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Antioxidant enzymes in ringed seal tissues: Potential protection against dive-associated ischemia/reperfusionB
Jose Pablo Va zquez-Medina a, Tania Zenteno-Sav n b,*, Robert Elsner c
b a Departamento de Biolog a Marina. Universidad Auto noma de Baja California Sur, La Paz, Baja California Sur, Me xico Programa de Planeacio n Ambiental y Conservacio n, Centro de Investigaciones Biolo gicas del Noroeste, S.C., Mar Bermejo 195, Playa Palo Santa Rita, La Paz, Baja California Sur, CP 23090Me xico 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 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 preferential 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 ZentenoSav n (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-Sav n). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.09.004

ischemia (restriction of blood flow) in the most hypoxiatolerant 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-Sav n and Elsner, 1998, 2000). We hypothesized that elevated antioxidant capacity balances the increases of ROS production in seal tissues (Zenteno-Sav n 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

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protective mechanisms against oxidative damage. Antioxidant enzymes play an important role in the ROS cascade reaction: superoxide dismutase (SOD) transforms superoxide radical 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 spectrophoU 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

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 U 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 (Flohe and Gu nzler, 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 monitoring 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 measured following the method of Bradford (1976) using the BioRad 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-

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Table 1 Antioxidant enzyme activities (U/mg protein) in ringed seal and domestic pig tissues Species Seal Tissue Heart Kidney Liver Lung Muscle Heart Kidney Liver Lung Muscle SOD 77.21 T 25.79 55.28 T 10.64a 71.72 T 36.70a 78.65 T 20.39a 14.57 T 3.99b 26.48 T 8.35abc 38.35 T 7.33a 24.74 T 3.70ab 8.92 T 2.28bc 15.35 T 4.42bc
a

CAT 1956.16 T 581.01 8245.23 T 960.78a 19196.85 T 4527.31a 1614.59 T 289.49bc 891.68 T 268.03d 7154.30 T 1900.34b 7717.12 T 1176.64c 11004.28 T 3583.43a 3644.76 T 630.01cd 3462.32 T 848.94b
bc

GST 13.40 T 3.76 7.18 T 1.57b 35.11 T 5.92a 3.54 T 1.51c 0.95 T 0.24d 4.24 T 1.59b 5.98 T 0.80c 70.00 T 12.88a 2.69 T 0.78b 0.91 T 0.28d
b

GPx 15.72 T 1.24bc 17.77 T 2.16bc 33.46 T 8.61ab 47.28 T 10.81a 4.91 T 0.86d 8.011 T 0.92b 36.12 T 13.14a 25.55 T 3.73a 16.11 T 2.14a 2.01 T 0.26c

Pig

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, Richmond, CA, USA) software. 3. Results 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
100 90 80

seal heart, lung and muscle (Fig. 2). Ringed seal liver had higher ( p < 0.05) CAT activity than pig liver (Fig. 2). 3.3. Glutathione peroxidase 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

20000

16000

U SOD/ mg protein

70 60 50 40 30 20 10 0 HEART KIDNEY LIVER

U CAT/ mg protein

+
12000

8000 4000

HEART KIDNEY LIVER

LUNG MUSCLE

0 LUNG MUSCLE

Fig. 1. Superoxide dismutase activity (SOD, U/mg protein) in pig ( , n = 10) and in ringed seal ( , n = 11) tissues. Data are shown as mean T SEM. * = p < 0.05; + = p < 0.1.

Fig. 2. Catalase 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; + = p < 0.1.

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201

60

50

40

30

20


HEART KIDNEY LIVER LUNG MUSCLE

10

Fig. 3. Glutathione peroxidase activity (GPx, U/mg protein) in pig ( , n = 10) and in ringed seal ( , n = 11) tissues. Data are shown as mean T SEM. * = p < 0.05; + = p < 0.1.

(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 reperfusion. 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; ZentenoSav n et al., 2002). Higher GST activity in ringed seal than in pig heart is probably related to the lower TBARS content reported by Zenteno-Sav n 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; Ha ggendal 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

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 that GSH accumulation in ringed seal heart may be an effective 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 mechanisms are strongly involved in lung protection from the potential H2O2 accumulation derived from elevated ROS generation and SOD activity after diving. In human patients, antioxidant vitamins play a very important role in maintaining lung function (Schunemann et al., 2002). Recent studies (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-Sav n 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

U GPX/ mg protein

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.

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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 peroxidation in ringed seal muscle (Zenteno-Sav n 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 contribute 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-Jime nez, 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-Sav n et al. (2002) reported low levels of lipid peroxidation in ringed seal kidney, probably due to non-enzymatic antioxidant protection. 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-Sav n 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-Jime nez, 2003) where this enzyme participates in detoxification processes 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 (Halliwell 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 mechaU 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-Sav n and Elsner, 2000). High GST activity intervenes in the removal of peroxidation-derived products and xenobiotics, which could be important in ROS reactions and ROS-induced damage (Mart nez-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.

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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. ZentenoSav n by Instituto Nacional de Ecolog a, Me xico. We also wish to thank Dr. Jose Mar a Acosta Far as 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-Limas lab is enormously appreciated. References
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