Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204

www.elsevier.com/locate/cbpc

Antioxidant enzymes in ringed seal tissues: Potential protection against

dive-associated ischemia/reperfusionB
José Pablo Vázquez-Medina a, Tania Zenteno-Savı́n b,*, Robert Elsner c
a
Departamento de Biologı́a Marina. Universidad Autónoma de Baja California Sur, La Paz, Baja California Sur, México
b
Programa de Planeación Ambiental y Conservación, Centro de Investigaciones Biológicas del Noroeste, S.C., Mar Bermejo 195, Playa Palo Santa Rita, La Paz,
Baja California Sur, CP 23090—México
c
Institute of Marine Science. University of Alaska Fairbanks, Fairbanks, AK, U.S.A

Received 7 June 2005; received in revised form 21 September 2005; accepted 22 September 2005
Available online 2 November 2005

Abstract

Diving seals experience heart rate reduction and preferential distribution of the oxygenated blood flow to the heart and brain, widespread
peripheral vasoconstriction, and selective ischemia in the most hypoxia-tolerant tissues. The first breath after the dive restores the oxygenated
blood flow to all tissues and raises the potential for the production of reactive oxygen species (ROS). We hypothesized that in order to counteract
the damaging effects of ROS and to tolerate repetitive cycles of ischemia/reperfusion associated with diving, ringed seal (Phoca hispida) tissues
have elevated activities of antioxidant enzymes. Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and
glutathione-S-transferase (GST) were measured by spectrophotometric techniques in heart, kidney, liver, lung, and muscle extracts of ringed seals
and domestic pigs (Sus scrofa). The results suggest that in ringed seal heart SOD, GPx and GST activities are an efficient protective mechanism for
counteracting ROS production and its deleterious effects. Apparently CAT activity in seal liver and GPx activity in seal muscle participate in the
removal of hydroperoxides, while seal lung appears to be protected from oxidative damage by SOD and GPx activities.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Antioxidant enzymes; Diving; Ischemia/reperfusion; Oxidative stress; Reactive oxygen species; Seals

1. Introduction ischemia (restriction of blood flow) in the most hypoxia-

Seals of the Phocidae family have developed a number of
mechanisms that allow them to tolerate long dives and to
survive progressive asphyxia (the combination of hypoxia,
hypercapnia and acidosis) (Elsner and Gooden, 1983). These
mechanisms include elevated hemoglobin and myoglobin
concentrations, high mitochondrial content in specific tissues,
elevated blood volumes, and a number of cardiovascular
adjustments during a dive, including bradycardia and prefer-
ential blood flow distribution to the central nervous system by
a widespread vasoconstriction, which results in selective
B This paper is part of a special issue of CBP dedicated to ‘‘The Face of Latin
American Comparative Biochemistry and Physiology’’ organized by Marcelo
Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Tania Zenteno-
Savı́n (Mexico) and the editors of CBP. This issue is in honour of Cicero Lima
and the late Peter W. Hochachka, teacher, friend and devoted supporter of Latin
American science.
* Corresponding author. Tel.: +52 612 123 8502; fax: +52 612 125 3625.
E-mail address: tzenteno04@cibnor.mx (T. Zenteno-Savı́n).
1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cbpc.2005.09.004
tolerant tissues, followed by prompt reperfusion (restoration of
blood flow) at the end of the dive (Kooyman and Ponganis,
1998; Elsner, 1999; Kanatous et al., 1999, 2002). Prolonged
ischemia results in ATP depletion, accumulation of purine
nucleotides, intracellular calcium increase and activation of the
oxidative enzyme xanthine oxidase (Elsner et al., 1998;
Hermes-Lima et al., 1998; Halliwell and Gutteridge, 2001).
In terrestrial organisms ischemia/reperfusion raises the
production of reactive oxygen species (ROS) and the potential
for oxidative damage (Halliwell and Gutteridge, 2001). In diving
seals, ischemia/reperfusion associated with a dive apparently
increases the production of ROS but not the oxidative damage
(Zenteno-Savı́n and Elsner, 1998, 2000). We hypothesized that
elevated antioxidant capacity balances the increases of ROS
production in seal tissues (Zenteno-Savı́n et al., 2002).
High antioxidant status in diving mammals seems to be the
key for tolerating repetitive ischemia/reperfusion episodes
(Elsner et al., 1995, 1998; Wilhem-Filho et al., 2002).
Enzymatic and non-enzymatic antioxidants are important
 J.P. Vázquez-Medina et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204
protective mechanisms against oxidative damage. Antioxidant
enzymes play an important role in the ROS cascade reaction:
superoxide dismutase (SOD) transforms superoxide radical
U U
(O2 ) into hydrogen peroxide (H2O2), catalase (CAT) and
glutathione peroxidase (GPx) remove hydrogen peroxide and
U
limit the hydroxyl radical (OH ) formation, and glutathione-S-
transferases (GST) removes toxic products of ROS damage
(Halliwell and Gutteridge, 2001).
To our knowledge, only a few studies of antioxidant enzyme
activities in diving mammal tissues have been reported (Elsner
et al., 1995, 1998; Wilhem-Filho et al., 2002), and, except for
the findings of Murphy and Hochachka (1981), of unusual
changes in glutathione levels in blood during and after
restrained dives in Weddell seals, no studies have formally
addressed changes in antioxidant defenses before, during and
after a dive. More investigation of antioxidant enzyme
activities is necessary for understanding how these animals
protect themselves against oxidative damage derived from
ischemia/reperfusion cycles associated with diving.
2. Materials and methods
2.1. Sample collection
Fresh tissue samples from 11 ringed seals (Phoca hispida)
of both sexes (7 males and 4 females), average body mass
31.98 T 3.47 kg, were obtained incidental to subsistence
hunting through the collaboration with the North Slope
Borough Department of Wildlife Management and Inupiat
Eskimo hunters, near Barrow, Alaska, U.S.A. For comparative
purposes fresh tissue samples from 10 pigs (Sus scrofa) (5
males and 5 females), average body mass 73.40 T 1.10 kg, were
obtained at a local slaughter-house in La Paz, Baja California
Sur, Mexico. Approximately 5 g of heart, kidney, liver, lung
and muscle from both species were collected and immediately
frozen by immersion in liquid nitrogen for transportation and
subsequently stored at 80 -C.
2.2. Tissue homogenates
Previous to enzyme activity determinations, each tissue
sample was homogenized in 2 volumes of phosphate buffer (50
mM, pH 7.5; EDTA 1 mM; PMSF 1 mM) and centrifuged at
2000  g for 20 min at 4 -C. The supernatant was taken and
used for the analysis.
2.3. Superoxide dismutase (EC 1.15.1.1)
Total superoxide dismutase activity was measured spectropho-
U
tometrically using xanthine/xanthine oxidase as a O2 generating
system and nitroblue tetrazolium (NBT) as a detector (Suzuki,
2000). Each sample was diluted 1 : 10 with phosphate buffer (50
mM, pH 7.5, EDTA 1 mM). Sodium-carbonate working solution
(50 mM, xanthine 0.1 mM, NBT 0.025 mM, EDTA 0.1 mM),
xanthine oxidase (0.1 U/mL in ammonium sulfate 2 M) and
sample or blank (phosphate buffer 50 mM, pH 7.5; EDTA 1 mM)
were mixed in a cuvette. The change in absorbance per minute at
199
560 nm (DA 560) was calculated. Enzymatic activity was expressed
in units of SOD activity per mg of protein. One unit of SOD
activity is defined as the amount of enzyme needed to inhibit the
reaction of O2 with NBT by 50%.
2.4. Catalase (EC 1.11.1.6)
Catalase activity was evaluated by measuring the decrease
in H2O2 concentration at 240 nm (Aebi, 1984). Working
solution (phosphate buffer 100 mM; H2O2 10 mM) and sample
were mixed in a cuvette. The change in absorbance per minute
at 240 nm (DA 240) was calculated. Enzyme activity was
expressed in units of CAT activity per milligram of protein.
One unit of CAT activity is defined as the amount of enzyme
needed to reduce 1 Amol H2O2/min.
2.5. Glutathione peroxidase (EC 1.11.1.9)
Selenium-dependent glutathione peroxidase activity was
measured by monitoring the continuous decrease in NADPH
concentration using H2O2 as a substrate (Flohé and Günzler,
1984). In a cuvette, potassium phosphate buffer (500 mM),
EDTA (50 mM), sodium azide (20 mM), glutathione reductase
(15 U/mL), NADPH (1.5 mM), reduced glutathione (250 mM),
sample and H2O2 (10 mM) were mixed; the absorbance was
followed at 340 nm, and the change in absorbance per minute
(DA 340) was calculated. Two blanks, one without H2O2 and
another without sample, were run simultaneously. Enzyme
activity was expressed in units of GPx activity per mg of
protein. One unit of GPx activity is defined as the amount of
enzyme that oxidizes 1 Amol of NADPH per min.
2.6. Glutahione-S-transferase (EC 2.5.1.18)
Glutathione-S-transferase activity was determined by mon-
itoring the formation of the thioether product from the reaction
between GSH and CNDB (1-chloro, 2,4-dinitrobenzene)
(Habig and Jakoby, 1981). Working solution (phosphate buffer
100 mM, GSH 1 mM, EDTA 60 mM), CNDB (10 mM) and
sample were placed in a cuvette and mixed. Absorbance was
followed at 340 nm, and the change in absorbance per minute
(DA 340) was calculated. One blank was run in absence of
sample. Enzyme activity was expressed in units of GST activity
per mg of protein. One unit of GST activity is defined as the
amount of enzyme that synthesizes 1 Amol of product/min.
2.7. Protein assay
Soluble protein content in tissue homogenates was mea-
sured following the method of Bradford (1976) using the Bio-
Rad prepared reagent and bovine serum albumin as standard.
2.8. Statistical analyses
Enzyme activities are reported as mean T SEM. Significant
differences between tissues and species were estimated using
Kruskal – Wallis and Mann – Whitney tests (Zar, 1999). Statis-
200 J.P. Vázquez-Medina et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204

Table 1
Antioxidant enzyme activities (U/mg protein) in ringed seal and domestic pig tissues
Species Tissue SOD CAT GST GPx
a bc b
Seal Heart 77.21 T 25.79 1956.16 T 581.01 13.40 T 3.76 15.72 T 1.24bc
Kidney 55.28 T 10.64a 8245.23 T 960.78a 7.18 T 1.57b 17.77 T 2.16bc
Liver 71.72 T 36.70a 19196.85 T 4527.31a 35.11 T 5.92a 33.46 T 8.61ab
Lung 78.65 T 20.39a 1614.59 T 289.49bc 3.54 T 1.51c 47.28 T 10.81a
Muscle 14.57 T 3.99b 891.68 T 268.03d 0.95 T 0.24d 4.91 T 0.86d
Pig Heart 26.48 T 8.35abc 7154.30 T 1900.34b 4.24 T 1.59b 8.011 T 0.92b
Kidney 38.35 T 7.33a 7717.12 T 1176.64c 5.98 T 0.80c 36.12 T 13.14a
Liver 24.74 T 3.70ab 11004.28 T 3583.43a 70.00 T 12.88a 25.55 T 3.73a
Lung 8.92 T 2.28bc 3644.76 T 630.01cd 2.69 T 0.78b 16.11 T 2.14a
Muscle 15.35 T 4.42bc 3462.32 T 848.94b 0.91 T 0.28d 2.01 T 0.26c
Data are shown as mean T SEM. Different letters denote significant differences, p < 0.05. SOD = superoxide dismutase, CAT = catalase, GST = glutathione S-
transferase, GPx = glutathione peroxidase.

tical analyses were performed using the SYSTAT 9.0 (SPSS, seal heart, lung and muscle (Fig. 2). Ringed seal liver had
Richmond, CA, USA) software. higher ( p < 0.05) CAT activity than pig liver (Fig. 2).

3. Results 3.3. Glutathione peroxidase

3.1. Superoxide dismutase
Results obtained for SOD activity in seal and pig tissues are
summarized in Table 1 and Fig. 1. SOD activity was lower
( p < 0.05) in ringed seal muscle than in other tissues (Table 1).
Ringed seal lung had higher ( p < 0.05) SOD activity than pig
lung (Fig. 1). In cardiac tissue, SOD activity was higher
( p < 0.1) in ringed seal than in pig.
3.2. Catalase
Table 1 and Fig. 2 show the results obtained for CAT
activity in seal and pig tissues. CAT activity was higher
( p < 0.05) in ringed seal kidney and liver, and lower ( p < 0.05)
in ringed seal muscle than in other tissues (Table 1). Pig heart,
lung and muscle had higher ( p < 0.05) CAT activity than ringed
Results obtained for GPx activity in seal and pig tissues are
presented in Table 1 and Fig. 3. GPx activity was higher
( p < 0.05) in ringed seal lung than in heart, kidney or muscle, and
lower ( p < 0.05) in muscle than in other tissues (Table 1). Ringed
seal heart, lung and muscle had higher ( p < 0.05) GPx activity
than pig heart, lung and muscle (Fig. 3). Pig kidney had higher
( p < 0.1) GPx activity than ringed seal kidney (Fig. 3).
3.4. Glutathione-S-transferase
Data obtained for GST activity in tissues from seal and pig
are summarized in Table 1 and Fig. 4. GST activity was higher
( p < 0.05) in ringed seal liver and lower in muscle ( p < 0.05)
than in other tissues (Table 1). Ringed seal heart and kidney
had higher ( p < 0.05) GST activity than lung (Table 1). Ringed
seal heart had higher ( p < 0.05) GST activity than pig heart

100 20000

90

80 16000
U SOD/ mg protein

U CAT/ mg protein

70
+
60 ∗ 12000
50 + ∗
40 8000
30

20
∗ ∗
4000
10

0 0
HEART KIDNEY LIVER LUNG MUSCLE HEART KIDNEY LIVER LUNG MUSCLE

Fig. 1. Superoxide dismutase activity (SOD, U/mg protein) in pig ( , n = 10) Fig. 2. Catalase activity (CAT, U/mg protein) in pig ( , n = 10) and in ringed

* = p < 0.05; + = p < 0.1.

˝
and in ringed seal ( , n = 11) tissues. Data are shown as mean T SEM.
˝
seal ( , n = 11) tissues. Data are shown as mean T SEM. * = p < 0.05;
+ = p < 0.1.
 J.P. Vázquez-Medina et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204
70
60
U GPX/ mg protein
50
∗ that GSH accumulation in ringed seal heart may be an effective
40
30
20 ∗ mechanisms are strongly involved in lung protection from the
10
∗ generation and SOD activity after diving. In human patients,
0 lung function (Schunemann et al., 2002). Recent studies
HEART KIDNEY LIVER LUNG MUSCLE
Fig. 3. Glutathione peroxidase activity (GPx, U/mg protein) in pig ( , n = 10)
* = p < 0.05; + = p < 0.1.
˝
and in ringed seal ( , n = 11) tissues. Data are shown as mean T SEM.
(Fig. 4). In hepatic tissue, GST activity was higher ( p < 0.05) in
pig than in ringed seal (Fig. 4).
4. Discussion
SOD activity was higher in ringed seal heart than in pig
heart. Elsner et al. (1998) reported similar results for both of
these species. Seal cardiac tissue receives intermittent blood
flow, resulting in decreased oxygen consumption while
maintaining reduced cardiac function (Elsner et al., 1985;
Elsner, 1999). Our results suggest that in seal cardiac tissue
elevated SOD, GPx and/or GST activities may contribute to
the defense against ROS generation associated with reperfu-
sion. In terrestrial mammals, experimental preconditioning
(intermittent reductions of blood flow and short ischemia/
reperfusion episodes) induces physiological protective
mechanisms associated with heat shock proteins (HSP) and
manganese-dependent SOD activity (Mn-SOD) (Murry et al.,
1986; Nao et al., 1990; Cohen et al., 1990; Flack et al.,
1999; Halliwell and Gutteridge, 2001). In phocid seals this
condition may be a natural defense mechanism directly
related to the diving response (Elsner et al., 1998; Zenteno-
Savı́n et al., 2002). Higher GST activity in ringed seal than
in pig heart is probably related to the lower TBARS content
reported by Zenteno-Savı́n et al. (2002) and to the lesser
accumulation of malondialdehyde (MDA), hydroxynonenal
and other residues from lipid peroxidation in this tissue
(Prohaska, 1980; Halliwell and Gutteridge, 2001).
CAT activity was significantly lower in ringed seal than in
pig heart. Catecholamine accumulation, possibly derived from
transportation and handling stress, is suspected to have induced
ROS production (Fraser et al., 1975; Sigal et al., 1982;
Häggendal et al., 1987; Sing, 1992) and activity of antioxidant
enzymes in pig heart. GPx activity was significantly higher in
ringed seal than in pig heart. CAT and GPx have an important
role in hydroperoxide removal; GPx participates when H2O2
concentration remains constant, whereas CAT activity rises
201
with an increase in H2O2 content (Chance et al., 1979). Under
basal conditions, skeletal muscle has higher GPx than CAT
activity (Halliwell and Gutteridge, 2001). Higher GST and GPx
activities in ringed seal compared to pig cardiac tissue suggest
antioxidant protective mechanism.
SOD and GPx activities were significantly higher in ringed
seal than in pig lung. CAT activity was significantly lower in
ringed seal than in pig lung. CAT activity was lower in ringed
seal than in pig lung. It is possible that GPx and other antioxidant
potential H2O2 accumulation derived from elevated ROS
antioxidant vitamins play a very important role in maintaining
(Johnson et al., 2004, 2005) suggest that Hypoxia-Inducible
Factor (HIF) expression plays a protective role against oxidative
damage in ringed seal lung. A number of reports conclude that
U
isolated lung exposure to high concentration of O2 results in
increased oxidized glutathione (GSSG) levels and GPx activity
(Halliwell and Gutteridge, 2001).
SOD activity in ringed seal muscle was significantly lower
than in the other ringed seal tissues and not significantly
different from pig muscle. Zenteno-Savı́n et al. (2002) reported
U
higher O2 production, higher levels of lipid peroxidation and
lower antioxidant capacity in ringed seal muscle than in renal
or cardiac ringed seal tissues. Seal muscle adaptations to diving
include high mitochondrial density, and high myoglobin and
polyunsaturated fatty acid (PUFA) concentrations (Kanatous et
al., 1999, 2002; Davis and Kanatous, 1999). These adaptations
contribute to maintaining aerobic metabolism during ischemia
(Kanatous et al., 1999, 2002), but raise the potential production
U
of O2 derived from respiration processes during repetitive
cycles of ischemia/reperfusion (Longo et al., 1996; Zwicker et
al., 1998). PUFA content makes seal muscle highly susceptible
to lipid peroxidation. SOD is involved in the transformation of
100
90
∗
80
U GST/ mg protein
70
60
50
40
30
20 ∗
10
0
HEART KIDNEY LIVER LUNG MUSCLE
Fig. 4. Glutathione-S-transferase activity (CAT, U/mg protein) in pig ( , n = 10)
˝
and in ringed seal ( , n = 11) tissues. Data are shown as mean T SEM.
* = p < 0.05.
202 J.P. Vázquez-Medina et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204
U U
O2 into H2O2 (McCord and Fridovich, 1969). O2 is unable
U
to start the lipid peroxidation chain reaction; however, O2
U
and H2O2 in the presence of iron or copper can generate OH , a
highly reactive oxygen species which can oxidize lipids,
proteins and nucleic acids (Halliwell and Gutteridge, 2001).
It is possible that, in order to control formation of H2O2 and
U
OH , and to protect ringed seal muscle from peroxidative
damage, SOD activity is lower in muscle than in the other
ringed seal tissues. SOD and GST activities were not
significantly different between ringed seal and pig muscle.
CAT activity was significantly lower in ringed seal than in pig
muscle and GPx activity was significantly higher in ringed seal
than in pig muscle. Diving seal muscle blood flow is generally
reduced during a dive (Elsner et al., 1966). Mitochondrial
metabolism, myoglobin oxidation and ischemia/reperfusion are
associated with an increase in ROS generation and peroxida-
tion in ringed seal muscle (Zenteno-Savı́n et al., 2002). In
garter snake (Thamnophis sirtalis parietalis) muscle CAT
activity increases during freezing episodes (equivalent to
ischemia/reperfusion episodes), but GSH content, which can
stimulate peroxidase activities, does not increase under anoxia
(Hermes-Lima and Storey, 1993). Resichl (1986) suggests that,
during reperfusion, superficial hemoglobin SH groups contrib-
ute to the removal of ROS and xenobiotics. It is possible that
low SOD activity, high GPx activity and elevated GSH levels
U
contribute to control H2O2 content and limit OH synthesis in
ringed seal muscle. CAT activity was significantly higher in
ringed seal kidney and liver than in the other ringed seal
tissues. Similar results have been reported in other vertebrate
species (Willmore and Storey, 1997; Ansaldo et al., 2000;
Lushcak et al., 2001; Valdivia-Jiménez, 2003). Metabolic
rate, iron content and detoxification function from liver
(Schmidt-Nielsen, 2001) can explain these results. Iron and
U
copper react with H2O2 to produce OH , thus, CAT has an
U
important role in removing H2O2 and preventing OH
synthesis in these tissues (Pippenger et al., 1998). Seal
kidney and liver are vigourously vasoconstricted during a
dive (Elsner, 1999). Seal kidney has been identified as an
ischemia-tolerant tissue with low hypoxanthine (HX) content
(Elsner et al., 1995, 1998). Zenteno-Savı́n et al. (2002)
reported low levels of lipid peroxidation in ringed seal
kidney, probably due to non-enzymatic antioxidant protec-
tion. Our results support this idea since SOD, CAT and GST
activities were not statistically different between ringed seal
and in pig kidney and GPx activity was higher in pig than in
ringed seal kidney.
Elsner et al. (1998) reported higher SOD activity in pig than
in ringed seal kidney. Zenteno-Savı́n et al. (2002) found higher
U
O2 production in ringed seal than in pig kidney, lower lipid
peroxidation in ringed seal than in pig kidney, and higher
antioxidant capacity in ringed seal kidney. In experimental
models, Chade et al. (2003) reported that antioxidant vitamins
counteract ischemia/reperfusion damage. These results support
the idea that in phocid seal kidney, non-enzymatic antioxidants
play an important role in counteracting ROS production and
damage. GST activity was significantly higher in ringed seal
liver than in other tissues. High GST concentrations have been
reported in vertebrate liver (Jakoby, 1985; Tsuchida and Sato,
1992; Hermes-Lima and Storey, 1993, 1996; Valdivia-Jiménez,
2003) where this enzyme participates in detoxification pro-
cesses catalyzing xenobiotic conjunction to the SH group of
GSH (Habig and Jakoby, 1981; Jakoby, 1985). In goldfish liver
(Carassius auratus) Lushcak et al. (2001) reported a decrease
in total protein content, probably because Ca2+ accumulation
activates proteases that transform xanthine dehydrogenase
U
(XDH) into XO, a potential O2 generator, when ATP
depletion results in HX accumulation during ischemia (Halli-
well and Gutteridge, 2001). Lack of elevated SOD activity in
U
ringed seal liver may suggest that O2 generation is not
extensive, probably because HX is efficiently recycled by the
enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT) (Elsner et al., 1998).
Higher CAT activity in ringed seal than in pig liver indicates
the existence of an efficient hydroperoxide protective mecha-
U
nism in this seal tissue that could regulate OH formation and
limit peroxidation damage (Halliwell and Gutteridge, 2001).
Pig hepatic tissue showed low CAT activity, which is probably
reflected in high lipid peroxidation levels (Zenteno-Savı́n and
Elsner, 2000).
High GST activity intervenes in the removal of peroxida-
tion-derived products and xenobiotics, which could be
important in ROS reactions and ROS-induced damage
(Martı́nez-Cayuela, 1998). Our results and other studies of
antioxidant enzymes in vertebrate liver (Hermes-Lima and
Storey, 1993; Willmore and Storey, 1997; Grundy and Storey,
1998; Lushcak et al., 2001) suggest that CAT activity is an
important protective mechanism against ROS damage in
hepatic tissue. On the other hand, it is possible that GSH
and GSSG participate actively in liver protection from ROS
derived from ischemia/reperfusion (Hermes-Lima and Storey,
1993; Willmore and Storey, 1997; Grundy and Storey, 1998;
Lushcak et al., 2001).
In summary, we found differences in SOD, CAT, GPx and
GST activities among ringed seal tissues which are probably
related to the specific function, aerobic metabolism and
perfusion maintenance of each tissue during a dive. We found
differences in SOD, CAT, GPx and GST activities between
ringed seal and pig tissues. These results suggest that SOD,
GST and GPx activities play an important role in protecting
ringed seal heart, lung and muscle from ROS damage derived
from ischemia/reperfusion cycles associated with diving. In
ringed seal liver, CAT apparently plays an important role in
removing hydroperoxides. Non-enzymatic antioxidants might
be involved in protection against ROS damage in ringed seal
kidney.
While this study suggest that ringed seal tissues are
endowed with an enhanced antioxidant capacity to tolerate
ischemia/reperfusion, further studies of antioxidant enzyme
activities in seal tissues before, during and after a dive are still
lacking. Tissue levels of GSH, the GSSG : GSH ratios and
enzymatic activity of GR, to provide information on the
substrate availability for GPX and GST, in ringed seals, as well
as comparative studies to include other phocid and otariid
species, are being undertaken at our laboratories.
 J.P. Vázquez-Medina et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 198 – 204
Acknowledgements
The authors wish to thank all members of the North Slope
Alaska Department of Wildlife Management, the Barrow Arctic
Science Consortium and Inupiat Eskimo hunters who provided
logistic support and assistance in obtaining ringed seal samples.
Sampling was performed under terms of a marine mammal
scientific permit issued to R. Elsner by the Office of Protected
Species, US National Marine Fisheries Service. Samples were
imported under terms of import permits issued to T. Zenteno-
Savı́n by Instituto Nacional de Ecologı́a, México. We also wish
to thank Dr. José Marı́a Acosta Farı́as and personnel of Rastro
Municipal de La Paz for the assistance in obtaining pig samples.
Two anonymous reviewers provided invaluable editorial
assistance. Research was funded by grants from the Office for
Naval Research, UC Mexus, CONACYT, UAF, CIBNOR and
CNPq. The instruction and advice in antioxidant enzyme
analyses provided by Gabriella Ramos, Luciano Cardoso,
Daniel Prado, Orlando V. Furtado-Filho and everyone at Dr.
Hermes-Lima’s lab is enormously appreciated.
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