Full text of DOI:10.1093/gerona/56.10.B426

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 10, B426–B431
Age-Related Increase of Brain Cyclooxygenase
Activity and Dietary Modulation of Oxidative Status
Bong Sook Baek,1,2 Jung Won Kim,1,2 Ji Hyeon Lee,2 Hyun Joo Kwon,1,2 Nam Deuk Kim,1,2
Ho Sung Kang,2 Mi Ae Yoo,2 Byung Pal Yu,3 and Hae Young Chung1
1College of Pharmacy and 2Research Institute of Genetic Engineering, Pusan National University, Pusan, South Korea.
3The University of Texas Health Science Center at San Antonio, Texas, USA.
Several studies have demonstrated that inhibitors of cyclooxygenase (COX) attenuate various
neuronal injuries and age-dependent demented conditions. From these findings, we proposed to
test the effect of age on COX activity and its possible suppression by the antiaging action of di-
etary restriction in the rat brain. The status of reactive oxygen species (ROS) was also assessed
to correlate with COX activity to delineate the underlying mechanism of the altered COX activ-
ity during aging. These results showed that COX activity significantly increased in 24-month-
old rats compared with 6-month-old rats in an ad libitum group. Interestingly, mRNA and pro-
tein levels of COX-2 showed little corresponding age-related change. The formation of ROS
was found to increase gradually with age in ad libitum fed rats. However, dietary restriction sup-
pressed the increase at the age of 24 months. To substantiate the relationship between ROS and
COX activity when the rats were 24 months of age, we conducted in vitro experiments with a C6
glioma cell line. Together, it is concluded that increased COX activity with age is due to the ac-
tivation of COX catalytic reaction by ROS without increased gene expression of COX-2 and
that it is related to the increased pro-oxidant status in aged rats.
YCLOOXYGENASE (COX) is the rate-limiting en-
zyme in prostaglandin (PG) synthetic pathway, which
all suppressed by DR (16). In addition, the decline of anti-
oxidant defense and detoxification systems during aging has
been effectively modulated by DR (17,18). From these find-
ings, Yu (19) proposed the broad antioxidative action exhib-
ited by DR as the underlying mechanism of its life-prolong-
ing effect.
However, to date, it is not known whether DR attenuates
the production of ROS and PG, and whether it modulates
COX activity in the brain. Thus, the aim of this study was to
investigate changes in COX activity with age and the modu-
lation by DR. We quantified ROS generation to correlate
with COX activity and its mRNA gene expression in the
cerebrums of aged rats. To substantiate these findings in
brain tissue, we further examined the in vitro effect of vari-
ous oxidants on COX activity in C6 glioma cells.
C
converts arachidonic acid to various PG metabolites (1).
Two isoforms of COX have been identified in eukaryotic
cells: constitutively expressed COX-1 (2,3) and inducible
COX-2 (4,5). COX-1 is expressed in most tissues and is re-
sponsible for maintaining prostanoids at physiological lev-
els (1). In contrast, COX-2 that shows undetectable levels
under normal physiological conditions can be induced by li-
popolysaccharides and cytokines (6). COX-2 catalyzes high
levels of prostanoid synthesis under various pathological
conditions. For example, in the brain, the induction of
COX-2 is pathologically responsible for an increased pros-
tanoid production (7). In astrocytes and microglial cells,
COX-2 has often been implicated in neurodegenerative dis-
eases (7–9) and cerebral inflammatory processes (10,11).
Several studies have shown that COX is activated by hy-
droperoxides for the initiation of the arachidonic acid cas-
cade pathway (12,13), and recently nonradical peroxynitrite
has been reported to be an effective initiator of COX activ-
ity (14); this suggests that a variety of oxidants may be
physiological activators of COX.
MATERIALS AND METHODS
Materials and Chemicals
2ꢀ,7ꢀ-dichlorofluorescein diacetate (DCFDA) was pur-
chased from Molecular Probes, Inc. (Eugene, OR). 32P-
adenosine triphosphate (250 ꢁCi) and an enzyme immu-
noassay (EIA) kit for prostaglandin E2 were purchased from
Amersham (Bucks, UK). RNAzol B was obtained from
TEL-TEST, Inc. (Friendwood, TX). Primers for reverse
transcription polymerase chain reaction (RT-PCR) were
synthesized by Bioneer (Daejeon, Korea). Primary and sec-
ondary antibodies for Western blotting were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA) and Serotec
Recent studies on the modulation of reactive oxygen spe-
cies (ROS) reactions by dietary restriction (DR) as an anti-
aging measure have established that this dietary interven-
tion can attenuate the age-related inflammatory process (15)
and oxidative stress (16–18). For instance, the production of
superoxide, hydroxyl radicals, and H2O2 and the lipid per-
oxidation of mitochondrial and microsomal membranes are
B426

ROS EFFECT ON AGE-RELATED INCREASE OF CYCLOOXYGENASE ACTIVITY
B427
(Oxford, England), respectively. Polyvinylidene difluoride
(PVDF) membranes were obtained from Millipore Corpora-
tion (Bedford, MA).
3ꢀ. The expected product sizes for COX-1 and COX-2 were
441 and 474 base pairs, respectively. The mRNA levels of
constitutively expressed glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) were used as positive control. The
primer pairs for GAPDH were sense, 5ꢀ-GGGTGATGCTG-
GTGCTGAGTATGT-3ꢀ and antisense, 5ꢀ-AAGAATGG-
GAGTTGCTGTTGAAGTC-3ꢀ. The GAPDH primer set
yields a PCR product of 700 base pairs in length. PCR con-
ditions were 33 cycles for COX-1, 35 cycles for COX-2,
and 20 cycles for GAPDH, respectively, consisting of 30-
second denaturation at 94ꢃC, 30-second annealing at 54ꢃC,
and 1-minute extension at 72ꢃC. Electrophoresis was per-
formed in 1.5% agarose gel. After the gel was stained with
ethidium bromide, the band was visualized and quantified
under a transilluminator.
Animals and Diet
Specific pathogen-free (SPF) male Fischer 344 rats from
the colony of the National Institute on Aging, part of the
National Institutes of Health, were raised at the University
of Texas Health Science Center at San Antonio and were
fed a diet of the following composition: 21% soybean pro-
tein, 15% sucrose, 43.65% dextrin, 10% corn oil, 0.15%
l-methionine, 0.2% choline chloride, 5% salt mix, 2% vita-
min mix, and 3% Solka-Floc (International Fiber Co., North
Tonawanda, NY). All rats were fed ad libitum (AL) until 6
weeks of age, and then they were divided into two groups:
an AL-fed control group and a DR group. Rats in the DR
group were restricted by 40% of the food intake of the AL
control group. Cerebrums from rats of 6, 12, 18, and 24
months of age were used in this study. Detailed information
and procedures on dietary modulation and the maintenance
of the SPF status of animals have been published by Yu and
colleagues (20).
Western Blotting
Western blotting was carried out by the method of Habib
and colleagues (23). Briefly, total protein equivalents (60 ꢁg)
for each sample were separated by sodium dodecyl sulfate
(SDS)-polyacrylamide minigel electrophoresis and trans-
ferred to a PVDF membrane at 15 V for 1.5 hours in a
semidry transfer system. Nonspecific binding to the mem-
brane was blocked by 1% nonfat milk blocking buffer of
10mM Tris (pH 7.5), 100mM NaCl, and 0.1% Tween 20 at
room temperature for 1 hour. The membrane was washed
and then probed with the polyclonal goat antibody against
COX-1 or COX-2 (Santa Cruz Biotechnology, 1:200) over-
night at 4ꢃC. Bands were visualized by using horseradish
peroxidase-conjugated donkey antigoat antibody (Serotec,
1:1000) and enhanced chemiluminescence assay (Amer-
Tissue Preparation
Rats were sacrificed by decapitation, and the brains were
immediately dissected to isolate the cerebrums. Isolated rat
cerebrums were homogenized in seven volumes of ice-cold
50mM phosphate buffer at a pH of 7.4; the buffer contained
0.5mM phenylmethylsulfonyl fluoride, 1mM ethylenedi-
amine tetra-acetic acid, 80 mg/L trypsin inhibitor, and 1ꢁM
leupeptin. It was centrifuged at 900 ꢂ g at 4ꢃC for 15 min-
utes. The supernatants were centrifuged at 12,000 ꢂ g at
4ꢃC for 15 minutes to yield pelletized mitochondrial frac-
tions, and these supernatants were referred to as postmito-
chondrial fractions.
Cyclooxygenase Activity
COX activity was measured by a modified method of Bos
and colleagues (21). The activity was determined by the
quantification of prostglanden E2 (PGE2). Briefly, postmito-
chondrial fractions were incubated in 50mM phosphate
buffer (pH 7.4) containing 0.6ꢁM arachidonate at 37ꢃC
for 30 minutes. Then, the mixture was boiled for 3 min-
utes to terminate the reaction, followed by centrifugation at
10,000 ꢂ g for 15 minutes. PGE2 in supernatant was assayed
for the measurement of COX activity with an EIA kit.
Measurement of COX mRNA
Total RNA was prepared by using RNAzol B (2 ml/100
mg of tissue) according to the manufacturer’s instruction.
After the first strand cDNA was synthesized from 2 ꢁg of
total RNA by reverse transcription, polymerase-chain reac-
tion was performed with the cDNA. The primer pairs used
for COX-1 detection (22) were sense, 5ꢀ-CTGCATGTG-
GCTGATGTCATC-3ꢀ and antisense, 5ꢀ-AGGACCCGT-
CATCTCCAGGGTAATC-3ꢀ, and those for COX-2 (22)
were sense, 5ꢀ-CAAGCAGTGGCAAAGGCCTCCATT-3ꢀ
and antisense, 5ꢀ-TAGTCTGGAGTGGGAGGCACTTGC-
Figure 1. Effects of age and dietary restriction on cyclooxygenase
(COX) activity in cerebrums. Each value is the mean ꢄ SE of six rats.
Statistical significance: *p ꢅ .05 vs 6 month-old rats of the ad libitum
group, ##p ꢅ .01 vs 24-month-old rats of the ad libitum group. PGE2 ꢆ
prostaglandin E2.

B428
BAEK ET AL.
sham). Prestained blue protein markers (Biorad, Hercules,
CA) were used for molecular weight determination.
Statistical Analysis
Results were analyzed statistically by post hoc tests of the
statistical package Super ANOVA. Values of p ꢅ .05 were
considered statistically significant.
Measurement of ROS
ROS generation was measured by the method of Thomas
and colleagues (24) as previously described. Briefly, 25ꢁM
DCFDA was mixed with postmitochondrial fractions iso-
lated from cerebrums and incubated at 37ꢃC. Changes in
fluorescence intensity were measured at an excitation wave-
length of 485 nm and an emission wavelength of 530 nm by
the Fluorescence Plate Reader (BIO-TEK Instruments, Wi-
nooski, VT) for 30 minutes. The concentrations of dichlo-
rofluorescein (DCF) formed were calculated according to a
DCF standard calibration curve.
RESULTS
Effects of Age and DR on COX Activity
The COX activity of AL-fed rats showed a significant
and age-related increase, more pronouncedly in 24-month-
old rats than in 6-month-old rats (Figure 1). It is worth not-
ing that the increase of COX activity in AL-fed rats was in
parallel to the increased ROS generation at 24 months of
age, as shown Figure 4 below. In contrast, DR suppressed
the increase at 24 months of age.
PGE2 Production by Glioma Cells
Glioma cells were plated in six-well plates and incubated
with various oxidants, H2O2, ꢇO2ꢈ, t-butyl hydroperoxide (t-
BHP), hydroxynonenal (HNE), hydroxyhexenal (HHE), and
malondialdehyde (MDA) for 6 hours. The generation of
ꢇO2ꢈ was done by the use of pyrogallol (25). The collected
supernatants from the culture medium were applied for de-
termination of PGE2 with an EIA kit (21).
Modulation of COX Gene Expression by Age and DR
As a way to measure mRNA levels of COX, both COX-1
and COX-2, total RNA extracts were analyzed by the RT-
PCR method. As shown in Figure 2, the mRNA expression
of the constitutive COX-1 and inducible COX-2 showed lit-
tle change either with age or by DR.
Figure 2. Effects of age and dietary restriction on the mRNA levels of cyclooxygenase (COX)-1 and COX-2; A is the ad libitum fed group and
B is the diet-restricted group. GAPDH ꢆ glyceraldehyde-3-phosphate dehydrogenase; 6A ꢆ 6-month-old rats of ad libitum group.

ROS EFFECT ON AGE-RELATED INCREASE OF CYCLOOXYGENASE ACTIVITY
B429
Figure 3. Effects of age and dietary restriction on the protein level of cyclooxygenase (COX)-1 and COX-2; A is the ad libitum fed group and
B is the diet-restricted group. 6A ꢆ 6-month-old rats of ad libitum group.
As a way to examine whether COX-2 activity was modu-
lated at the protein synthesis step, Western blotting was car-
ried out. The COX-2 protein levels were similar to mRNA
levels (Figure 3), indicating that the age-associated increase
of COX-2 activity was not accompanied by newly synthe-
sized COX protein. Therefore, it is concluded that the age-
related increase in COX activity is most likely due to post-
translational modulation, the catalytic activation of the
COX-2 molecule per se rather than the involvement of in-
creased COX-2 gene expression.
cantly stimulated by H2O2 (two-fold), t-BHP (six-fold), and
ꢇO2 (1.5-fold), whereas HNE, HHE, and MDA provided
no discernable stimulation. These results are consistent with
evidence, albeit indirect, that the age-related increase of
COX activity might closely be related to the increased lev-
els of oxygen-derived ROS during aging.
ꢈ
DISCUSSION
Increased COX activity and PGE2 production have been
implicated as active participants in the pathogenesis of sev-
eral age-related diseases, such as cancer, Alzheimer’s dis-
ease, and autoimmune diseases (26). More specifically,
COX-2 is shown to be associated with acute exudative in-
flammation, granuloma formation, bone resorption, and
pain in rheumatoid arthritis (27). These evidences clearly
indicate a significant role of COX-2 in these age-related
pathophysiological processes.
ROS Generation in the Aging Process
Because the increase of COX activity with age may be af-
fected by changes in oxidative status, we examined ROS
generation with age and its suppression by DR (Figure 4).
Data clearly showed that the ROS formation increased with
age in AL-fed rats, whereas the antioxidative DR consis-
tently suppressed this age-dependent increase (Figure 4).
In the present study, we obtained data for the first time
that cerebral COX activity significantly increases in 24-
month-old rats compared with 6-month-old rats. More sig-
nificantly, we were able to show that DR rats suppress the
increases found at 24 months. As a way to further define the
molecular insights on whether this increased activity was
modulated at a gene level, COX mRNA and protein levels
were analyzed. Our data showed no indication that the
amounts of either COX mRNA or protein were involved.
Effect of Oxidants on COX Activity in C6 Glioma cells
As a way to obtain further clues on the nature of ROS on
the COX activation observed in the rat brain tissue, C6
glioma cells were challenged in vitro with various pro-oxi-
dant reactive species. For this purpose, H2O2, ꢇO2ꢈ, t-BHP,
HNE, HHE, and MDA were used. As shown in Figure 5, the
COX activity measured by PGE2 production was signifi-

B430
BAEK ET AL.
expression. Our results are in line with those of Wu and col-
leagues (28), who showed the age-associated increase in
COX activity and PGE2 production of macrophage without
a change of COX gene expression. The influence of redox
status including various pro-oxidants on COX activity has
been shown by several studies (29). For instance, some an-
tioxidants and antioxidant enzymes such as glutathione
peroxidase can inhibit COX activity (29). These studies in-
dicated that the full activation of COX under pathophysi-
ological conditions may require the presence of pro-oxi-
dants as possible regulators. In this context, it is worth
pointing out that Kulmacz and Wang (30) showed that
COX-2 activation is more receptive to ROS activation
than COX-1. Thus, it can be speculated, based on our
present data and others, that prostanoid hydroperoxides syn-
thesized from the arachidonate cascade can exacerbate the
inflammatory condition by further activation of COX-2.
This possibility is consistent with previous findings on age-
related increased proinflammatory processes often observed
with age and the attenuation by the antiaging action of DR
(31,32).
In summary, our results showed that COX activity in-
crease with age, and this age-associated increase in COX
activity is due to COX activation by ROS rather than in-
duced gene expression of COX-2. This was confirmed in
vitro by showing the stimulating effect of ROS on COX ac-
tivity in C6 glioma cells. Furthermore, we were able to es-
tablish that DR can reverse the increase found when the rats
were at 24 months of age, suggesting a mechanistic basis for
the DR’s known anti-inflammatory efficacy.
Figure 4. Effects of age and dietary restriction on reactive oxygen spe-
cies (ROS) generation in postmitochondrial fraction of cerebrum. Each
value is the mean ꢄ SE of six rats. Statistical significance: *p ꢅ .05;
***p ꢅ .001 vs 6-month-old rats of the ad libitum group; ###p ꢅ .001 vs
24-month-old rats of the ad libitum group. DCF ꢆ dichlorofluorescein.
Thus, it is concluded that the increase of COX activity and
its suppression by DR observed is due to the posttransla-
tional modulation responsible for the increase in COX ac-
tivity rather than the involvement of increased COX-2 gene
Acknowledgments
This work was supported by the Korean Research Foundation under
Grant KRF-99-0005-F00030/F00037. We are grateful to the Aging Tissue
Bank for the supply of the aging tissue.
Address correspondence to Hae Young Chung, Department of Phar-
macy, College of Pharmacy, Pusan National University, Gumjung-ku,
Pusan 609-735, Korea. E-mail: hyjung@hyowon.pusan.ac.kr
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Received February 26, 2001
Accepted April 19, 2001
Decision Editor: John Faulkner, PhD