Int. J. Aquat. Biol. (2017) 5(6): 360-369 DOI: ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology Original Article Evaluation of oxidative stress induced by cadmium and comparative antioxidant effects of Shirazi thyme (Zataria multiflora Boiss) and vitamin E in common carp (Cyprinus carpio) Mohammad Mohiseni*1, Dara Bagheri2, Mahdi Banaee1, Behzad Nematdust Haghi1 1Department of Fisheries, Natural Resources and Environmental Faculty, Behbahan Khatam Alanbia University of Technology, Behbahan, Iran. 2Department of Fisheries, Persian Gulf University, Bushehr, Iran. Article history: Received 11 September 2017 Accepted 20 November 2017 Available online 2 5 December 2017 Keywords: Fish Antioxidant system Heavy metal Medicinal plant Stress Abstract: Shirazi thyme is an active phytobiotc contains phenolic compounds and flavonoids which have strong antioxidant properties. This study was conducted to investigate the potential protective effects of Shirazi thyme compared to that of vitamin E against cadmium toxicity. Common carp juveniles (34±3 g) were divided into four groups and fed by three different diets, including commercial diet without any additive (for control and metal only group) and supplemented with either 1% ground Shirazi thyme or 100 mg/kg vitamin E. All treatments except the control were exposed to sublethal concentration of waterborne cadmium (1.5 mg/L free ion) for 15 days and liver, kidney and gill were sampled 3, 7, 10 and 15 days after the exposure. The results showed that treatment of the fish with cadmium for 15 days resulted in a significant reduction in glutathione reductase (GR), glutathione-S-transferase (GST) and catalase (CAT) and led to liver, kidney and gills dysfunction. On the other hand, the level of malondialdehyde (MDA) significantly increased during metal exposure. Supplementation of diets with Shirazi thyme and vitamin E led to a significant protection against metal exposure in different tissues. Moreover, Shirazi thyme was found to be as effective as vitamin E. The current finding can provide a useful reference for stress protective effects of thyme and its beneficial role in aquaculture. Introduction Due to industrialization, the application of heavy metals in a variety of industrial and agricultural fields has been expanded (Tan et al., 2010). Heavy metals cannot be eliminated through biological degradation, therefore, potentially can accumulate in the tissues of aquatic animals making this toxicant deleterious to the aquatic environment. Accumulation of heavy metals in the tissues may intensify the generating of reactive oxygen species (ROS) which may lead to oxidative stress resulting in lipid peroxidation, DNA damage and alteration in ion homeostasis (Kalay et al., 1999; Farombi et al., 2007). The toxicity generated by this contaminant generally involves neurotoxicity, hepatotoxicity and nephrotoxicity (Stohs and Bagchi, 1995). Cells have equipped by enzymatic systems which have an ability to convert oxidants into non- toxic molecules, thus protecting the organism from the harmful effects of the xenobiotics. Glutathione *Corresponding author: Mohammad Mohiseni DOI: https://doi.org/10.22034/ijab.v5i6.318 E-mail address: mohiseni@ut.ac.ir reductase (GR), Glutathione-S-transferase (GST), superoxide dismutase and catalase (CAT) are the main cell enzymatic defense systems (Gate et al., 1999). Cadmium is one of the most biologically toxic heavy metals with a high environmental concern (Ruangsomboon and Wongrat, 2006; Mohiseni et al., 2016). Cadmium can actively be accumulated and result in toxicity to liver, kidneys, brain and heart. Several studies showed that using free radical scavengers and antioxidants can protect the organism against cadmium toxicity (Ochi et al., 1987; Fariss, 1991; Mohiseni et al., 2017a). In this context, protective effects of some natural antioxidant including vitamins (especially E and C) are well documented (Chaurasia and Kar, 1997; Gupta et al., 2004; Cinar et al., 2010; Mekkawy et al., 2011; Harabawy and Mosleh, 2014). Phenolic compounds in phytochemicals are believed to promote optimal health relatively through 361 Int. J. Aquat. Biol. (2017) 5(6): 360-369 their antioxidant and free radical scavenging effects, thereby, protecting cellular components against free radical-induced damages. But due to their diverse chemical structures, they are likely to possess different antioxidant capacities (Hamzawy et al., 2012; Soleimany et al., 2016; Mohiseni et al., 2017b). Thyme is a phytobiotic and used since ancient times as a medicinal herb. It has strong antimicrobial and antioxidant activity due to its very high contents of thymol, p-cymene, carvacrol, eugenol and 4- allylphenol (Alçiçek, 2011; Yılmaz et al., 2012). Shirazi thyme (Zataria multiflora Boiss) is a thyme- like plant belonging to the Lamiaceae family that geographically grows in central and southern parts of Iran, Pakistan and Afghanistan (Hosseinzadeh et al., 2000). The essential oil of Shirazi thyme contains significant quantities of phenolic compounds such as thymol, carvacrol and monoterpenes, which have an antioxidant, antibacterial and antifungal activities (Ehsani et al., 2014). There are some evidences about the positive effects of thyme on growth and feed utilization in broiler (Hosseini et al., 2013; Sadek et al., 2014) and fish (Dorojan et al., 2014; Sönmez et al., 2015), but despite the strong antioxidant capacity of these components, a little information are exist about using of thyme to improve stress resistance in fish (Antache et al., 2014). Due to the presence of active ingredients, medicinal herbs possess antioxidant properties and can be used as anti-stress in aquaculture. This will reduce the cost and side effects of synthetic or chemical products. They are also eco-friendly compounds and hence will not affect the environment. Although various medicinal plants contain numerous antioxidant compounds, however, a few of them have been investigated so far. Therefore, this study was conducted to evaluate the potential protective effects of Shirazi thyme compared to vitamin E (a strong and well-known natural antioxidant) against cadmium exposure in juvenile common carp based on oxidative stress responses. Materials and Methods Diet preparation: Three experimental diets were prepared and used for feeding trial during the experiment. A commercial diet (Table 1) for carp (Naghshin Kermanshah, Iran) was milled by feed producer, feed additives were added, mixed thoroughly and finally, the diets re-pelletized with kitchen grinder using 3mm die (Montero et al., 1999). For control and metal only (MO), milled commercial diet re-pelletized without any supplementation whereas in diets 2 and 3 the commercial diet was supplemented with either 10 g/kg dry feed ground Shirazi thyme (T) (Yılmaz et al., 2013) or 100 mg/kg of dry feed vitamin E (E) (Kaushik, 1995; Ortuño et al., 2001). They were placed through natural air flow and after drying were kept in the refrigerator (4°C). Acclimation condition and experimental design: In autumn 2014, 180 healthy juveniles of common carp (average weight of 34±3 g) obtained from Persian fish hatchery (Ahvaz, Iran) and transferred to the Khatam Alanbia, University of Technology (Behbahan, Iran). Two weeks before the experiment, juveniles were randomly divided into four groups (in triplicates) and transferred to the separate 300-L tanks, each containing 15 juveniles and individually equipped with air stone and heater (25±2°C, pH 7.3). The oxygen level was monitored daily (8±1.5 mg/l) and the fish were kept under photoperiod cycle of 12L:12D. Initially, all fish were fed 3% of biomass per day with the commercial diet. Water exchanging during both acclimation and the whole experiment periods was done daily at the rate of 30%. After the acclimation period, all groups were fed only by their own specially-prepared diets. Except for control, all experimental groups were exposed to the sublethal waterborne concentration of cadmium (1.5 mg/L free ion) for 15 days (Vinodhini and Narayanan, 2009). Table 1. Proximate composition of experimental diet (Naghshin, Kermanshah, Iran). Chemical analysis Proportion (%) Crude Protein 32 Crude Lipid 5.5 Crude ash 10 Carbohydrate 47-49 Crude fiber 4 Total Phosphor 1.2 362 Mohiseni et al./ Shirazi thyme, a natural antioxidant in fish feed Sampling was done on 3, 7, 10 and 15 days after cadmium exposure. 3 fish were caught from each tank (9 fish per treatment) at each sampling time. The fish were sacrificed by spinal cord dislocation, then they were dissected and liver, kidney and gills were removed and washed in an ice-cold KCl solution (1.15%). The samples were homogenized in 5 volumes of 100 mM phosphate buffer (pH=7.4). Homogenate was centrifuged at 12000×g for 15 min at 4°C. The supernatant was separated and stored at - 70°C until antioxidant analysis (Oliveira et al., 2010). Enzyme activity and MDA assay: GR activity was determined using reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxidized glutathione (GSSG) as substrates by a technique described by Cohen Duvel (1988). GST activity was evaluated according to Habig et al. (1974) using 1chloro 2,4 dinitrobenzene as substrate. CAT activity was measured spectrophotometrically based on H2O2 decomposition as substrate (Aebi, 1984). Total protein content of aliquots was measured according to the Bradford method (Kruger, 1994). Lipid peroxidation assay was carried out in terms of MDA by measuring the thiobarbituric acid reacting substances (TBARS) and expressed as nmol MDA/g wet weight (Ringwood et al., 2003). Statistical analysis: All data were statistically analyzed by one-way analysis of variance (ANOVA). Turkey’s test was used to evaluate the mean difference among experimental groups (P<0.05). Statistical analyses were performed using IBM SPSS Statistics for Windows (Version 19). The data presented as mean±SE. Results The specific activity of liver GR, GST and CAT, and MDA levels are presented in Figure 1. The results showed that cadmium exposure induced GR activity at the 7th day of exposure approximately in all treatments but after 15 days of exposure, the lowest and highest enzyme activity were recorded in the MO and T groups, respectively. Although the GST in the Figure 1. Effect of experimental diets on enzyme activity and lipid peroxidation in liver of common carp during different times of metal exposure. Different letters shows significant differences between groups (P<0.05). 363 Int. J. Aquat. Biol. (2017) 5(6): 360-369 MO group showed the highest activity at the 3rd day, the enzyme level decreased by time and reached the lowest level at the end of the experiment. The T and E treatments had a significant higher GST activities compared to the control and MO groups. CAT showed slight changes in all treatments during the experiment, however, the enzyme activity for the MO decreased significantly compared to the other treatments (except for the E) at the end of the experiment (the 15th day). A significant elevation in MDA level was obtained for the MO group at all sampling times; however, there were not significant differences between the control and the other treatments. The increase in MDA level in the MO group was more than 200% compared to the control in the most cases. Despite the cadmium exposure, the levels of MDA in the T and E groups did not exceed the normal range (P>0.05). Except for 7th day that the MO group showed the highest activity, kidney GR significantly decreased compared to the control and T groups at the 10th and both 10th and 15th days after cadmium exposure, respectively (Fig. 2). Cadmium led to a significant elevation in kidney GST activity at the 3rd, 7th and 10th day in all the metal exposed groups. Although the GST levels for the T and E groups tended to remain in normal range, the enzyme activity significantly decreased in the MO group at the final sampling time. The same pattern was observed in CAT at the 15th day of cadmium exposure, where the lowest enzyme activity was observed in the MO group (P<0.05), although, the CAT activity remained unchanged at the previous sampling times (3rd, 7th and 10th). Based on lipid peroxidation analysis, thyme and vitamin E kept the kidney MDA levels within the normal range in the cadmium-exposed fish. On the other hand, the MO group had the highest levels of kidney MDA during the experiment. Figure 3 shows the gills enzyme activity and lipid peroxidation at different times of the metal exposure. There was some slight fluctuations in gill GR activity in all cadmium exposed fish during the initial times post exposure (3rd, 7th and 10th), but at the final Figure 2. Effect of experimental diets on enzyme activity and lipid peroxidation in kidney of common carp during different times of metal exposure. Different letters shows significant differences between groups (P<0.05). 364 Mohiseni et al./ Shirazi thyme, a natural antioxidant in fish feed sampling, the highest and lowest enzyme activity were recorded for the T and MO groups, respectively. Similar trends were obtained about GST approximately. Shirazi thyme seems to have more ameliorating effects than vitamin E on the gill CAT activity and the highest enzyme activities were related to the T group at 10th and 15th days. Similar to the MDA levels in liver and kidney (Figs. 1, 2), the gill MDA levels of the MO group were significantly higher than that of the control at the most times (3rd, 10th and 15th), while the rate of gill lipid peroxidation in the T and E groups was not exceeded the control levels (P>0.05). Discussion Cadmium is a toxic metal that can promote an oxidative stress and because of its long retention in vital tissues (such as liver and kidney), subsequently contributes to the development of serious pathological effects (El-Demerdash et al., 2004). The present study has clearly demonstrated the capability of cadmium to induce oxidative stress in different tissues of common carp. Based on the results, although GR, GST and CAT activities initially tend to increase after cadmium exposure, finally the most enzyme activity showed a clear reduction in the MO group in all experimental tissues. Although the mechanism of the cadmium- induced oxidative stress is still not completely clarified, some hypotheses have been proposed. Cadmium potentially can inhibit the mitochondrial electron-transfer chain reaction and leading to the accumulation of semiubiquinones. This unstable component would transfer one electron to molecular oxygen to form superoxide anion, the high energy free radical. It has also suggested that cadmium could directly weaken some antioxidant enzymes including CAT, GR and superoxide dismutase. This two phenomenon will intensify the cadmium toxicity and leading to oxidative stress (Cinar et al., 2010). Figure 3. Effect of experimental diets on enzyme activity and lipid peroxidation in gill of common carp during different times of metal exposure. Different letters shows significant differences between groups (P<0.05). 365 Int. J. Aquat. Biol. (2017) 5(6): 360-369 Although the CAT activities showed significant decrease in the MO group at 10th and 15th days, the significant differences were only recorded at the latter day for liver and kidney. The iron deficiency may explain the dissimilar CAT activities in different tissues. Jurczuk et al. (2004) stated that cadmium has a potential effects to impair the intestinal absorption of iron which is required for the CAT activity. As the fish were challenged by waterborne cadmium in the current study, the higher recorded fluctuation in gill CAT activity for the MO group may be related to their direct exposure to the metal. Several investigations have shown that the formation of oxygen free radicals or ROS increased as a result of metal exposure (Stohs and Bagchi, 1995; Almeida et al., 2002; Tan et al., 2010). ROS can elicit widespread damage to cell components mainly as lipid peroxidation of membrane lipids (Almeida et al., 2002). The cell’s antioxidant system in response will increase the rate of antioxidant enzymes production to cope with the stressful condition. When the metal concentration or exposing duration increased and overcame the capacity of the natural detoxifying system, enzyme activities will eventually decrease and several detrimental effects may occur (Stohs and Bagchi, 1995; Sevcikova et al., 2011). In agreement with our results, the potentially harmful effects of heavy metals in cell antioxidant depletion has been previously reported in several investigations (Stohs and Bagchi, 1995; Romeo et al., 2000; El-Demerdash et al., 2004; Farombi et al., 2007; Tan et al., 2010; Sevcikova et al., 2011). Our data on cadmium exposure in fish have shown that the rate of lipid peroxidation (MDA) in the MO group was increased in different tissues which directly results in free radical-mediated toxicity. Lipid peroxidation is one of the main indicators of oxidative damage incidence and has been found to have an important role in toxicity and carcinogenicity (El- Nekeety et al., 2011). The rate of lipid peroxidation is followed by the balance between the production of ROS and the elimination potential of those radicals by an antioxidant (Sayeed et al., 2003). The increased level of MDA along with the decreased activity of GR, GST and CAT in the MO group may be attributed to the free radical formation that initiated chain reaction of bond formation with vital macromolecules (such as nucleic acid, protein, lipids and carbohydrates), impairing crucial cellular process that may disrupt the normal metabolism of the cells and ultimately organs (El-Nekeety et al., 2011). Similar to the vitamin E, Shirazi thyme was found to highly potentiate against cadmium toxicity. Based on the results, Shirazi thyme stimulated antioxidant enzyme activities after cadmium exposure. It can be implied that the antioxidant compounds in Shirazi thyme may enhance or reserved the existing antioxidant system. The suggested role of thyme compounds in the prevention of cadmium toxicity can be explained by their ability in radical scavenging (Miura et al., 2002) and also the promotion of antioxidant enzyme activities (Sengül et al., 2008). Flavonoids and phenolic compounds are the main components in many medicinal herbs (Kandaswami and Middleton, 1994; Rice-Evans et al., 1997). These biologically active chemicals are thought to promote optimal health via their antioxidant and radical scavenging properties, thereby keeping cellular organelles from induced damage by free radicals (Ündeğer et al., 2009). Shirazi thyme is mainly composed of monoterpenes and aromatic compounds that have antibacterial, antiviral and antifungal activities. The plants essential oil also contains considerable amounts of phenolic compounds, mainly including carvacrol, thymol and р-cymene (Kavoosi et al., 2012). The antioxidant activity of these compounds has been previously reported in several investigations (Miura et al., 2002; Ündeğer et al., 2009; Kavoosi et al., 2012; Sajed et al., 2013). These antioxidants are able to protect or amend the liver health and function in unfavorable environmental conditions and therefore resulted in improved metabolism and better animal growth performance. Our data on common carp support conclusions of researchers who attributed physiological protecting effects of thyme in animal diets. Accordingly, Hassan Barakat (2008) was found that thyme pretreatment in mice leads protection against nickel toxicity more 366 Mohiseni et al./ Shirazi thyme, a natural antioxidant in fish feed effective than basil. El-Nekeety et al. (2011) also reported that the essential oil of common thyme (Thymus vulgaris) has potential antioxidant activities and may induce protective effects against aflatoxicosis in the male rat in dose-dependent manner. The same results on hepatorenoprotective effects of common thyme against aflatoxicosis in the rat have been previously reported (Hamzawy et al., 2012). Vitamin E has also shown inhibitory properties over harmful effects of cadmium. Considering the lipid solubility of vitamin E, the biological function of the compound is interpreted by many reports as support for prevention of lipid peroxidation (McCay, 1985; Chow, 1991; Harabawy and Mosleh, 2014). Although this is considered as a primary role of vitamin E, however, there is some evidence that scavenging of lipid-based radicals may not be attributed the only form of its activity (Chow, 1991). The oxidation of some amino acid-based radicals including radicals of tryptophan, tyrosine, methionine and histidine is also decreased in the presence of this antioxidant (McCay, 1985). Supporting with this documentation, we also found the efficient role of vitamin E against lipid peroxidation as there was not a significant increase in liver, kidney and gill MDA levels in vitamin E treated group. Generally, the current results revealed that cadmium induced stressful effects on liver, kidney and gills function. By inducing antioxidant enzymes activities and reduction in lipid peroxidation of the tissues, Shirazi thyme (10 mg/kg diet) showed high protective effects as those observed for vitamin E (100 mg/kg diet) in common carp juveniles. Generally, the current finding can provide a useful reference for stress protective effects of thyme and its beneficial role in aquaculture. References Aebi H. (1984). Catalase in vitro. Methods in enzymology, 105: 121-126. Alçiçek Z. (2011). The effects of thyme (Thymus vulgaris L.) oil concentration on liquid-smoked vacuum-packed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) fillets during chilled storage. Food chemistry, 128(3): 683-688. Almeida J., Diniz Y., Marques S., Faine L., Ribas B., Burneiko R., Novelli E. (2002). The use of the oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environment International, 27(8): 673- 679. Antache A., Cristea V., Grecu I., Creţu M. (2014). The synergistic influence of thymus vulgaris and vitamin E on growth performance and oxidative stress at Oreochromis niloticus species. Seria Zootehnie, 62: 85- 90. Chaurasia S.S., Kar A. (1997). Protective effects of vitamin E against lead-induced deterioration of membrane associated type-I iodothyronine 5′- monodeiodinase (5′ DI) activity in male mice. Toxicology, 124(3): 203-209. Chow C.K. (1991). Vitamin E and oxidative stress. Free Radical Biology and Medicine, 11(2): 215-232. Cinar M., Yigit A., Eraslan G. (2010). Effects of vitamin C or vitamin E supplementation on cadmium induced oxidative stress and anaemia in broilers. Revue de Médecine Vétérinaire, 161(10): 449-454. Cohen M.B., Duvel D.L. (1988). Characterization of the inhibition of glutathione reductase and the recovery of enzyme activity in exponentially growing murine leukemia (11210) cells treated with 1, 3-bis (2- chloroethyl) 1-nitrosourea. Biochemical Pharmacology, 37(17): 3317-3320. Dorojan O.G., Placinta S., Petrea S. (2014). The influence of some phytobiotics (thyme, seabuckthorn) on growth performance of stellate sturgeon (A. stellatus, Pallas, 1771) in an industrial recirculating aquaculture system. Scientific Papers Animal Science and Biotechnologies, 47(1): 205-210. Ehsani A., Jasour M.S., Hashemi M., Mehryar L., Khodayari M. (2014). Zataria multiflora Boiss essential oil and sodium acetate: how they affect shelf life of vacuum‐packaged trout burgers. International Journal of Food Science and Technology, 49(4): 1055-1062. El-Demerdash F.M., Yousef M.I., Kedwany F.S., Baghdadi H.H. (2004). Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and β-carotene. Food and chemical Toxicology, 42(10): 1563-1571. El-Nekeety A.A., Mohamed S.R., Hathout A.S., Hassan N. S., Aly S.E., Abdel-Wahhab M.A. (2011). Antioxidant 367 Int. J. Aquat. Biol. (2017) 5(6): 360-369 properties of Thymus vulgaris oil against aflatoxin- induce oxidative stress in male rats. Toxicon, 57(7): 984-991. Fariss M.W. (1991). Cadmium toxicity: unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes. Toxicology, 69(1): 63-77. Farombi E., Adelowo O., Ajimoko Y. (2007). Biomarkers of oxidative stress and heavy metal levels as indicators of environmental pollution in African cat fish (Clarias gariepinus) from Nigeria Ogun River. International Journal of Environmental Research and Public Health, 4(2): 158-165. Gate L., Paul J., Ba G.N., Tew K., Tapiero H. (1999). Oxidative stress induced in pathologies: the role of antioxidants. Biomedicine and Pharmacotherapy, 53(4): 169-180. Gupta R.S., Gupta E.S., Dhakal B.K., Thakur A.R., Ahnn J. (2004). Vitamin C and vitamin Е protect the rat testes from cadmium-induced reactive oxygen species. Molecules and Cells, 17(1): 132-139. Habig W.H., Pabst M.J., Jakoby W.B. (1974). Glutathione S-transferases the first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry, 249(22): 7130-7139. Hamzawy M.A., El-Denshary E., Hassan N.S., Mannaa F., Abdel-Wahhab M.A. (2012). Antioxidant and hepatorenoprotective effect of thyme vulgaris extract in rats during aflatoxicosis. Global Journal Pharmacology, 6(2): 106-117. Harabawy A.S., Mosleh Y.Y. (2014). The role of vitamins A, C, E and selenium as antioxidants against genotoxicity and cytotoxicity of cadmium, copper, lead and zinc on erythrocytes of Nile tilapia, Oreochromis niloticus. Ecotoxicology and Environmental Safety, 104: 28-35. Hassan A., Barakat I. (2008). Assessment of oxidative stress induced by nickel chloride and antioxidant effects of Basil (Ocimum basilicum L.) and Thyme (Tymus vulgaris L.). Journal of Genetic Engineering and Biotechnology, 6: 29-38. Hosseini S.A., Meimandipour A., Alami F., Mahdavi A., Mohiti-Asli M., Lotfollahian H., Cross D. (2013). Effects of ground thyme and probiotic supplements in diets on broiler performance, blood biochemistry and immunological response to sheep red blood cells. Italian Journal of Animal Science, 12(1): 116-120. Hosseinzadeh H., Ramezani M., Salmani G.-A. (2000). Antinociceptive, anti-inflammatory and acute toxicity effects of Zataria multiflora Boiss extracts in mice and rats. Journal of Ethnopharmacology, 73(3): 379-385. Jurczuk M., Brzóska M.M., Moniuszko-Jakoniuk J., Gałażyn-Sidorczuk M., Kulikowska-Karpińska E. (2004). Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol. Food and Chemical Toxicology, 42(3): 429-438. Kalay M., Ay Ö., Canli M. (1999). Heavy metal concentrations in fish tissues from the Northeast Mediterranean Sea. Bulletin of Environmental Contamination and Toxicology, 63(5): 673-681. Kandaswami C., Middleton E. (1994). Free Radicals in Diagnostic Medicine. In: D. Armstrong (Ed.). Free Radicals in Diagnostic Medicine. New York: Springer pp: 351-376. Kaushik S. (1995). Nutrient requirements, supply and utilization in the context of carp culture. Aquaculture, 129(1): 225-241. Kavoosi G., Teixeira da Silva J.A., Saharkhiz M. J. (2012). Inhibitory effects of Zataria multiflora essential oil and its main components on nitric oxide and hydrogen peroxide production in lipopolysaccharide‐stimulated macrophages. Journal of Pharmacy and Pharmacology, 64(10): 1491-1500. Kruger N.J. (1994). The Bradford method for protein quantitation. Basic protein and peptide protocols: 9-15. McCay P.B. (1985). Vitamin E: interactions with free radicals and ascorbate. Annual review of nutrition, 5(1): 323-340. Mekkawy I.A., Mahmoud U.M., Wassif E.T., Naguib M. (2011). Effects of cadmium on some haematological and biochemical characteristics of Oreochromis niloticus (Linnaeus, 1758) dietary supplemented with tomato paste and vitamin E. Fish physiology and biochemistry, 37(1): 71-84. Miura K., Kikuzaki H., Nakatani N. (2002). Antioxidant activity of chemical components from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) measured by the oil stability index method. Journal of Agricultural and Food chemistry, 50(7): 1845-1851. Mohiseni M., Asayesh S., Shafiee Bazarnoie S., Mohseni F., Moradi N., Matouri M., Mirzaee N. (2016). Biochemical alteration induced by cadmium and lead in common carp via an experimental food chain. Iranian Journal of Toxicology, 10(4): 25-32. Mohiseni M., Farhangi M., Agh N., Mirvaghefi A., Talebi K. (2017a). Toxicity and Bioconcentration of Cadmium 368 Mohiseni et al./ Shirazi thyme, a natural antioxidant in fish feed and Copper in Artemia Urmiana Nauplii. Iranian Journal of Toxicology, 11(1): 33-41. Mohiseni M., Sepidnameh M., Bagheri, D., Banaee, M., Nematdust Haghi, B. (2017b). Comparative effects of Shirazi thyme and vitamin E on some growth and plasma biochemical changes in common carp (Cyprinus carpio) during cadmium exposure. Aquaculture Research, 48(9): 4811-4821. Montero D., Marrero M., Izquierdo M., Robaina L., Vergara J., Tort L. (1999). Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress. Aquaculture, 171(3): 269-278. Ochi T., Takahashi K., Ohsawa M. (1987). Indirect evidence for the induction of a prooxidant state by cadmium chloride in cultured mammalian cells and a possible mechanism for the induction. Mutation Research, 180(2): 257-266. Oliveira M., Ahmad I., Maria V., Pacheco M., Santos M. (2010). Antioxidant responses versus DNA damage and lipid peroxidation in golden grey mullet liver: a field study at Ria de Aveiro (Portugal). Archives of Environmental Contamination and Toxicology, 59(3): 454-463. Ortuño J., Cuesta A., Esteban M.A., Meseguer J. (2001). Effect of oral administration of high vitamin C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system. Veterinary Immunology and Immunopathology, 79(3): 167-180. Rice-Evans C., Miller N., Paganga G. (1997). Antioxidant properties of phenolic compounds. Trends in Plant Science, 2(4): 152-159. Ringwood A.H., Hoguet J., Keppler C., Gielazyn M., Ward B., Rourk A. (2003). Cellular biomarkers (lysosomal destabilization, glutathione & lipid peroxidation) in three common estuarine species: a methods handbook. South Carolina: Marine Resources Research Institute, South Carolina Department of Natural Resources Charleston. Romeo M., Bennani N., Gnassia-Barelli M., Lafaurie M., Girard J. (2000). Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax. Aquatic Toxicology, 48(2): 185-194. Ruangsomboon S., Wongrat L. (2006). Bioaccumulation of cadmium in an experimental aquatic food chain involving phytoplankton (Chlorella vulgaris), zooplankton (Moina macrocopa), and the predatory catfish Clarias macrocephalus× C. gariepinus. Aquatic Toxicology, 78(1): 15-20. Sadek K., Ahmed H., Ayoub M., Elsabagh M. (2014). Evaluation of Digestarom and thyme as phytogenic feed additives for broiler chickens. European Poultry Science, 78: 1-12. Sajed, H., Sahebkar, A., Iranshahi, M. (2013). Zataria multiflora Boiss (Shirazi thyme) - an ancient condiment with modern pharmaceutical uses. Journal of Ethnopharmacology, 145(3): 686-698. Sayeed I., Parvez S., Pandey S., Bin-Hafeez B., Haque R., Raisuddin S. (2003). Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotoxicology and Environmental Safety, 56(2): 295-301. Sengül T., Yurtseven S., Cetin M., Kocyigit A., Sögüt B. (2008). Effect of thyme (T. vulgaris) extracts on fattening performance, some blood parameters, oxidative stress and DNA damage in Japanese quails. Journal of Animal and Feed Sciences, 17(4): 608-620. Sevcikova M., Modra H., Slaninova A., Svobodova Z. (2011). Metals as a cause of oxidative stress in fish: a review. Vet Med, 56(11): 537-546. Soleimany V., Banaee M., Mohiseni M., Nematdoost Hagi B., Mousavi Dehmourdi L. (2016). Evaluation of pre- clinical safety and toxicology of Althaea officinalis extracts as naturopathic medicine for common carp (Cyprinus carpio). Iranian Journal of Fisheries Sciences, 15(2): 613-629. Sönmez A.Y., Bilen S., Alak G., Hisar O., Yanık T., Biswas, G. (2015). Growth performance and antioxidant enzyme activities in rainbow trout (Oncorhynchus mykiss) juveniles fed diets supplemented with sage, mint and thyme oils. Fish physiology and biochemistry, 41(1): 165-175. Stohs S., Bagchi D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radical Biology and Medicine, 18(2): 321-336. Tan X.Y., Luo Z., Zhang G.Y., Liu X.J., Jiang M. (2010). Effect of dietary cadmium level on the growth, body composition and several hepatic enzymatic activities of juvenile yellow catfish, Pelteobagrus fulvidraco. Aquaculture Research, 41(7): 1022-1029. Ündeğer Ü., Başaran A., Degen G., Başaran N. (2009). Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol. Food and Chemical Toxicology, 47(8): 369 Int. J. Aquat. Biol. (2017) 5(6): 360-369 2037-2043. Vinodhini R., Narayanan M. (2009). The impact of toxic heavy metals on the hematological parameters in common carp (Cyprinus carpio L.). Iranian Journal of Environmental Health, Science and Engineering, 6(1): 23-28. Yılmaz S., Ergün S., Çelik E.Ş. (2012). Effects of herbal supplements on growth performance of sea bass (Dicentrarchus labrax): Change in body composition and some blood parameters. Journal of BioScience and Biotechnology, 1(3): 217-222. Yılmaz S., Ergün S., Çelik E.Ş. (2013). Effect of dietary herbal supplements on some physiological conditions of sea bass Dicentrarchus labrax. Journal of Aquatic Animal Health, 25(2): 98-103. Int. J. Aquat. Biol. (2017) 5(6): 360-369 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2017 Iranian Society of Ichthyology چکیده فارسی ازیشیر آویشن اکسیدانی آنتی اثر مقایسه و کادمیم سنگین فلز از ناشی اکسیداسیونی آسیب بروز ارزیابی (Zataria multiflora Boiss )ویتامین و E معمولی کپور در (Cyprinus carpio) 1حقی دوست نعمت بهزاد ،1بنایی مهدی ،2باقری دارا ،*1محیسنی محمد 1گروه شیالت، دانشکده منابع طبیعی، دانشگاه صنعتی خاتم االنبیاء بهبهان، بهبهان، ایران. .ایران برازجان، فارس، خلیج دانشگاه طبیعی، منابع و کشاورزی دانشکده شیالت، گروه2 چکیده: با مطالعه نای. دهندمی نشان خود از باالیی اکسیدانیآنتی خصوصیات ترکیبات این که است فالوونوئیدی و فنلی ترکیبات حاوی شیرازی آویشن با هایماهی. است شده انجام شده شناخته طبیعی اکسیدانآنتی یک عنوانبه E ویتامین با مقایسه در شیرازی آویشن حفاظتی اثر بررسی هدف راکخو از استفاده با غذایی هایجیره. گرفتند قرار تغذیه مورد مشخص غذایی جیره سه با و شده بندی تقسیم گروه چهار به( 34±3) وزنی میانگین . شدند ساخته جیره کیلوگرم در گرممیلی 100 یا و شیرازی آویشن درصد 1 مکمل حاوی و( تنهایی به فلز و کنترل گروه برای) افزودنی بدون تجاری با مواجهه از پس 15 و 10 ،7 ،3 روزهای در و گرفته قرار کادمیم زیرکشنده غلظت معرض در روز 15 مدت به تیمارها کلیه کنترل، گروه از غیر به غلظت در داریمعنی کاهش کادمیم با مواجهه روز 15 طول در که داد نشان نتایج. گرفتند قرار بردارینمونه مورد آبشش و کلیه کبد، سنگین، فلز مواجهه اثر در آلدهیددیمالون سطح دیگر سوی از. شد دیده بررسی مورد هایبافت در کاتاالز و ترانسفرازاسگلوتاتیون ردوکتاز، گلوتاتیون هایآنزیم مواجهه در را داریمعنی حفاظتی اثر E ویتامین و شیرازی آویشن مکمل از استفاده. داد نشان داریمعنی افزایش شده یاد هایبافت در سنگین فلز با نقش بر بررسی این از حاصل هاییافته. بود بهتر E ویتامین از شیرازی آویشن حفاظتی اثر که داد نشان نتایج زمینه این در. داد نشان کادمیم با .کندمی تاکید پروریآبزی در آن کاربردی مزایای همچنین و استرس با مواجهه در شیرازی آویشن حفاظتی .استرس دارویی، گیاه سنگین، فلز ،اکسیدانیآنتی سیستم، ماهی :کلمات کلیدی