Heat stress-induced alterations of antioxidants in the freshwater fish Channa punctata Bloch

 Sports

 2 views
of 13
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Description
The effect of elevated temperature on the antioxidants in the freshwater fish Channa punctata was investigated. Fish stressed with an elevated temperature of 12° C, range ± 1° C over the ambient temperature for 3 h showed a significant (P 
Share
Tags
Transcript
  Heat stress-induced alterations of antioxidants in thefreshwater fish  Channa punctata  Bloch M. K AUR , F. A TIF , M. A LI , H. R EHMAN AND  S. R AISUDDIN * Ecotoxicology Laboratory, Department of Medical Elementology & Toxicology,Jamia Hamdard (Hamdard University), New Delhi 110 062, India(Received 30 March 2004, Accepted 13 June 2005) The effect of elevated temperature on the antioxidants in the freshwater fish  Channa punctata was investigated. Fish stressed with an elevated temperature of 12  C, range    1  C over theambient temperature for 3 h showed a significant ( P  <  0  05–0  01) reduction in the levels of antioxidants: reduced glutathione (GSH) and non-protein thiols. Activity of glutathione reduc-tase was also reduced in all the tissues (liver, kidney and gills) after 3 h of heat stress and 24 hrecovery. Catalase (CAT) showed enhanced activity in liver in both the conditions while gillsand kidney showed a decreased CAT activity. Glutathione S-transferase (GST) activity inkidney and liver decreased significantly ( P  <  0  05–0  01) after 3 h of heat stress. At 24 h GSTactivity showed a tendency to normalize in all the tissues along with a concomitant increase inthe GSH level in the kidney. Total and protein thiols in heat stressed fish when matched withcontrols, showed significant ( P  <  0  05) reduction in the kidney only with a transient increase inliver and gills. Heat shock also induced lipid peroxidation in 3 h heat-treated and recoverygroups when compared with controls. Elevated temperature therefore resulted in tissue specificand time-dependent alterations of antioxidants in the fish. It also induced lipid peroxidation invarious tissues.  # 2005 The Fisheries Society of the British Isles Key words: antioxidants; biomarkers; heat stress; modulation; thiols. INTRODUCTION All the aerobic organisms have a well developed antioxidant system whichincludes low molecular mass free radical scavengers such as glutathione andother thiols, ascorbate, vitamin E and enzymes such as superoxide dismutase(SOD), catalase (CAT) and an array of glutathione-dependent enzymes (Benzie,2000; Suntres, 2002). An important feature of the antioxidant enzymes is thatthey can be induced by a slight oxidative stress. Severe oxidative stress, however,suppresses the activities of these enzymes due to damage and loss in the com-pensatory mechanisms (Zhang  et al  ., 2004). Antioxidants can be modulated by anumber of environmental variables (Wilhelm  et al  ., 2001; Martinez-Alvarez et al  ., 2002). Amongst these, temperature is an important variable that has asignificant influence on aquatic ectotherms. Water temperature influences *Author to whom correspondence should be addressed. Tel.:  þ 91 11 26059688 ext. 5568, fax: þ 91 11 26059663; email: sraisuddin@jamiahamdard.ac.in Journal of Fish Biology  (2005)  67,  1653–1665doi:10.1111/j.1095-8649.2005.00872.x,availableonlineathttp://www.blackwell-synergy.com 1653 #  2005TheFisheriesSocietyoftheBritishIsles  oxygen concentration, metabolism, reproduction and growth of fishes (Morgan et al  ., 1999; Fraser  et al  ., 2002; Langston  et al  ., 2002). Induction of stressresponses including those by environmental temperature variation determineswhether an organism adapts to changed conditions, and survives or suffers fromphysiological disturbances (Matthews & Berg, 1997; Peuranen  et al  ., 2003). Inthis regard, induction or suppression of antioxidants has been recognized as oneof the most important preliminary responses of organisms in stressful environ-ments (Hermes-Lima & Zenteno-Savin, 2002; Abele & Puntarulo, 2004;Bocchetti  et al  ., 2004).The environmental stress resulting from temperature variation is also reportedto modulate the antioxidants and induce production of reactive oxygen species(ROS) leading to lipid peroxidation (LPO) (Flanagan  et al  ., 1998; Altan  et al  .,2003; Guderley, 2004). Since production of ROS is linked with the response of antioxidants, some studies have been conducted in birds and mammals on themodulatory role of heat on antioxidants (Altan  et al  ., 2003; Mahmoud & Edens,2003). The study of the influence of elevated temperature as one of the environ-mental stressors on the antioxidant profile of aquatic organisms including fishesbecomes important as in the aquatic environment they are often exposed tomultiple stressors (Bocchetti  et al  ., 2004). In fishes information on this aspect isvery scanty. Therefore, the present study was undertaken to determine the effectof elevated temperature on antioxidant profile in spotted snakehead  Channa punctata  Bloch, a tropical freshwater fish. The effect of elevated temperatureon lipid peroxidation was also investigated. MATERIALS AND METHODS EXPERIMENTAL FISH The spotted snakehead is a common freshwater fish widely distributed in warm waterbodies of India and neighbouring countries (Jain & Garg, 1984). Fish were maintained inglass aquaria (60 l capacity) following standard fish maintenance procedures (Clesceri, et al  ., 1998). Each aquarium held eight fish. The range in mass was 70–80 g and in totallength ( L T ) was 15–17 cm. They were acclimatized for 15 days at ambient temperature(20  C, range    2  C) before experimental use. The water was oxygen saturated andprovided with constant illumination. The whole aquarium water was replaced with a freshvolume of water every other day to minimize contamination from the metabolic wastes. EXPERIMENTAL DESIGN The control group (group A) comprised of 16 fish maintained at ambient temperature(20  C, range    2  C). Sixteen acclimatized fish were transferred to another aquarium,which contained preheated water at 32  C (12  C above the average ambienttemperature). These fish remained exposed to the elevated temperature for 3 h. After3 h, half of these fish ( n  ¼  8) were sacrificed (group B) for measurement of variousvariables. The remaining fish ( n  ¼  8) were transferred to another aquarium containingwater at ambient temperature and were sacrificed after 24 h of recovery from the 3 hheat treatment (group C). The transfer was without any time lapse. The dissolvedoxygen (DO) content in the tank water at ambient temperature and 32  C was 7  2and 5  4 mg l  1 , respectively. LPO, CAT, glutathione S-transferase (GST), glutathionereductase (GR), reduced glutathione (GSH), total thiols (T-SH), non-protein thiols(NP-SH) and protein thiols (P-SH) were measured in liver, kidney (whole mass) and gills. 1654  M. KAUR  ET AL. #  2005 The Fisheries Society of the British Isles,  Journal of Fish Biology  2005,  67,  1653–1665  PREPARATION OF POSTMITOCHONDRIAL SUPERNATANT(PMS) Fish were dissected to remove liver, kidney and gills. Gills were excised out, gill rakersremoved and lamellae were homogenized. Tissue homogenates were prepared in chilledphosphate buffer (0  1 M, pH 7  4) using a Potter-Elvehjem homogenizer and centrifugedin a refrigerated centrifuge (Hermle, Model Z 32 K) at 10500  g  for 30 min at 4  C toobtain PMS (10%). MEASUREMENT OF LIPID PEROXIDATION Lipid peroxidation was measured by the procedure of  Uchiyama & Mihara (1978).Briefly, 0  25 ml of homogenate was mixed with 25  m l of 10 mM butylated hydroxyltoluene (BHT, Sigma, U.S.A.), 3 ml 1% orthophosphoric acid (E. Merck, India) and1 ml of 0  67% thiobarbituric acid (TBA, Sigma). The mixture was incubated at 90  C for45 min. The absorbance was measured at 535 nm. The rate of lipid peroxidation wascalculated as nmol of thiobarbituric acid reactive substance (TBARS) formed h  1 g  1 of tissue using a molar extinction coefficient of 1  56    10 5 M  1 cm  1 . MEASUREMENT OF ANTIOXIDANT ENZYMES Catalase activity was assayed by the method of Claiborne (1985). The assay mixture(3 ml) consisted of 1  95 ml phosphate buffer (0  05 M, pH 7  0), 1 ml hydrogen peroxide(0  019 M) and 0  05 ml PMS. Change in absorbance was recorded at 240 nm. CATactivity was calculated as nmol H 2 O 2  consumed min  1 mg protein  1 . The method of Habig  et al  . (1974) with some modification was used to measure the GST activity. ForGST activity measurement, the reaction mixture of 2 ml consisted of phosphate buffer(0  1 M, pH 6  5), 1 mM GSH (Sigma), 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB,Sigma) and PMS. The enzyme activity was calculated as nmol CDNB conjugateformed min  1 mg protein  1 using a molar extinction coefficient of 9  6    10 3 M  1 cm  1 . The GR activity was measured by the method of  Carlberg & Mannervik(1975). The assay mixture consisted of 1  65 ml phosphate buffer (0  1 M, pH 7  6),0  1 ml 0  5 mM ethylene diamine tetraacetatic acid (EDTA, Sigma), 0  1 ml oxidizedglutathione (GSSG, Sigma), 0  1 ml 0  1 mM nicotinamide adenine dinucleotide phos-phate reduced (NADPH, Sigma) and 0  1 ml PMS in the total volume of 2 ml. Theenzyme activity was calculated as nmol NADPH oxidized min  1 mg protein  1 using amolar extinction coefficient of 6  22    10 3 M  1 cm  1 . MEASUREMENT OF THIOLS Reduced GSH was measured in PMS according to the method of  Jollow  et al  . (1974).Sulphosalicylic acid (S.D. Fine-Chem Ltd., India) 4% in the ratio of 1 : 1 was used toprecipitate PMS. The samples were kept at 4  C for 1 h followed by centrifugation at1200  g  for 15 min at 4  C. The assay mixture consisted of supernatant, phosphate buffer(0  1 M, pH 7  4) and dithio-bis-2-nitrobenzoic acid (DTNB, stock  ¼  100 mM in 0  1 Mphosphate buffer, Sigma) in the total volume of 3 ml. The optical density of the reactionproduct was measured spectrophotometrically at 412 nm. The GSH values were calcu-lated as nmol GSH  1 g  1 tissue. The T-SH, NP-SH and P-SH in the PMS of all thetissues were measured using the method of Sedlak & Lindsay (1968). Briefly, for T-SH1  5 ml tris buffer (0  2 M, pH 8  2), 0  1 ml 0  01 M DTNB, PMS and methanol weremixed in the total volume of 10 ml. After 10 min, the mixture was centrifuged at3000  g  at 4  C for 10 min and the absorbance of the supernatant was measured at412 nm. For NP-SH, 0  4 ml PMS was precipitated with 0  1 ml 40% trichloroaceticacid (TCA) and 0  5 ml distilled water. After 10 min, the mixture was centrifuged at3000  g . The absorbance of supernatant with tris buffer (0  4 M, pH 8  9) and 0  01 M HEAT STRESS MODULATES ANTIOXIDANTS OF FISH  1655 #  2005 The Fisheries Society of the British Isles,  Journal of Fish Biology  2005,  67,  1653–1665  DTNB in the total volume of 1  5 ml was read at 412 nm. The level of P-SH wascalculated by subtracting the values of NP-SH from T-SH content. The molar extinctioncoefficient of 13000 M  1 cm  1 was used to measure the various thiols. MEASUREMENT OF PROTEIN Protein content in different preparations was estimated by the method of Lowry  et al  .(1951) using Folin’s reagent with bovine serum albumin (BSA) as protein standard. STATISTICAL ANALYSIS Significance of differences relative to controls was assessed using one-way ANOVA.The differences showing significance level of   P  <  0  05 were further tested by Dunnett’smultiple comparison  t -test to resolve the difference among group means. The values of  P  <  0  05 and  P  <  0  01 were used as significance levels. RESULTS EFFECT OF HEAT STRESS ON LIPID PEROXIDATION Heat shock treatment increased lipid peroxidation in all the tissues (Fig. 1).The increase was statistically significant ( P  <  0  01–0  05) in group B when com-pared with group A (control fish). Even after 24 h of 3 h heat shock (group C)the values were significantly higher in comparison to control fish in all the tissues( P  <  0  05–0  01). The effect was more pronounced in kidney and gills in group C 0100200300400500600700GillTissue ********* Liver Kidney   n  m  o   l   T   B   A   R   S   f  o  r  m  e   d   h  –   1     g   t   i  s  s  u  e  –   1 F IG . 1. Mean    S . E . ( n  ¼  6) lipid peroxidation values in liver, kidney and gills of   Channa punctata  ingroups A (control) ( & ), B ( & ) and C ( & ). *,  P  <  0  05 and **,  P  <  0  01, significant change whencompared with group A values. 1656  M. KAUR  ET AL. #  2005 The Fisheries Society of the British Isles,  Journal of Fish Biology  2005,  67,  1653–1665  after 24 h of heat stress. A comparison of groups B and C revealed lower LPO inliver after 24 h than after 3 h heat treatment (insignificant decrease). But in gillsand kidney LPO values remained significantly elevated in both groups. EFFECT OF HEAT STRESS ON ANTIOXIDANT ENZYMES Heat stress modulated the activity of catalase in all the tissues. While itsignificantly increased in liver (Table I,  P  <  0  05), a significant decrease wasrecorded in kidney (Table II,  P  <  0  05) and gills (Table III,  P  <  0  01). Theeffect was persistent even after 24 h. Difference of CAT activity between groupsB and C fish was not significant for any of the tissues.Glutathione S-transferase activity decreased significantly ( P  <  0  01) in liver(Table I) and kidney (Table II,  P  <  0  05) after 3 h of heat treatment whencomparison was made with control fish. In gills (Table III), however, a signifi-cant increase ( P  <  0  05) was recorded. Liver GST activity remained significantlylow ( P  <  0  05) and there was no significant difference between the values of groups B and C but in case of kidney (Table II), GST values in the 24 h recoverygroup increased significantly ( P  <  0  05) in comparison to the 3 h heat treatmentgroup.Glutathione reductase activity decreased significantly ( P  <  0  05–0  01) in allthe tissues immediately after heat stress of 3 h (Tables I to III). Even after24 h, the reduction was significant in liver (Table I,  P  <  0  05), kidney(Table II,  P  <  0  05) and gills (Table III,  P  <  0  01) when compared with T ABLE  I. Mean    S . E . ( n  ¼  6) concentrations of catalase, glutathione S-transferase, glu-tathione reductase, total thiols, non-protein thiols and protein thiols in the liver of  Channa punctata VariableGroup A(Control)Group B(3 h heat stress)Group C(24 h post-heat stress)Catalase (nmol H 2 O 2 consumed min  1 mgprotein  1 )83  0    5  2 114  7    9  7* 121  2    8  4*Glutathione S-transferase(nmol CDNB conjugatesmin  1 mg protein  1 )265  7    9  8 220  8    1  1** 228  0    13  5*Glutathione reductase(nmol NADPH oxidizedmin  1 mg protein  1 )184  0    14  4 115  7    7  8** 136  1    7  0*Total thiols ( m M SHcontent g wet tissue  1 )68  0    2  0 80  0    6  7 71  7    6  2Non-protein thiols ( m MSH content g wet tissue  1 )1  6    0  2 0  8    0  1** 0  9    0  1**Protein thiols ( m M SHcontent g wet tissue  1 )66  4    1  9 79  1    6  6 70  9    6  1 *, ( P  <  0  05) and **,  P  <  0  01, when compared with group A (control) values. HEAT STRESS MODULATES ANTIOXIDANTS OF FISH  1657 #  2005 The Fisheries Society of the British Isles,  Journal of Fish Biology  2005,  67,  1653–1665
Related Search
Similar documents
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks