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The Journal of Neuroscience, August 15, 2001, 21(16):5993-5999
Interactions between Apolipoprotein E Gene and Dietary
 -Tocopherol Influence Cerebral Oxidative Damage in Aged Mice
Erin E.
Reich1,
Kathleen S.
Montine1,
Myron D.
Gross2,
L. Jackson
Roberts II1,
Larry L.
Swift1,
Jason D.
Morrow1, and
Thomas J.
Montine1
1 Departments of Pathology, Medicine, and Pharmacology
and Center for Molecular Neurosciences, Vanderbilt University Medical
Center, Nashville, Tennessee 37232, and 2 Department of
Pathology and Laboratory Medicine, University of Minnesota,
Minneapolis, Minnesota 55455
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ABSTRACT |
Cerebral oxidative damage is a feature of aging and is increased in
a number of neurodegenerative diseases. We pursued the gene-environment interaction of lack of apolipoprotein E (apoE) and modulation of dietary  -tocopherol on cerebral oxidative damage in aged male and female mice by quantifying the major isomers of
cerebral isoprostanes, derived from arachidonic acid (AA) oxidation, and neuroprostanes, derived from docosahexaenoic acid (DHA) oxidation. Mice fed -tocopherol-deficient, normal, or -supplemented diet had
undetectable, 4486 ± 215, or 6406 ± 254 ng of
-tocopherol per gram of brain tissue (p < 0.0001), respectively. Two factors, male gender and lack of apoE,
combined to increase cerebral AA oxidation by 28%, whereas three
factors, male gender, lack of apoE, and deficiency in -tocopherol,
combined to increase cerebral DHA oxidation by 81%. -Tocopherol
supplementation decreased cerebral isoprostanes but not neuroprostanes
and enhanced DHA, but not AA, endoperoxide reduction in
vivo and in vitro. These results demonstrated
that the interaction of gender, inherited susceptibilities, and dietary
-tocopherol contributed differently to oxidative damage to cerebral
AA and DHA in aged mice.
Key words:
aging; mouse; brain; apolipoprotein E; -tocopherol; oxidative damage; isoprostanes; neuroprostanes
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INTRODUCTION |
Aging is a complex trait that
derives critical input from genetic and environmental factors. A
substantial body of data support a pivotal role for oxidative stress as
a key effector in both genetic and environmental aspects of aging
(Finkel and Holbrook, 2000 ). Increased oxidative stress contributes to
aging through mechanisms that involve structural damage to cellular
macromolecules (Halliwell, 1992). These irreversible modifications can
have several deleterious effects on cell function, including oxidative
modification of nucleic acids, that are thought to contribute to
altered gene expression in aging (Beckmam and Ames, 1998).
Recently, others quantified age-related change in expression of over
11,000 genes in mouse cerebral cortex and hypothalamus; apolipoprotein
E (apoE) was among the few genes that showed significantly reduced expression in both regions of brain (Jiang et al., 2001). Importantly, lack of apoE is associated with increased oxidative damage
in aged mouse brain (Montine et al., 1999a; Pratico et al., 1999).
Moreover, inheritance of the  4 allele of human apolipoprotein E gene
(APOE4) is associated with poorer cognitive
performance with age and an increased risk of developing Alzheimer's
disease (AD) (Mahley and Huang, 1999). The isoform encoded by
APOE4, apoE4, may be deficient in some critical function(s)
relative to the other common isoforms of apoE (Mahley and Huang, 1999)
or may have dominant negative effects (Buttini et al., 2000). Some
groups have associated homozygosity for APOE4 with increased
markers of brain oxidative damage (Montine et al., 1998; Pratico et
al., 2000); however, this association has not been consistent among laboratories or methods used to detect oxidative damage (Sayre et al.,
1997; Montine et al., 1999c). Together, these data suggest that reduced
apoE activity, from either decreased expression with advancing age or
inheritance of APOE4, may contribute to oxidative damage to brain.
Vitamin E is a family of tocopherols, of which -tocopherol is the
most biologically active antioxidant in vivo (Marcus and Coulston, 1996). At physiologic concentrations, -tocopherol acts as
a lipophilic radical scavenger and thereby limits lipid peroxidation, although it may have other actions (Thomas and Stocker, 2000). Dietary
supplementation with -tocopherol appears to impede the progression
of some age-related diseases that are associated with increased
oxidative damage, including atherosclerosis (for review, see Jialal et
al., 2001) and possibly AD (Sano et al., 1997); however, the effects of
-tocopherol dietary supplementation in patients with established AD
were modest. These results raise the possibilities that -tocopherol
may not be an effective antioxidant in brain or that -tocopherol may
have activity other than radical scavenging in those regions of brain
affected by AD.
We showed previously that free radical damage to arachidonic acid (AA)
and docosahexaenoic acid (DHA) in brain can be quantified by measuring
isoprostanes (IsoPs) and neuroprostanes (NPs), respectively, highly
accurate and specific markers of free radical-mediated damage (Roberts
et al., 1998; Roberts, 2000). Additionally, because the major isomers
of IsoPs and NPs, F-ring or D/E-ring compounds, derive from common
endoperoxide intermediates by reduction or isomerization, respectively,
the ratio of F- to D/E-ring compounds is a measure of the reducing
environment in which oxidation occurred (Reich et al., 2000, 2001).
Here we used these quantitative endpoints to test the hypothesis that
there is a gene-environment interaction between apoE and
-tocopherol with respect to age-related oxidative damage to mouse cerebrum.
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MATERIALS AND METHODS |
Mice and tissue acquisition. The primary study group
consisted of 63 mice purchased from The Jackson Laboratory (Bar Harbor, ME) at 3 weeks of age. Mice were either C57BL/6J [wild type (wt)] or
apoE  / mice (C57BL/6J-Apoetm1Unc) backcrossed six to
eight generations with C57BL/6L mice and were weighed every 4-8 weeks. These mice were divided among three different diets (vida
infra). A second study group of eight wt and eight apoE
/ mice was purchased from the same vendor at the same age and
housed under the same conditions as the primary study group; these mice
were fed standard mouse food. Three mice, two wt and one
apoE /, died within a month of purchase. The remaining
60 mice were aged 9 months on the different diets and were
killed at 39 weeks of age. While under deep anesthesia, 0.5-1
ml of blood was collected. Brains were removed immediately and
sectioned into two cerebral hemispheres and cerebellum. Tissue and
plasma were flash frozen in liquid nitrogen and were kept frozen at
80°C until analyzed.
Diets. All diets for the primary study group were purchased
from Harlan Teklad (Madison, WI). Mice were reared on one of three different diets from the age of 3 weeks: a normal diet containing 50 IU
of -tocopherol per kilogram of food, a deficient diet containing 0 IU of -tocopherol per kilogram of food, and a supplemented diet
containing 500 IU of -tocopherol per kilogram of food. The -tocopherol-deficient diet was formulated from tocopherol-stripped corn oil as the base material, with remaining dietary requirements, excluding -tocopherol, added to this base. For the normal and supplemented diets, all-rac--tocopherol acetate was added to the
-tocopherol-deficient diet at levels of 50 and 500 IU per kilogram
of food, respectively. Fresh food was received every 3 weeks and was
changed in all cages biweekly to prevent oxidation. All mice received
food and water ad libitum.
Quantitative methods. F-Ring and D/E-ring IsoPs, NPs, and
fatty acids were measured in one cerebral hemisphere as described previously (Reich et al., 2001). Briefly, each cerebrum was weighed and
homogenized in Folch solution. Subsequently, lipids were extracted by
the method of Folch.
D2/E2-IsoPs and
D4/E4-NPs esterified in tissue were converted to O-methyloxime derivatives in Folch
solution, and all compounds were hydrolyzed by chemical saponification. F-Ring and D/E-ring IsoPs and NPs were analyzed using gas
chromatography (GC) with negative ion chemical ionization mass
spectrometry and selective ion monitoring as described previously
(Reich et al., 2001). Data are presented for the separate
compounds, total IsoPs or NPs (sum of F-ring and D/E-ring compounds),
or the ratio of F-ring to D/E-ring compounds. Cerebral AA and DHA
concentrations were determined as described previously (Montine et al.,
1999a). Briefly, an aliquot of Folch extract from each tissue sample
was transmethylated, and the total fatty acid composition was
quantified using GC with flame ionization detection.
-Tocopherol was quantitated in plasma and cerebellum using a
published HPLC-electrochemical detection method (Gross et al.,
1995).
Synaptosomes. Rat cerebral synaptosomes were prepared and
oxidized with AAPH as described previously (Reich et al., 2000). Briefly, synaptosomes were preincubated with varying concentrations of
-tocopherol for 1 hr and then oxidized by incubation with 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) at 37°C for 2 hr.
Reactions were terminated by placing samples at 80°C.
Quantification of F-ring and D/E-ring IsoPs and NPs were performed as
described above for mouse cerebrum.
Statistical analysis. Statistical analyses were performed
using Graph Pad Prism 3.0 software (San Diego, CA)
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RESULTS |
For the primary study group, we obtained 63 mice immediately after
weaning (3 weeks of age) and maintained them on a diet of mouse food
with normal levels of -tocopherol (50 IU/kg), food supplemented with
-tocopherol (500 IU/kg), or food lacking -tocopherol for the rest
of their lives. As expected, male mice weighed more than female mice at
all time points (Fig. 1). All groups of
mice gained weight over the first 6 months and then either gained
weight or maintained weight from 6-8 months of age. From 8-9 months, some groups of wt and apoE / mice showed a slight
reduction in body weight, although it was not statistically
significant. Therefore, we terminated the study at 39 weeks of age,
after 9 months on the different diets. Body weights in males or females were analyzed by two-way ANOVA at 39 weeks of age. In both male and female mice, apoE genotype was weakly related to lower
body weight (p < 0.05 for both males and
females). Diet was not related to body weight in male or female
mice.
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Figure 1.
Body weights of the 60 mice in the primary study
group versus age. Male and female mice are represented with
filled and open symbols, respectively.
Genotypes are either wt (solid lines) or
apoE / (dashed lines). Diets are
either -tocopherol-deficient (inverted triangles),
normal (squares), or supplemented
(triangles). Data are mean ± SEM weights for all
mice in each group. Male mice weighed significantly more than female
mice at all ages. Two-way ANOVAs for genotype versus diet for weight at
39 weeks of age had p < 0.05 for genotype in males
and p < 0.05 for genotype in females. Diet was not
significantly related to body weight in males or female.
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Previously, we reported that cerebral F2-IsoPs
are modestly elevated in aged apoE / mice, a finding
confirmed by others (Montine et al., 1999a; Pratico et al., 1999).
apoE / mice in the present study reared on the normal
diet had total IsoP levels of 16.8 ± 1.2 ng/gm, whereas wt mice
on the normal diet had levels of 13.5 ± 0.5 ng/gm
(t24 = 2.41; p < 0.05). Total IsoP levels were stratified into four groups according to
both gender and genotype and compared by ANOVA
(F(3,24) = 4.14; p < 0.05). Interestingly, corrected repeated-pairs comparisons showed that
total IsoP levels were significantly elevated in apoE /
male mice compared with wt males (p < 0.05) but
not apoE / female mice compared with wt females.
This novel observation of a gender-specific effect of lack of apoE on
cerebral oxidative damage was also present in a separate set of eight
wt and eight apoE / mice (equally divided between
genders) aged to 9 months under the same conditions and fed standard
mouse food (data not shown). The combined results from these two
experiments are presented in Figure 2.
One-way ANOVA for the combined data set was highly significant
(F(3,40) = 5.88; p < 0.002), and corrected repeated-pairs comparisons showed significantly
elevated cerebral total IsoP levels in apoE / male mice
compared with wt male mice (p < 0.01) and
apoE / female mice (p < 0.01);
wt females did not differ from apoE / females. In
contrast to total IsoPs, ANOVA for total NPs was not statistically significant for the primary group alone
(F(3,24 = 1.8; p = 0.19) or when combined with the second group of mice
(F(3,40) = 1.5; p = 0.22).
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Figure 2.
Cerebral total (sum of F-ring and D/E-ring) IsoPs
in mice from both the primary study group maintained on diet with
normal -tocopherol levels and the secondary group raised on normal
mouse food stratified by genotype and gender. Data are mean ± SEM
for each group. One-way ANOVA (F(3,40) = 5.88) had p < 0.002 with corrected
repeated-pairs comparisons showing significant differences between wt
male versus apoE / male mice
(p < 0.01) and apoE /
male versus apoE / female mice
(p < 0.01).
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We determined the concentrations of -tocopherol in plasma and
cerebellum to assess the efficacy of the different diets to modulate
endogenous -tocopherol levels. Plasma -tocopherol levels were
0.01 ± 0.00, 0.50 ± 0.10, or 1.47 ± 0.26 mg/dl for
mice on the deficient, normal, or supplemented diet, respectively
(F(2,54) = 7.93; p < 0.0001; corrected repeated-pairs comparisons had p < 0.05 for normal vs deficient diets and p < 0.01 for
normal vs supplemented diets). Brain -tocopherol levels also varied
significantly with diet (F(2,55) = 280; p < 0.0001) (Fig.
3); repeated-pairs comparisons showed
that brain -tocopherol levels were significantly greater in mice
reared on the supplemented diet (p < 0.001) and significantly less in mice reared on the deficient diet
(p < 0.001) compared with mice reared on the
normal diet. There was a significant correlation between plasma and
brain -tocopherol concentrations in all mice
(p < 0.0001;
R2 = 0.24). One-way ANOVAs for
brain or plasma -tocopherol levels were not significant when data
were stratified by gender and genotype into four groups.
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Figure 3.
Brain -tocopherol levels were quantified in
cerebellum from each mouse using HPLC with electrochemical detection,
and the mean ± SEM values for mice on the three different diets
are presented (one-way ANOVA had p < 0.0001). Mice
were reared on one of three different diets from 3 to 39 weeks of age.
Diets were prepared from tocopherol-stripped base material with no
(deficient), 50 IU/kg (normal), or 500 IU/kg (supplemented)
all-rac--tocopherol acetate added.
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We analyzed the effects of the different diets on total IsoPs or NPs by
two-way ANOVA for wt versus apoE / mice of each gender.
In male mice, two-way ANOVA for total IsoP was significant for genotype
only (p < 0.05) but not for diet or interaction
between diet and genotype (Fig.
4A). Post
hoc analysis with one-way ANOVA revealed that total IsoPs were
significantly related to diet but only in wt male mice
(F(2,17) = 7.89; p < 0.005). Corrected repeated-pairs comparisons showed that wt male mice
on the supplemented diet had on average 37% lower total IsoPs compared
with wt male mice on normal diet (p < 0.05);
there was no difference in total IsoPs between normal and deficient
diets. Two-way ANOVA for total IsoPs in female mice was not significant
for genotype or diet (data not shown).
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Figure 4.
A, Cerebral total (sum of F-ring
and D/E ring) IsoPs in male mice were stratified according to genotype
and diet, and the mean ± SEM values are presented for each group.
Two-way ANOVA was significant for genotype
(p < 0.05) but not diet; there was no
significant interaction between genotype and diet. Post
hoc one-way ANOVAs showed that cerebral total IsoPs were
significantly different among male wt
(F(2,17) = 7.89; p < 0.005), but not apoE /, mice. B,
Cerebral total (sum of F-ring and D/E ring) NPs in male mice were
stratified according to genotype and diet, and the mean ± SEM
values are presented for each group. Two-way ANOVA was significant for
diet (p < 0.05) but not genotype alone;
however, there was a significant interaction between genotype and diet
(p < 0.05). Post hoc one-way
ANOVAs showed that cerebral total NPs were significantly different
among male apoE /
(F(2,17) = 6.61; p < 0.01), but not wt, mice.
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In contrast to total IsoPs in which genotype most strongly influenced
variance in data in male mice, two-way ANOVA showed that total NPs in
male mice were significantly influenced by diet (p < 0.05) but not genotype (Fig.
4B). Importantly, there was significant interaction
between diet and genotype (p < 0.05). Post hoc one-way ANOVA showed that the combination of lack
of apoE plus -tocopherol deficiency led to a near doubling of NPs in
apoE / male mice
(F(2,17) = 6.61; p < 0.01); corrected repeated-pairs comparisons were significant for normal
versus deficient diets (p < 0.05 for both
genotypes) but not normal versus supplemented diets. Post
hoc one-way ANOVA for total NPs in wt males on the different diets
was not significant (Fig. 4B). Two-way ANOVA for cerebral total NP levels in female mice was not significantly related
to diet or genotype (data not shown).
The ratio of F- to D/E-ring IsoPs and NPs can be used as a measure of
reducing capacity of the environment in which oxidation of AA
and DHA, respectively, occurred. Two-way ANOVAs for the F- to D/E-IsoP
ratio was not significant for diet or genotype in male or female mice
(data not shown). In contrast, two-way ANOVA for the F- to D/E-NP ratio
in male mice was significant for diet (p < 0.01) but not genotype or interaction between diet and genotype.
Post hoc one-way ANOVA for the effects of diet on the NP
ratio in wt male mice was significant
(F(2,18) = 17.59; p < 0.0001) (Fig. 5). Corrected
repeated-pairs comparisons showed that the F- to D/E-NP ratio was
significantly increased in wt male mice on the
-tocopherol-supplemented versus normal diet (p < 0.01) but that there was no significant
difference in NP ratio between deficient and normal diet. The
relationship of F- to D/E-NP ratio to diet was remarkably similar in
apoE / male mice compared with wt male mice (Fig. 5).
One-way ANOVA for the effects of diet on NP ratio in male
apoE / mice was
F(2,18) = 3.65 and p < 0.05, with p < 0.05 for normal versus supplemented diets. Two-way ANOVA for the F- to D/E-NP ratio in female mice was not
significant for diet or genotype (data not shown).
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Figure 5.
The ratios of cerebral F-ring to D/E-ring NPs in
male mice were stratified according to genotype and diet, and the
mean ± SEM values presented for each group. Two-way ANOVA was
significant for diet (p < 0.05) but not
genotype or interaction between diet and genotype. Post
hoc one-way ANOVAs with corrected repeated-pairs comparisons
showed significantly increased ratios in mice of both genotypes on the
-tocopherol-supplemented diet (sup vitE) compared
with normal diet. There was no significant difference in the F-ring to
D/E-ring ratio between mice on a normal diet and a
-tocopherol-deficient diet (no vitE).
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We next determined the concentrations of cerebral AA and DHA in each
mouse. Two-way ANOVA showed that cerebral DHA levels were significantly
related to diet in male (p < 0.0001) and female (p < 0.0001) mice but not related to the
presence or absence of apoE, nor was there an interaction between diet
and genotype. Two-way ANOVA showed that cerebral AA levels were not
significantly related to either diet or genotype. Post hoc
one-way ANOVA showed that cerebral DHA levels were significantly
increased in male and female mice on -tocopherol-deficient diets
(p < 0.05 for both genders) and tended to be
lower for mice of both genders on the supplemented diet compared with
mice on the normal diet. Because of this association with diet
alone in both genders, cerebral DHA levels were further analyzed by
regression analysis with brain -tocopherol level; results for AA are
included for comparison (Fig. 6).
Cerebral DHA, but not AA, levels were significantly inversely related
to brain -tocopherol concentrations
(R2 = 0.51;
p < 0.0001). This unexpected association between
deficiency of -tocopherol and increased cerebral DHA raised the
possibility that the increased total NP levels observed in male
apoE / mice may be attributable to increased substrate
and not increased oxidative stress. Therefore, IsoP and NP levels were
calculated per microgram of DHA or AA in the same sample, respectively,
rather than per milligram of protein. When the data were reanalyzed,
there was no change in the statistically significant relationships
reported above. A summary of the statistically significant changes in
mice on the deficient and supplemented diets relative to mice of
the same gender and genotype on a normal diet is presented in Table 1.
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Figure 6.
Cerebral AA and DHA concentrations as a percentage
of total fatty acids were determined by GC and plotted versus brain
-tocopherol concentrations. Linear regression analysis was
performed, and the best-fit lines plus 95% confidence intervals also
are plotted. There was a highly significant negative linear correlation
between the percentage of cerebral fatty acids as DHA and the
concentration of -tocopherol in brain
(R2 = 0.51;
p < 0.0001). There was no significant relationship
between cerebral AA levels and -tocopherol concentrations.
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Table 1.
Statistically significant percentage of changes relative to
mice of the same gender and genotype raised on a normal diet
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We further investigated the ability of -tocopherol supplementation
to promote reduction of the endoperoxide of DHA but not AA in rat brain
synaptosomes oxidized ex vivo with AAPH. Previously, we
showed that oxidation of rat brain synaptosomes under these conditions
leads to time- and concentration-dependent increases in F-ring and
D/E-ring IsoPs and NPs (Reich et al., 2000). Plasma levels of
-tocopherol vary among individuals, but normal values are ~10-50
µM (Elin, 1996). Therefore, rat brain
synaptosomes were exposed to a constant oxidative stress with 5 mM AAPH plus 0, 1, 10, or 100 µM -tocopherol for 24 hr and then assayed
for F- and D/E-ring IsoPs and NPs to model our in vivo
experiments with -tocopherol-deficient, normal, and -supplemented
diets. Similar to what has been observed with plasma low-density
lipoprotein oxidized under similar conditions, -tocopherol displayed
concentration-dependent pro-oxidant activity in this in
vitro system, but only with DHA and not AA (Fig.
7A). One-way ANOVA showed that
-tocopherol significantly increased total NP formation
(F(3,8) = 57.4; p < 0.0001) in the presence of AAPH and that this was significantly
different from AAPH alone for only 10 (p < 0.05) and 100 (p < 0.001)
µM -tocopherol. In contrast, one-way ANOVA
for total IsoPs was not significantly different among the different
concentrations of -tocopherol. The increased NPs formed in
synaptosomes by the addition of -tocopherol with AAPH were not
proportionately distributed between F- and D/E-ring forms but rather
displayed an -tocopherol concentration-dependent shift toward F-ring
NPs. One-way ANOVA showed that increasing concentrations of
-tocopherol significantly increased the F- to D/E-NP ratio
(F(3,8) = 87.2; p < 0.0001) and that this increase was significantly different from AAPH
alone at 10 and 100 µM -tocopherol (p < 0.001 for both concentrations) (Fig.
7B). In contrast, there was no change in the ratio of
F2-IsoP to D2/E2-IsoP with increasing concentrations of
-tocopherol.
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Figure 7.
F- and D/E-ring NPs and IsoPs were determined in
rat cerebral synaptosomes oxidized with AAPH in the presence of
increasing concentrations of -tocopherol. A,
Concentration-response relationship for total (F-ring plus D/E-ring)
IsoPs and NPs. B, F- to D/E-ring ratio for IsoPs and
NPs.
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DISCUSSION |
Oxidative damage to cerebrum may be a central mechanism that
contributes to age-related decline in cognitive function as well as a
number of degenerative diseases of brain, including AD (Markesbery and
Carney, 1999). Cerebral oxidative damage is not uniform throughout the
population (Montine et al., 1999b,c), likely because of differences in
inherited susceptibilities and environmental factors among individuals;
however, little data are available on the specifics of these
gene-environment interactions. We pursued the effects of
gene-environment interactions from inherited susceptibility attributable to lack of apoE and the modulation of dietary
-tocopherol on quantitative indices of cerebral oxidative damage in
aged mice of both genders. The major findings from this investigation
were as follows: (1) gender had a pervasive effect on cerebral
oxidative damage and response to -tocopherol; (2) diet or genotype
influenced oxidative damage to AA and DHA differently, strongly
implying that quantification of oxidative damage to these two fatty
acids reflects events in two different biochemical compartments in
cerebrum, and (3) -tocopherol may exert its antioxidant activity in
cerebrum through a combination of radical scavenging and enhanced
reducing capacity.
Our results indicated that male gender should be considered a
susceptibility factor for cerebral oxidative damage in this strain of
mouse. Several studies have investigated behavioral and brain
structural changes in aged apoE / mice. Although some groups have consistently observed differences between aged
apoE / mice and wt controls, several groups
have been unable to replicate these findings (Masliah et al., 1995;
Fagan et al., 1998; Montine et al., 1999a; Buttini et al., 2000). The
basis for this discrepancy has not been clear. Our data suggest that
gender composition of the study groups may be a key factor. Indeed, our
results add to those of others showing significant differences between
adult male and female apoE / mice (Raber et al., 1998).
Contrary to male-specific effects observed in our study, women who have
inherited an APOE4 allele are at greater risk for developing
AD than men (Corder et al., 1993; Bretsky et al., 1999), and female
transgenic mice that express a mutant form of the human amyloid
precursor protein have greater senile plaque formation in brain at 15 months of age or older than males (Callahan et al., 2001). Although
direct comparison among these studies is limited by the use of
different models and different endpoints, they suggest a complex
relationship among age, gender, age-related hormonal changes, and the
different facets of AD pathogenesis. One study has shown that
-tocopherol dietary supplementation ameliorates brain structural
changes associated with aging in apoE / mice, although
these investigators did not specify any gender difference in the
response to -tocopherol (Veinbergs et al., 2000). Our results also
demonstrated coincident biochemical changes in brain that accompany
-tocopherol supplementation. Thus, differences in diets, in addition
to gender, may contribute to variation in results among different
laboratories that have investigated aged apoE / mice.
Our studies showed that at least two risk factors are needed to
increase cerebral AA oxidation (male gender and lack of apoE), whereas
three risk factors (male gender, lack of apoE, and deficiency in
-tocopherol) must combine to increase oxidative damage to DHA. As
mentioned, AA is evenly distributed throughout gray matter and white
matter, whereas DHA is concentrated in neurons (Salem et al., 1986;
Moore, 1993). These data imply that cerebral AA exists in biochemical
environments that are more prone to oxidative damage than are the
environments that contain DHA. Neuron plasma membranes contain a much
higher level of DHA than other cell types, presumably serving an
important role in the membrane characteristics of neurons. Therefore,
it is perhaps not surprising that additional mechanisms exist to
protect DHA from oxidation in vivo. Our in vivo
results showing increased total NP in male apoE / mice with deficiency in -tocopherol suggest that one mechanism to protect
neuronal membranes is radical scavenging by -tocopherol.
The extent of AA and DHA oxidation responded differently to dietary
supplementation with -tocopherol. The inability of increased brain
-tocopherol to decrease basal total NPs suggests that the lower
levels of brain -tocopherol achieved on the normal diet fully
protected DHA. In contrast, -tocopherol supplementation did reduce
AA oxidation in male wt mice, suggesting that normal diet did not
provide full antioxidant protection to AA and providing additional
evidence that -tocopherol preferentially protects cerebral DHA over
AA in vivo. However, the results with cerebral AA oxidation
on the supplemented diet are more complex because there was no
reduction in total IsoPs in male apoE/ mice. One interpretation of these data are that male apoE / mice
may have reached a maximum plateau of IsoP levels that does not show
response to increased brain -tocopherol, whereas the lower cerebral
IsoP levels in male wt mice may still be in a range that is responsive to dietary supplementation with -tocopherol. Regardless of the mechanisms involved, an important public health consideration from
these results is that normal dietary levels of -tocopherol provided
maximum free radical scavenging protection for cerebral DHA but that
long-term dietary supplementation achieved significantly higher
-tocopherol levels in brain and augmented free radical protection
for AA in wt male mice.
In addition to -tocopherol acting as an apparent radical scavenging
agent in cerebrum, our results also suggested that, in vivo,
-tocopherol may act as a reductant of the DHA endoperoxide but not
the AA endoperoxide. Other antioxidants have both radical scavenging
and reducing activity, the best example being glutathione. In
vitro, -tocopherol has been shown to possess reducing activity for Cu(II) (Kontush et al., 1996), and we confirmed similar
activity in the synaptosome experiments; however, we are unaware of any previous demonstration of this action of -tocopherol in
vivo. The mechanisms by which -tocopherol had a selective
reducing effect on DHA endoperoxide in vivo and in
vitro are not clear. Possibilities include a preferential
biochemical interaction between -tocopherol and DHA endoperoxide or
possibly a higher concentration of -tocopherol in DHA-containing
compartments in vivo. Similar to others (Thomas and Stocker,
2000), we observed an in vitro pro-oxidant effect of
-tocopherol, again selective for DHA over AA. Although the
physiological significance of this in vitro pro-oxidant effect is not clear, these data do reinforce the conclusion of a
preferential interaction of -tocopherol with DHA over AA.
In summary, we investigated the interaction of apoE and -tocopherol
on cerebral oxidative damage in aged mice. By all measures, male mice
were more vulnerable to cerebral oxidative damage than female mice.
Multiple stressors were required to increase oxidative damage to
cerebrum, although DHA appeared better protected from oxidative damage
than was AA. The effects of -tocopherol in vivo were
complex, demonstrating radical scavenging activity for both AA and DHA,
depending on its concentration in brain, and reducing activity for the
DHA endoperoxide, the latter activity being reproducible in
vitro. Finally, we defined a significant interaction between lack
of apoE and deficiency of -tocopherol in enhancing DHA but not AA
oxidative damage. These results demonstrated that the interaction of
gender, inherited susceptibilities, and diet contributed to age-related
oxidative damage to cerebrum.
| |
FOOTNOTES |
Received April 26, 2001; revised June 1, 2001; accepted June 6, 2001.
This work was supported by National Institutes of Health Grants
AG00774, AG16835, and AG05114, as well as a grant from the Alzheimer's
Association (T.J.M.) and a Burroughs-Welcome Clinical Scientist Award
in Translational Research (J.D.M.). We thank Bill Zackert, Ling Gao,
Stephanie Sanchez, and Erin Terry for their expert assistance.
Correspondence should be addressed to Dr. Thomas J. Montine, Department
of Pathology, Vanderbilt University Medical Center, C-3321A Medical
Center North, Nashville, TN 37232. E-mail:
tom.montine{at}mcmail.vanderbilt.edu.
| |
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ABSTRACT
INTRODUCTION
