Effect of temperature acclimation on the liver antioxidant defence system of the Antarctic...

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Comparative Biochemistry and Physiology, Part B xxx (2014) xxx–xxx

CBB-09782; No of Pages 8

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part B

j ourna l homepage: www.e lsev ie r .com/ locate /cbpb

Effect of temperature acclimation on the liver antioxidant defence systemof the Antarctic nototheniids Notothenia coriiceps and Notothenia rossii

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FCintia Machado a, Tania Zaleski a, Edson Rodrigues b, Cleoni dos Santos Carvalho c,Silvia Maria Suter Correia Cadena d, Gustavo Jabor Gozzi d, Priscila Krebsbach a,Flávia Sant'Anna Rios a, Lucélia Donatti a,⁎a Adaptive Biology Laboratory, Department of Cell Biology, Federal University of Parana, Curitiba, Paraná, Brazilb Basic Bioscience Institute, Taubaté University, Taubaté, São Paulo, Brazilc Department of Biology, Federal University of Sao Carlos, Sorocaba, Sao Paulo, Brazild Department of Biochemistry, Federal University of Paraná, Curitiba, Paraná, Brazil

⁎ Corresponding author at: Universidade Federal do PaBiologia Celular, Av. Cel. Francisco H. dos Santos, s/n, Jard19031, CEP: 81531-970, Curitiba, PR, Brazil. Tel.: +55 411682.

E-mail address: [email protected] (L. Donatti).

http://dx.doi.org/10.1016/j.cbpb.2014.02.0031096-4959/© 2014 Published by Elsevier Inc.

Please cite this article as: Machado, C., etnototheniids Notothenia coriiceps and Nototh

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Article history:Received 18 November 2013Received in revised form 25 February 2014Accepted 27 February 2014Available online xxxx

Keywords:Antarctic fishThermal acclimationOxidative stress

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CTED The aim of this study was to determine whether endemic Antarctic nototheniid fish are able to adjust their liver

antioxidant defence system in response to the temperature increase. The activity of the superoxide dismutase(SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase(GR) enzymes as well as the content of non-enzymatic oxidative stress markers such as reduced glutathione(GSH), lipid peroxidation (LPO) and protein carbonyl (PC) were measured in the liver of two Antarctic fish spe-cies, Notothenia rossii and Notothenia coriiceps after 1, 3 and 6 days of exposure to temperatures of 0 °C and 8 °C.The GST activity showed a downregulation inN. rossii after 6 days of exposure to the increased temperature. Theactivity profiles of GST and GR in N. rossii and of GPx in N. coriiceps also changed as a consequence of heating to8 °C. The GSH content increased by heating to 8 °C after 3 days inN. coriiceps and after 6 days inN. rossii. The con-tent of malondialdehyde (MDA), a LPO marker, showed a negative modulation by the heating to 8 °C in N. rossiiafter 3 days of exposure to temperatures. Present results show that heating to 8 °C influenced the levels and pro-files of the antioxidant enzymes and defences over time in the nototheniid fish N. rossii and N. coriiceps.

© 2014 Published by Elsevier Inc.
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1. Introduction

Temperature is an abiotic factor that influencesmany biological pro-cesses and can determine and delimit the geographic distribution of liv-ing organisms (Windisch et al., 2011). In fish, the temperature canfunction as a stressing agent (Wedemeyer et al., 1990), and the meta-bolic rate of an ectothermic organism is strongly related to environmen-tal temperature (Clarke and Johnston, 1996; Clarke and Fraser, 2004).Studies indicate that the thermal tolerance of an organism is closely re-lated to its aerobic ability because both cooling and heating may alteroxygen balance in the tissues, promoting the generation of reactive ox-ygen species (ROS). These oxygen species react with lipids and nucleicacids, causing oxidative stress and damage to the cells (Abele et al.,1998; Stadtman and Levine, 2000; Martínez-Álvarez et al., 2005;

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raná (UFPR), Departamento deim das Américas, Caixa Postal:3361 1752; fax: +55 41 3361

al., Effect of temperature acenia rossii, Comp. Biochem. P

Halliwell and Gutteridge, 2007). To prevent the increase of ROS levelscells use their antioxidant defences (Abele et al., 1998, 2001; Martínez-Álvarez et al., 2005; Heise et al., 2006),which can be either enzymatic ornon-enzymatic (Sies, 1985; Martínez-Álvarez et al., 2005; Yilmaz et al.,2006).

The Southern Ocean is considered to be the coldest and most ther-mally stable marine environment on Earth. Sea temperature remainsconstant at approximately −1.9 °C in the areas close to the continentand can vary from +1.5 °C in summer to −1.8 °C in the winter in theAntarctic Peninsula and islandsnorth of it (Sidell, 2000). The current en-vironmental pressure on Antarctica (Clarke et al., 2007; Hellmer et al.,2012; Ross et al., 2012) indicates that the water temperature of theAustral Ocean has increased approximately 0.2 °C since 1950 at depthsof 700 to 1100m, between 35 and 65°S. Studies show that this increasein temperature extends to superficial waters (King, 1994; King andHarangozo, 1998; Renwick, 2002; Turner et al., 2005).

The fish that inhabit the Southern Ocean have developed adaptationmechanisms to extremely low temperatures that are responsible fortheir survival in this environment (Crossley, 1995; Somero et al.,1996; Chen et al., 1997; Jin and DeVries, 2006; diPrisco et al., 2007;Brodte et al., 2008; Crockett, 2011). Attributed to the higher oxygen

climation on the liver antioxidant defence system of the Antarctichysiol. B (2014), http://dx.doi.org/10.1016/j.cbpb.2014.02.003

http://dx.doi.org/10.1016/j.cbpb.2014.02.003

mailto:[email protected]


http://www.sciencedirect.com/science/journal/10964959


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solubility in cold sea water, a high concentration of oxygen in tissues ofpolar fish and consequently high levels of ROS can be expected (Ansaldoet al., 2000; Abele and Puntarulo, 2004). Fish species in the Antarctic, thehigh concentration of solutes in the aqueous cytosol, and increased sur-face area and density of mitochondria may facilitate the adaptation ofthese animals to such changes in tissue oxygen conductance influencedby low temperatures (Sidell andHazel, 1987; Sidell, 1998; Guderley andSt-Pierre, 2002; Abele and Puntarulo, 2004). Although the polar animalsare adapted to cold and balanced environment in relation to antioxidantprocesses resulting in this adaptation, these animals are sensitive to ox-idative stress caused by other stressors such as increased temperature,intoxication radiation, starvation, etc. (Abele and Puntarulo, 2004;Abele, 2012).

The acclimation and thermal tolerance of Antarctic organisms tohigh temperatures have been studied, and studies on this topic are in-creasing due to global climate change (Somero and DeVries, 1967;Brauer et al., 2005; Lannig et al., 2005; Lowe and Davison, 2005; Jinand DeVries, 2006; Clarke et al., 2007; Franklin et al., 2007; Brodteet al., 2008; Robinson and Davison, 2008; Clark and Peck, 2009; Bilykand DeVries, 2011; Windisch et al., 2011; Jayasundara et al., 2013).The activity and expression of antioxidant enzymes and other oxidativestressmarkers have been used for the evaluation of Antarctic organismssubjected to thermal stress. Mueller et al. (2012) tested the hypoth-esis that the lower thermal tolerance of icefishes from the familyChannichthyidae (white-blooded) compared with those from thefamily Nototheniidae (red-blooded) (Bilyk and DeVries, 2011) maystem from a larger vulnerability of the white-blooded fish to oxida-tive damage as a result of the exposure to a maximum critical tempera-ture (CTMax). Thorne et al., 2010 evaluated, among other markers, theexpression profiles of some antioxidant enzymes in the liver ofHarpagiferantarcticus (the Antarctic plunderfish) exposed to a temperature of 6 °Cin 48 h and no differences were observed in gene regulation of antioxi-dants “classics”.

The present study aimed at investigating whether the hepatic tissueof the nototheniids (Notothenia rossii and Notothenia coriiceps) adaptsits oxidative metabolism and its antioxidant defence in response to anincrease of environmental temperature despite the fish live in a coldand thermally stable environment. These two species are phylogeneti-cally very close (Near and Cheng, 2008) and found at the same placesin many Antarctic regions (Bellisio and Tomo, 1974), but they have dif-ferent geographical distributions (Gon and Heemstra, 1990) and are,therefore, subjected to different environmental temperatures.

2. Materials and methods

The experiments were performed at the Brazilian Antarctica StationComandante Ferraz (EACF) located at Admiralty Bay (61°S and 63°30′Sand 53°55′W and 62°50′W), King George Island, in the South ShetlandIslands, Antarctic Peninsula. The environmental licences were grantedby the Environmental Assessment Group of the BrazilianMinistry of En-vironment. The experiments were approved by the Ethics Committee ofthe Federal University of Paraná (UFPR) under the number 496.

2.1. Temperature bioassays

N. coriiceps (n=60; TL=35±3 cm;W=775±182 g) andN. rossii(n= 60; TL= 37± 3 cm;W=791± 145 g) were collectedwith hookand line at 10–25 m depth. After collection, fish were maintained for3 days, according to Ryan (1995), in 1000-L tankswith controlled abiot-ic conditions, a salinity of 35 ± 1.0, a temperature of 0 ± 0.5 °C and aphotoperiod of 12 h of light and 12 h of dark. The fish were fed withepaxial muscle every two days, alternating with the change of waterin the tanks.

For each species, the fish were randomly selected and transferredto 1000-L tanks containing sea water with a temperature of 8 ± 0.5 °C(experimental) for 1, 3 or 6 days or 0 ± 0.5 °C (control) for 1, 3 or

Please cite this article as: Machado, C., et al., Effect of temperature acnototheniids Notothenia coriiceps and Notothenia rossii, Comp. Biochem. P

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6 days. Ten individuals were used for each experiment. The watertemperature of the tanks was rigorously maintained with Aquatherm09-01T-11457 thermostats (Full Gauge) coupled to heaters (Altman).

The temperature of 0 °C was chosen as the control and is withinthe thermal variation range found in the areas close to the AntarcticPeninsula and South Shetland Islands (Sidell, 2000), where the fishwere captured. The experimental temperature of 8 °C approaches thethermal tolerance of Antarctic fish as reported by Somero and DeVries(1967), Pörtner et al. (2004), Lowe and Davison (2005), Franklin et al.(2007) and Strobel et al. (2013).

Behavioural observations were made during the experiments basedon previous studies of Antarctic fishes (Fanta et al., 2001a, 2001b; Fantaet al., 2002; Donatti and Fanta, 2007, 2002). Qualitatively, themotility ofanimals (resting or swimming), the agonistic interactions betweenspecimens and food consumption were observed. N. rossii andN. coriiceps survived the times (1, 3 and 6 days) and temperatures(0 °C and 8 °C). Higher swimming speed and greater competition forfood in N. rossiiwere observed to 8 °C.

After the bioassays were performed, the animals were anesthetisedwith 1% (p/v) benzocaine and killed by spinal transection. Subsequent-ly, liver sampleswere removed, frozen in liquid nitrogen and stored in afreezer at−80 °C.

2.2. Analytical methods

The liver samples from N. coriiceps and N. rossii were homogenisedin phosphate-buffered saline (PBS), pH 7.2, and then centrifuged at12,000 g at 4 °C for 20min. The supernatantwas separated for the deter-mination of protein concentration, the activity of antioxidant enzymesand the non-enzymatic marker levels.

Total protein concentrationwas determined according to the Bradfordmethod (1976), using bovine serum albumin (BSA) as the standard. Theabsorbance of the samples was measured at 545 nm.

2.3. Assay of antioxidant enzyme activities

CAT (EC 1.11.1.6) activitywas evaluated bymeasuring the consump-tion of hydrogen peroxide using a spectrophotometer at 240 nm(Beutler, 1975). The reaction medium contained potassium phosphatebuffer (50 mM, pH 7.0) and 1.0 mg/mL of protein. The reaction was ini-tiated by the addition of 10 mM H2O2 and monitored for 60 s at 25 °C.

The activity of SOD (EC 1.15.1.1) was determined according to themethodology described by Crouch et al. (1981), which is based on theability of this enzyme to inhibit the reduction of nitroblue tetrazolium(NBT) into blue formazan by the O2

− generated by the hydroxylaminein the alkaline solution. The reaction system was maintained at 25 °Cand consisted of NBT solution (100 μM) and hydroxylamine in alkalinesolution (36.85 mM). This inhibition was spectrophotometrically mea-sured at 560 nm.

The activity of GR (EC 1.8.1.7)was evaluated spectrophotometricallybymeasuring the oxidation of NADPH and the concomitant reduction ofglutathione disulphide (GSSG) at 340 nm (Carlberg and Mannervik,1985). The reaction medium contained potassium phosphate buffer(100 mM, pH 7.0), 5.0 mM EDTA, 0.5 mM NADPH and 5 mM GSSG.The reaction was started by the addition of GSSG and monitored for10 min.

The activity of GPx (EC 1.11.1.9) was determined by measuring theoxidation of NADPH at 340 nm. The enzyme uses GSH to reduce organicperoxide, resulting in GSSG. GSSG is reduced by the enzyme GR usingelectrons donated by NADPH (Wendel, 1981). The reaction systemconsisted of 100 mM phosphate buffer (pH 7.0), 2 mM reduced gluta-thione (GSH), 0.2 mM NADPH, 0.2 U of purified glutathione reductase(GR), 0.5 mM t-butyl hydroperoxide and 1 mg/mL of protein. The reac-tion occurred at 25 °C and was initiated by the addition of 0.5 mM hy-drogen peroxide and monitored for 3 min at 340 nm.



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The activity of GST (EC 2.5.1.18) was determined according to themethodology of Keen et al. (1976) using 1-chloro-2,4-dinitrobenzene(CDNB) as a substrate. The glutathionylation of CDNB was measured ina spectrophotometer at 340 nm. The final reaction contained 100 mMNa-phosphate buffer (pH 6.5), 1mMGSHand 1.5mMCDNB (in ethanol).GST activitywas triggered by the addition of CDNB, and the appearance ofthe thioether product was followed at 340 nm.

The antioxidant enzyme activitieswere expressed as units (U)/mgofprotein. One unit of SOD activity is defined as the amount of enzymethat inhibits the NBT oxidation reaction by 50% of the maximal inhibi-tion. One unit of CAT, GPx, GST and GR activity is defined as the amountof enzyme consuming 1 μmol of substrate or generating 1 μmol of prod-uct per minute.

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2.4. Indices of oxidative stress

The concentration of GSH and other thiols was determined using themethod described by Sedlak and Lindsay (1968). Thismethod is based onthe precipitation of proteins and subsequent reaction of non-proteinthiols with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), resulting in aproduct that absorbs light at 415 nm. The GSH values are expressed asnmol of thiols per mg of protein−1.

The LPO indexwas evaluated by LOOH levels and by the levels of theend products of lipid peroxidation, including malondialdehyde (MDA),by the TBARS reaction. The LOOH content was measured by the FOXmethod, described by Jiang et al. (1991),which is based on theoxidationof Fe2+ mediated by peroxides under acidic conditions and the forma-tion of the Fe3+-xylenol orange complex in the presence of the stabiliserbutylated hydroxytoluene, which absorbs light at 560 nm. The MDAlevels were determined in a spectrophotometer at 535 nm bythe TBARS reaction, using malondialdehyde-(bis)-acetate (Merck,Darmstadt, Germany) as a standard (Uchiyama and Mihara, 1978).LOOH and TBARS values are expressed as nmol of hydroperoxides permg of protein−1 and nmol of MDA/g wet mass (gwm), respectively.

The concentration of oxidative damage in proteins (PC) was deter-mined by the measurement of carbonyl groups as described by Levine

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Fig. 1. Activity of superoxide dismutase (SOD) and catalase (CAT) in the liver of Notothenia corifilled square, respectively) for 1, 3 and 6 days. The vertical lines indicatemeans± SEM. Differen(0 °C and 8 °C).


et al. (1990), in which the obtained samples were precipitated and dis-solved with dinitrophenylhydrazine. The carbonyl groups were mea-sured by absorbance at 362 nm. The results are expressed as nmol ofcarbonyls per mg of protein−1.

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2.5. Data analysis

The data were previously tested for normality (Kolmogorov–Smirnov test) and homogeneity of variances (Levene's test). The enzy-matic activities of CAT, SOD, GR, GPx and GST and the GSH, LOOH,MDA and CP levels of the control groupwere compared with the exper-imental groups using a bifactorial analysis of variance (two-wayANOVA), in which the exposure time (1, 3 or 6 days) and the tempera-ture (0 or 8 °C) were the categorical variables. Next, the Tukey test wasperformed for the comparison of averages. The data are shown as themean ± standard error of the mean (SEM), and the differences wereconsidered significant at P b 0.05.

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3.1. Antioxidant enzyme activities

The increase in temperature to 8 °C did not affect the SOD activity inthe two species analysed (Fig. 1A and B). However, differences in theSOD activity between the species were observed over the exposuretime. In N. coriiceps, the SOD activity decreased in the groups exposedfor 3 or 6 days in comparisonwith the groups exposed for 1 day to either0 °C or 8 °C (Fig. 1A). In contrast, SODactivity inN. rossiiwas higher after3 days compared with the groups exposed for 1 or 6 days (Fig. 1B).

Catalase activity was not affected by the increase in temperature to8 °C in both species (Fig. 1C and D). The CAT activity was similar forboth species throughout the exposure time. In the control groups(0 °C), the CAT activity significantly decreased after 3 and 6 days oftreatment, whereas a decrease in CAT activity in the experimentalgroups (8 °C) was observed at 6 days.

iceps (A and C) and Notothenia rossii (B and D) exposed to 0 °C and 8 °C (empty circle andt lowercase letters indicate differences between the exposure times at a given temperature



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Fig. 2. Activity of glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) in the liver of Notothenia coriiceps (A, C and E) and Nototheniarossii (B, D and F) exposed to 0 °C and 8 °C (empty circle and filled square, respectively) for 1, 3 and 6 days. The vertical lines indicate means ± SEM. Different lowercase letters indicate dif-ferences between the exposure times at a given temperature (0 °C and8 °C). Asterisks indicate significant differences (P≤ 0.05) between the temperatures (0 °C and8 °C) at a given timepoint.

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The increase in temperature to 8 °C did not change the GST activityin N. coriiceps compared with fish exposed to 0 °C (Fig. 2A). In contrast,after 6 days of exposure to 8 °C, the GST activity in N. rossii showed anegative modulation compared with fish maintained at 0 °C (Fig. 2B).An increase in GST levels in N. rossiiwas observed in the control groups(0 °C) after 3 and 6 days of exposure; this increase was not observed inthe experimental groups (8 °C) (Fig. 2B).

GR activity did not change by increasing of temperature to 8 °C inboth species (Fig. 2C and D). However, the activity of GR was differentfor the species over the exposure time. In N. coriiceps, the obtainedvalues were not dependent on the exposure times and temperatures

Table 1Significance levels of the two-way ANOVA testing effects of temperature (TE) (0 and 8 °C) and t(SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathi(MDA) and protein carbonylation (PC).

SOD CAT GST GR G

F p F p F p F p F

Temperature 0.55 0.46 0.085 0.77 0.004 0.95 0.087 0.77Time 90.16 b0.001* 18.13 b0.001* 2.39 0.10 0.27 0.76 4TE × TI 3.69 0.03* 4.22 0.020* 2.49 0.09 0.58 0.56


tested (Table 1). In contrast, the GR activity in N. rossii did not changewithin the control groups, but the values were decreased in the experi-mental groups after 3 and 6 days of exposure (Fig. 2D). (See Table 2.)

The increase in temperature to 8 °C did not alter GPx activity com-pared with fish maintained at 0 °C in the two species analysed (Fig. 2Eand F). However, the GPx activity was different for the species overthe exposure times. In N. coriiceps, an increase in GPx was observedafter 6 days at 8 °C, whereas the levels did not vary throughout expo-sure to 0 °C water (Fig. 2E). In N. rossii, a slight decrease in GPx activityin the experimental and control groupswas observed after 3 days of ex-posure. A subsequent increase in GPx levels occurred after 6 days,

ime of exposure (TI) (1, 3 and 6 days) of theNotothenia coriiceps for superoxide dismutaseone reductase (GR) reduced glutathione (GSH), lipid peroxides (LOOH), malondialdehyde

Px GSH LOOH MDA PC

p F p F p F p F p

0.15 0.70 9.36 b0.001* 8.03 0.001* 0.69 0.41 3.86 0.007*3.04 b0.001* 0.14 0.71 0.01 0.91 5.6 0.007* 1.01 0.323.66 0.033* 0.69 0.51 0.09 0.91 6.6 0.003* 2.32 0.11



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t2:1 Table 2t2:2 Significance levels of the two-way ANOVA testing effects of temperature (TE) (0 and 8 °C) and time of exposure (TI) (1, 3 and 6 days) of the s for superoxide dismutase (SOD), catalaset2:3 (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) reduced glutathione (GSH), lipid peroxides (LOOH), malondialdehyde (MDA) and pro-t2:4 tein carbonylation (PC).

SOD CAT GST GR GPx GSH LOOH MDA PCt2:5

F p F p F p F p F p F p F p F p F pt2:6

Temperature 1.58 0.21 13.44 b0.001* 5.92 b0.018* 1.01 0.32 0.30 0.58 6.35 0.01* 0.16 0.69 0.68 0.42 0.06 0.81t2:7

Time 60.14 b0.001* 23.76 b0.001* 9.90 b0.001* 11.19 b0.001* 17.12 b0.001* 37.92 b0.001* 2.25 0.11 5.70 0.006* 0.65 0.52t2:8

TE × TI 0.075 0.93 0.22 0.81 1.31 0.28 2.25 0.11 1.16 0.32 4.74 0.013* 0.23 0.79 6.65 0.003* 0.09 0.91t2:9

Fig. 3.Reduced glutathione (GSH),malondialdehyde (MDA), lipid peroxides (LOOH), and protein carbonylation (PC) in the liver ofNotothenia coriiceps (A, C, E and G) andNotothenia rossii(B, D, F and H) exposed to 0 °C and 8 °C (empty circle and filled square, respectively) for 1, 3 and 6 days. The vertical lines indicate means± SEM. Different lowercase letters indicate dif-ferences between the exposure times at a given temperature (0 °C and 8 °C).


Please cite this article as: Machado, C., et al., Effect of temperature acclimation on the liver antioxidant defence system of the Antarcticnototheniids Notothenia coriiceps and Notothenia rossii, Comp. Biochem. Physiol. B (2014), http://dx.doi.org/10.1016/j.cbpb.2014.02.003


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returning GPx levels to those observed in groupswith 1 day of exposurefor both temperatures tested (Fig. 2F).

3.2. Indices of oxidative stress

GSH levels increased in the experimental group after 3 days com-pared to 1 and 6 days, however, the GSH levels at 0 °C were constantthroughout the exposure times (Fig. 3A). In N. rossii the temperatureof 8 °C produced an increase in GSH levels when compared with thoseobserved at 0 °C. These levels were significantly higher in the experi-mental groups than in the control groups after 6 days of exposure. Inthis species the GSH levels significantly decreased after 6 days at bothtemperatures studied compared with the levels observed after 1 and3 days of exposure (Fig. 3B).

The increase in temperature to 8 °C did not affect MDA levels inN. coriiceps compared with fish maintained at 0 °C and also did notchange with exposure time (Fig. 3C). TheMDA content showed a nega-tivemodulation after the increase in temperature to 8 °C inN. rossii after3 days. MDA levels increased only after 3 days of exposure, which didnot occur in the experimental groups (8 °C) (Fig. 3D).

Significant differences were not observed in LOOH and PC levels atthe different exposure times or temperatures tested for both species(Figs. 3E, F, G and H).

4. Discussion

The activity and expression of antioxidant enzymes have been usedto evaluate the response of Antarctic organisms under thermal stress(Park et al., 2008a,2008b;Mueller et al., 2012). Indeed, some studies in-dicate that an increase in the environmental temperature increases theconsumption of oxygen, which in turn promotes the generation of ROS(Abele et al., 1998, 2001). In this context, we testedwhether an increasein environmental temperature affects the activities of enzymes involvedin oxidative metabolism and antioxidant defences in the liver of twoAntarctic nototheniids, N. rossii and N. coriiceps, that are adapted to astable and low water temperature conditions.

SOD and CAT are important enzymes for oxidative metabolism. SODcatalyses the dismutation of superoxide ion into oxygen and hydrogenperoxide that will then be decomposed into water and oxygen by CAT(Halliwell and Gutteridge, 2007). Some SOD isoforms have been wellcharacterised in fish, including Antarctic fish (Cassini et al., 1993; Kenet al., 2003; Lesser, 2006; Santovito et al., 2006), and it is thought thatthese proteins evolved to maintain catalytic function over a wide rangeof temperatures (Cassini et al., 1993; Abele and Puntarulo, 2004; Lesser,2006). In the present study, the increase in temperature from 0 °Cto 8 °C did not alter SOD and CAT activities in the two speciesanalysed. This finding may be due to the organ chosen, in this case theliver, and to the fact that other organsmay be involved in antioxidant de-fence in the species analysed. Mueller et al. (2012) also did not observechanges in CAT activity in the heart and muscle tissues of Antarcticfish, includingN. coriiceps, in response to exposure to their CTMax. How-ever, a decrease in SOD transcription levels in icefish (white-blooded)and Gobionotothen gibberifrons (red-blooded) hearts was observed, sug-gesting that these organisms may have a reduced ability to upregulateantioxidant levels in response to thermal stress. In N. rossii collected innature, SOD values were found to be approximately 200 times higherin the blood than in the liver (Ansaldo et al., 2000). Studies haveshown that erythrocytes have mechanisms responsible for defenceagainst the formation of superoxide anions as a consequence of the highconcentration of oxygen that is transported by these cells (Holeton,1970; Cassini et al., 1993).

The SOD activity in N. coriiceps and the CAT activity in both speciesstudied decreased over time, indicating an inhibition or decrease inexpression of these enzymes. In N. rossii, the SOD activity was higherafter 3 days than after 1 or 6 days of exposure. The change in activityof these enzymes has been observed in fish exposed to increasing


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temperature, but at times less than 24 h (Lushchak and Bagnyukova,2006; Bagnyukova et al., 2007).

The GSH-dependent antioxidant enzymes (GPx, GR and GST) arealso markers used to distinguish between oxidative and thermal stressin tropical and temperate fish (Heise et al., 2006; Bagnyukova et al.,2007; Leggatt et al., 2007; Grim et al., 2013). However, the behaviourof this antioxidant system in response to an increase in environmentaltemperature in Antarctic fish has not been reported in the literature.These markers have been evaluated in Antarctic fish under other stressconditions, such as exposure to pesticides (Ghosh et al., 2013) andheavy metals (Benedetti et al., 2007). GPx acts to consume H2O2 andthen transforms reduced glutathione (GSH) into oxidised glutathione(GSSG) (Wendel, 1981). In turn, GR acts by regenerating reduced gluta-thione (Halliwell and Gutteridge, 2007). The general function of theGSTenzymes is the detoxication of electrophile compounds by glutathioneconjugation (Halliwell and Gutteridge, 2007). In the present study, theincrease in temperature to 8 °C produced a downregulation of GST ac-tivity compared with fish at 0 °C only in N. rossii. However, the increasein temperature did not change the GR and GPx activities in the two spe-cies studied. Accordingly, the levels of the GSH-dependent enzymes indifferent tissues of temperate fish under thermal stresswere also not af-fected by an increase in temperature (Lushchak and Bagnyukova, 2006;Leggatt et al., 2007; Grim et al., 2013).

InN. coriiceps, only GPx activity changed over time in response to theincrease in temperature. An increase in the activity levels was observedafter 6 days in the experimental group (8 °C). In N. rossii, an increase inGST activity was observed in the control groups (0 °C) over time,whereas a decrease in GR activity was observed in the experimentalgroups (8 °C) over time.

The GSH content present in cells is used as a non-enzymatic markerof oxidative stress (Sedlak and Lindsay, 1968; Halliwell and Gutteridge,2007; Grim et al., 2013). InN. coriiceps, higher GSH levelswere observedafter 3 days at 8 °C than after 1 or 6 days, and there was no change at0 °C. In N. rossii, an increase in GSH levels was observed in response tothe increase in temperature (8 °C) compared with control fish after6 days. Leggatt et al. (2007) reported similar findings in the liver andbrain of Fundulus heteroclitus (killifish), and Lushchak and Bagnyukova(2005) reported an increase in GSH levels in the liver and muscle ofC. auratus. However, there was a significant decrease in GSH levels inN. rossii at 0 °C after 6 days of exposure. We believe that the decreaseinGSH levels over timemay be related to the increase inGST activity ob-served after 3 days in fish maintained at 0 °C, as this enzyme uses GSHto promote the association of glutathione with metabolites that are po-tentially dangerous to cells, aiding the degradation of these substances(Leaver and George, 1998). It is important to note that glutathione canneutralise ROS in its reduced form (GSH) and also plays an importantrole as a cofactor for various glutathione-dependent antioxidant en-zymes (Halliwell and Gutteridge, 2007). However, in addition to theconsumption of GSH through the aforementioned metabolic pathways,the synthesis of GSH in response to an increase in temperature shouldbe considered. Leggatt et al. (2007) reported a decrease in GSH levelsin hepatic cell lines of rainbow trout (Oncorhynchus mykiss) that wereexposed to the temperatures of 10, 18 and 26 °C over time. However,a larger decrease in GSH levels was observed at 26 °C. The decrease ob-served by Leggatt et al. (2007) is due to the use of inhibitors of GSH syn-thesis and suggests that the positive regulation found in our study maystem from an increase in the synthesis of GSH in response to the in-crease in temperature to 8 °C in N. rossii.

An increase in oxidative stress is evidenced by changes in the levelsof oxidative damage markers that can be the products of protein andlipid oxidation (Stadtman and Levine, 2000; Hermes-Lima, 2004;Halliwell and Gutteridge, 2007; Lushchak, 2011). LPO is initiated bythe reaction of a free radical with an unsaturated fatty acid and propa-gated by the formation of peroxyl radicals (Lima and Abdalla, 2001;Halliwell andGutteridge, 2007). PC levels (Shacter, 2000) are importantnon-enzymatic markers for oxidative stress.



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LOOH levels did not change over time or when fish were exposed todifferent temperatures. The FOX method used in the present study al-lows the determination of hydroperoxides (Jiang et al., 1991), whichare products of the initial oxidation of lipids and are unstable moleculesthat break down or conjugate to other molecules soon after beingformed, resulting in secondary products of lipoperoxidation (Halliwelland Gutteridge, 2007). Because LPO is a complex process, it needs tobe evaluated using different methodological approaches (Lima andAbdalla, 2001). In this work, we also determined MDA concentration,with the aim of obtainingmore consistent information about LPO levelsin response to thermal stress in Antarcticfish. TheMDA levels inN. rossiihad a negative modulation by the increase in temperature to 8 °C after3 days of exposure. The increase of MDA levels and GST activity inN. rossii after 3 days of exposure at 0 °C indicates a possible up-regulation of this enzyme by increasing the levels of MDA, since GST isinvolved in the detoxification of lipoperoxidation products (Hermes-Lima, 2004; Bagnyukova et al., 2006), supported by decreased levels ofMDA and maintaining GST activity in 6 days at 0 °C. In N. coriiceps, wedid not observe any effect of temperature or exposure time on MDAlevels.

In response to exposure to CTMax, Mueller et al. (2012) reportedincrease in MDA and PC levels in the hearts of icefish but not innototheniids. In the present study PC levels did not vary with the in-crease in temperature in the two species studied. PC level is closely re-lated to ROS generation, which occurs through irreversible oxidativemodifications; these modifications are fairly difficult to induce becausemechanisms other than the antioxidant system, such as chaperones, areused to protect cells against this damage (Dalle-Donne et al., 2003; Parket al., 2008a).

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5. Conclusions

Taking into account the increasing trend of temperature in Antarctica,especially in the Antarctic Peninsula, the need formetabolic adaptation ofAntarctic organisms in response to oscillations in temperature and theimportance of understanding the cell mechanisms bywhich the Antarcticorganisms defend themselves against the oxidative stress imposed by theincrease in temperature, we conclude that, in liver, the increase intemperature to 8 °C induced responses affecting the activity and pro-files of antioxidant enzymes and antioxidant defences over time inthe nototheniids N. rossii and N. coriiceps. Studies focused on otherorgans must be undertaken to enable a better understanding of theeffects of temperature on the antioxidant system of Antarctic fish.

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UNCOAcknowledgments

We are grateful to the following for their support: Brazilian Ministryof Environment (MMA), Brazilian Ministry of Science, Technology andInnovation (MCTI), National Council for the Development of Scientificand Technological Research (CNPq), Brazilian Federal Agency for Sup-port and Evaluation of Graduate Education (CAPES) and the Secretariatof the Inter‐ministerial Commission for the Resources of the Sea(SeCIRM). The authors would like to thank Dr Edith Susana ElisabethFanta (in memoriam), coordinator of the projects Evolution and Bio-diversity in the Antarctic: The response of life to change (EBA), andDr Yocie Yoneshigue Valentin, coordinator of the National Instituteof Antarctic Science and Technology of Environmental Research(INCT APA), for all the help and incentive given during execution ofthe present work. The study was supported by CAPES, CNPq andFAPERJ through the projects PNPD (CAPES Process Auxpe No. 2443/2011), EBA (MCTI/CNPq Process No. 52.0125/2008‐8, InternationalPolar Year), Productivity in Research granted to L. Donatti (CNPq.Process No. 305562/2009-6 and 305969/2012-9) and INCT‐APA(CNPq Process No. 574018/2008‐5, FAPERJ E‐26/170.023/2008).


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