently
repaired.4 However, this second line of defense against
oxidative damage of DNA may be compromised in the setting of
ischemia-reperfusion.5 Individual bases in DNA may be oxidized to form adducts such as 8-hydroxyguanine (8-OH-Gua), 8-hydroxyadenine (8-OH-Ade), 4,6-diamino-5-formamidopyrimidine (FapyAde), 2-hydroxyadenine (2-OH-Ade), 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 5-hydroxy-5-methylhydantoin (5-OH-5-MeHyd), and 5-hydroxyhydantoin (5-OH-Hyd).6 These oxidative DNA adducts are potentially mutagenic or lethal. The XRCC1 protein is involved in contributes to base excision repair and therefore, may contribute to enhance genome integrity and stability after ischemic insult. In the preceding article by Fujimura et al, the expression of the XRCC1 protein was reduced in the ischemic region in a mouse MCAO model. Suppressed XRCC1 expression preceded DNA fragmentation and cell death. This finding raises the possibility that a defective DNA repair machinery caused by reduced expression of DNA repair proteins such as XRCC1 may contribute to irreversible DNA damage and ultimate cell demise after focal cerebral ischemia-reperfusion.
Received May 25, 1999; revision received August 2, 1999; accepted August 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Beckman KB, Ames BN. The free radical theory of aging matures. Physiol Rev.. 1998;78:547–581.
2. Chan PH. Role of oxidants in ischemic brain damage. Stroke.. 1996;27:1124–1129.
3. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. Oxford, UK: Clarendon Press; 1989.
4. Chopp M, Chan PH, Hsu CY, Cheung ME, Jacobs TP. DNA damage and repair in central nervous system injury: National Institute of Neurological Disorders and Stroke Workshop Summary. Stroke.. 1996;27:363–369.
5. Liu PK, Hsu CY, Dizdaroglu M, Floyd RA, Kow YW, Karakaya A, Rabow LE, Cui JK. Damage, repair, and mutagenesis in nuclear genes after mouse forebrain ischemia-reperfusion. J Neurosci.. 1996;16:6795–6806.
6. Jaruga P, Dizdaroglu M. Repair of products of oxidative DNA base damage in human cells. Nucleic Acids Res.. 1996;24:1386–1394.
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