DOI: 10.1111/jpn.12162
ORIGINAL ARTICLE
Effect of ellagic acid on some haematological, immunological and antioxidant parameters of rainbow trout (Oncorhynchus mykiss) € ntu € rk1 and A. Pala2 S. Misße Yonar1, M. E. Yonar1, Y. Yo 1 Department of Aquaculture, Fisheries Faculty, Firat University, Elazig, Turkey, and 2 Department of Aquaculture, Fisheries Faculty, Tunceli University, Tunceli, Turkey
Summary In this study, effect of ellagic acid on some haematological, immunological and antioxidant parameters in the blood and various tissues of rainbow trout (Oncorhynchus mykiss) were examined. Four groups of rainbow trout were fed experimental diets containing either no ellagic acid (control) or supplemented with ellagic acid at 50 mg/kg diet (EA-50), 100 mg/kg diet (EA-100) or 150 mg/kg diet (EA-150) for 21 days. Samples of the blood and tissue (liver, kidney and spleen) were collected at the end of the experiment and analysed for their haematological profile (the red blood cell count, the haemoglobin concentration and the haematocrit level), immune response (the white blood cell count, the oxidative radical production (NBT activity), the total plasma protein and total immunoglobulin level) and oxidant/antioxidant status (the malondialdehyde level, the superoxide dismutase, catalase and glutathione peroxidase activity as well as the reduced glutathione concentration). The findings of this study demonstrated that ellagic acid had a positive effect on the haematological parameters, the immune response and the antioxidant enzyme activities of the fish. Keywords rainbow trout, ellagic acid, haematology, immunity, oxidative stress Correspondence S. Misße Yonar, Department of Aquaculture, Fisheries Faculty, Firat University, 23119 Elazig, Turkey. Tel: +90 424 2370000/4560; Fax: +90 424 2386287; E-mail: [email protected] Received: 14 October 2013; accepted: 10 December 2013
Introduction Ellagic acid is a polyphenolic compound present in fruits and berries such as pomegranates, strawberries, raspberries and blackberries (Malik et al., 2011). Important biological activities, such as radical scavenging activities, chemopreventive, antimicrobial, oestrogenic/ anti-oestrogenic, anti-inflammatory, anticarcinogenic, antifibrosis, and antiviral activities have been ascribed to ellagic acid (Losso et al., 2004; Landete, 2011). It contains four hydroxyl groups and two lactone groups in which hydroxyl group is known to increase antioxidant activity in lipid peroxidation and protect cells from oxidative damage (C ß eribasßı et al., 2012). Blood is a pathophysiological reflector of the whole body, and therefore, blood parameters are important in diagnosing the structural and functional status of fish exposed to toxicants (Adhikari et al., 2004). In addition, haematological studies provide quite frequently and routinely accepted procedures in diagnosis of mammal research diseases and in aquaculture to evaluate the interactions between dietary levels of nutrients (Oliveira Ribeiro et al., 2006). 936
The use of antibiotics to treat bacterial infections has resulted in a global increase in resistance. For this reason, studies on immunostimulants are becoming more frequent. Immunostimulants comprise a group of biological or synthetic compounds that rapidly activate non-specific defence mechanisms such as vitamin combinations, trace minerals and products derived from either plants or animals that prove effective in the prevention of diseases. However, their mode of action has not been well known. Therefore, substances can be injected or mixed with the food, and results can be tested by means of experimental models with suitable animals for this purpose, such as the determination of immune parameters or challenge with pathogens (Garcia et al., 2007). Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants, the excessive accumulation of the ROS or both. Despite the potential danger of the ROS, cells have a variety of defence mechanisms to neutralise the harmful effect of free radicals (Monteiro et al., 2006). In all organisms, the main antioxidative enzymes that serve to detoxify reactive
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oxygen species are superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione S-transferase (GST) and other low molecular weight scavengers such as reduced glutathione (GSH) (Storey, 1996; Dr€ oge, 2002). The aim of this study was to determine effect of ellagic acid on levels of haematological, immunological and antioxidant parameters in rainbow trout fed with ellagic acid-supplemented diet.
The effect of ellagic acid in rainbow trout
Sample collection and preparation
Ellagic acid is hardly dissolved under natural condition. Therefore, it was dissolved in alkaline solution (0.01 M NaOH; approximately pH 12). The pH of the final solution after the addition of EA was approximately 8 (T€ urk et al., 2010). This final solution (pH 8) was added to diets. Forty fish were divided randomly into four groups and received one of four different diets prepared in the laboratory. The normal pellet diet was crushed, mixed with the final solution containing the adequate amount of ellagic acid and made again into pellets, thus obtaining diets supplemented with 50 mg (EA50), 100 mg (EA-100) or 150 mg (EA-150) ellagic acid/kg diet. The diets were reformed into pellets, spread to dry and stored at +4 °C for the feeding experiment. The remade pellets were given to the fish manually at a rate of approximately 2% fish body weight per day for 21 days. Control group received the normal pellet diet, which not contained ellagic acid. No mortality was observed during the experiment. The entire experiment was repeated two independent times; each replicate for each group contained ten fish, for a total of 80 fish. The doses of ellagic acid used in this study were selected on the basis of the previous study (Harttig et al., 1996).
At the end of the experiment, blood was collected from the caudal vein of individual fish after anaesthetisation with benzocaine. These blood samples were collected in anticoagulated (K3-EDTA) tubes for haematologic and immunological evaluation. The haematologic and immunological analyses were determined on the same day the blood samples were taken from the fish. An aliquot of the whole blood sample was used to determine the haematocrit (Ht) level, red blood cell (RBC) and white blood cell (WBC) counts, haemoglobin (Hb) concentration and oxidative radical production (NBT activity). The remaining blood was spun down at 1500 9g for 5 min at 4 °C to prepare plasma, which was used to analyse the total plasma protein (TP) and total immunoglobulin (TI) level. The RBC and WBC counts were performed using a haemocytometer and Natt and Herrick (1952) solution. The Hb concentration was determined with Drabkin’s reagent read at 540 nm (Drabkin, 1946), and the Ht level was determined by a microhaematocrit centrifugation technique. The NBT were measured with the method described by Siwicki et al. (1994). The levels of TP and TI were measured according to Siwicki et al. (1994). Immediately after the blood samples were collected, the liver, kidney and spleen were carefully removed, washed with physiological saline (0.9% NaCl) and stored at 40 °C until the biochemical assays, which were performed within 1 month after extraction. The tissue was homogenised in a teflon–glass homogeniser in buffer containing 1.15 % KCl at a 1:10 (w/v) ratio to the whole homogenate. The homogenate was centrifuged at 18000 9g at 4 °C for 30 min before the determination of the malondialdehyde (MDA) and GSH levels as well as the SOD, CAT and GSH-Px activities. Lipid peroxidation levels were measured according to the concentration of thiobarbituric acid-reactive substances (TBARs). The amount of produced MDA was used as an index of lipid peroxidation (C ß eribasßı et al., 2012). The method described by Placer et al. (1966) was used to determine the MDA levels in all tissues. The measurements were taken in accordance with the method described by Sun et al. (1988) for the determination of the tissue SOD activity. The CAT activity measurements were taken in accordance with the method described by Aebi (1983). The GSH-Px activity measurements were taken in compliance with the method described by Beutler (1975). The GSH concentration was measured by an assay using the dithionitrobenzoic acid recycling method described by Ellman (1959). The protein levels in the tissues were
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Materials and methods Chemicals and fish
Ellagic acid was purchased from Fluka (Steinheim, Germany). All other chemicals were supplied from Sigma-Aldrich Chemical (St Louis, MO, USA). Rainbow trouts (Oncorhynchus mykiss) were obtained from local fish farming (Elazig, Turkey). The fish (236.17 13.16 g) were transported to the laboratory and acclimatised in the aquariums (capacity 540 L; 80 9 75 9 90 cm) for 2 weeks at 14.2 °C, pH 7.3, with a dissolved oxygen content of 7.9 mg/l and a 12:12 light:dark photoperiod. During this period, the fish were supplied with commercial fish food twice daily. Feed preparation and experimental set-up
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The effect of ellagic acid in rainbow trout
determined by the method described by Lowry et al. (1951).
ellagic acid significantly increased the concentration of GSH and the SOD, CAT and GSH-Px activities (p < 0.05).
Statistical analysis
The results are expressed as the mean standard deviation. The statistical significance of the differences between the data obtained from the control and experimental groups was analysed via analysis of variance (one-way ANOVA) and Duncan’s test using the SPSS 21.0 computer program (IBM Corporation, Armonk, NY, USA). P-values 0.05). The groups that received ellagic acid had a significantly different WBC count, TP and TI levels compared with the control group (p < 0.05). An increase in NBT activity was also found in the groups that were administered ellagic acid, but it was insignificant (p > 0.05). The effect of ellagic acid on the lipid peroxidation and antioxidant enzyme activities are summarised in Table 2. The MDA level was significantly decreased in the liver, kidney and spleen samples of the groups that were administered ellagic acid. In addition,
Discussion The haematological parameters are widely used to evaluate the toxic stress of environmental contaminants (Saravanan et al., 2010). In addition, blood is an excellent indicator of toxic stress, and analysis of haematological parameters in fish is widely used to assess the toxic stress and functional status of the animal health. In this study, the RBC count increased significantly in the ellagic acid-treated fish. This result agrees with the previous observation by Harikrishnan et al. (2009), who significantly higher value of red blood cells existed in Carassius auratus that were treated with dietary supplementation of mixed herbal extracts-enriched diets. Furthermore, the Ht and Hb values slightly increased near control after feeding with 50, 100 and 150 mg/kg of ellagic acid-enriched diets. Our data here show that ellagic acid had a positive effect on the haematological parameters. Leucocytes play an important role in non-specific or innate immunity, and the leucocyte count/activity can indicate the health status of a fish (Secombes, 1996). The present study indicates that the WBC count was significantly higher in the groups that were treated with ellagic acid. A similar result of increased leucocyte count has been reported in kelp grouper, Epinephelus bruneus that were fed Inonotus obliquus which contains phenolic compounds, such as melanins, and lanostane-type triterpenoids for antioxidant, antitumour, antiviral, anticancer, antimicrobial, and anti-inflammatory properties and enhanced the immunity (Harikrishnan et al., 2012). The adherence/ Table 1 Some haematological and immunological values in the control and experimental groups
Groups Parameters
Control
Ht (%) Hb (g/dl) RBC (9106) WBC (9103) TP (mg/ml) TI (mg/ml) NBT (mg/ml)
33.45 6.91 1.54 33.75 26.43 11.86 1.08
EA-50 5.29a 0.86a 0.10a 4.29a 4.83a 3.22a 0.13a
35.96 7.12 1.68 39.78 32.51 15.73 1.13
EA-100
6.72a 0.93a 0.13b 5.47b 6.11b 5.20b 0.11a
34.19 7.07 1.72 41.86 33.49 15.92 1.11
EA-150 6.20a 1.10a 0.11bc 6.10b 7.24b 4.46b 0.09a
36.75 7.10 1.77 40.75 38.69 16.09 1.12
8.12a 0.91a 0.12c 5.34b 6.37c 6.33b 0.15a
Ht, haematocrit level; Hb, haemoglobin concentration; RBC, erythrocyte counts; WBC, leucocyte counts; TP, total plasma protein level; TI, total immunoglobulin level; NBT, oxidative radical production (NBT activity). EA-50: the group administered 50 mg ellagic acid; EA-100: the group administered 100 mg ellagic acid; EA-150: the group administered 150 mg ellagic acid. The groups in the same line with different letters are statistically significant (p < 0.05).
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Table 2 The MDA levels, the GSH concentrations and the SOD, CAT and GSH-Px activities in the control and experimental groups Tissues Liver
Kidney
Spleen
Groups Control EA-50 EA-100 EA-150 Control EA-50 EA-100 EA-150 Control EA-50 EA-100 EA-150
MDA (nmol/g protein) 7.20 5.83 5.49 5.68 11.83 8.15 7.89 7.55 8.01 5.36 6.51 5.43
a
2.36 1.88b 1.96b 2.13b 3.71a 4.20b 5.17b 4.39b 2.93a 2.38b 1.76c 1.44b
SOD (U/mg protein) 3.75 4.42 4.66 4.81 3.01 3.62 3.98 4.19 2.95 3.58 3.86 3.82
a
0.68 0.73b 0.60b 0.94b 0.53a 0.62b 0.38bc 0.69c 0.44a 0.73b 0.61b 0.90b
CAT (k/mg protein) 4.22 4.89 4.92 5.03 3.89 4.20 4.23 4.38 3.56 4.16 4.20 4.32
a
0.86 0.73b 0.66b 1.10b 0.78a 0.92b 0.80b 0.76b 0.72a 0.63b 0.88b 0.63b
GSH-Px (U/mg protein) 3.45 4.29 4.31 4.40 2.73 3.30 3.41 3.35 2.26 2.78 3.10 3.06
a
0.47 0.63b 0.86b 0.71b 0.69a 0.90b 0.84b 0.77b 0.52a 0.69b 0.95b 0.87b
GSH (lmol/g protein) 202.58 230.19 236.41 233.42 59.91 89.78 91.50 92.18 83.14 95.28 92.51 98.76
24.73a 22.10b 18.76b 25.49b 12.09a 15.37b 23.12b 19.10b 12.03a 10.79b 14.49b 18.71b
MDA, malondialdehyde level; SOD, superoxide dismutase activity; CAT, catalase activity; GSH-Px, glutathione peroxidase activity. EA-50: the group administered 50 mg ellagic acid; EA-100: the group administered 100 mg ellagic acid. EA-150: the group administered 150 mg ellagic acid. k: the first-order rate constant. The groups in the same column with different letters are statistically significant (p < 0.05).
NBT and respiratory burst process assay assesses the non-specific immune response and the antibacterial mechanisms of the tested substances (Ibrahem et al., 2010). The present study revealed that the administration of ellagic acid insignificantly enhanced NBT activity. Several humoural elements of the non-specific immune system are present in plasma, such as immunoglobulins, transferrin, agglutinins and precipitins (Magnad ottir, 2006). The present study revealed that the levels of TP and TI were significantly higher in the experimental groups compared with the control group. This finding agrees with a previous report by Yonar et al. (2011) with rainbow trout that were fed a diet that contained propolis, a natural polyphenolic product derived from plant resins, exhibits valuable pharmacological and biological properties, such as antibacterial, antifungal, antiviral, anti-inflammatory, antitumour, local-anaesthetic, antioxidant, immunostimulant and cytostatic effect. Consequently, ellagic acid administration stimulated specific and nonspecific immune responses. Factors that protect against lipid peroxidation, such as enzymes, carotenoids, vitamins and low molecular weight scavengers, were found in all fish tissues (Matkovics et al., 1977). The specifically adapted enzymes SOD, CAT and GSH-dependent enzymes have been detected in a number of fish species. Both oxidative responses and the antioxidant potential of fish differ according to species habitat and feeding behaviour (Radi and Matkovics, 1988; Winston and Di Giulio, 1991). The response of the antioxidant system to oxidative stress in various tissues shows differences that are due to tissue-specific antioxidant potentials
(Ahmad et al., 2000). In our study, significant differences were similarly found in the oxidative responses and antioxidant capacities of tissues. The tissue differences could be due to different rates of free radical generation, differences in susceptibility to oxidative damage or different antioxidant capacities of the tissues, but ellagic acid might have changed the oxidative sensitivity of tissues to free radicals. Oxidative damage is related to the formation of reactive oxygen species (ROS). Oxidative damage can occur when the antioxidant and detoxifying systems are deficient and not able to neutralise the active intermediates that are produced by xenobiotics and their metabolites. Lipid peroxidation is considered to be a valuable indicator of the oxidative damage of the cellular components (Ferreira et al., 2005). The enzymes SOD, CAT and GSH-Px play significant roles in vivo due to their antioxidant function, and their elevated expression and activity are indicative of oxidative stress (Lushchak, 2011). SOD is a group of metalloenzymes that are crucial antioxidants. This group of enzymes constitutes the primary defence system against the toxic effect of superoxide radicals (O2 ) in aerobic organisms (Kappus, 1985; Kohen and Nyska, 2002). CAT is an enzyme located in peroxisomes that facilitates the removal of hydrogen peroxide (H2O2), which is metabolised to molecular oxygen and water (Aebi, 1983; van der Oost et al., 2003; Yilmaz et al., 2006). GSH-Px catalyses the reduction in both H2O2 and lipid peroxides (Bruce et al., 1982; Winston and Di Giulio, 1991). In our study, administration of ellagic caused statistically significant reduction in the levels of MDA and resulted in a significant
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increase in the tissue SOD, CAT and GSH-Px activities. This status may be explained with possible enhancement of antioxidant capacity in the tissues. Glutathione is a main non-protein thiol and is a primary reductant that is present in cells (Siegers, 1989; Yilmaz et al., 2006). In this study, the GSH level was significantly increased in the groups that were administered ellagic acid. This effect of ellagic acid might be related to increased biosynthesis of GSH or increased levels of other antioxidants. References Adhikari, S.; Sarkar, B.; Chatterjee, A.; Mahapatra, C. T.; Ayyappan, S., 2004: Effects of cypermethrin and carbofuran on certain hematological parameters and prediction of their recovery in a freshwater teleost, Labeo rohita (Hamilton). Ecotoxicology and Environmental Safety 58, 220–226. Aebi, H., 1983: Catalase. In: H. U. Bergmeyer (ed.), Methods in Enzymatic Analysis. Academic Press, New York, pp. 27–86. Ahmad, I.; Hamid, T.; Fatima, M.; Chand, H. S.; Jain, S. K.; Athar, M.; Raisuddin, S., 2000: Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochimica et Biophysica Acta 1523, 37–48. Beutler, E., 1975: Glutathione peroxidase (GSH-Px). In: E. Beutler (ed.), Red Cell Metabolism. A manual of Biochemical Methods, Grune and Straton, New York, pp. 71–73. Bruce, A.; Freeman, D.; James, C., 1982: Biology of disease free radicals and tissue injury. Laboratory Investigation 47, 412–426. C urk, G.; ß eribasßı, O. A.; Sakin, F.; T€ S€ onmez, M.; Atesßßs ahin, A., 2012: Impact of ellagic acid on adriamycin induced testicular histopathological lesions apoptosis lipid peroxidation and sperm damages. Experimental and Toxicologic Pathology 64, 717–724. Drabkin, D. L., 1946: The crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. Journal of Biological Chemistry 164, 703–723. Dr€ oge, W., 2002: Free radicals in the physiological control of cell function. Physiological Reviews 82, 47–95. Ellman, G. L., 1959: Tissue sulphydryl groups. Archives of Biochemistry and Biophysics 82, 70–77.
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To summarise, the present study demonstrated that (i) ellagic acid had a positive effect on the haematological parameters, (ii) ellagic acid administration activated the specific and non-specific immune response, and (iii) ellagic acid enhanced antioxidant capacity in the tissues. Therefore, ellagic acid may be used as an immunostimulant and antioxidant in fish. However, further investigations are necessary to elucidate the potential usefulness of this compound as an immunostimulant and antioxidant in fish.
Ferreira, M.; Moradas-Ferreira, P.; ReisHenriques, M. A., 2005: Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal. Aquatic Toxicology 71, 39–48. Garcia, F.; Pilarski, F.; Onaka, M. E.; de Moraes, F. R.; Martins, M. L., 2007: Hematology of Piaractus mesopotamicus fed diets supplemented with vitamins C and E, challenged by Aeromonas hydrophila. Aquaculture 271, 39–46. Harikrishnan, R.; Balasundaram, C.; Kim, M. C.; Kim, J. S.; Han, Y. J.; Heo, M. S., 2009: Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts. Fish and Shellfish Immunology 27, 508–515. Harikrishnan, R.; Balasundaram, C.; Heo, M. S., 2012: Effect of Inonotus obliquus enriched diet on hematology, immune response, and disease protection in kelp grouper, Epinephelus bruneus against Vibrio harveyi. Aquaculture 344, 48–53. Harttig, U.; Hendricks, J. D.; Stoner, D. G.; Bailey, S. G., 1996: Organ specific, protocol dependent modulation of 7,12dimethylbenz[a]anthracene carcinogenesis in rainbow trout (Oncorhynchus mykiss) by dietary ellagic acid. Carcinogenesis 17, 2403–2409. Ibrahem, M. D.; Fathi, M.; Mesalhy, S.; Abdel-Aty, A. M., 2010: Effect of dietary supplementation of inulin and vitamin C on the growth, hematology, innate immunity, and resistance of Nile tilapia (Oreochromis niloticus). Fish and Shellfish Immunology 29, 241–246. Kappus, H., 1985: Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance. In: H. Sies (ed.), Oxidative Stress; Oxidants and Antioxidants. Academic Press, London, pp. 273–310. Kohen, R.; Nyska, A., 2002: Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions,
and methods for their quantification. Toxicologic Pathology 30, 620–650. Landete, J. M., 2011: Ellagitannins, ellagic acid and their derived metabolites: a review about source, metabolism, functions and health. Food Research International 44, 1150–1160. Losso, J. N.; Bansode, R. R.; Trappey, A. II; Bawadi, A. H.; Truax, R., 2004: In vitro anti-proliferative activities of ellagic acid. Journal of Nutritional Biochemistry 15, 672–678. Lowry, O. H.; Rosenberough, N. J.; Farr, A. L.; Randal, R. J., 1951: Protein measurement with folinphenol reagent. Journal of Biochemistry 193, 265–275. Lushchak, V. I., 2011: Environmentally induced oxidative stress in aquatic animals. Aquatic Toxicology 101, 13–30. Magnad ottir, B., 2006: Innate immunity of fish (overview). Fish and Shellfish Immunology 20, 137–151. Malik, A.; Afaq, S.; Shahid, M.; Akhtar, K.; Assiri, A., 2011: Influence of ellagic acid on prostate cancer cell proliferation: a caspase–dependent pathway. Asian Pacific Journal of Tropical Medicine 4, 550–555. Matkovics, B.; Navak, R.; Hoang Due, H.; Szabo, L.; Varga, S. I.; Zalesna, G., 1977: A comparative study of some more important experimental animal peroxide metabolism enzymes. Comparative Biochemistry and Physiology. B, Comparative Biochemistry 56, 31–34. Monteiro, D. A.; Alves de Almeida, J.; Rantin, A. F. T.; Kalinin, L., 2006: Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion). Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP 143, 141–149. Natt, M. P.; Herrick, C. A., 1952: New blood diluents for counting the
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S. Misße Yonar et al.
erythrocytes and leukocytes of the chicken. Poultry Science 31, 735–738. Oliveira Ribeiro, C. A.; Filipak Neto, F.; Mela, M.; Silva, P. H.; Randi, M. A. F.; Rabitto, I. S.; Alves Costa, J. R. M.; Pelletier, E., 2006: Hematological findings in neotropical fish Hoplias malabaricus exposed to subchronic and dietary doses of methylmercury, inorganic lead, and tributyltin chloride. Environmental Research 101, 74–80. van der Oost, R.; Beyer, J.; Vermeulen, N. P., 2003: Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environmental Toxicology and Pharmacology 13, 57–149. Placer, Z. A.; Cushman, L.; Johnson, B. C., 1966: Estimation of products of lipid peroxidation (Malonyl dialdehyde) in biological fluids. Analytical Biochemistry 16, 359–364. Radi, A. A.; Matkovics, B., 1988: Effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues. Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP 90, 69–72.
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Saravanan, M.; Kumar, K. P.; Ramesh, M., 2010: Haematological and biochemical responses of freshwater teleost fish Cyprinus carpio (Actinopterygii: Cypriniformes) during acute and sublethal exposure to lindane. Pesticide Biochemistry and Physiology 100, 206–211. Secombes, C. J., 1996: The nonspecific immune system: cellular defences. In: G. Iwama, T. Nakanish (eds), The fish Immune system, Organism, Pathogen and Environment. Academic Press, Toronto, pp. 63–103. Siegers, C. P., 1989: Glutathione and glutathione dependent enzymes. Progress in Pharmacology and Clinical Pharmacology 7, 171–180. Siwicki, A. K.; Anderson, D. P.; Dixon, O. W., 1994: Dietary intake of immunostimulants by rainbow trout affects nonspecific immunity and protection against furunculosis. Veterinary Immunology and Immunopathology 41, 125–139. Storey, K. B., 1996: Oxidative stress: animal adaptations in nature. Brazilian Journal of Medical and Biological Research 29, 1715–1733.
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Sun, Y.; Oberley, L. W.; Li, Y., 1988: A simple method for clinical assay of superoxide dismutase. Clinical Chemistry 34, 497–500. T€ urk, G.; S€ onmez, M.; C ß eribasßı, A. O.; Y€ uce, A.; Atesßßs ahin, A., 2010: Attenuation of cyclosporine A-induced testicular and spermatozoal damages associated with oxidative stress by ellagic acid. International Immunopharmacology 10, 177–182. Winston, G. W.; Di Giulio, R. T., 1991: Prooxidant and antioxidant mechanisms in aquatic organisms. Aquatic Toxicology 19, 137–161. Yilmaz, S.; Atessahin, A.; Sahna, E.; Karahan, I.; Ozer, S., 2006: Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology 218, 164–171. Yonar, M. E.; Misße Yonar, S.; Silici, S., 2011: Protective effect of propolis against oxidative stress and immunosuppression induced by oxytetracycline in rainbow trout (Oncorhynchus mykiss, W.). Fish and Shellfish Immunology 31, 318–325.
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ORIGINAL ARTICLE
Effect of ellagic acid on some haematological, immunological and antioxidant parameters of rainbow trout (Oncorhynchus mykiss) € ntu € rk1 and A. Pala2 S. Misße Yonar1, M. E. Yonar1, Y. Yo 1 Department of Aquaculture, Fisheries Faculty, Firat University, Elazig, Turkey, and 2 Department of Aquaculture, Fisheries Faculty, Tunceli University, Tunceli, Turkey
Summary In this study, effect of ellagic acid on some haematological, immunological and antioxidant parameters in the blood and various tissues of rainbow trout (Oncorhynchus mykiss) were examined. Four groups of rainbow trout were fed experimental diets containing either no ellagic acid (control) or supplemented with ellagic acid at 50 mg/kg diet (EA-50), 100 mg/kg diet (EA-100) or 150 mg/kg diet (EA-150) for 21 days. Samples of the blood and tissue (liver, kidney and spleen) were collected at the end of the experiment and analysed for their haematological profile (the red blood cell count, the haemoglobin concentration and the haematocrit level), immune response (the white blood cell count, the oxidative radical production (NBT activity), the total plasma protein and total immunoglobulin level) and oxidant/antioxidant status (the malondialdehyde level, the superoxide dismutase, catalase and glutathione peroxidase activity as well as the reduced glutathione concentration). The findings of this study demonstrated that ellagic acid had a positive effect on the haematological parameters, the immune response and the antioxidant enzyme activities of the fish. Keywords rainbow trout, ellagic acid, haematology, immunity, oxidative stress Correspondence S. Misße Yonar, Department of Aquaculture, Fisheries Faculty, Firat University, 23119 Elazig, Turkey. Tel: +90 424 2370000/4560; Fax: +90 424 2386287; E-mail: [email protected] Received: 14 October 2013; accepted: 10 December 2013
Introduction Ellagic acid is a polyphenolic compound present in fruits and berries such as pomegranates, strawberries, raspberries and blackberries (Malik et al., 2011). Important biological activities, such as radical scavenging activities, chemopreventive, antimicrobial, oestrogenic/ anti-oestrogenic, anti-inflammatory, anticarcinogenic, antifibrosis, and antiviral activities have been ascribed to ellagic acid (Losso et al., 2004; Landete, 2011). It contains four hydroxyl groups and two lactone groups in which hydroxyl group is known to increase antioxidant activity in lipid peroxidation and protect cells from oxidative damage (C ß eribasßı et al., 2012). Blood is a pathophysiological reflector of the whole body, and therefore, blood parameters are important in diagnosing the structural and functional status of fish exposed to toxicants (Adhikari et al., 2004). In addition, haematological studies provide quite frequently and routinely accepted procedures in diagnosis of mammal research diseases and in aquaculture to evaluate the interactions between dietary levels of nutrients (Oliveira Ribeiro et al., 2006). 936
The use of antibiotics to treat bacterial infections has resulted in a global increase in resistance. For this reason, studies on immunostimulants are becoming more frequent. Immunostimulants comprise a group of biological or synthetic compounds that rapidly activate non-specific defence mechanisms such as vitamin combinations, trace minerals and products derived from either plants or animals that prove effective in the prevention of diseases. However, their mode of action has not been well known. Therefore, substances can be injected or mixed with the food, and results can be tested by means of experimental models with suitable animals for this purpose, such as the determination of immune parameters or challenge with pathogens (Garcia et al., 2007). Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants, the excessive accumulation of the ROS or both. Despite the potential danger of the ROS, cells have a variety of defence mechanisms to neutralise the harmful effect of free radicals (Monteiro et al., 2006). In all organisms, the main antioxidative enzymes that serve to detoxify reactive
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oxygen species are superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), glutathione S-transferase (GST) and other low molecular weight scavengers such as reduced glutathione (GSH) (Storey, 1996; Dr€ oge, 2002). The aim of this study was to determine effect of ellagic acid on levels of haematological, immunological and antioxidant parameters in rainbow trout fed with ellagic acid-supplemented diet.
The effect of ellagic acid in rainbow trout
Sample collection and preparation
Ellagic acid is hardly dissolved under natural condition. Therefore, it was dissolved in alkaline solution (0.01 M NaOH; approximately pH 12). The pH of the final solution after the addition of EA was approximately 8 (T€ urk et al., 2010). This final solution (pH 8) was added to diets. Forty fish were divided randomly into four groups and received one of four different diets prepared in the laboratory. The normal pellet diet was crushed, mixed with the final solution containing the adequate amount of ellagic acid and made again into pellets, thus obtaining diets supplemented with 50 mg (EA50), 100 mg (EA-100) or 150 mg (EA-150) ellagic acid/kg diet. The diets were reformed into pellets, spread to dry and stored at +4 °C for the feeding experiment. The remade pellets were given to the fish manually at a rate of approximately 2% fish body weight per day for 21 days. Control group received the normal pellet diet, which not contained ellagic acid. No mortality was observed during the experiment. The entire experiment was repeated two independent times; each replicate for each group contained ten fish, for a total of 80 fish. The doses of ellagic acid used in this study were selected on the basis of the previous study (Harttig et al., 1996).
At the end of the experiment, blood was collected from the caudal vein of individual fish after anaesthetisation with benzocaine. These blood samples were collected in anticoagulated (K3-EDTA) tubes for haematologic and immunological evaluation. The haematologic and immunological analyses were determined on the same day the blood samples were taken from the fish. An aliquot of the whole blood sample was used to determine the haematocrit (Ht) level, red blood cell (RBC) and white blood cell (WBC) counts, haemoglobin (Hb) concentration and oxidative radical production (NBT activity). The remaining blood was spun down at 1500 9g for 5 min at 4 °C to prepare plasma, which was used to analyse the total plasma protein (TP) and total immunoglobulin (TI) level. The RBC and WBC counts were performed using a haemocytometer and Natt and Herrick (1952) solution. The Hb concentration was determined with Drabkin’s reagent read at 540 nm (Drabkin, 1946), and the Ht level was determined by a microhaematocrit centrifugation technique. The NBT were measured with the method described by Siwicki et al. (1994). The levels of TP and TI were measured according to Siwicki et al. (1994). Immediately after the blood samples were collected, the liver, kidney and spleen were carefully removed, washed with physiological saline (0.9% NaCl) and stored at 40 °C until the biochemical assays, which were performed within 1 month after extraction. The tissue was homogenised in a teflon–glass homogeniser in buffer containing 1.15 % KCl at a 1:10 (w/v) ratio to the whole homogenate. The homogenate was centrifuged at 18000 9g at 4 °C for 30 min before the determination of the malondialdehyde (MDA) and GSH levels as well as the SOD, CAT and GSH-Px activities. Lipid peroxidation levels were measured according to the concentration of thiobarbituric acid-reactive substances (TBARs). The amount of produced MDA was used as an index of lipid peroxidation (C ß eribasßı et al., 2012). The method described by Placer et al. (1966) was used to determine the MDA levels in all tissues. The measurements were taken in accordance with the method described by Sun et al. (1988) for the determination of the tissue SOD activity. The CAT activity measurements were taken in accordance with the method described by Aebi (1983). The GSH-Px activity measurements were taken in compliance with the method described by Beutler (1975). The GSH concentration was measured by an assay using the dithionitrobenzoic acid recycling method described by Ellman (1959). The protein levels in the tissues were
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Materials and methods Chemicals and fish
Ellagic acid was purchased from Fluka (Steinheim, Germany). All other chemicals were supplied from Sigma-Aldrich Chemical (St Louis, MO, USA). Rainbow trouts (Oncorhynchus mykiss) were obtained from local fish farming (Elazig, Turkey). The fish (236.17 13.16 g) were transported to the laboratory and acclimatised in the aquariums (capacity 540 L; 80 9 75 9 90 cm) for 2 weeks at 14.2 °C, pH 7.3, with a dissolved oxygen content of 7.9 mg/l and a 12:12 light:dark photoperiod. During this period, the fish were supplied with commercial fish food twice daily. Feed preparation and experimental set-up
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The effect of ellagic acid in rainbow trout
determined by the method described by Lowry et al. (1951).
ellagic acid significantly increased the concentration of GSH and the SOD, CAT and GSH-Px activities (p < 0.05).
Statistical analysis
The results are expressed as the mean standard deviation. The statistical significance of the differences between the data obtained from the control and experimental groups was analysed via analysis of variance (one-way ANOVA) and Duncan’s test using the SPSS 21.0 computer program (IBM Corporation, Armonk, NY, USA). P-values 0.05). The groups that received ellagic acid had a significantly different WBC count, TP and TI levels compared with the control group (p < 0.05). An increase in NBT activity was also found in the groups that were administered ellagic acid, but it was insignificant (p > 0.05). The effect of ellagic acid on the lipid peroxidation and antioxidant enzyme activities are summarised in Table 2. The MDA level was significantly decreased in the liver, kidney and spleen samples of the groups that were administered ellagic acid. In addition,
Discussion The haematological parameters are widely used to evaluate the toxic stress of environmental contaminants (Saravanan et al., 2010). In addition, blood is an excellent indicator of toxic stress, and analysis of haematological parameters in fish is widely used to assess the toxic stress and functional status of the animal health. In this study, the RBC count increased significantly in the ellagic acid-treated fish. This result agrees with the previous observation by Harikrishnan et al. (2009), who significantly higher value of red blood cells existed in Carassius auratus that were treated with dietary supplementation of mixed herbal extracts-enriched diets. Furthermore, the Ht and Hb values slightly increased near control after feeding with 50, 100 and 150 mg/kg of ellagic acid-enriched diets. Our data here show that ellagic acid had a positive effect on the haematological parameters. Leucocytes play an important role in non-specific or innate immunity, and the leucocyte count/activity can indicate the health status of a fish (Secombes, 1996). The present study indicates that the WBC count was significantly higher in the groups that were treated with ellagic acid. A similar result of increased leucocyte count has been reported in kelp grouper, Epinephelus bruneus that were fed Inonotus obliquus which contains phenolic compounds, such as melanins, and lanostane-type triterpenoids for antioxidant, antitumour, antiviral, anticancer, antimicrobial, and anti-inflammatory properties and enhanced the immunity (Harikrishnan et al., 2012). The adherence/ Table 1 Some haematological and immunological values in the control and experimental groups
Groups Parameters
Control
Ht (%) Hb (g/dl) RBC (9106) WBC (9103) TP (mg/ml) TI (mg/ml) NBT (mg/ml)
33.45 6.91 1.54 33.75 26.43 11.86 1.08
EA-50 5.29a 0.86a 0.10a 4.29a 4.83a 3.22a 0.13a
35.96 7.12 1.68 39.78 32.51 15.73 1.13
EA-100
6.72a 0.93a 0.13b 5.47b 6.11b 5.20b 0.11a
34.19 7.07 1.72 41.86 33.49 15.92 1.11
EA-150 6.20a 1.10a 0.11bc 6.10b 7.24b 4.46b 0.09a
36.75 7.10 1.77 40.75 38.69 16.09 1.12
8.12a 0.91a 0.12c 5.34b 6.37c 6.33b 0.15a
Ht, haematocrit level; Hb, haemoglobin concentration; RBC, erythrocyte counts; WBC, leucocyte counts; TP, total plasma protein level; TI, total immunoglobulin level; NBT, oxidative radical production (NBT activity). EA-50: the group administered 50 mg ellagic acid; EA-100: the group administered 100 mg ellagic acid; EA-150: the group administered 150 mg ellagic acid. The groups in the same line with different letters are statistically significant (p < 0.05).
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The effect of ellagic acid in rainbow trout
Table 2 The MDA levels, the GSH concentrations and the SOD, CAT and GSH-Px activities in the control and experimental groups Tissues Liver
Kidney
Spleen
Groups Control EA-50 EA-100 EA-150 Control EA-50 EA-100 EA-150 Control EA-50 EA-100 EA-150
MDA (nmol/g protein) 7.20 5.83 5.49 5.68 11.83 8.15 7.89 7.55 8.01 5.36 6.51 5.43
a
2.36 1.88b 1.96b 2.13b 3.71a 4.20b 5.17b 4.39b 2.93a 2.38b 1.76c 1.44b
SOD (U/mg protein) 3.75 4.42 4.66 4.81 3.01 3.62 3.98 4.19 2.95 3.58 3.86 3.82
a
0.68 0.73b 0.60b 0.94b 0.53a 0.62b 0.38bc 0.69c 0.44a 0.73b 0.61b 0.90b
CAT (k/mg protein) 4.22 4.89 4.92 5.03 3.89 4.20 4.23 4.38 3.56 4.16 4.20 4.32
a
0.86 0.73b 0.66b 1.10b 0.78a 0.92b 0.80b 0.76b 0.72a 0.63b 0.88b 0.63b
GSH-Px (U/mg protein) 3.45 4.29 4.31 4.40 2.73 3.30 3.41 3.35 2.26 2.78 3.10 3.06
a
0.47 0.63b 0.86b 0.71b 0.69a 0.90b 0.84b 0.77b 0.52a 0.69b 0.95b 0.87b
GSH (lmol/g protein) 202.58 230.19 236.41 233.42 59.91 89.78 91.50 92.18 83.14 95.28 92.51 98.76
24.73a 22.10b 18.76b 25.49b 12.09a 15.37b 23.12b 19.10b 12.03a 10.79b 14.49b 18.71b
MDA, malondialdehyde level; SOD, superoxide dismutase activity; CAT, catalase activity; GSH-Px, glutathione peroxidase activity. EA-50: the group administered 50 mg ellagic acid; EA-100: the group administered 100 mg ellagic acid. EA-150: the group administered 150 mg ellagic acid. k: the first-order rate constant. The groups in the same column with different letters are statistically significant (p < 0.05).
NBT and respiratory burst process assay assesses the non-specific immune response and the antibacterial mechanisms of the tested substances (Ibrahem et al., 2010). The present study revealed that the administration of ellagic acid insignificantly enhanced NBT activity. Several humoural elements of the non-specific immune system are present in plasma, such as immunoglobulins, transferrin, agglutinins and precipitins (Magnad ottir, 2006). The present study revealed that the levels of TP and TI were significantly higher in the experimental groups compared with the control group. This finding agrees with a previous report by Yonar et al. (2011) with rainbow trout that were fed a diet that contained propolis, a natural polyphenolic product derived from plant resins, exhibits valuable pharmacological and biological properties, such as antibacterial, antifungal, antiviral, anti-inflammatory, antitumour, local-anaesthetic, antioxidant, immunostimulant and cytostatic effect. Consequently, ellagic acid administration stimulated specific and nonspecific immune responses. Factors that protect against lipid peroxidation, such as enzymes, carotenoids, vitamins and low molecular weight scavengers, were found in all fish tissues (Matkovics et al., 1977). The specifically adapted enzymes SOD, CAT and GSH-dependent enzymes have been detected in a number of fish species. Both oxidative responses and the antioxidant potential of fish differ according to species habitat and feeding behaviour (Radi and Matkovics, 1988; Winston and Di Giulio, 1991). The response of the antioxidant system to oxidative stress in various tissues shows differences that are due to tissue-specific antioxidant potentials
(Ahmad et al., 2000). In our study, significant differences were similarly found in the oxidative responses and antioxidant capacities of tissues. The tissue differences could be due to different rates of free radical generation, differences in susceptibility to oxidative damage or different antioxidant capacities of the tissues, but ellagic acid might have changed the oxidative sensitivity of tissues to free radicals. Oxidative damage is related to the formation of reactive oxygen species (ROS). Oxidative damage can occur when the antioxidant and detoxifying systems are deficient and not able to neutralise the active intermediates that are produced by xenobiotics and their metabolites. Lipid peroxidation is considered to be a valuable indicator of the oxidative damage of the cellular components (Ferreira et al., 2005). The enzymes SOD, CAT and GSH-Px play significant roles in vivo due to their antioxidant function, and their elevated expression and activity are indicative of oxidative stress (Lushchak, 2011). SOD is a group of metalloenzymes that are crucial antioxidants. This group of enzymes constitutes the primary defence system against the toxic effect of superoxide radicals (O2 ) in aerobic organisms (Kappus, 1985; Kohen and Nyska, 2002). CAT is an enzyme located in peroxisomes that facilitates the removal of hydrogen peroxide (H2O2), which is metabolised to molecular oxygen and water (Aebi, 1983; van der Oost et al., 2003; Yilmaz et al., 2006). GSH-Px catalyses the reduction in both H2O2 and lipid peroxides (Bruce et al., 1982; Winston and Di Giulio, 1991). In our study, administration of ellagic caused statistically significant reduction in the levels of MDA and resulted in a significant
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increase in the tissue SOD, CAT and GSH-Px activities. This status may be explained with possible enhancement of antioxidant capacity in the tissues. Glutathione is a main non-protein thiol and is a primary reductant that is present in cells (Siegers, 1989; Yilmaz et al., 2006). In this study, the GSH level was significantly increased in the groups that were administered ellagic acid. This effect of ellagic acid might be related to increased biosynthesis of GSH or increased levels of other antioxidants. References Adhikari, S.; Sarkar, B.; Chatterjee, A.; Mahapatra, C. T.; Ayyappan, S., 2004: Effects of cypermethrin and carbofuran on certain hematological parameters and prediction of their recovery in a freshwater teleost, Labeo rohita (Hamilton). Ecotoxicology and Environmental Safety 58, 220–226. Aebi, H., 1983: Catalase. In: H. U. Bergmeyer (ed.), Methods in Enzymatic Analysis. Academic Press, New York, pp. 27–86. Ahmad, I.; Hamid, T.; Fatima, M.; Chand, H. S.; Jain, S. K.; Athar, M.; Raisuddin, S., 2000: Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochimica et Biophysica Acta 1523, 37–48. Beutler, E., 1975: Glutathione peroxidase (GSH-Px). In: E. Beutler (ed.), Red Cell Metabolism. A manual of Biochemical Methods, Grune and Straton, New York, pp. 71–73. Bruce, A.; Freeman, D.; James, C., 1982: Biology of disease free radicals and tissue injury. Laboratory Investigation 47, 412–426. C urk, G.; ß eribasßı, O. A.; Sakin, F.; T€ S€ onmez, M.; Atesßßs ahin, A., 2012: Impact of ellagic acid on adriamycin induced testicular histopathological lesions apoptosis lipid peroxidation and sperm damages. Experimental and Toxicologic Pathology 64, 717–724. Drabkin, D. L., 1946: The crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. Journal of Biological Chemistry 164, 703–723. Dr€ oge, W., 2002: Free radicals in the physiological control of cell function. Physiological Reviews 82, 47–95. Ellman, G. L., 1959: Tissue sulphydryl groups. Archives of Biochemistry and Biophysics 82, 70–77.
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To summarise, the present study demonstrated that (i) ellagic acid had a positive effect on the haematological parameters, (ii) ellagic acid administration activated the specific and non-specific immune response, and (iii) ellagic acid enhanced antioxidant capacity in the tissues. Therefore, ellagic acid may be used as an immunostimulant and antioxidant in fish. However, further investigations are necessary to elucidate the potential usefulness of this compound as an immunostimulant and antioxidant in fish.
Ferreira, M.; Moradas-Ferreira, P.; ReisHenriques, M. A., 2005: Oxidative stress biomarkers in two resident species, mullet (Mugil cephalus) and flounder (Platichthys flesus), from a polluted site in River Douro Estuary, Portugal. Aquatic Toxicology 71, 39–48. Garcia, F.; Pilarski, F.; Onaka, M. E.; de Moraes, F. R.; Martins, M. L., 2007: Hematology of Piaractus mesopotamicus fed diets supplemented with vitamins C and E, challenged by Aeromonas hydrophila. Aquaculture 271, 39–46. Harikrishnan, R.; Balasundaram, C.; Kim, M. C.; Kim, J. S.; Han, Y. J.; Heo, M. S., 2009: Innate immune response and disease resistance in Carassius auratus by triherbal solvent extracts. Fish and Shellfish Immunology 27, 508–515. Harikrishnan, R.; Balasundaram, C.; Heo, M. S., 2012: Effect of Inonotus obliquus enriched diet on hematology, immune response, and disease protection in kelp grouper, Epinephelus bruneus against Vibrio harveyi. Aquaculture 344, 48–53. Harttig, U.; Hendricks, J. D.; Stoner, D. G.; Bailey, S. G., 1996: Organ specific, protocol dependent modulation of 7,12dimethylbenz[a]anthracene carcinogenesis in rainbow trout (Oncorhynchus mykiss) by dietary ellagic acid. Carcinogenesis 17, 2403–2409. Ibrahem, M. D.; Fathi, M.; Mesalhy, S.; Abdel-Aty, A. M., 2010: Effect of dietary supplementation of inulin and vitamin C on the growth, hematology, innate immunity, and resistance of Nile tilapia (Oreochromis niloticus). Fish and Shellfish Immunology 29, 241–246. Kappus, H., 1985: Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance. In: H. Sies (ed.), Oxidative Stress; Oxidants and Antioxidants. Academic Press, London, pp. 273–310. Kohen, R.; Nyska, A., 2002: Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions,
and methods for their quantification. Toxicologic Pathology 30, 620–650. Landete, J. M., 2011: Ellagitannins, ellagic acid and their derived metabolites: a review about source, metabolism, functions and health. Food Research International 44, 1150–1160. Losso, J. N.; Bansode, R. R.; Trappey, A. II; Bawadi, A. H.; Truax, R., 2004: In vitro anti-proliferative activities of ellagic acid. Journal of Nutritional Biochemistry 15, 672–678. Lowry, O. H.; Rosenberough, N. J.; Farr, A. L.; Randal, R. J., 1951: Protein measurement with folinphenol reagent. Journal of Biochemistry 193, 265–275. Lushchak, V. I., 2011: Environmentally induced oxidative stress in aquatic animals. Aquatic Toxicology 101, 13–30. Magnad ottir, B., 2006: Innate immunity of fish (overview). Fish and Shellfish Immunology 20, 137–151. Malik, A.; Afaq, S.; Shahid, M.; Akhtar, K.; Assiri, A., 2011: Influence of ellagic acid on prostate cancer cell proliferation: a caspase–dependent pathway. Asian Pacific Journal of Tropical Medicine 4, 550–555. Matkovics, B.; Navak, R.; Hoang Due, H.; Szabo, L.; Varga, S. I.; Zalesna, G., 1977: A comparative study of some more important experimental animal peroxide metabolism enzymes. Comparative Biochemistry and Physiology. B, Comparative Biochemistry 56, 31–34. Monteiro, D. A.; Alves de Almeida, J.; Rantin, A. F. T.; Kalinin, L., 2006: Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion). Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP 143, 141–149. Natt, M. P.; Herrick, C. A., 1952: New blood diluents for counting the
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S. Misße Yonar et al.
erythrocytes and leukocytes of the chicken. Poultry Science 31, 735–738. Oliveira Ribeiro, C. A.; Filipak Neto, F.; Mela, M.; Silva, P. H.; Randi, M. A. F.; Rabitto, I. S.; Alves Costa, J. R. M.; Pelletier, E., 2006: Hematological findings in neotropical fish Hoplias malabaricus exposed to subchronic and dietary doses of methylmercury, inorganic lead, and tributyltin chloride. Environmental Research 101, 74–80. van der Oost, R.; Beyer, J.; Vermeulen, N. P., 2003: Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environmental Toxicology and Pharmacology 13, 57–149. Placer, Z. A.; Cushman, L.; Johnson, B. C., 1966: Estimation of products of lipid peroxidation (Malonyl dialdehyde) in biological fluids. Analytical Biochemistry 16, 359–364. Radi, A. A.; Matkovics, B., 1988: Effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues. Comparative Biochemistry and Physiology. Toxicology & Pharmacology : CBP 90, 69–72.
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Saravanan, M.; Kumar, K. P.; Ramesh, M., 2010: Haematological and biochemical responses of freshwater teleost fish Cyprinus carpio (Actinopterygii: Cypriniformes) during acute and sublethal exposure to lindane. Pesticide Biochemistry and Physiology 100, 206–211. Secombes, C. J., 1996: The nonspecific immune system: cellular defences. In: G. Iwama, T. Nakanish (eds), The fish Immune system, Organism, Pathogen and Environment. Academic Press, Toronto, pp. 63–103. Siegers, C. P., 1989: Glutathione and glutathione dependent enzymes. Progress in Pharmacology and Clinical Pharmacology 7, 171–180. Siwicki, A. K.; Anderson, D. P.; Dixon, O. W., 1994: Dietary intake of immunostimulants by rainbow trout affects nonspecific immunity and protection against furunculosis. Veterinary Immunology and Immunopathology 41, 125–139. Storey, K. B., 1996: Oxidative stress: animal adaptations in nature. Brazilian Journal of Medical and Biological Research 29, 1715–1733.
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Sun, Y.; Oberley, L. W.; Li, Y., 1988: A simple method for clinical assay of superoxide dismutase. Clinical Chemistry 34, 497–500. T€ urk, G.; S€ onmez, M.; C ß eribasßı, A. O.; Y€ uce, A.; Atesßßs ahin, A., 2010: Attenuation of cyclosporine A-induced testicular and spermatozoal damages associated with oxidative stress by ellagic acid. International Immunopharmacology 10, 177–182. Winston, G. W.; Di Giulio, R. T., 1991: Prooxidant and antioxidant mechanisms in aquatic organisms. Aquatic Toxicology 19, 137–161. Yilmaz, S.; Atessahin, A.; Sahna, E.; Karahan, I.; Ozer, S., 2006: Protective effect of lycopene on adriamycin-induced cardiotoxicity and nephrotoxicity. Toxicology 218, 164–171. Yonar, M. E.; Misße Yonar, S.; Silici, S., 2011: Protective effect of propolis against oxidative stress and immunosuppression induced by oxytetracycline in rainbow trout (Oncorhynchus mykiss, W.). Fish and Shellfish Immunology 31, 318–325.
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