206

Maturation increases superoxide radical

production without increasing oxidative damage in
the skeletal muscle of hooded seals
(Cystophora cristata)
J.P. Vázquez-Medina, N.O. Olguı́n-Monroy, P.D. Maldonado, A. Santamarı́a,
M. Königsberg, R. Elsner, M.O. Hammill, J.M. Burns, and T. Zenteno-Savı́n

Abstract: Diving vertebrates represent unique models for the study of the physiological responses to reactive oxygen spe-
cies (ROS) production and oxidative stress because of their adaptability to cope with dive-derived ROS production. We
hypothesized that in the skeletal muscle of a diving mammal, the hooded seal (Cystophora cristata (Erxleben, 1777)),
ROS production increases with maturation but the accumulation of oxidative damage does not. To test this, we analyzed
the tissue capacity to produce ROS, the accumulation of oxidative damage, and the activity and protein content of the
cooper, zinc, and manganese dependent superoxide dismutases (Cu,ZnSOD, MnSOD) in skeletal muscle from neonates,
weaned pups, and adult hooded seals. Our results showed higher tissue capacity to produce ROS, higher Cu,ZnSOD and
MnSOD activities, and higher MnSOD protein content in adult seals than in pups. No differences in oxidative damage to
lipids, proteins, or DNA were detected among groups. Results suggest that increased SOD activity likely counters the oxi-
dative damage commonly associated with increased ROS production. These findings highlight the unusual tolerance of
skeletal muscle of seals to increased ROS production.
Résumé : Les vertébrés plongeurs sont des modèles uniques pour l’étude des réactions physiologiques à la production
d’espèces d’oxygène réactif (ROS) et du stress oxydatif à cause de leur adaptabilité pour faire face à la production de
ROS produits durant la plongée. Nous émettons l’hypothèse selon laquelle, dans le muscle squelettique du phoque à capu-
chon (Cystophora cristata (Erxleben, 1777)), la production de ROS augmente en fonction de la maturation, mais non l’ac-
cumulation de dommages oxydatifs. Afin de tester l’hypothèse, nous analysons la capacité du tissu à produire des ROS,
l’accumulation de dommages oxydatifs, ainsi que l’activité et le contenu protéinique des superoxydes dismutases dépen-
dantes du cuivre, du zinc et du manganèse (Cu,ZnSOD, MnSOD) dans le muscle squelettique chez des phoques à capu-
chon nouveau nés, des petits sevrés et des adultes. Les tissus ont une plus forte capacité de production de ROS et
possèdent des activités plus élevées de Cu,ZnSOD et de MnSOD et des contenus protéiniques plus importants de MnSOD
chez les phoques adultes que chez les petits. Aucune différence n’a pu être décelée entre les groupes dans les dommages
oxydatifs aux lipides, aux protéines et à l’ADN. Nos résultats laissent croire que l’activité accrue des SOD bloque vrai-
semblablement les dommages oxydatifs couramment associés à la production plus élevée de ROS. Ces observations souli-
gnent la tolérance exceptionnelle du muscle squelettique des phoques à la production accrue de ROS.
[Traduit par la Rédaction]

Received 9 June 2010. Accepted 1 December 2010. Published on the NRC Research Press Web site at cjz.nrc.ca on 12 February 2011.
J.P. Vázquez-Medina,1 N.O. Olguı́n-Monroy, and T. Zenteno-Savı́n.2 Planeación Ambiental y Conservación, Centro de
Investigaciones Biológicas del Noroeste, S.C, Mar Bermejo 195, Playa Palo de Santa Rita, La Paz, B.C.S., 23090, Mexico.
P.D. Maldonado. Laboratorio de Patologı́a Vascular Cerebral, Instituto Nacional de Neurologı́a y Neurocirugı́a ‘‘Dr. Manuel Velasco
Suárez’’, Insurgentes sur 3877, Colonia La Fama, Tlalpan, Mexico City, 14269, Mexico.
A. Santamarı́a. Laboratorio de Aminoácidos Excitadores, Instituto Nacional de Neurologı́a y Neurocirugı́a ‘‘Dr. Manuel Velasco
Suárez’’, Insurgentes sur 3877, Colonia La Fama, Tlalpan, Mexico City, 14269, Mexico.
M. Königsberg. Laboratorio de Bioenergética y Envejecimiento Celular, Departamento de Ciencias de la Salud, Universidad Autónoma
Metropolitana-Iztapalapa, Avenida San Rafael Atlixco No. 186, Colonia Vicentina, Delegación Iztapalapa, Mexico City, 09340, Mexico.
R. Elsner. Institute of Marine Sciences, University of Alaska Fairbanks, P.O. Box 757220, Fairbanks, AK 99775-7220, USA.
M.O. Hammill. Maurice Lamontagne Institute, Department of Fisheries and Oceans, 850, route de la Mer, Mont-Joli, QC G5H 3Z4,
Canada.
J.M. Burns. Department of Biological Sciences, University of Alaska Anchorage, 3211 Providence Drive, Anchorage, AK 99508, USA.
1Present address: School of Natural Sciences, University of California Merced, 5200 North Lake Road, Merced, CA 95343, USA.
2Corresponding author (e-mail: tzenteno04@cibnor.mx).

Can. J. Zool. 89: 206–212 (2011) doi:10.1139/Z10-107 Published by NRC Research Press
Vázquez-Medina et al.
Introduction
Oxygen consumption by animal cells is essential for the
production of the energy needed to maintain cellular func-
tions and metabolic activity. Paradoxically, oxygen-mediated
ATP production via the electron transport chain is accompa-
nied by the production of potentially damaging reactive oxy-
gen species (ROS) (Halliwell and Gutteridge 2007). During
basal metabolic conditions, 0.1% of the oxygen consumed is
transformed into superoxide radical (O2–) (Fridovich 2004).
O2– is dismutated in the mitochondria by the action of the
enzyme manganese-dependent superoxide dismutase
(MnSOD), and in the cytosol, by the copper and zinc con-
taining superoxide dismutase (Cu,ZnSOD) (Weisiger and
Fridovich 1973). O2– dismutation results on hydrogen per-
oxide (H2O2) generation and the potential production of
highly reactive hydroxyl (OH) radicals (Boveris and
Chance 1973).
Excessive ROS generation and (or) inefficient ROS scav-
enging by enzymatic and nonenzymatic antioxidants lead to
the pathophysiological condition of oxidative stress (Sies
1985). During this condition, oxidative damage is promoted
by the reaction of ROS with lipids, proteins, and DNA (Hal-
liwell and Gutteridge 2007). ROS production, oxidative
damage accumulation, and antioxidant enzymes loss of func-
tion increase with age progression in a variety of animal
systems (Fraga et al. 1990; Kaneko et al. 1996; Starke-Reed
and Oliver 1989; Sohal et al. 1993; Barja 2002).
To our knowledge, no information on maturation-related
increases in ROS production, oxidative damage accumula-
tion, or antioxidant enzymes in seals or in any other marine
mammal is yet available. Although there is a considerable
body of literature dealing with oxidative stress and aging in
several animal species, there is very little information avail-
able on oxidative stress and development. Seals represent a
unique model for the study of developmental-related
changes in oxidative stress variables because seals, like
other diving vertebrates, have an enhanced antioxidant sys-
tem that protects them against the potentially damaging ef-
fects of diving-induced ischemia–reperfusion, a condition
that increases ROS production (Elsner et al. 1998; Zenteno-
Savı́n et al. 2002; Wilhem Filho et al. 2002; Hermes-Lima
and Zenteno-Savı́n 2002; Vázquez-Medina et al. 2006,
2007, 2010; Valdivia et al. 2007; Zenteno-Savı́n et al.
2010). In this study, we compared muscle ROS production,
oxidative damage accumulation, and SOD activities and pro-
tein content among three age groups of hooded seals (Cysto-
phora cristata) (Erxleben, 1777). We hypothesize that
because seals are adapted to tolerate diving-induced ROS
production, muscle of hooded seals will not accumulate oxi-
dative damage with maturation. Moreover, because diving
capacity and diving behavior of hooded seals develop with
maturation (Burns et al. 2007, Folkow et al. 2010), we hy-
pothesize that the development of hooded seals is accompa-
nied by an increase in SOD activity and protein content.
Materials and methods
Animal handling and sample collection
Animal capture and handling protocols were reviewed and
approved by Fisheries and Oceans Canada and the Institu-
tional Animal Care and Use Committee of the University of
207
Alaska Anchorage. Samples were imported into the United
States under marine mammal permits 782-1399 and 782-
1694-02, and into Mexico under the terms of permits issued
by Instituto Nacional de Ecologı́a and Procuradurı́a Federal
de Protección al Ambiente.
Fourteen healthy hooded seals were captured on the pack
ice near the Magdalen Islands, Gulf of St. Lawrence, Can-
ada (approximately 47823’N, 61852’W). Animals were cate-
gorized visually as adults (5 reproductive females; body
mass (mean ± SE) = 249.3 ± 25.2 kg) or pups. Pups were
categorized as neonates (3 males and 1 female; body
mass = 21.6 ± 3.3 kg; age = 1 day) or weaners (3 females
and 2 males; body mass = 48.1 ± 4.5 kg; age = 5–14 days)
following Bowen et al. (1987). Seals were physically re-
strained, drugged with diazepam (0.2–0.3 mg
kg–1) or
tiletamine–zolazepam (0.7–1.0 mg
kg–1), and sacrificed us-
ing methods approved for scientific collection by Fisheries
and Oceans Canada. Tissue sample collection was per-
formed in parallel with other studies (Burns et al. 2007,
2010; Lestyk et al. 2009) to maximize the use of the sam-
ples obtained from the sacrificed animals. Samples of long-
isimus dorsi muscle (approximately 2 g) were obtained by
dissection, placed in cryogenic vials, and stored at –80 8C
until analyzed.
Biochemical assays
Superoxide radical production
Endogenous O2– production was measured following the
procedures proposed by Drossos et al. (1995) in tissue slices
(approximately 50 mg) placed in test tubes containing
Krebs–Henseleit buffer. O2– production was calculated by
monitoring the change of ferricytochrome c to ferrocyto-
chrome c as previously described (Zenteno-Savı́n et al.
2002). Results were expressed as nanomoles of O2– per mi-
nute per gram of wet tissue.
Oxidative damage
Oxidative damage to lipids was measured by calculating
the tissue content of thiobarbituric acid reactive substances
(TBARS). Briefly, samples were homogenized in two vol-
umes of 0.9% NaCl as described previously (Zenteno-Savı́n
et al. 2002; Vázquez-Medina et al. 2007). TBARS content
(nanomoles per gram of wet tissue) was calculated from a
standard curve of malondialdehyde bis-(diethylacetal) run in
parallel with the samples. Oxidative damage to proteins was
determined by quantifying the tissue content of protein car-
bonyls. Samples were homogenized (1:20 m/v) in ice-cold
5% sulfosalicylic acid and centrifuged at 19 000 g at 4 8C
for 5 min. Protein carbonyls content was measured by using
10 mmol
L–1 DNPH as previously described (Vázquez-
Medina et al. 2007). Results were expressed as nanomoles
of protein carbonyls per gram of wet tissue. Oxidatively
modified DNA content was measured by quantifying the
concentration of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-
oxodGuo). DNA was isolated by following the chaotropic-
NaI method (Wang et al. 1994) with modifications (Matos
et al. 2001). Frozen tissue (approximately 500 mg) was ho-
mogenized in 5 mL of lysis buffer (0.32 mol
L–1 sucrose,
5 mmol
L–1 EDTA, 1% Triton X-100, 5 mmol
L–1 MgCl2,
10 mmol
L–1 Tris-HCl, 0.1 mmol
L–1 desferroxamine,
Published by NRC Research Press
208
pH 7.5) and centrifuged at 1500g for 10 min. Pellets were
resuspended in 10 mmol
L–1 Tris-HCl buffer (5 mmol
L–1
EDTA, 10% SDS, 0.15 mmol
L–1 desferroxamine, pH 8.0).
RNAase A (10 mg
mL–1) and RNAase T1 (1000 U
mL–1)
were added. Samples were incubated for 1 h at 37 8C. Sam-
ples were incubated again under the same conditions after
the addition of proteinase K (20 mg
mL–1). Samples were
then centrifuged at 5000g for 15 min at 4 8C. Liquid phases
were collected and a 7.6 mol
L–1 NaI solution (40 mmol
L–1
Tris-HCl, 20 mmol
L–1 EDTA-Na2, 0.3 mmol
L–1 desferrox-
amine, pH 8.0) was added. Samples were mixed with isopro-
panol and stored overnight at –70 8C. Precipitates were
collected by centrifugation at 9000g for 15 min and washed
with 60% isopropanol and 70% ethanol. Samples were then
centrifuged at 9000g for 15 min. DNA pellets were dis-
solved in 0.1 mmol
L–1 desferroxamine. DNA concentrations
were measured by spectrophotometry at 260 nm. DNA pu-
rity was assessed by calculating the A260/A280 ratio. Only
sample extractions with ratios >1.75 were used. For each
sample, 100 mg of DNA were diluted in Milli-Q water and
1 mol
L–1 sodium acetate with nuclease P1 (5 U
mL–1) was
added. Samples were incubated at 37 8C for 30 min, after
which 1 mol
L–1 Tris-HCl buffer (pH 7.4) and acid phospha-
tase (3 U
mL–1) were added and samples were incubated at
37 8C for 1 h. Aqueous layers were obtained by centrifuga-
tion and analyzed for 8-oxodGuo content by using high-
performance liquid chromatography (HPLC) with electro-
chemical (EC) detection as previously described (López-
Diazguerrero et al. 2005). Digested DNA was injected into
an HPLC–EC system (Waters 600 C pump, Supelco supel-
cosil LC-18 reverse-phase column, 250 mm long  4.6 mm
inner diameter, particle size 5 mm), using 50 mmol
L–1 PBS
buffer (pH 5.5, 8% methanol) as isocratic eluent at a flow
rate of 1 mL
min–1. EC detection was performed using an
INTRO detector (Antec Leyden, Leiden, the Netherlands)
operated at 290 mV. Elution of unmodified nucleosides was
simultaneously detected at 254 nm by using a 486 Waters
UV spectrometer set. The molar ratio of 8-oxodGuo to de-
oxy-guanosine (dGuo) was determined. Results were ex-
pressed as the ratio of 8-oxodGugo to 106 dGuo.
SOD activity and protein expression
Total SOD (MnSOD + Cu,ZnSOD) and MnSOD activities
were measured following the method of Oberley and Spitz
(1984). Samples were homogenized in lysis buffer
(10 mmol
L–1 Tris-HCl, 15 mmol
L–1 NaCl, 0.25 mmol
L–1
sucrose, 1% Triton X-100, pH 7.0) containing protease in-
hibitors. To measure MnSOD activity, Cu,ZnSOD was in-
hibited with diethyldithiocarbamate. Assays were run as
described previously (Herrera-Mundo et al. 2006).
Cu,ZnSOD activity was calculated by subtracting MnSOD
activity from total SOD activity. Results were expressed as
units of total SOD, Cu,ZnSOD, or MnSOD activity per
milligram of protein. One unit of activity is defined as the
amount of protein that inhibits 50% of NBT reduction.
Cu,ZnSOD and MnSOD protein expression was evaluated
by Western blot. Samples were homogenized 1:10 (m/v) in
50 mmol
L–1 potassium phosphate buffer containing
1 mmol
L–1 EDTA, 1% Triton X-100, 1% PMSF, and 1%
protease inhibitor cocktail. Total protein content was deter-
mined by using the Bio-Rad Bradford Protein Assay (Bio-
Can. J. Zool. Vol. 89, 2011
Rad Laboratories, Hercules, California, USA). Twenty mi-
crograms of total protein were resolved in 4%–12% SDS
gels under denaturing conditions. Proteins were electroblot-
ted using the Bio-Rad Trans-Blot SD semi-dry cell onto
0.45 mm nitrocellulose membranes. Membranes were
blocked for 1 h at room temperature with 3% BSA in phos-
phate buffer saline containing 0.05% Tween-20 and incu-
bated overnight with rabbit polyclonal antibodies against
mammalian MnSOD, Cu,ZnSOD (Assay Designs, Ann Ar-
bor, Michigan, USA; catalogue Nos. SOD-111 and SOD-
100) diluted 1:4000 and a goat polyclonal antibody against
actin (Santa Cruz Biotechnology, Santa Cruz, California,
USA; catalogue No. sc-1616) diluted 1:1000. Membranes
were washed, incubated with HRP-conjugated secondary
antibodies (Santa Cruz Biotechnology), rewashed, and de-
veloped using the Bio-Rad Immun-Star WesternC kit. Inten-
sities of blot bands were quantified using Bio-Rad’s
Quantity One software. Values were expressed as percentage
of change from neonates after band intensities were normal-
ized against actin.
Statistics
Differences in responses among age groups were detected
by one-way analysis of variance (ANOVA) with Bonferroni
post hoc tests. Groups were considered statistically different
at p < 0.05. Statistical analyses were performed using the
SYSTAT# 12.0 software (Cranes Software International
Ltd., Richmond, California, USA). Results are presented as
means ± SE.
Results
O2– production increases with maturation in the muscle
of hooded seals
Endogenous O2– production was measured as an index of
the tissue capacity to produce ROS to evaluate whether ROS
production increases with development in hooded seal
muscle. O2– production was higher in adults than in neo-
natal pups (F = 23.065, p = 0.037). Values in weaned pups
were intermediate between those of neonates and adults
(Fig. 1). These results show that tissue capacity to produce
ROS is higher in the muscle of adult hooded seals than in
the muscle of developing animals.
Oxidative damage does not increase with maturation in
the muscle of hooded seals
Muscle content of TBARS, protein carbonyls, and 8-
oxodGuo was measured in neonates, weaned pups, and adult
seals to evaluate whether maturation increases oxidative
damage accumulation in the skeletal muscle of hooded seals.
There were no significant differences in the content of oxi-
dative damage to lipids, proteins, or DNA among the three
groups (Table 1) despite the increased O2– production ob-
served in the muscle of adult seals. These results suggest
that hooded seals possess mechanisms to avoid the accumu-
lation of oxidative damage commonly associated with in-
creased ROS production.
SOD activity and MnSOD protein content increase with
maturation in the muscle of hooded seals
MnSOD, Cu,ZnSOD, and total SOD activities along with
Published by NRC Research Press
Vázquez-Medina et al.
Table 1. Oxidative damage accumulation does not increase with maturation in the skeletal
muscle of hooded seals (Cystophora cristata).
TBARS
Age class (nmol
g wet tissue–1)
Neonatal pups 0.05±0.02
Weaned pus 0.08±0.009
Adults 0.05±0.01
Note: TBARS, thiobarbituric acid reactive substances; 8-oxodGuo, 8-oxo-7,8-dihydro-2’-
deoxyguanosine; dGuo, deoxy-guanosine.
Fig. 1. Superoxide radical (O2–) production increases with matura-
tion in the skeletal muscle of hooded seals (Cystophora cristata).
O2– production in skeletal muscle from neonatal pups, weaned
pups, and adult hooded seals. Results are expressed as means ± SE.
*, p < 0.05.
MnSOD and Cu,ZnSOD protein content were measured in
the muscle of neonatal, weaned pups, and adult hooded seals
to evaluate the potential role of these antioxidant enzymes in
counteracting the observed increases in ROS production. To-
tal SOD, MnSOD, and Cu,ZnSOD activities were higher
(total SOD, F = 80.298, p < 0.001; MnSOD, F = 14.534,
p = 0.001; Cu,ZnSOD, F = 88.167, p < 0.001) in adults
than in neonatal or weaned pups, which did not differ signif-
icantly (Figs. 2A–2C). In the same way, MnSOD protein
content was higher in adults than in neonantes or weaned
pups (F = 5.059, p = 0.03) (Fig. 3A), whereas Cu,ZnSOD
protein content did not differ among groups (F = 3.943, p <
0.071) (Fig. 3B). These results show that SOD activity in-
creases with maturation in the muscle of hooded seals and
likely helps to counteract the observed increases in ROS
production contributing to the avoidance of oxidative dam-
age accumulation.
Discussion
Mitochondria play a primary role in the production of
ROS in animal cells, and it has been widely documented
that this mitochondria-derived free radical production in
skeletal muscle increases with age, even during unperturbed
metabolic conditions (Nohl and Hegner 1978; Sohal et al.
1995; Wei 1998; Mecocci et al. 1999; Cadenas and Davies
2000; Marzani et al. 2004; Halliwell and Gutteridge 2007;
Figueiredo et al. 2008). There is also evidence for the accu-
mulation of oxidized molecules with age progression in sev-
eral terrestrial mammal species (Cutler 1985; Garland et al.
1988; Mecocci et al. 1999; Davies and Delsignore 2001;
209
Protein carbonyls DNA damage
(nmol
g wet tissue–1) (8-oxodGuo/106 dGuo)
106.61±32 1.0±0.2
103.76±31 1.5±0.3
96.87±33 0.75±0.3
Hamilton et al. 2001; Pamplona 2008). In the present study,
we show that ROS production in the skeletal muscle of
hooded seals increases with maturation, but lipid peroxida-
tion, protein oxidation, and oxidatively modified DNA con-
tent do not.
O2– production in the skeletal muscle of adult hooded
seals is within the range of values reported for the muscle
of adult ringed seals (Pusa hispida (Schreber, 1775)),
whereas O2– production in the muscle of pups was closer
to that reported for the muscle of adult pigs (Zenteno-Savı́n
et al. 2002). Protein carbonyls levels found in the muscle of
hooded seals are generally lower than the levels reported for
the muscle of adult pigs and ringed seals (Vázquez-Medina
et al. 2007), whereas TBARS levels are within the same
range as those of pigs but lower than those of ringed seals
(Zenteno-Savı́n et al. 2002). Age-related accumulation of
oxidized proteins is a complex function involving the rates
of ROS formation, the antioxidant systems, and the rates of
degradation of oxidized proteins (Starke-Reed and Oliver
1989). Tissues from adult ringed seals showed higher rates
of ROS production during in vitro experiments that simulate
dive-induced ischemia–reperfusion than tissues from non-
diving adult pigs (Zenteno-Savı́n et al. 2002). However, rel-
ative to terrestrial species, tissues from ringed seals also
showed enhanced activities of key antioxidant enzymes and
high glutathione content, which likely counteract the poten-
tially negative effects of dive-derived ROS production by
preventing the accumulation of oxidative damage (Vázquez-
Medina et al. 2006, 2007). Li et al. (2005), as observed in
the current study, failed to find any increases in the accumu-
lation of 8-oxodGuo when comparing mature vs. developing
individuals of several cetacean species. In contrast, Gauthier
et al. (1999) reported an age-related increase in micronuclei
cells (MNC) of developing bottlenose dolphins (Tursiops
truncatus (Montagu, 1821)), indicating that oxidative dam-
age may accumulate with development among some marine
mammals.
Studies in rats have shown that the activity of the DNA-
repairing enzyme 8-oxoguanine DNA glycosylase (OGG1),
which is responsible for 8-oxodGuo repair, can be stimu-
lated by ROS derived from exercise training protocols
(swimming included), resulting in a reduction of 8-oxodGuo
accumulation (Ogonovszky et al. 2005; Radák et al. 2007).
Considering that the diving lifestyle of adult hooded seals
exposes them to repetitive cycles of ischemia–reperfusion
(Elsner 1999), which promote ROS generation (Zenteno-
Savı́n et al. 2002), it is possible that dive-derived ROS pro-
duction stimulates increases in OGG1 or other DNA repair
mechanisms, preventing the accumulation of oxidatively
modified DNA content.
Published by NRC Research Press
210
Fig. 2. Superoxide dismutase (SOD) activity increases with matura-
tion in the muscle of hooded seals (Cystophora cristata). (A) Total
SOD, (B) MnSOD, and (C) Cu,ZnSOD activities in muscle from
neonatal pups, weaned pups, and adult hooded seals. One unit of
SOD activity is defined as the amount of enzyme that inhibits 50%
of NBT reduction. Results are expressed as means ± SE. *, p <
0.05.
Can. J. Zool. Vol. 89, 2011
Fig. 3. MnSOD protein expression increases with maturation in the
muscle of hooded seals (Cystophora cristata). (A) MnSOD and
(B) Cu,ZnSOD relative protein content in muscle from neonatal
pups, weaned pups and adult hooded seals. Results are expressed as
means ± SE of the percent change from neonatal pups. Percent
changes were obtained after calculating the ratios of MnSOD to ac-
tin or Cu,ZnSOD to actin. *, p < 0.05.
Both diving capacity and diving behavior of hooded seals
develop with maturation (Burns et al. 2007, Folkow et al.
2010). Because growing seals possess an increased anti-
oxidant capacity (Vázquez-Medina et al. 2010), we hypothe-
size that the development of hooded seals’ diving ability is
accompanied by the development of an enhanced anti-
oxidant capacity. This would allow them to tolerate both
dive-derived and maturation-related increases in ROS pro-
duction, and to prevent the accumulation of oxidatively
modified DNA, lipids, and proteins.
Our results support the former hypothesis, as we found
higher SOD activity and MnSOD protein content in adult
seals than in pups. Even though Cu,ZnSOD protein content
is not increased in adult animals, its sustained expression
seems to be enough to promote a maturation-related increase
in Cu,ZnSOD activity. Moreover, total SOD activity values
found in the muscle from pups of hooded seals are similar
to those reported from the skeletal muscle of sea turtles
(Valdivia et al. 2007), whereas the values in muscle from
adult hooded seals are more than two times higher than
those reported for adult pigs or the short-diving ringed seal
(Vázquez-Medina et al. 2006), suggesting that the
antioxidant system may also be increased in long- and
Published by NRC Research Press
Vázquez-Medina et al.
deep-diving seals, such as the hooded seal, than in short and
shallow divers.
In summary, our results show that tissue capacity to pro-
duce ROS increases with maturation in the muscle of
hooded seals, but these maturation-related increases in ROS
production are not correlated with increases in oxidative
damage accumulation. The observed increases in SOD activ-
ity and MnSOD protein content seem to counteract the in-
creased ROS production in adult seals and to prevent, at
least in part, the accumulation of oxidative damage. These
observed increases in SOD activity and MnSOD protein
content also seem to be correlated with the rapid physiolog-
ical development of hooded seals. More work is needed to
elucidate the participation of other antioxidant mechanisms
(enzymatic and nonenzymatic) and repair systems in coun-
teracting ROS production in seals, and to corroborate the
link between the antioxidant protection against dive- and
maturation-related ROS production.
Acknowledgements
A. Luna, V. Pérez, O. Lugo, and D. Rojas provided in-
struction and advice on analytic techniques. The Canadian
Coast Guard helicopter pilots Harrison McRae and Bruce
Kendall provided logistic support in sample collection. The
Château Madelinot and Roger Simon provided laboratory
space. Lena Measures, Stephan Pillet, and Sam Turgeon
assisted with sample collection. Comments and suggestions
from Rudy Ortiz and two anonymous reviewers helped to
improve the manuscript. J.P.V.-M. is supported by
Programa de Estudios de Posgrado (CIBNOR), UC-Mexus,
CONACYT, and Secretarı́a de Educación Pública
fellowships. This research was funded by grants from
SEMARNAT-CONACYT and CIBNOR.
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