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Copperzinc superoxide dismutase transgenic brain accumulates hydrogen peroxide after perinatal hypoxia ischemia.

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Lopper/Linc 3uDeroxide Uismutase
Transgenic Brain Accumulates €3ydrogen
Peroxide after Perinatal Hypox.ia Ischemia
Heather J. Fullerton, MD,* Jeremy S. Ditelberg, MD, t Sylvia F. Chen, PhD,* Dean P. Sarco, BS,*
Pak H. Chan, PhD,S Charles J. Epstein, MD,O and Donna M. Ferriero, MD*S
Unlike the mature animal, immature mice transgenic for copperhinc superoxide dismutase (SOD1) have greater brain
injury after hypoxia-ischemia than their wild-type nontransgenic littermates. To assess the role of oxidative stress in the
pathogenesis of this injury, we measured histopathological damage, lipid peroxidation products, enzymatic activities of
catalase and glutathione peroxidase, and hydrogen peroxide (H,O,) concentration in these animals before and after
hypoxic-ischemic injury. Lipid peroxidation products were significantly increased 2 hours after the insult in both transgenic and nontransgenic brains in hippocampus, the most damaged brain region. Catalase activity did not increase in
response to SODl overexpression or injury in either group. However, glutathione peroxidase activity, unchanged in
response to overexpression, decreased significantly 24 hours after injury in both groups. At 24 hours after injury, greater
H,O, accumulation was observed in transgenic brains. Because SODl dismutates superoxide to H,O,, overexpression of
SODl in the presence of developmentally low activities of the catalytic enzymes glutathione peroxidase and catalase leads
to an increased production of H,O,, and may explain the increased brain injury observed after hypoxia-ischemia in
neonatal SODl mice.
Fullerton HJ, Ditelberg JS, Chen SF, Sarco DP, Chan PH, Epstein CJ, Ferriero DM.
Copperhinc superoxide dismutase transgenic brain accumulates hydrogen peroxide
after perinatal hypoxia ischemia. Ann Neurol 1778;44:357-364
Perinatal hypoxic-ischemic injury causes devastating
brain damage associated with significant morbidity and
mortality. The pathogenesis of this injury is complex,
because interrelated toxic events such as energy depletion, release of excitatory amino acids, initiation of apoptosis, and accumulation of reactive oxygen species
occur simultaneously and contribute to cellular malfunction and death.'-3 Oxygen free radicals, which include the superoxide anion (.02-)
and hydroxyl radical ('OH) and other reactive oxygen species such as
hydrogen peroxide (H,O,) and nitric oxide, are generated by a number of metabolic reactions in the brain,
particularly those occurring during cellular respiration.
Under normal conditions, cellular defense mechanisms
that lower levels of reactive oxygen species are able to
compensate for oxidative stress. During hypoxia-ischemia, however, oxygen radicals are overproduced, the
defense mechanisms are altered, and the radicals reach
toxic levels and disrupt cellular activity by attacking
membrane lipids, proteins, and
The cell's most important defense against the exces-
sive production of free radicals is the concerted action
of three enzymes (Fig l), superoxide dismutase (SOD;
EC, glutathione peroxidase (EC,
and catalase (EC SOD catalyzes the dismutation of the superoxide radical to H20,. Three different forms have been described-cytosolic copper/zinc
(Cu/Zn) SOD (SODI), mitochondrial manganese
SOD, and extracellular Cu/Zn SOD.' Catalase and
glutathione peroxidase (GPx) catalyze the reduction of
H202 to water and oxygen, and, in the presence of
ferrous ions, H,O, can also be rapidly converted to the
mote toxic hydroxyl radical via the Fenton reaction.8
We have previously described that, in a neonatal
model of hypoxic-ischemic injury, mice transgenic (tg)
for SODl had greater brain damage than their nontransgenic (ntg) littermates when exposed to a longduration hypoxic event.' In adult models of ischemia,
however, increased S O Dl activity has been associated
with neuroprote~tion,'~-'~
presumably by improving
the brain's ability to detoxify superoxide radicals.
SOD1 has been associated with enhanced cellular dam-
From the Departments of 'Neurology and §Pediatrics, University of
California-San Francisco, San Francisco, and $Department of Neurosurgery, Stanford University, Stanford, CA; and tDepartment of
Pathology, Harvard University, Boston, MA.
Address correspondence to Dr Ferriero, Department of Neurology,
BOX01 14, University of California-San Francisco, San Francisco,
CA 94143-01 14.
Received Feb 2, 1998, and in revised form Mar 19. Accepted for
publication Mar 19, 1998.
Copyright 0 1798 by the American Neurological Association 357
mates. All animal research was approved by the University of
California at San Francisco Committee on Animal Research.
Hypoxic-Ischemic Injuly and
Histopathological Analysis
0 2
Fig 1. A schematic drawing of the relationship between superoxide dismutase, catahe, and glutathione peroxidase (GPx).
The superoxide anion is produced via several metabolic pathways, predominantly tbrougb the electron transport chain (1).
Superoxide anion is dismutated to hydrogen peroxide (H,OJ
by superoxide dismutase (SOD) (2). H,O, can be converted
to H,O and 0, by catalase (3) or GPx (4), the latter using
glutathione as a reducing agent. Catahe binds to H,O, in
the process of decomposing it, to form compound I. The catah e inhibitor aminotriazole acts by irreversibly binding and
inhibiting compound I (5). H,O, can also be reduced by ferric ions to form the highly toxic hydroyl radical via the Fenton reaction (6).
age and abnormal cellular function in neurons and
other cell systerns.l5-” A possible explanation for the
variable effect of SOD in the brain during different
stages of development is that, although there is a compensatory increase in catalase activity in the mature
SODl tg brain,20 an increase in catalase or GPx activities does not occur in the neonatal brain. T h e consequent imbalance in antioxidant enzyme activities
would allow for greater production of H,O, after perinatal hypoxia-ischemia and thus lead to greater injury.
To address this hypothesis, we measured neuropathological damage, lipid peroxidation products, catalase
and GPx activities, and relative H,O, concentrations
in the brains of neonatal SODl tg and ntg mice under
normal conditions and after a moderate hypoxic-ischemic insult.
Subjects and Methods
Tg mice of the strain TgHS/SF-218/3 were derived from the
founder stock described by Epstein and colleagues.21 This
strain carries several copies of the human SODl gene in its
genome; brain SODl activity in this strain is 2.3 times that
of control. The founder mice were bred with wild-type
CD-1 mice to produce 261 pups; 200 pups were used in
experiments. SODl tg pups were identified by qualitative gel
electrophoresis of the human SOD1.21 There was no appreciable phenotypic difference between the tg and ntg litter-
358 Annals of Neurology Vol 44 No 3 September 1998
A model of perinatal hypoxic-ischemic injury was used
in this study, originally described by Rice and co-workersz2
for the neonatal rat and modified by us for the neonatal
On postnatal day 7, mouse pups were anesthetized and their right common carotid artery was ligated by
electrical coagulation; after a 2-hour recovery period, they
were placed in a hypoxic chamber (8% oxygen) in a 37°C
water bath for 30 minutes. A duration of 30 minutes of hypoxia was chosen to minimize mortality, thus allowing for a
reproducible moderate degree of injury. Pups were killed either 2 or 24 hours after removal from hypoxia, and cortices
and hippocampi were dissected free on ice. Samples were
flash frozen in methylbutane on dry ice and stored at -70°C
until ready for assay. Brains of uninjured control “zero time
point” postnatal day 7 pups were similarly collected.
In a separate group of postnatal day 7 pups (SOD1 tg,
n = 21; ntg, n = 26), brains were analyzed for histopathological damage 2 hours, 24 hours, and 7 days after hypoxiaischemia. These animals were exposed to hypoxia-ischemia
as described above, anesthetized with 50 mg/kg pentobarbita1
intraperitoneally, and perfused with cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were removed, postfmed in paraformaldehyde for 4 hours, and
transferred to 30% sucrose. Coronal forebrain sections were
cut at 20-pm intervals. Sections were srained with cresyl violet and evaluated blindly for histopathological damage,
based on a previously described scoring method in which
0 = no damage, 1 = mild (focal areas of edema and cell
loss), 2 = moderate (confluent areas of edema and hippocampal neuronal loss), and 3 = severe (diffuse cortical
edema and cell loss in cortex and hippocamp~s).~
Lipid Peroxidation Assay
Lipid peroxidation products malondialdehyde (MDA) and
4-hydroxy-2(E,honenal (4-HNE) were measured by using a
commercial colorimetric assay based on previously described
methods.’* Individual right and left cortices and hippocampi
were dissected on dry ice, homogenized in 20 mM Tris-HCI
(pH 7.4; 4”C), and centrifuged (3,000 g for 10 minutes).
Lipid peroxidation products were assayed by coupling MDA
and 4-HNE to a chromogenic compound, N-methyl-2phenylindole, to form a stable chromophore. Absorbance of
the chromophore was measured at 586 nm. All samples were
measured in a single assay. The total protein in each sample
solution was determined by using a protein assay kit based
on the method of Lowry and collaborator^^^ and others?‘
Results were expressed as nanomoles of MDA equivalents
(MDA and 4-HNE) per milligram of protein.
Glutathione Peroxihe Assay
GPx assay was performed by using previously described
methods with minor modification^.^' Samples prepared as
above were maintained at 4“C, homogenized in phosphate
buffer (0.1 M potassium phosphate, 2 mM sodium azide;
pH 7.0), and centrifuged at 5,000 g for 5 minutes. GPx
activity was determined in duplicate by kinetic colorimetric
assay, following the decrease in absorbance of NADPH at
340 nm.28 Activity was expressed as units per milligram of
protein (U/mg of protein), where 1 unit was defined as 1
nmol NADPH consumed per minute.
Cataiuse Assay
Catalase assay was performed by using previously described
methods with slight m o d i f i c a t i ~ n .Brain
~ ~ , ~samples
~ ~ ~ ~were
homogenized in 200 pl of cold phosphate buffer (0.1 M
sodium phosphate buffer, 0.1 mM EDTA, 0.1% Triton
X-100; pH 7.8), and separated over glass wool columns.
Samples, maintained at 4"C, were diluted 1:10 (cortex) or
1:5 (hippocampus) with buffer. Catalase activity was determined in duplicate samples by kinetic colorimetric assay, following the decrease in absorbance of a known amount of
H20, at 240 nm.30 Catalase activity was then expressed as
units per milligram of protein, and one unit was defined as 1
pmol of H202 reduced per minute.20 All values were normalized to an internal control that consisted of the combined
homogenate of several cortices or hippocampi. As previous
studies have found blood contamination to have a negligible
effect on measurements of catalase and GPx activities in similarly prepared brain homogenates, such contamination was
not considered in this s t ~ d y . ~ ~ , ~ ~
Aminotriazole Treatment: A n indirect Measure of
Hydrogen Peroxide Concentration
Mice were injected intraperitoneally with the catalase inhibitor, aminotriazole (200 mg/kg of body weight, normal saline
vehicle; n = 46), or an equivalent volume of vehicle (n =
44), 2 hours before they were killed. This time point was
chosen based on a time curve of inhibition of brain catalase
after intraperitoneal injection of aminotriazole demonstrating
about 50% inhibition at 2 hours (Fig 2). Catalase activity
was measured in the brains of these mice as described above.
Aminotriazole selectively and irreversibly inhibits catalase
Therethat is bound to H 2 0 2 (compound 1; see Fig l).31,32
fore, inhibition of catalase activity in the presence of aminotriazole is directly proportional to the H 2 0 2 concentration
at the time of aminotriazole exposure.29
Tissue levels of aminotriazole were assayed by the colorimetric method of Green and F e i n ~ t e i n ,using
~ ~ a protocol
previously described in detail." With this method, aminotriazole is first diazotized by sodium nitrate, then coupled
to a chromotropic acid to form a colored derivative, the absorbance of which is read at 525 nm. All samples were run in
a single assay. Results were expressed as micrograms of aminorriazole per milligram of protein.
The following reagents were obtained from Sigma Chemical
(St Louis, MO): 3-amino-l,2,4-triazole (aminotriazole), 30%
H20,, yeast glutathione reductase (GSSG-R), and chromotropic acid. Commercial kits were used for protein assay
(Pierce, Rockford, IL) and lipid peroxidation assay (Calbiochem, San Diego, CA).
Statistical Analysis
For the H202 experiments, differences between tg and ntg
were tested by a one-tail Student's t test, because the data
8 30-
g 20-
Time after AT (hours)
Fig 2. Time curve for the inhibition of catalase by aminotriazole. Pups (copper/zinc superoxide dismutase [SODl] transgenic [td and nontransgenic [ntd)were injected intraperitoneally with aminotriazole 07;.200 mg/kg of body weight)
and killed at variow time points ajier injection. Zero time
pups were injected with an equivalent volume of normal saline instead of aminotriazole. Catahe activiq was then measured in the cortex and hippocampw and expressed in units
per milligram ofprotein, where 1 unit equals I pmol of
H202 reduced per minute. Data are mean ? SEM values.
DataJFom SODl tg and ntgpups were combined, as they
were not significantly different. 9 = 0.0001, compared with
zero time point.
could only change in one direction. Differences over time
were tested by repeated-measures analysis of variance
(ANOVA). For the lipid peroxidarion assay, aminotriazole
tissue levels, and enzyme assays, differences between tg and
ntg mice, and between right and left brain segments, were
tested by factorial two-way ANOVA. Differences in time
points (before, 2 hours, and 24 hours after injury) were
tested by repeated-measures ANOVA. All ANOVAs were
followed by Fisher post hoc testing. Histopathological scoring was analyzed by Mann-Whitney U test. The null hypothesis was rejected at the level of 0.05. All continuous data
were expressed as mean t- SEM, categorical data as median
with range.
Histopathological Analysis
As in the model of severe injury (90 minutes hypoxia
right common carotid artery ligation),' this moderligation) produced
ate insult (30 minutes of hypoxia
more injury in the SODl tg brain than in the ntg
brain (median injury score for tg = 3 vs ntg = 2; p <
0.01). Injury was present in both SODl tg and ntg
brains as early as 2 hours after hypoxia-ischemia, with
edema formation present in the more severely affected
brains. While cortical injury remained patchy and con-
Fullerton et al: H202 in Neonatal Hypoxic-Ischemic Brain Injury
fined to the parietal cortex, hippocampal injury involved all sectors, with the greatest injury seen in CA1
through (243. It is noteworthy that there were small
foci of edema observed in the contralateral (to ligation)
hemisphere, which were no longer apparent at 24
hours after the injury. Seven days after the insult (postnatal day 14), the edema had resolved and was replaced
by patchy infarction of tissue in the parietal cortex and
entire hippocampus (with minimal sparing of dentate
gyrus) .
Lipid Peroxidztion Products
To evaluate whether the histopathological damage seen
was associated with oxidative stress, we measured the
lipid peroxidation products MDA and 4-HNE. As
shown in Figure 3A, MDA equivalents increased significantly 2 hours after hypoxia-ischemia in the hippocampus, that is, from 5.23 k 0.66 nmol/mg of protein in SODl tg hippocampus before injury, to
3.85 nmol/mg of protein in the right (ipsilateral to carotid ligation) SODl tg hippocampus 2
hours after injury ( p < 0.005), correlating well with
the histopathological regions of damage. Similar results
were seen in the brains of damaged ntg mice ( p <
0.05). MDA levels were significantly greater in the
right hippocampus (injured) compared with left hippocampus (uninjured) in the SODl tg brain at 2 hours
( p < 0.05). Elevations in lipid peroxidation products
were no longer appreciable by 24 hours after hypoxiaischemia, when the MDA levels decreased to values
similar to those before injury, indicating that the generation of lipid peroxidation products had ceased.
There were no significant changes in MDA levels in
the whole cortical homogenates (see Fig 3B) of tg or
ntg mice at any time point, consistent with the patchy
pattern of injury observed in the cortex.
Time after HI (hrs)
Enzyme Activities
Catalase activity remained unchanged after hypoxicischemic injury in the hippocampus (Fig 4A) and cortex (44.1 k 3.9 vs 40.6 2 1.5 U/mg of protein) from
both ntg and tg brains. Likewise, catalase activity did
not increase in a compensating manner in response to
SODl overexpression in tg brains under normal conditions (noninjected animals = zero time point). As
seen in Figure 4B, GPx activity decreased precipitously
after injury, from 692.5 2 99.8 U/mg of protein in
SODl tg hippocampus before injury, to 151.2 2 60.3
U/mg of protein 24 hours after injury ( p < 0.0001).
Similar findings were observed in the hippocampus of
injured ntg brains. There was no significant difference
between SODl tg and ntg brains in baseline GPx activity (692.5 ? 99.8 vs 975.4 ? 200.1 U/mg of protein).
360 Annals of Neurology Vol 44
No 3
September 1998
N LCortex
Time after HI (hrs)
Fig 3. Lipid peroxidation products accumulate after perinatal
hypoxia-ischemia (HI) in superoxide dismutase (SODl) transgenic (tgl and nontransgenic (ntd h+pocampus (A), but not
cortex (B). Seven-day-old SODl tg pups and their ntg littermates were subjected to hypoxia-ischemia and killed 2 or 24
hours afzer injuty. Malondialdehyde (MDA) equivalents (lipid
peroxidation products) were measured in homogenates of the
right (R) and lefz (L) cortex and hippocampus (Hippo), and
expressed as nanomoles of MDA equivalents per milligram of
protein. Data are mean 2 SEM values of 5 pups. a p <
0.005, compared with zero and 24-hour time points. bp <
0.05, compared with zero time point, 'p < 0.05, compared
with lefz hippocampus.
H,O, Production
We used the aminotriazole method to identify H,O,
production in vivo. We first demonstrated that there
was no significant difference in brain tissue levels of
aminottiazole measured 2 hours after a single intraperitoneal injection (200 mg/kg of body weight) between
SODl tg and ntg mice (Fig 5A), concurring with find-
Time after HI (hours)
" 0
Time after HI (hrs)
3 1000
SODl transgenic
2 750
Time after HI (hrs)
Fig 4. Catalytic enzyme activities in noninjured and injured
copper/zinc superoxide dismutase (SODl) transgenic (tg) and
wild-type (nontransgenic (ntgi, animals. Seven-ahy-old superoxide dismutase (SOD) tg pups and their ntg littermates were
subjected to hypoxia-ischemia and killed 2 or 24 hours afzer
injury. Data from right hippocampal samples are shown.
(A) Catahe actiuity is not altered by overexpression of the
SODl protein, nor does it change sip$cantly in wild-type or
tg brains after hypoxic-ischemic (HI) injury. Catalase activity
is expressed as units per milligram of protein, where I unit
equals 1 pmol of H202 reduced per minute. Zero time animals are uninjured controls. (B) Glutathione peroxidme (GPx)
activity falls afzer perinatal hypoxia-ischemia (HI) in SOD tg
and ntg brains. GPx activity is expressed as units per milligram ofprotein. Data are expressed as mean ? SEM values.
ap = 0.005; bp = 0.0001, compared with 0 hours (no hypoxia-ischemia).
ings in a study of adult SODl tg mice injected with
aminotriazole 1 g/kg.20 Therefore, the time points chosen for measurement of hydrogen peroxide, by using
Time after HI (hours)
Fig 5. H202 accumulation, as measured indirectly by the
aminotriazole inhibition of endogenous catalase activity, occurs
after hypoxic-ischemic injury in postnatal &y 7 mouse brain.
(A) Aminotriazole concentrations in ipsilateral to ligation
(ischemic, right) cortex and hippocampus 2 and 24 hours after
hypoxia-ischemia (HI). Data were combined for copper/zinc
superoxide dismutase (SOD1) transgenic (td and nontransgenic (ntd hemispheres, as concentration ranges were not
sign+cantly different between wild types and transgenics.
(B) H202 accumulates in hippocampus 24 hours after
hypoxic-ischemic (HI) injury and is greater in the SODl
tg brain compared with wild type. Data are expressed as
percentages of inhibition of catahe activity by aminotriawle
at 0, 2, and 24 hours afzer injuy. #p = 0.003.
the aminotriazole inhibition assay, were justified based
on the appearance and the half-life of the drug in brain
(see Fig 2).
Hydrogen peroxide accumulation as measured by
aminotriazole inhibition of catalase activity was similar
between tg and ntg animals in uninjured brains (zero
time point; see Fig 5B). However, 24 hours after injury, catalase activity after aminotriazole injection was
significantly lower in tg hippocampus ( p < 0.02) and
cortex ( p = 0.003), indicating greater inhibition of the
enzyme (see Fig 5B) and, thus, higher H202 concentrations in those brains.
Fullerton et al: H,O, in Neonatal Hypoxic-Ischemic Brain Injury
This study demonstrates that oxidative stress is an important factor in the response of the immature brain to
hypoxic-ischemic injury. Overexpression of SOD 1 is
deleterious because the neonatal brain does not compensate by increasing the activities of downstream enzymes, GPx and catalase. This imbalance results in
H 2 0 2 accumulation, which contributes to cell death
and the exacerbated damage seen in the neonatal
SOD 1 tg brain after hypoxia-ischemia. These findings
have significant implications on the design of therapeutic strategies, as treatments that protect the adult brain
from hypoxia-ischemia may have the opposite effect
on the brain of the neonate.
Our histopathological data support the hypothesis
that H202 accumulation in regions of damage is toxi ~Indeed,
. ~ the accumulation of H20, appears to correspond well with the region of brain most severely affected by this insult, the hippocampus. To further link
oxidative stress with cellular toxicity, we found that
lipid peroxidation products increased significantly soon
after hypoxia-ischemia in the hippocampus as well.
The early appearance and subsequent disappearance of
lipid peroxidation products correlates with the expected
time course of an initial phase of apoptosis, which is
later replaced by necrosis. The greater degree of injury
seen in the tg brains is not represented by a higher
level of lipid peroxidation products, indicating that the
histopathological injury cannot be estimated by the absolute amount of lipid peroxidation products. A possible explanation for the return of lipid peroxidation
products to baseline 24 hours after injury is that the
superoxide radical may serve as either an initiator or
terminator of lipid p e r ~ x i d a t i o n .Other
~ ~ studies have
reported an association between increased SODl activity and an increase in lipid peroxidation products, presumably due to accumulation of H202 and other
downstream p r o d ~ c t s . ' " ~ ~ - ~ ~
To explain the cause of H20, accumulation after
hypoxic-ischemic injury, we demonstrated that SOD 1
overexpression leads to an imbalance between H202
production and consumption. We found that there was
no measurable compensatory increase in catalase or
GPx activity in normal, noninjured SODl tg mice
compared with their ntg littermates, concurring with
findings in Down syndrome fetal brains.35 In contrast,
a study in adult mice reported an increase in catalase
activity in S O D l tg brains, which may explain, in part,
the beneficial effects of SOD overexpression found in
adult brain after hypoxia-ischemia.20 Injury did not result in an up-regulation in enzymatic activity either.
There was no change in catalase activity in either tg or
ntg brains, and GPx activity, similarly unchanged immediately after injury, fell dramatically 24 hours later.
It is unlikely that the decline in GPx activity represents
decreased GPx concentrations from cell death, because
362 Annals of Neurology
Vol 44 No 3
Seprember 1998
there was no change in catalase in the same brains.
Rather, inactivation of the enzyme, which has been
demonstrated in the presence of the superoxide radical,
is the more plausible
These findings lead us to several conclusions. A tenuous balance exists between the H202-producing and
consuming enzymes in the uninjured state both in the
wild-type and the tg neonatal brain. After hypoxiaischemia, catalase and GPx initially function at levels
sufficient to detoxify the excess H202that is produced,
but as injury continues, and GPx activity falls, the preexisting imbalance is exaggerated and H202 accumulates. Numerous investigators have reported on the deleterious effects of an imbalance between S O Dl and
catalase/GPx in brain and in other cell types, suggesting that the ratio between these enzymes may be more
important than the absolute levels of activity of the individual enzymes.35-38,4'-45
Evidence that profound differences exist between the
antioxidant enzyme systems of the adult and the neonate could explain the dichotomous effect of SODl
overexpression. SOD1 levels are low embryonically, triple to reach their maximum level during the neonatal
period, and then decrease minimally with adulth00d.~"'~ Catalase levels are also low embryonically,
triple to reach their maximum levels during the neonatal period, but then fall dramatically in adulthood to
slightly below the embryonic
GPx levels are
low embryonically and neonatally, and then gradually
increase to reach their maximum levels during adulthood.46-48,50 The response of these enzymes to oxidative stress also appears to be different in the neonate
than the adult. In one study, although there was no
change in GPx activity after SODl overexpression, an
increase in catalase activity was seen in all regions of
the tg brains compared with wild type.20 This contrasts
with our neonatal study, where SODl tg brains had no
adaptive rise in the activity of either enzyme. In an
adult rat model of ischemidreperfusion, GPx activity
remained constant for 6 days,51 whereas GPx activity
fell dramatically 24 hours after injury in the neonatal
Recent evidence suggests that H202is toxic to neurons. Fetal Down syndrome-derived neurons containing excess S O Dl generate more reactive oxygen species
(including H202) causing cell death, which is prevented by the addition of ~ a t a l a s e .In
~ ~primary neuronal cultures, brief exposure to H202 induces cell
death in a dose-dependent manner53354and H,02 mediates amyloid P protein n e u r o t ~ x i c i t yAlthough
mechanism of H20, toxicity is not well established,
one possibility is that H202triggers apoptosis. Studies
in model systems of apoptosis have implicated H202as
an early signal in programmed cell death.56 It is noteworthy that SODl tg neurons show a higher susceptibility to kainic acid-induced apoptotic cell death com-
pared with ntg neurons.57 Another potential mechanism of H20, toxicity is the conversion of H,02 to
the highly toxic hydroxyl radical in the presence of
Fez+. Mounting evidence implicates this reaction as a
crucial step in the pathogenesis of H202 toxicity, on
both cellular and molecular level^.*^^'
This study was supported in part by NS01692, NS33997,
NS35902, NS32553, and AG08938.
We thank Wyatt Tellis, R. Ann Sheldon for technical assistance,
and Ray Swanson, MD, for helpful discussions.
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hydrogen, perinatal, peroxide, ischemia, transgenic, superoxide, copperzinc, dismutase, brain, hypoxia, accumulated
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