Journal of Gerontology: BIOLOGICAL SCIENCES
Cite journal as: J Gerontol A Biol Sci Med Sci
2009 Vol. 64A, No. 11, 1114–1125
Overexpression of Mn Superoxide Dismutase Does Not
Increase Life Span in Mice
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Advance Access publication on July 24, 2009
of physiological function caused by the progressive accu-
mulation of oxidative damage. In 1972, Harman ( 1 ) modi-
fi ed his original theory to incorporate the contribution of
mitochondria to oxidative stress and proposed the mitochon-
drial theory of aging. The core principle of the theory is that
the generation of reactive oxygen species (ROS) and the sub-
sequent oxidative damage to key mitochondrial components,
such as protein, lipids, and DNA, lead to an overall decline
in cellular function and ultimately determines the life span
of an organism ( 1 ). Since the original proposal by Harman,
signifi cant amounts of correlative data have been accumu-
lated in support of the oxidative stress theory of aging.
Mn superoxide dismutase (MnSOD) is an essential mito-
chondrial antioxidant enzyme that scavenges superoxide
product of mitochondrial respiration ( 2 , 3 ). MnSOD is located
in the mitochondrial matrix and, because of its rapid reac-
tion with O2
mitochondrial oxidative damage. The potential importance
of MnSOD in aging has been highlighted by studies showing
that mutations in the insulin or insulin-like growth factor-1
(IGF-1) signaling pathway lead to increased life span in
invertebrates ( 4 , 5 ). Early studies showed that the daf-2
mutants in Caenorhabditis elegans upregulate the expres-
sion of the sod-3 gene, that is, the gene for MnSOD, through
an insulin-like signaling pathway ( 6 ). Using DNA microarray
analysis, Kenyon ’ s laboratory ( 7 ) subsequently found that
HE oxidative stress theory of aging proposes that the life
span of an organism is determined by the age-related loss
?−) generated in the respiratory chain as a by-
?− , is thought to be a fi rst-line defense against
the expression of the sod-3 and the ctl-1 and ctl-2 genes,
which code for catalase in C. elegans , was upregulated in
these long-lived mutants and met their criteria for playing a
role in extended life span. There is also evidence that some
mouse models with mutations in the insulin or IGF-1 sys-
tem show alterations in the expression of MnSOD and resis-
tance to oxidative stress. For example, Yamamoto and
colleagues ( 8 ) reported that Klotho transgenic mice, which
have increased life span, showed increased expression of
MnSOD that was correlated with increased resistance to
oxidative stress. Taguchi and colleagues ( 9 ) reported that
mice in which the gene for insulin receptor substrate 2 ( Irs2 )
was conditionally knocked out and lived longer and had
higher levels of MnSOD in their brain. Ames dwarf mice,
which have low circulating levels of IGF-1, also show in-
creased superoxide dismutase (SOD) activity in several tis-
sues ( 10 ), and Baba and colleagues ( 11 ) showed that mice
defi cient in the insulin receptor have increased activity of
MnSOD that correlates with their increased resistance to
oxidative stress. Therefore, these observations have been
often used to argue that the upregulation of MnSOD by re-
duced insulin or IGF-1 signaling may play an important role
in the mechanism(s) responsible for the increased resistance
to oxidative stress and increased life span observed in ani-
mal models with mutations in the insulin or IGF-1 signaling
Studies using genetic manipulations to alter the level of
MnSOD activity have demonstrated that regulating O2
levels is critical for maintenance of cellular function and
Youngmok C. Jang , 1 , 2 Viviana I. Pérez , 1 , 2 Wook Song , 1 , 2 Michael S. Lustgarten , 2 , 3 Adam B. Salmon , 2
James Mele , 2 , 3 Wenbo Qi , 1 , 2 Yuhong Liu , 1 , 2 Hanyu Liang , 1 , 2 Asish Chaudhuri , 2 , 4 , 5 Yuji Ikeno , 1 , 2 , 5
Charles J. Epstein , 6 Holly Van Remmen , 1 , 2 , 5 and Arlan Richardson 1 , 2 , 5
1 Department of Cellular and Structural Biology , 2 The Sam and Ann Barshop Institute for Longevity and Aging Studies , 3 Department of
Physiology , and 4 Department of Biochemistry, University of Texas Health Science Center at San Antonio .
5 GRECC, South Texas Veterans Health Care System, San Antonio .
6 Institute of Human Genetics and Department of Pediatrics, University of California, San Francisco .
Genetic manipulations of Mn superoxide dismutase (MnSOD), SOD2 expression have demonstrated that altering the
level of MnSOD activity is critical for cellular function and life span in invertebrates. In mammals, Sod2 homozygous
knockout mice die shortly after birth, and alterations of MnSOD levels are correlated with changes in oxidative damage
and in the generation of mitochondrial reactive oxygen species. In this study, we directly tested the effects of overexpress-
ing MnSOD in young (4 – 6 months) and old (26 – 28 months) mice on mitochondrial function, levels of oxidative damage
or stress, life span, and end-of-life pathology. Our data show that an approximately twofold overexpression of MnSOD
throughout life in mice resulted in decreased lipid peroxidation, increased resistance against paraquat-induced oxidative
stress, and decreased age-related decline in mitochondrial ATP production. However, this change in MnSOD expression
did not alter either life span or age-related pathology.
Key Words : Oxidative damage — Mn superoxide dismutase — Pathology — Aging .
EFFECT OF MNSOD OVEREXPRESSION ON OXIDATIVE STRESS AND AGING
limits the life span of an organism. For example, in Saccha-
romyces cerevisiae , deletion of the gene MnSOD dramati-
cally accelerates chronological aging and overexpression of
MnSOD increases chronological life span ( 12 , 13 ). Tower ’ s
group reported a similar extension of life span by overex-
pressing MnSOD in Drosophila melanogaster ( 14 ). In mice,
two independent laboratories have shown that the deletion
of MnSOD is lethal ( 15 , 16 ); mice exhibit oxidative damage
and die shortly after birth ( 15 , 16 ). Conversely, overexpress-
ing MnSOD has been reported to protect against mitochon-
drial apoptosis and oxidative damage by generating less
mitochondrial ROS ( 17 , 18 ).
The purpose of this study was to test the effects of over-
expressing MnSOD on mitochondrial function, levels of
oxidative stress or damage, and life span in mice. We show
that an approximately twofold increase in MnSOD expres-
sion throughout life resulted in a slight decrease in oxida-
tive damage and enhanced resistance against oxidative
stress. However, overexpressing MnSOD did not alter either
life span or age-related pathology in these mice.
M aterials and M ethods
The Sod2 transgenic (Tg) mice used in this study were
obtained from Dr. Epstein ’ s laboratory as described by
Raineri and colleagues ( 19 ). A 13-kb genomic Sod2 clone
isolated from C57BL/6J mice, which encompassed 2 kb of
the native Sod2 promoter, was used to generate Sod2 Tg
mice. Subsequent generations of Sod2 Tg mice were bred
by mating male Sod2 Tg mice to female wild-type (WT)
C57BL/6J mice purchased from Jackson Laboratories (Bar
Harbor, ME). The mice were genotyped at 4 – 5 weeks of age
by polymerase chain reaction analysis of DNA obtained
from tail clips as previously described ( 16 ). The mice were
maintained under pathogen-free barrier conditions in a tem-
perature-controlled environment and fed a commercial
mouse chow (Teklad Diet LM485) ad libitum. For tissue
collection, mice were euthanized by CO 2 inhalation fol-
lowed by cervical dislocation. Hind limb skeletal muscle
was used for mitochondrial isolation and enzymatic assays.
For the life span and all other analyses, male WT and
Sod2 Tg mice were housed four per cage following weaning
and fed commercial mouse chow (Teklad Diet LM485) ad
libitum. WT and Tg mice were assigned to survival groups
at 2 months of age and allowed to live out their entire life
span. There was no censoring of the WT or the Sod2 Tg
mice when measuring survival. The life span for individual
Sod2 Tg mice and WT mice was determined by recording
the age of spontaneous death; and the median, mean, 10th
percentile, and maximum survivals were calculated for each
group. All procedures followed the guidelines approved by
the Institutional Animal Care and Use Committee at the
University of Texas Health Science Center at San Antonio
and South Texas Veterans Health Care System, Audie L.
Isolation of the Skeletal Muscle Mitochondria
Mitochondria were purifi ed from whole-hind limb skele-
tal muscle according to Chappell and Perry ( 20 ) and Ernster
and Nordenbrand ( 21 ), as described previously ( 22 ). Hind
limb skeletal muscles, gastrocnemius, tibialis anterior, and
vastus lateralis were excised, weighed, bathed in 150 mM
KCl, and placed in Chappell – Perry buffer with the protease
nagarse. The minced skeletal muscle was homogenized, the
homogenate was centrifuged for 10 minutes at 600 g , and
the supernatant passed through cheesecloth and centrifuged
at 14,000 g for 10 minutes. The resulting pellet was washed
once in modifi ed Chappell – Perry buffer with 0.5% bovine
serum albumin (BSA) and once in modifi ed Chappell – Perry
buffer without BSA. Mitochondria were used immediately.
Protein concentration was measured by the Bradford method
(Bio-Rad, Richmond, CA ).
MnSOD Activity Assay
MnSOD was measured in tissue extracts from brain, kid-
ney, liver, and heart of male WT and Sod2 Tg mice using
native gels as previously described by Van Remmen and col-
leagues ( 23 ). Extracts containing 40 – 80 m g protein were
separated on a 10% polyacrylamide gel, and the gel was
soaked in a solution containing nitroblue tetrazolium, ribofl a-
vin, and N,N,N,N, tetramethyl ethylene diamine (TEMED).
In the presence of SOD, the ribofl avin is activated to oxidize
an electron donor (TEMED). The gel image was captured
with a digital-camera imager system (ImageMaster VDS;
Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed
using ImageQuant Software (Sunnyvale, CA) to quantify the
intensity of the regions representing the MnSOD activity.
Mitochondrial H 2 O 2 Release
Mitochondrial ROS production was measured with the
Amplex Red-horseradish peroxidase method (Molecular
Probes, Eugene, OR) ( 24 ). Horseradish peroxidase (HRP, 2
U/mL) catalyses the H 2 O 2 -dependent oxidation of nonfl uo-
rescent Amplex Red (80 m M) to fl uorescent resorufi n red
( 24 ). Thirty-seven units per milliliter CuZnSOD was added
to convert all O2
very rapidly with HRP and HRP-Compound I, resulting in
an underestimation of the actual rate of H 2 O 2 production
( 25 , 26 ). Therefore, our results refl ect the sum of both
superoxide and H 2 O 2 production and are referred to as
ROS rather than H 2 O 2 production ( 27 ). Fluorescence was
followed at an excitation wavelength of 545 nm and an
emission wavelength of 590 nm using a Fluoroskan Ascent
type 374 multiwell plate reader (Labsystems, Helsinki,
Finland). The slope of the increase in fl uorescence is con-
verted to the rate of H 2 O 2 production with a standard curve.
?− into H 2 O 2 , a necessity because O2
JANG ET AL.
All assays were performed at 37°C in black 96-well plates
with 5 mM succinate plus rotenone and 5 mM glutamate
plus malate as complex I- and complex II-linked substrates,
respectively. For each assay, one reaction well contained
buffer only and another contained buffer with mitochondria
to estimate the mitochondria without substrate (state 1) ( 28 ).
The reaction buffer consisted of 125 mM KCl, 10 mM 2-
hydroxyethyl-1-piperazineethanesulfonic acid (HEPES),
5 mM MgCl 2 , and 2 mM K 2 HPO 4 , pH 7.4. Data are ex-
pressed in pmole H 2 O 2 /min/mg mitochondrial protein.
Superoxide production was measured indirectly by inhi-
bition of aconitase activity, as described by Gardner ( 29 , 30 )
and Muller and colleagues ( 22 ). Briefl y, aconitase activity
was quantifi ed by measuring the reduction of NADP + . Mi-
tochondria ( ~ 0.4 mg of protein/mL) were aliquoted in 96-
well plates in 100 m L at pH 7.44 in 125 mM KCl, 10 mM
HEPES, 5 mM MgCl 2 , and 2 mM K 2 HPO 4 and incubated at
30°C up to 40 minutes. Substrate (5 mM glutamate or
malate) was then added as indicated. For each experiment,
200 U/mL SOD (from bovine erythrocytes; Sigma, St.
Louis, MO) and 900 U/mL catalase were also added to
eliminate extramitochondrial H 2 O 2 and superoxide. Incuba-
tion was stopped, and aconitase activity measurements were
begun by the addition of 1 volume (100 m L) of 50 mM Tris,
0.6 mM MnCl 2 , 60 mM citrate, 0.2% Triton X-100, 100 m M
NADP + , and 1 U of isocitrate dehydrogenase. Fluoromet-
ric measurements (excitation at 355 nm and emission at
460 nm) were then started immediately using a microplate
reader. The “ control or blank ” to measure aconitase-inde-
pendent NADP + reduction consisted of the same buffer
with isocitrate dehydrogenase omitted. The slope of the
increase in NADPH fl uorescence was taken as the amount
of aconitase activity.
ATP production by isolated mitochondria was measured
using a luminometric assay (ATP Bioluminescence Assay
CLS II; Roche, Indianapolis, IN) that follows the change in
luminescence at 560 nm. The assay utilizes the dependence
of ATP in the light-emitting luciferase-catalyzed oxidation
of luciferin. Mitochondrial proteins, in the presence of com-
plex II-linked substrates (succinate with rotenone to inhibit
reverse electron transfer through complex I), were mixed in
the assay buffer (125 mM KCl, 10 mM HEPES, 5 mM
MgCl 2 , and 2 mM K 2 HPO 4 , pH 7.44) and added to a 96-
well plate. Luciferase (Roche) and 0.3 mM ADP were then
added to start the reaction, and the rates of ATP production
were determined at 560 nm using a Fluoroskan Ascent mul-
tiwell plate reader (Labsystems). The slope of the increase
in luminescence is converted to the rate of ATP production
with a standard curve.
Mitochondrial oxygen consumption was measured using
a Clark electrode as originally described by Estabrook ( 31 ).
The respiratory buffer consisted of 125 mM KCl, 10 mM
HEPES, 5 mM MgCl 2 , and 2 mM K 2 HPO 4 , pH 7.44, with
0.3% BSA. State 3 respiration was induced with the addi-
tion of 0.3 mM ADP. The respiratory control ratio (RCR)
was ~ 10 with glutamate or malate as substrate, indicating
intactness of the inner mitochondrial membrane.
Measurements of Oxidative Damage
Lipid peroxidation. — F 2 -isoprostanes were measured in
plasma and tissues using the gas chromatography – mass
spectrometry method of Roberts and Morrow ( 32 ) as previ-
ously described ( 33 ). The levels of F 2 -isoprostanes are ex-
pressed as nanograms F 2 -isoprostanes per milliliter of
plasma or per gram tissue.
Protein oxidation. — Protein oxidation in tissues was de-
termined by the level of protein carbonyls using an advanced
variant ( 34 ) of the original method described by Ahn and
colleagues ( 35 ). In brief, fresh tissue was homogenized in
degassed 20 mM sodium phosphate buffer pH 6.0 con-
taining 0.5 mM MgCl 2 , 1 mM ethylenediaminetetraacetic
acid (EDTA), and protease cocktail inhibitors (500 m M
4-(2-Aminoethyl)benzenesulfonyl fl uoride-hydrochloride,
HCl, 150 nM aprotinin, 0.5 mM EDTA, disodium salt, and 1
m M leupeptin hemisulfate ). For the cytoplasmic fraction, the
homogenate was centrifuged at 100,000 g at 4°C for 1 hour
and the supernatant was saved for further processing. One
percent streptomycin sulfate was added to remove nucleic
acids. The protein samples were then bubbled with nitrogen
for 15 seconds at 25 kPa, followed by treatment with 0.3 M
guanidine HCl for partial unfolding of the proteins in the
sample. The hydrazine reagent, fl uorescein-5-thiosemicar-
bazide (FTC; Molecular Probes; 1 mM), was added and in-
cubated for 2 hours at 37°C in the dark. The excess
unreacted FTC was removed by precipitation with equal vol-
ume of 20% trichloroacetic acid followed by washing four
times with ethanol or ethyl acetate (1:1; v/v). Equal amounts
of protein were loaded on a 12% sodium dodecyl sulfate – -
polyacrylamide gel electrophoresis (SDS-PAGE) to resolve
the FTC-labeled proteins. A fl uorescence scan of the gel was
then taken, which measures the amount of bound FTC, that
is, the amount of protein carbonyls. The gel was then stained
with Coomassie blue, and the protein concentration was de-
termined. The carbonyl content of the protein samples was
expressed as the ratio of FTC fl uorescence (carbonyls) to
Coomassie blue absorption (protein concentration).
Western Blot Analysis
CuZnSOD , glutathione peroxidase 1 (GPX1), and cata-
lase levels were determined by Western blot analysis. Protein
EFFECT OF MNSOD OVEREXPRESSION ON OXIDATIVE STRESS AND AGING
(40 – 80 m g) samples were separated on a 4% – 20% SDS-
PAGE gel and transferred onto a nitrocellulose membrane.
Equal loading was confi rmed visually with Ponceau S stain-
ing. Membranes were blocked in 5% nonfat milk in Tris-
buffered saline (TBS) with 0.1% Tris-buffered saline
Tween-20 (TBST) and incubated at 4°C in TBST overnight
with the appropriate primary antibodies: CuZnSOD (1 m g/
mL; Stressgen, Canada), GPX1 (1:1,000; Santa Cruz Bio-
technology, Santa Cruz, CA), and catalase (1:5,000; Re-
search Diagnostics, Minneapolis, MN). Following three
washings using TBST, membranes were incubated with
HRP-conjugated secondary antibodies (1:2,000; Santa Cruz
Biotechnology). The protein band was visualized using the
ECL Plus Kit (Amersham Pharmacia Biotech), and the in-
tensities of the bands were quantifi ed using ImageQuant v5.0
(Molecular Dynamics, Amersham, UK). Western blot data
were analyzed by normalizing protein bands to levels of
skeletal muscle actin (Sigma) as loading control.
Sensitivity of Murine Embryonic Fibroblasts and
Mice to Paraquat
Timed mating of male Sod2 Tg mice and female WT
mice was carried out to generate Sod2 Tg and WT embryos.
Murine embryonic fi broblasts (MEFs) were derived from
13- to 14-day mouse embryos, and the sensitivity of MEFs
to paraquat was determined as previously described ( 36 ).
MEFs were seeded (2 × 10 4 cells/well) in 48-well plates in
Dulbecco’s modifi ed eagle medium/F12 supplemented with
10% fetal bovine serum and incubated at 37°C with 5%
CO 2 . After 24 hours, the cells were treated with various
concentrations of paraquat (50 – 400 m M) and cell viability
was assessed by the neutral red assay ( 37 ). All treatments
were performed in triplicate, and each experiment was re-
peated with cells isolated from three animals.
The sensitivity of whole animals to oxidative stress
was determined using paraquat. Paraquat was dissolved
in 0.9% saline (25 mg/mL) and injected intraperitoneally
at the dosage of 50 mg/kg body weight. The mice were
then followed periodically over 7 days and deaths re-
corded. At the end of 7 days, all the remaining mice were
Pathological Assessment of Mice
Mice found dead in the survival study were removed im-
mediately from the cage to minimize autolysis, and necropsy
was performed. Organs and tissues were excised and pre-
served in 10% buffered formalin: brain, pituitary gland,
heart, lung, trachea, thymus, aorta, esophagus, stomach,
small intestine, colon, liver, pancreas, spleen, kidneys, uri-
nary bladder, reproductive system (prostate, testes,
epididymis, and seminal vesicles), thyroid gland, adrenal
glands, parathyroid glands, psoas muscle, knee joint, ster-
num, and vertebrae. Any other tissue with gross lesions was
also excised. The fi xed tissues were processed convention-
ally, embedded in paraffi n, sectioned at 5 m m, and stained
with hematoxylin – eosin. Although autolysis of varying
severity occurred, it did not prevent the histopathological
evaluation of lesions. Diagnosis of each histopathological
change was made with histological classifi cations in aging
mice described by Bronson and Lipman ( 38 ). A list of
pathological lesions was constructed for each mouse that
included both neoplastic and nonneoplastic diseases.
The probable cause of death for each mouse was deter-
mined by the severity of diseases found by necropsy as as-
sessed independently by two pathologists. For neoplastic
diseases, cases that had grades 3 and 4 lesions were catego-
rized as “ death by neoplastic lesions. “ For nonneoplastic
diseases, cases that had a severe lesion, for example,
grade 4, associated with other histopathological changes
(pleural effusion, ascites, congestion, and edema in lung)
were categorized as death by the nonneoplastic lesion. In
more than 90% of the cases, there was agreement by the two
pathologists. In cases in which the pathologists disagreed or
in which no disease was considered severe enough, the
cause of death was categorized as unknown.
Characterization of Sod2 Tg Mice
To show that MnSOD is overexpressed throughout the
life span of the transgenic mice, we measured the enzymatic
activity of MnSOD in tissues from both young (4 – 6 months)
and old (26 – 28 months) WT and Sod2 Tg mice. As shown
in Figure 1 , the activity of MnSOD is increased approxi-
mately twofold in the brain, kidney, liver, heart, and skeletal
muscle of Sod2 Tg mice. We measured MnSOD activity
and protein content in mitochondria isolated from a few tis-
sues in WT and Sod2 Tg mice and found a similar level of
overexpression in the Sod2 Tg mice. Furthermore, we found
no evidence that mitochondrial content was altered in the
Sod2 Tg mice as measured by marker proteins (i.e., cyto-
chrome c, COX IV, citrate synthase) that correlate with mi-
tochondrial content (data not shown). To determine whether
overexpression of MnSOD had an effect on other compo-
nents of the antioxidant defense system, we measured the
levels of CuZnSOD, GPX1, and catalase in skeletal muscle
from young and old Sod2 Tg and WT mice. As shown in
Table 1 , the levels of all three proteins were similar regard-
less of age or genotype. Therefore, overexpression of Mn-
SOD did not downregulate the expression of other
We compared body weight and food consumption of
Sod2 Tg and age-matched WT mice and found no changes
in either body weight ( Table 2 ) or food consumption (data
not shown). Additionally, no differences in the tissue
weights were observed with age or genotype in liver, spleen,
kidney, and brain between the two groups. However, age-
associated changes were seen in heart and skeletal muscle
JANG ET AL.
( Table 2 ). The decrease in skeletal muscle mass with age is
a common biomarker of aging that is believed to occur be-
cause of increased oxidative damage or stress that the data
to be presented subsequently demonstrate ( 39 ). As can be
seen in Table 2 , a signifi cant decline in muscle mass of gas-
trocnemius or plantaris and tibialis anterior was observed
with age. These data are consistent with other reports show-
ing that gastrocnemius and tibialis anterior, which contain
a high content of fast twitch fi bers, are more susceptible to
age-related muscle loss ( 27 ). In contrast, we found that the
soleus, which is predominately slow fi ber, showed no age-
related decline in muscle mass, which is consistent with
previous reports ( 40 , 41 ). However, despite evidence for an
increase in ROS, overexpressing MnSOD had no effect on
the age-related decline in mass in the gastrocnemius or
plantaris and tibialis anterior muscles ( Table 2 ).
ROS Production in Skeletal Muscle Mitochondria
To test whether an increased level of MnSOD decreases
mitochondrial ROS generation, we measured the O2
and H 2 O 2 release in skeletal muscle mitochondria from young
and old mice ( Figure 2 ). Aconitase activity in the mitochon-
dria was used as an indirect measure of O2
Fe-S clusters in aconitase are highly sensitive to reaction of
nitase activity in Sod2 Tg mice ( Figure 2A ) was signifi cantly
higher in both young and old animals, and this increase was
greater in the old ( ~ 40%) than in young ( ~ 13%) mice.
?− levels because the
?− ( 29 , 30 ). Compared with WT mice, mitochondrial aco-
Figure 1. MnSOD activity in various tissues of wild-type (WT) and Sod2 Tg mice. The activity of MnSOD was measured in tissue homogenates isolated from
brain, kidney, liver, skeletal muscle, and heart of young and old WT (open bar) and Sod2 Tg (solid bar) mice, determined using native gels described in the Materials
and Methods section . The data were obtained from four to six mice per group and expressed as mean ± SEM . The MnSOD activity for young WT mice was normalized
to 1. Data were statistically analyzed using two-way analysis of variance with the Bonferroni test mice; an asterisk denotes those values that are signifi cantly different
from young WT mice at the p < .05 level.
EFFECT OF MNSOD OVEREXPRESSION ON OXIDATIVE STRESS AND AGING
The rate of H 2 O 2 release from mitochondria was measured
in the absence of added respiratory substrates (state 1) and
in the presence of 5 mM glutamate plus malate. In state 1
( Figure 2B ), H 2 O 2 production from skeletal muscle mito-
chondria was increased ~ 90% with age, consistent with what
we have previously observed ( 27 ). MnSOD overexpression
did not alter the rate of H 2 O 2 release in either age group.
Similarly, using complex I-linked substrate glutamate and
malate ( Figure 2C ), H 2 O 2 levels were signifi cantly increased
( ~ 50%) with age, but no difference in H 2 O 2 was observed
between Sod2 Tg and age-matched WT mice.
We next assessed mitochondrial function by measuring
the RCR and ATP production from mitochondria isolated
from skeletal muscle. The RCR decreased signifi cantly with
age (10%) but was unaffected by MnSOD overexpression
( Figure 3A ). ATP production by isolated mitochondria
was measured in the presence of mitochondrial complex
II-linked substrates, succinate or rotenone ( Figure 3B ). In
agreement with our previous report ( 27 ), ATP production
from the skeletal muscle mitochondria showed a signifi cant
decline with age using complex II-linked substrate. Similar
results were observed using glutamate or malate (data not
shown). In contrast, mitochondria from Sod2 Tg mice did
not show a signifi cant decrease with age in ATP production,
and there was no signifi cant difference in ATP production
by mitochondria from Sod2 Tg and WT at the two ages
Table 2. Total BW and Tissue Weights in Young and Old WT and Sod2 Tg Mice
BW Liver SpleenKidney HeartBrain
Young Sod2 Tg
Old Sod2 Tg
26.2 ± 0.4
25.9 ± 0.5
31.1 ± 0.9*
29.1 ± 1.0*
1.24 ± 0.03
1.32 ± 0.06
1.98 ± 0.30
1.41 ± 0.15
0.09 ± 0.01
0.07 ± 0.00
0.10 ± 0.01
0.11 ± 0.02
0.32 ± 0.01
0.32 ± 0.01
0.44 ± 0.03
0.42 ± 0.02
0.11 ± 0.001
0.12 ± 0.001
0.14 ± 0.001*
0.15 ± 0.01*
0.45 ± 0.00
0.43 ± 0.00
0.45 ± 0.01
0.46 ± 0.01
0.013 ± 0.002
0.017 ± 0.000
0.013 ± 0.001
0.014 ± 0.002
0.291 ± 0.005
0.282 ± 0.012
0.191 ± 0.010*
0.203 ± 0.017*
0.172 ± 0.003
0.172 ± 0.005
0.134 ± 0.009*
0.142 ± 0.009*
Note : The BW and the weight of various tissues from young (4 – 5 months) and old (26 – 28 months) WT and Sod2 Tg mice were collected from fi ve to six mice
per group and were analyzed using analysis of variance with Bonferroni ’ s post hoc test. Asterisks denote a statistically signifi cant difference between young and old
mice at the p < .05 level. BW = body weight; Gast/pln = gastrocnemius/plantaris, TA/EDL = tibialis anterior/extensor digitorum longus; WT = wild type.
Oxidative Damage in Skeletal Muscle of Young and Old
WT and Sod2 Tg Mice
Several groups, including ours, have shown that oxida-
tive damage to protein, lipid, and DNA in skeletal muscle
increases with age ( 27 ). To test whether the Sod2 Tg mice
are protected against protein oxidation with age, we mea-
sured the level of protein carbonyls from the cytosolic frac-
tion of skeletal muscle. Protein carbonyls were signifi cantly
elevated with age in WT mice ( Figure 4A ). In contrast, we
did not observe a statistically signifi cant increase in carbo-
nyls with age in the Sod2 Tg mice. Although there was a
trend toward a decrease in protein carbonyl levels in the old
Sod2 Tg compared with old WT mice ( p = .08), the protein
carbonyl levels were not statistically different between the
WT and the Sod2 Tg mice in either age group ( Figure 4A ).
Next, we assessed oxidative damage to lipids by measuring
F 2 -isoprostanes in the skeletal muscle ( Figure 4B ). Unlike
other lipid peroxidation derivatives, F 2 -isoprostanes are
stable and provide excellent biomarkers of lipid peroxida-
tion. We found that with age, skeletal muscle from WT mice
exhibited a twofold increase in F 2 -isoprostane levels. How-
ever, the F 2 -isoprostane levels did not increase signifi cantly
with age in the Sod2 Tg mice, and in old mice, the levels of
F 2 -isoprostanes were reduced signifi cantly (43%) in the
Sod2 Tg mice compared with WT mice.
Sensitivity of WT and Sod2 Tg Mice to Oxidative Stress
To determine whether the overexpression of MnSOD af-
fected the sensitivity of the mice to oxidative stress, we tested
the sensitivity of MEFs and whole animals to paraquat.
Paraquat (1,1 ′ -dimethyl-4,4 ′ -bipyridinium dichloride) is a
bipyridyl compound that is widely used as a redox cycler
to stimulate superoxide production in organisms, cells, and
mitochondria. A recent study demonstrated that complex I
toward the mitochondrial matrix is the major site of paraquat-
induced superoxide generation ( 44 , 45 ). Figure 5A shows the
sensitivity of MEFs from WT and Sod2 Tg mice to paraquat
at concentrations of 100 – 400 m M. The cell viability of MEFs
from the Sod2 Tg mice was signifi cantly higher than MEFs
isolated from WT mice at all doses studied, and at the highest
paraquat concentration tested, MEFs from Sod2 Tg mice
were fourfold more resistant to paraquat toxicity than MEFs
Table 1. Antioxidant Enzyme Levels in Young and Old WT and Sod2
Young Sod2 Tg
Old Sod2 Tg
556 ± 29
523 ± 25
557 ± 22
538 ± 28
0.127 ± 0.008
0.121 ± 0.010
0.116 ± 0.007
0.121 ± 0.003
1.15 ± 0.10
1.26 ± 0.13
1.25 ± 0.08
1.67 ± 0.17
Note : CuZnSOD, GPX1, and catalase levels were determined in skeletal
muscle of young (4 – 5 months) and old (26 – 28 months) WT and Sod2 Tg mice
by Western blot analysis as described in the Materials and Methods section
(Unit: OD/mg protein). Data shown are the mean ± SEM for four animals per
group and were analyzed using analysis of variance with Bonferroni ’ s post
JANG ET AL.
from WT mice. Figure 5B shows the sensitivity of WT and
Sod2 Tg mice to a lethal dose of paraquat. Over the 7-day
period following the administration of paraquat, the deaths in
the Sod2 Tg mice were 10% – 30% less than in the WT mice,
and the log-rank test showed that the survival curves for the
WT and Sod2 Tg mice were signifi cantly different. Thus,
both at the level of the cell and the whole organism, we found
that overexpressing MnSOD approximately twofold had a
signifi cant effect on resistance to oxidative stress.
Life Span and the End-of-Life Pathology of Sod2 Tg Mice
To determine whether overexpressing MnSOD throughout
life span has any effect on aging, we fi rst measured the sur-
vival of the WT and Sod2 Tg mice housed under barrier con-
ditions. Figure 6 shows the survival data and curves for the
Sod2 Tg and WT mice. The mean survival of the male WT
mice in our study was well over 32 months (982 days). This
is extremely long lived for C57BL/6 mice but is not unex-
pected because we measure life span under optimal condi-
tions that minimize potential complications from a poor
environment ( 47 ). We observed no signifi cant differences be-
tween the survival curves for the Sod2 Tg and WT mice or
between the mean, median, and 10% (when 90% of the mice
died) survivals of these mice. Thus, overexpression of Mn-
SOD had no signifi cant effect on the life span of the mice.
Figure 2. Aconitase activity and H 2 O 2 generation in skeletal muscle mito-
chondria from wild-type (WT) and Sod2 Tg mice. Aconitase activity ( A ) and
H 2 O 2 production ( B ) were determined in mitochondria isolated from skeletal
muscle of young and old WT (open bars) and Sod2 Tg mice (solid bars). The data
are the mean of four to six animals ± SEM and were analyzed by the nonparametric
test of analysis of variance (with Bonferroni ’ s post hoc). The asterisk denotes
a statistically signifi cant difference between WT and Sod2 Tg mice (aconitase
activity) and young and old mice (H 2 O 2 production) at the p < .05 level.
To determine whether overexpressing MnSOD has an
impact on the pathologic lesions leading to death, we con-
ducted a comprehensive end-of-life pathological analysis of
the WT and Sod2 Tg mice in the survival groups. As shown
in Table 3 , the probable causes of death for the WT and
Sod2 Tg mice were similar. As expected for mice in the
C57BL/6 background ( 48 , 49 ), the majority of fatal tumors
in both WT and Sod2 Tg mice was lymphoma. The propor-
tions of mice that died from neoplastic diseases were ~ 62%
for WT and 50% for the Sod2 Tg mice; however, this differ-
ence was not statistically signifi cant. There were no changes
in the average of age of tumor development between geno-
types, 933 and 922 days for WT and Sod2 Tg mice, respec-
tively; and the percentage of tumor bearing mice was 62%
for WT and 65% for Sod2 Tg mice. We also studied the ef-
fect of overexpressing MnSOD on the incidence of multiple
tumors in the mice. The WT mice showed an average of
0.73 tumors per mouse compared with 0.71 for the Sod2 Tg
mice. The major nonneoplastic pathology observed in the
WT and Sod2 Tg mice was glomerulonephritis, which was
similar in frequency in WT and Sod2 Tg mice. Although the
Figure 3. Mitochondrial respiration and ATP production in skeletal muscle
mitochondria from wild-type (WT) and Sod2 Tg mice. The mitochondrial respi-
ration control ratio ( A ) and ATP production ( B ) were determined in mitochon-
dria isolated from skeletal muscle of young and old WT (open bars) and Sod2
Tg (solid bars) mice using complex II-linked substrate. The data are the mean of
six animals ± SEM and were analyzed by the nonparametric test of analysis
of variance (with Bonferroni ’ s post hoc). The asterisk denotes a statistically
signifi cant difference between young and old mice at the p < .05 level.
EFFECT OF MNSOD OVEREXPRESSION ON OXIDATIVE STRESS AND AGING
Sod2 Tg mice showed a higher incidence of nonneoplastic
pathology, 50% versus 38%, this difference was not statisti-
cally signifi cant.
Our rationale for studying the effect of overexpressing
MnSOD on life span of mice originated from three areas.
First, Tower ’ s laboratory showed that overexpressing Mn-
SOD up to 75% in Drosophila increased life span by 15%
( 14 ). Second, Rabinovitch ’ s group showed that overex-
pressing catalase in mitochondria increased the life span of
mice by 21% ( 50 ), suggesting that an enhanced mitochon-
drial antioxidant defense system is important in increasing
longevity. Third, studies with C. elegans mutants in insulin
or IGF-1 signaling suggest that the induction of MnSOD,
one of the target genes that are upregulated through a reduc-
tion in insulin or IGF-1 signaling, is important in the in-
creased resistance to stress and increased life span ( 8 , 9 ).
In the current study, we used Sod2 Tg mice generated by
Epstein ’ s laboratory that overexpress MnSOD ( 19 ). We
show here that the Sod2 Tg mice expressed MnSOD approx-
imately twofold more than the WT mice in all tissues exam-
Figure 4. Oxidative damage in skeletal muscle from young and old wild-
type (WT) and Sod2 Tg mice. Protein carbonyl ( A ) and F 2 -isoprostane ( B ) lev-
els were measured in skeletal muscle from young and old mice of Sod2 Tg
(solid bars) and WT (open bars) mice. Protein carbonyl levels were determined
as described by ( 42 ) and F 2 -isoprostanes determined as described by ( 43 ). Data
shown are the mean ± SEM for six animals per group and were analyzed using
analysis of variance with Bonferroni ’ s post hoc test. The asterisks denote a
statistically signifi cant difference at the p < .05 level.
ined over the life span of the transgenic mice. Because there
was no signifi cant decrease in any of the other major anti-
oxidant enzymes (GPX1, catalase, and CuZnSOD), the Sod2
Tg mice would be expected to have an enhanced antioxidant
defense system with an increased ability to detoxify ROS,
type, and their body and tissue weights and food consump-
tion were similar to those of their WT littermates. Because
MnSOD is a key component of the mitochondrial antioxi-
dant defense system, we also were interested in how overex-
pressing MnSOD affects various functions of mitochondria
isolated from the skeletal muscle of young and old mice. As
expected, we observed a reduction in superoxide anion lev-
els (as measured indirectly by reduction in aconitase activ-
ity) in the mitochondria isolated from Sod2 Tg mice
compared with WT mice. However, no changes were seen in
?−) . The Sod2 Tg mice showed no overt pheno-
Figure 5. Effect of overexpressing MnSOD on sensitivity to oxidative stress.
( A ) Primary cultures of murine embryonic fi broblasts (MEFs) isolated from
Sod2 Tg and wild-type (WT) mice were treated with various doses of paraquat
for 48 hours. Cell viability was measured by the neutral red assay as described
in the Materials and Methods section. All values represent the mean ± SEM from
three different animals. The data were analyzed by a two-way analysis of vari-
ance with a follow-up Tukey ’ s multiple range test. The asterisk denotes those
values that are signifi cantly different at p < .05 between MEFs isolated from
Sod2 Tg compared with WT mice. ( B ) Paraquat (50 mg/kg) was administered to
12 WT (open diamonds) and 13 Sod2 Tg (solid triangles) mice, and the survival
of the mice was followed over 7 days. The survival curves were statistically ana-
lyzed using the log-rank test and shown to be signifi cant at the p < .05 level.
JANG ET AL.
mitochondrial H 2 O 2 generation. In an agreement with these
observations, in our previous report showed that, Sod2
heterozygous knockout mice ( Sod2 +/ − ) which have an ~ 50%
reduction in MnSOD activity, mitochondrial H 2 O 2 produc-
tion was not signifi cantly different in both young (3 – 5
months) and old (26 – 29 months) skeletal muscle. Although
it is logical to think that the majority of mitochondrial H 2 O 2
production is directly proportional to levels of MnSOD ac-
tivity, several possible reasons may explain why we did not
observe any differences in H 2 O 2 production between WT
and Sod2 Tg mice. Because the probe we used to detect
H 2 O 2 (Amplex Red) is membrane impermeable and because
H 2 O 2 can freely diffuse in and out of membranes, we may be
detecting only a small portion of H 2 O 2 released from the
mitochondria. For example, if there were an approximately
twofold increase in H 2 O 2 at the matrix side of mitochondria
due to MnSOD overexpression, other mitochondrial H 2 O 2
detoxifying enzymes such as peroxiredoxin 3 (PRX3), glu-
tathione peroxidase 1 (GPX1), and GPX4 may act prior to
the release of H 2 O 2 from the mitochondria. Additionally, we
assessed the level of superoxide in vivo by reduction in aco-
nitase activity, whereas the rate of H 2 O 2 production was
Figure 6. Life span of Sod2 Tg and WT mice. The survival curves of wild-type (WT; open symbols) and Sod2 Tg (solid symbols) mice were obtained from 50
male Sod2 Tg and 47 male WT mice. The survival data in the table are expressed in days. Mean survival ( ± SEM ) for each group was compared with the WT group
by performing a Student ’ s t test upon log-transformed survival times from the respective groups. The mean, median, 10%, and maximum survivals for each group
were compared with the WT group using a score test adapted from Wang and colleagues ( 67 ).
Table 3. The Probable Causes of Death for WT and Sod2 Tg Mice
WT (%) Sod2 Tg (%)
Acidophilic macrophage pneumonia
Note : Pathological analyses were determined in mice that died in the life
span study as described in the Materials and Methods section. The data were
obtained from 47 WT and 52 Sod2 Tg mice. WT = wild type.
measured solely in vitro in isolated mitochondria. Further-
more, it is also feasible to think that the mitochondrial sub-
strates we add to enhance electron transfer may not be
ideally mimicking the in vivo mitochondrial environment.
Previous studies with long-lived invertebrates ( 42 , 51 ),
dwarf mice ( 43 ), and caloric-restricted rodents ( 46 ) have
shown a strong correlation between resistance to oxidative
stress and increased longevity. We found that MEFs from
Sod2 Tg mice were more resistant to paraquat treatment
than cells from WT mice. In support of this observation,
when cadmium-induced toxicity was measured in similar
fashion, Sod2 Tg MEFs showed approximately twofold
higher resistance compared with WT MEFs (data not
shown). Moreover, when a lethal dose of paraquat was in-
jected intraperitoneally to assess toxicity at an organism
level, Sod2 Tg mice exhibited a higher survival rate com-
pared with age-matched WT littermates. It is noteworthy
that most of the differences in survival occur within 24
hours after injection, and survival rates were similar in both
groups after 24 hours. In our previous study, we did not ob-
serve acute early deaths in same strain of WT mice
(C57BL/6J) ( 52 ). This discrepancy could be due to the gen-
der difference in two studies as Holzenberger and colleagues
( 53 ) reported that survival in paraquat-induced toxicity is
signifi cantly different in male and female mice.
Hu and colleagues ( 54 ) also reported that overexpressing
MnSOD in another transgenic mouse reduced the age-related
increase in ROS levels in brain using dihydroethidium. We
also measured the effect of overexpressing MnSOD on the
age-related increase in oxidative damage in skeletal muscle,
using protein carbonyls as a marker for protein oxidation
and F 2 -isoprostanes as an indicator of lipid peroxidation. In
agreement with the previous fi ndings ( 55 ), we found that
although protein carbonyls were signifi cantly elevated with
age in the skeletal muscle of WT mice, they were not in
Sod2 Tg mice. Moreover, MnSOD overexpression had a
EFFECT OF MNSOD OVEREXPRESSION ON OXIDATIVE STRESS AND AGING
dramatic effect on lipid peroxidation. In WT mice, we ob-
served an approximately twofold increase in F 2 -isoprostanes
with age in skeletal muscle that was completely attenuated
in the Sod2 Tg mice.
Because Sod2 Tg mice are more resistant to oxidative
stress and show reduced oxidative damage, one would pre-
dict that transgenic mice overexpressing MnSOD would
show an increase in life span. Recently, Hu and colleagues
( 54 ) reported that overexpression of MnSOD increased the
life span of transgenic mice. In this study, the complemen-
tary DNA to Sod2 was expressed using a b -actin promoter
(twofold to fourfold increase, except liver) in B6C3 mice.
Using 30 WT and 24 transgenic mice, Hu and colleagues
( 54 ) reported that the transgenic mice overexpressing Mn-
SOD showed a 18% increase in maximum life span (1,095
vs 1,290 days). However, there was no statistical analysis of
the survival curves or survival data, and the mean survival
for the transgenic mice overexpressing MnSOD was only
4% longer than the WT mice (828 vs 864 days). In our pres-
ent study, we showed no statistical difference in the survival
between WT and Sod2 Tg mice; for example, neither the
mean, median, nor 10% survival were signifi cantly different
for WT and Sod2 Tg mice. Although the maximum survival
of the Sod2 Tg mice (1,245 days) in our study was similar
to the maximum life span of the transgenic mice (1,290
days) in the study by Hu and colleagues ( 54 ), the mean sur-
vival of WT and Sod2 Tg mice in our study (982 and 997
days, respectively) was ~ 15% longer than the mean survival
of the mice in the study by Hu and colleagues ( 54 ). In our
study, we used more than 45 mice per group to measure life
span, which gives us suffi cient power to detect a 10% differ-
ence in mean life span ( 47 ). As can be seen from our sur-
vival data, the mice in our study were long lived, for
example, a mean life span of 982 days for the male WT
mice, which is longer than historically reported for C57BL/6
mice ( 56 , 57 ) and longer than reported for the animal colonies
maintained by National Institute on Aging ( 58 ) or the
C57BL/6 mice maintained by Jackson Laboratories (59 ).
By maximizing the life span of the mice, potential genotype
and environmental interactions are minimized, and the
results should refl ect a more accurate measurement of the
effect of the manipulation, in this case the overexpression of
MnSOD, on aging. Our survival data show that overexpress-
ing MnSOD had no effect on life span, one important mea-
sure of aging. Our pathological data on the WT and Sod2 Tg
mice also show that overexpressing MnSOD had no effect
These data, when combined with an earlier study in which
we showed that the life span of Sod2 +/ − mice was not al-
tered ( 60 ), show that varying MnSOD levels fourfold (from
50% in the Sod2 +/ − mice to 200% in the Sod2 Tg mice) has
no detectable effect on the life span of mice maintained un-
der barrier conditions. Nevertheless, the level of MnSOD
expression is correlated with how well the mouse can han-
dle acute oxidative stress such as is caused by paraquat:
Sod2 +/ − mice are more sensitive ( 60 ) and Sod2 Tg mice are
more resistant ( 61 ). Our survival data in mice differed from
those from Drosophila that showed that overexpressing
MnSOD increased life span ( 14 ). It also should be noted
that observations of overexpressing CuZnSOD increased
the life span of Drosophila ( 14 , 62 ) were not replicated in
mice ( 63 ) (data from our laboratory submitted for publica-
tion). Therefore, it is likely that the effect of overexpressing
the SODs is species dependent, with overexpression of ei-
ther MnSOD or CuZnSOD increasing the life span of Dros-
ophila but not of mice. These data point to the importance
of genetic manipulations that extend life span in inverte-
brates may not be applicable in a mammalian system.
It is interesting to note that Schriner and colleagues ( 50 )
reported that transgenic mice overexpressing catalase in mi-
tochondria showed an increase in life span in contrast to our
study showing that overexpressing endogenous mitochon-
drial MnSOD had no affect on life span. In addition, a re-
cent report by Treuting and colleagues ( 64 ) reported that the
mitochondrial-targeted catalase transgenic mice showed re-
duced malignant nonhematopoetic tumor burden and re-
duced cardiac lesions at end-of-life pathology. In contrast,
we did not observe a signifi cant difference in tumor burden
in the Sod2 Tg mice compared with the WT. These data sug-
gest that mitochondrial H 2 O 2 levels are more important than
levels of mitochondrial H 2 O 2 may play an important role
in aging as a signaling molecule ( 65 ). Although a small
portion of mitochondrial O2
dria ( 66 ), the majority of O2
the mitochondrial electron transport chain are rapidly dis-
mutated by SOD to H 2 O 2 . In contrast, H 2 O 2 has relatively
long half-life and can easily diffuse across the mitochon-
drial membrane making it a potentially important molecule
for cell signaling ( 65 ).
?− levels in aging. For example, the reduced
?− can leak out of the mitochon-
?− anions that are generated by
National Institutes of Health grants ( P01AG19316 , P01AG020591 ,
R37AG026557 to A.R.), the San Antonio Nathan Shock Aging Center
( 1P30-AG13319 ), and VA Merit grants (A.R. and H.V.R.) and the Reserve
Educational Assistance Program from the Department of Veteran Affairs .
The authors would like to acknowledge the assistance of Marian Sabia,
Jay Cox, Vivian Diaz, and Amanda Jernigan for excellent mouse hus-
bandry. We would also like to express our thanks to Alex Bokov for assis-
tance in analyzing the survival data.
Address correspondence to Arlan Richardson, PhD, The Sam and Ann
Barshop Institute for Longevity and Aging Studies, University of Texas
Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio,
TX 78245-3207. Email: email@example.com
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Received November 13 , 2008
Accepted July 1 , 2009
Decision Editor: Huber R. Warner, PhD