Protective Effect of Lycopene on Oxidative Stress and Antioxidant Status in Cyprinus carpio during Cypermethrin Exposure M. Enis Yonar Department of Aquaculture and Fish Diseases, Faculty of Fisheries, Firat University, Elazig 23119, Turkey
Received 27 November 2010; revised 8 June 2011; accepted 20 June 2011 ABSTRACT: The aim of this study was to investigate the ameliorative properties of lycopene against the toxic effects of cypermethrin (CYP) by examining oxidative damage markers such as lipid peroxidation and the antioxidant defense system components in carp (Cyprinus carpio). The fish were divided into seven groups of 10 fish each and received the following treatments: group 1, no treatment; group 2, orally administered corn oil; group 3, oral lycopene (10 mg/kg body weight); group 4, exposure to 0.202 lg/L CYP; group 5, exposure to 0.202 lg/L CYP plus oral administration of 10 mg/kg lycopene; group 6, exposure to 0.404 lg/L CYP; and group 7, exposure to 0.404 lg/L CYP plus oral administration of 10 mg/kg lycopene. Treatment was continued for 28 days, and at the end of this period, blood and tissue (liver, kidney, and gill) samples were collected. Levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were determined in blood and tissues for measurement of oxidant-antioxidant status. MDA level, as an index of lipid peroxidation, increased in blood and tissues. Antioxidant enzyme activities in blood and tissues were modified in CYP groups compared with controls. Administration of lycopene ameliorated these parameters. The present results suggest that administration of lycopene might alleviate CYP-induced oxidative stress. # 2011 Wiley Periodicals, Inc. Environ Toxicol 21: 000–000, 2011. Keywords: pesticides; pyrethroid insecticide; cypermethrin; lycopene; oxidative stress; antioxidant enzymes; fish
INTRODUCTION Pesticides have become an increasingly serious source of chemical pollution of the environment due to their extensive usage in agriculture. In natural aquatic environments, alterations in the chemical composition, such as those caused by pesticide contamination, can affect the freshwater fauna, particularly fish. Indeed, fish have been extensively used as bioindicators for environmental pollutants in evaluations of the water quality of aquatic systems. Recent studies indicate that pesticide toxicity in fish may be related Correspondence to: M.E. Yonar; e-mail: [email protected] Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20757
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to an increased production of reactive oxygen species (ROS), leading to oxidative damage. ROS are products of electron transport chains, enzymes, and redox cycling and their production may be enhanced by exposure to xenobiotics. Oxidative stress occurs when ROS overwhelm the cellular defenses, causing damage to proteins, membranes, and DNA and is defined as a disruption of the pro-antioxidant balance, which leads to potential damage (Winston and Di Giulio, 1991; Kelly et al., 1998; Adams and Greeley, 2000). Fish, like many other vertebrates, try to reduce the damage from oxidative stress by using an antioxidant defense system; the first line of defense consists of antioxidant molecules, such as glutathione (GSH), vitamin C and E, carotenoids (Alvarez et al., 2005). Antioxidant enzymes comprise another defense
2011 Wiley Periodicals, Inc.
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mechanism, including the following radical-scavenging enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) (Storey, 1996; Droge, 2002; Valavanidis et al., 2006). Synthetic pyrethroid insecticides are extensively used in place of organochlorine, organophosphorus insecticides and carbamates to control pests. These insecticides are more likely to be toxic to fish and other aquatic organisms (Casida et al., 1983; Moore and Waring, 2001; Polat et al., 2002; Smith and Stratton, 1986). Cypermethrin (CYP) is a synthetic pyrethroid insecticide used to control many pests, such as moth pests attacking cotton, fruit and vegetable crops, including structural pest control, or landscape maintenance. This has resulted in its discharge into the aquatic environment and consequently several laboratory studies have been performed, which have shown that CYP is extremely toxic to fish and aquatic invertebrates at very low concentrations. Fish sensitivity to pyrethroids may be explained by their relatively slow metabolism and elimination of these compounds (David et al., 2003). Carotenoids are a family of fat-soluble pigments that are prevalent in numerous fruits and vegetables, and many studies have investigated their potential at ameliorating oxidative stress (Cohen, 2002; Visioli et al., 2003; Tapiero et al., 2004). Lycopene, a naturally occurring carotenoid in tomatoes and tomato products, has attracted considerable attention as a potential chemopreventive agent. Recently, lycopene has become the focus of much interest because of its highly efficient antioxidant scavenging activity against singlet-oxygen and free radicals (Cohen, 2002; Heber and Lu, 2002; Jonker et al., 2003; Michael and Bausch, 2003; Stahl and Sies, 2003; Tapiero et al., 2004; Velmurugan et al., 2004; Wertz et al., 2004). This antioxidant activity is a potential mechanism by which lycopene may contribute to the prevention of a range of oxidative damage, toxicity, and disease. Among the carotenoids, lycopene is one of the more effective antioxidants against biological ROS and may contribute to the prevention or amelioration of oxidative damage to cells and tissues both in vivo and in vitro (Matos et al., 2000; Velmurugan et al., 2002; Gupta et al., 2003; Reifen et al., 2004). In view of the known antioxidant effects of lycopene, the aim of this study was to evaluate the possible protective or ameliorative effects of lycopene on CYP-induced oxidative stress in Cyprinus carpio.
MATERIALS AND METHODS Chemicals The chemicals used in this study were obtained from SigmaAldrich (St. Louis, MO). CYP, (R,S)-alphacyano-3-phenoxybenzyl (1RS)-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcy
Environmental Toxicology DOI 10.1002/tox
clopropanecarboxylate, was obtained from Novartis, in form of Polytrin 200 EC, (purity 20%, dissolved in 80% acetone).
Fish Cyprinus carpio (72.59 6 13.30 g) were obtained from fish culture pools of the State Hydraulic Works, Elazig, Turkey. The fish were transported to the Fish Diseases Laboratory in the Fisheries Faculty and acclimatized in stock tanks (540 L capacity; 80 3 75 3 90 cm3) to laboratory conditions for 2 weeks. During this period, the fish were fed ad libitum with pellet feedstuff twice a day. The use of fish and the experimental protocol were approved by the Animal Experimentation Ethics Committee of the Firat University (FUAEEC) (Elazig, Turkey).
Water Composition Water composition was as follows: temperature 5 228C 6 28C; total hardness 5 161.3 6 15 mg/L CaCO3; pH 5 7.08 6 0.04; dissolved oxygen 5 7.62 6 0.5 mg/L; alkalinity 5 124 6 16 mg/L. Water used was aerated dechlorinated tap water; moreover, temperature, dissolved oxygen and pH were analyzed daily and a 12:12 h photoperiod was used.
Feed Preparation A commercial basal diet (Ecobio, Elazig/Turkey; including 45% crude protein, 20% crude fat, 11% ash, 3% crude fiber, 8.5% moisture, 12.5% nitrogen free extract, and 5124 kcal/ kg gross energy) was crushed and divided into two parts. The first part was mixed with 10 mg lycopene per kg fish weight; the second part was mixed with corn oil. The diets were thoroughly mixed, and water was added with a commercial food mixer; the diets were then repelleted with a mincer and subsequently spread to dry in a current of air at room temperature for 48 h. After that, the feeds were broken up and sieved into convenient pellet sizes for the fish and stored at 148C for the feeding experiment. Remade pellets were given to the fish manually at a rate of~2% fish body weight per day.
Experimental Setup and Exposure After 2 weeks of acclimation, the fish were randomly divided into seven groups that each contained 10 fish. The first group was held in tap water as a control group. Fish in group 2 received corn oil orally for 28 days. Fish in group 3 received lycopene orally for 28 days. Fish in group 4 were exposed to 0.202 lg/L CYP in their environment for 28 days. Fish in group 5 were exposed to 0.202 lg/L CYP, while lycopene was simultaneously administered for 28 days. Fish in group 6 were exposed to 0.404 lg/L CYP for 28 days. Fish in group 7 were exposed to 0.404 lg/L CYP while lycopene was simultaneously administered for 28
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
days. Control and experimental units were prepared with three replicates. Sublethal concentrations were selected based on 96-h LC50 value for C. carpio. According to Aydın et al. (2005), the 96-h LC50 of CYP for C. carpio was 0.809 lg/L. Fish were exposed to 0.202 lg/L (~1/4 of 96-h LC50) and 0.404 lg/L (~1/2 of 96-h LC50) of CYP for 28 days. Experimental aquaria were aerated and test media were replaced every day. No fish mortality occurred during these exposures.
Sample Collection and Preparation At the end of the experiments, blood was collected with heparinized syringes from the caudal vein of individual fish after anesthetization with benzocaine (25 mg/L). Tissue samples (liver, kidney, and gill) were collected from the individual fish after the blood samples. The levels of malondialdehyde (MDA) and reduced glutathione (GSH) and the catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities were determined in blood, liver, kidney, and gill tissues for oxidant-antioxidant status measurements. Blood samples were centrifuged at 1500 3 g for 10 min at 48C for the separation of plasma. The washing and hemolysis of erythrocytes were performed in accordance with the method described by Winterbourn et al. (1975). The washed erythrocytes were hemolyzed by ice cold distilled water and the hemolysate was used for the biochemical measurements. Immediately after the collection of blood samples from the fish, the liver, kidney, and gills were removed. Tissues were homogenized with a Teflon-glass homogenizer, in a buffer containing 1.15% KCl, to obtain 1:10 (w/v) whole homogenate. The homogenates were centrifuged at 18,000 3 g for 30 min at 48C, then used for determination of malondialdehyde (MDA) and reduced glutathione (GSH) concentrations, and catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities.
Biochemical Analysis MDA Concentration in Plasma and Tissues Concentrations of MDA, as indices of lipid peroxidation in the plasma and tissue samples, were measured using the thiobarbituric acid reaction (Placer et al., 1966). The quantification of the thiobarbituric acid reactive substances was determined by comparing the absorption with the standard curve of malondialdehyde equivalents, generated by the acid-catalyzed hydrolysis of 1,1,3,3-tetramethoxypropane.
SOD Activity in Blood and Tissues SOD activity was determined according to the method described by Sun et al. (1988), based on the principle
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wherein xanthine reacts with xanthine oxidase to generate superoxide radicals that react with nitrobluetetrazolium to form a colored formazan dye. For this purpose, 600 lL SOD in a reaction mixture containing 0.1 mM xanthine, 0.1 mM EDTA, 50 mg of bovine serum albumin, and 25 lmol of nitrobluetetrazolium per liter, was added to 125 lL of the supernatant or 125 lL SOD standard solution. A 25 lL volume of 9.9 nM xanthine oxidase solution was added to each tube at 30 s intervals. Each tube was incubated for 20 min at 258C and the reaction was terminated by adding 0.5 mL of 0.8 mM CuCl2 solution per tube every 30 s. The amount of formazan product was determined by measurement of absorbance at 560 nm.
CAT Activity in Blood and Tissues CAT activity was determined according to the method of Aebi (1983), and the principle of the assay was based on the determination of the rate constant of hydrogen peroxide decomposition by the CAT enzyme. In the experiment, 2 mL of the sample was added to 1 mL of 40 mM H2O2 in phosphate buffer (50 mM, pH 7.0 prepared by mixing the solutions 0.681 g KH2PO4 in 100 mL and 1.335 g Na2HPO4.2H2O in 150 mL) and the decrease in H2O2 was measured spectrophotometrically at 240 nm for 2 min.
GSH-Px Activity in Blood and Tissues GSH-Px activity was determined using the method of Beutler (1975), which records the disappearance of NADPH. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide (NaN3), 0.2 mM NADPH, 1 EU/mL GSH-Px, 1 mM GSH, and 0.25 mM H2O2. An enzyme source (0.1 mL) was added to 0.8 mL of this mixture, and this mixture was incubated at 258C for 5 min before the initiation of the reaction by the addition of 0.1 mL peroxide solution. The absorbance at 340 nm was recorded for 5 min. The activity was then calculated from the slope of the lines as micromoles of NADPH oxidized per minute.
GSH Concentration in Blood and Tissues GSH concentration was measured by an assay using the dithionitrobenzoic acid recycling method described by Ellman (1959). In this method, the chromophoric product resulting from the reaction of the reagent DTNB and erythrocyte-free sulfhydryl groups possessed a molar absorption at 412 nm. One milliliter samples were deproteinated by addition of solution containing 1.67 g metaphosphoric acid, 0.2 g Na2EDTA, and 30 g NaCl, in distilled water. Na2HPO4 of 2.4 mL, and 0.3 mL DTNB were added to the supernatants and cleared by centrifugation (10 min, 3000 3 g/min). The formation of 5-thio-2-
Environmental Toxicology DOI 10.1002/tox
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TABLE I. Plasma MDA level, blood SOD, CAT, GSH-Px activities, and blood GSH concentration in the control and experimental groups Groupsa 1 2 3 4 5 6 7
MDA (nmol/ml)
SOD (U/mg Hb)
CAT (k/g Hb)
GSH-Px (U/g Hb)
GSH (lmol/g Hb)
3.11 6 0.39b 3.20 6 0.61b 3.06 6 0.40b 9.17 6 1.15c 4.89 6 0.74b 12.07 6 0.46d 5.91 6 0.82b
1.96 6 0.18c 2.02 6 0.24c 1.99 6 0.31c 3.11 6 0.83d 2.18 6 0.37c,d 3.59 6 0.62d 2.38 6 0.55c,d
58.42 6 10.51b 57.21 6 9.76b 63.63 6 10.37c 66.14 6 8.64c 55.47 6 11.45b 71.23 6 12.71d 62.27 6 10.40c
35.20 6 6.64d 34.19 6 9.33d 37.08 6 10.44d 28.11 6 8.22c,d 33.47 6 9.10d 24.36 6 8.29c 31.52 6 10.41c,d
4.31 6 0.46d 4.16 6 0.30d 4.29 6 0.52d 3.32 6 0.29c,d 4.01 6 0.63d 2.71 6 0.55c 3.88 6 0.38c,d
k, the first-order rate constant; Hb, Haemoglobin. a Group 1, control; Group 2, corn oil; Group 3, lycopene (10 mg/kg fish/day); Group 4, CYP (0.202 lg/L); Group 5, CYP (0.202 lg/L) plus lycopene (10 mg/kg fish/day); Group 6, CYP (0.404 lg/L); Group 7, CYP (0.404 lg/L) plus lycopene (10 mg/kg fish/day). b,c,d The groups in the same column with different letters are statistically significant (p \ 0.05).
TABLE II. Liver MDA level, SOD, CAT, GSH-Px activities, and GSH concentration in the control and experimental groups Groups
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
42.38 6 7.49 40.27 6 9.22a 39.02 6 6.61a 71.14 6 12.43c 54.29 6 9.85d 68.47 6 9.50c 49.03 6 11.09a,d
2.76 6 0.61 2.88 6 0.47a 2.91 6 0.55a 4.65 6 0.89d 3.29 6 0.72a,d 5.38 6 0.78c 3.47 6 0.68a,d
166.29 6 34.09 161.88 6 27.51b 163.62 6 37.75b 197.34 6 42.32d 180.20 6 50.16a 212.06 6 46.41c 174.71 6 61.34a,b
56.29 6 7.42 54.37 6 9.11c 57.30 6 10.27c 43.18 6 8.89a,d 49.06 6 10.17d 37.29 6 10.91a 51.22 6 8.72d
3.58 6 0.66c 3.69 6 0.49c 3.86 6 0.53c 2.39 6 0.71a 3.02 6 0.82d 2.07 6 0.72a 2.94 6 0.90d
a
1 2 3 4 5 6 7
a
b
c
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
nitrobenzoic acid, which was proportional to GSH concentration, was monitored at 412 nm, at 258C, against reagent controls. Protein concentrations were measured according to Lowry et al. (1951). The concentration of hemoglobin (Hb) was determined using the method of Drabkin (1946).
Statistical Analysis The SPSS 10.1 package program was used in analyses. Study data were given as arithmetic means with standard deviations. The one way analysis of variance and Duncan’s test were used for the determination of the significance of differences among the groups. A value of p \ 0.05 was considered statistically significant.
RESULTS Fish Behavioral During the experiment, control and experimental carp showed normal feeding behavior. Furthermore, there were no signs of respiratory distress such as rapid ventilation, increased rate of gill cover movements, or floating at the
Environmental Toxicology DOI 10.1002/tox
surface of water. During the experiment, there were no mortalities in the groups.
MDA Concentration in Plasma and Tissues Statistically significant differences were observed in the plasma MDA levels of the treatment groups compared with the control (Table I). The MDA levels increased in the groups exposed to CYP alone and decreased in the groups administered lycopene in association with CYP exposure. The group that was administered lycopene alone showed no statistically significant difference from the control group (Table I). Significant increases in MDA were seen in the liver, kidney, and gill samples of the groups exposed to CYP alone. Cotreatment with lycopene resulted in a decrease in tissue MDA levels when compared with the CYP-treated groups. In the group that was administered lycopene alone, no statistically significant difference was seen in tissue MDA levels compared with the control group (Tables II–IV).
SOD Activity in Blood and Tissues Lycopene alone had no significant effect on blood SOD activity compared with the control. Exposure to CYP
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
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TABLE III. Kidney MDA level, SOD, CAT, GSH-Px activities, and GSH concentration in the control and experimental groups Groups 1 2 3 4 5 6 7
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
61.36 6 11.28a 64.59 6 10.37a 60.41 6 10.26a 87.13 6 14.59d 70.08 6 12.63b 96.62 6 16.44c 75.70 6 12.39b
2.38 6 0.34a 2.69 6 0.41c,a 2.32 6 0.25a 3.87 6 0.56c,d 2.96 6 0.75b 4.51 6 0.87c 3.12 6 0.62d
67.42 6 7.11b 65.09 6 9.34b 69.36 6 9.49b 81.11 6 12.63d 72.19 6 9.31b,d 88.57 6 12.76c 76.40 6 14.22d
34.39 6 2.44c 32.51 6 4.08c 33.14 6 3.57c 21.89 6 5.34b 29.27 6 5.79c,d 23.41 6 7.88d 27.00 6 8.11c,d
4.19 6 1.38c,d 4.02 6 0.63c,d 5.29 6 1.52c 3.40 6 0.76d 3.96 6 0.80c,d 3.08 6 1.41d 4.11 6 1.24c,d
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
TABLE IV. Gill MDA level, SOD, CAT and GSH-Px activities, and GSH concentration in the control and experimental groups Groups 1 2 3 4 5 6 7
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
53.24 6 8.14a 52.71 6 9.89a 50.45 6 7.26a 104.92 6 12.43b 76.53 6 14.38d 117.33 6 20.11c 79.61 6 15.66d
1.65 6 0.26b 1.67 6 0.21b 1.64 6 0.39b 2.94 6 0.42c 1.97 6 0.53b,c 2.82 6 0.64c 1.77 6 0.60b
17.39 6 1.52b 15.60 6 1.89b 16.01 6 0.74b 24.52 6 0.53c 20.08 6 0.68b,c 26.41 6 1.25c 18.79 6 0.98b
19.51 6 3.34c 18.84 6 2.36c 18.22 6 3.51c 12.07 6 4.09b 14.63 6 3.95b,c 10.14 6 5.18b 14.26 6 4.63b,c
3.10 6 0.66c 3.39 6 0.73c 3.58 6 0.97c 2.77 6 0.51b,c 3.41 6 0.45c 2.29 6 0.83b 2.63 6 0.36b,c
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
alone resulted in a statistically significant increase in the blood SOD activity compared with the control group. The CYP-exposed groups treated with lycopene had a lower SOD activity than the groups exposed to CYP alone, but a higher SOD activity than the control group (Table I). SOD activity was significantly increased in the liver, kidney, and gill of the groups exposed to CYP alone. Cotreatment with lycopene provided a marked normalization of tissue SOD activities when compared with the CYP groups. In the group that was administered lycopene alone, no statistically significant difference was found in tissue SOD activities when compared with the control group (Tables II–IV).
GSH-Px Activity in Blood and Tissues Blood GSH-Px activity was significantly decreased by CYP exposure. Cotreatment with lycopene caused a significant increase in erythrocyte GSH-Px activity when compared with the CYP groups (Table I). A statistically significant inhibition was also demonstrated in GSH-Px activities the tissue in the groups which were administered both doses of CYP. Treatment with lycopene provided a marked normalization of the tissue GSH-Px activities. Administration of lycopene alone had no significant effect when compared with the control group (Tables II–IV).
GSH Concentration in Blood and Tissues CAT Activity in Blood and Tissues Blood CAT activity was significantly increased by exposure to CYP compared with the control. However, the CAT activities of the groups treated with lycopene were closer to the control group and significantly lower than the groups exposed to CYP alone. Blood CAT activity was significantly increased by administration of lycopene alone compared with the control group (Table I). Tissue CAT activities showed a statistically significant increase in the groups that were exposed to CYP alone, compared with control group tissues. Tissues from fish cotreated with lycopene had lower CAT activities than the CYP groups, but higher activities than the control group (Table II–IV).
Administration of lycopene alone resulted in blood GSH values that were close to the values of the control group. Exposure to CYP alone resulted in a statistically significant decrease in GSH level compared with the control. Cotreatment with lycopene resulted in increased GSH level, but the values were closer to those of the control group and were significantly higher than those from blood from fish exposed to CYP alone (Table I). Compared with the control group, a statistically significant decrease in GSH was seen in tissues of the groups which were administered both doses of CYP. Treatment with lycopene provided a marked normalization of tissue GSH levels when compared with the CYP groups. No statistically significant difference was
Environmental Toxicology DOI 10.1002/tox
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determined between the groups that were administered lycopene alone and the control group, except for the kidney GSH level (Tables II–IV).
DISCUSSION Currently, synthetic pyrethroid insecticides pose a risk to humans especially those professionally involved in their production, those who use them in agriculture and the general population who consume contaminated food products. These compounds are more hydrophobic than other classes of insecticides (Michelangeli et al., 1990) and therefore their general site of action is biological membranes which cause deleterious effects. Pesticides may induce oxidative stress, leading to the generation of free radicals and causing lipid peroxidation, and may be the underlying molecular mechanism that gives rise to pesticide-induced toxicity (Agrawal et al., 1991; Khrer, 1993; Almeida et al., 1997; Ko¨pru¨cu¨ et al., 2008). Increased lipid peroxidation and oxidative stress can affect the activities of a number of protective enzymatic and nonenzymatic antioxidants that are known to be sensitive indicators of increased oxidative stress. In the present study, the protective effect of lycopene against CYP-induced oxidative damage has been evaluated in the blood and tissues of fish. In this study, the high levels of antioxidant enzymes (SOD and CAT) demonstrate an CYP-induced adaptive response in attempting to neutralize the generated ROS. However, the enhanced lipid peroxidation in blood and tissues of C.carpio shows that CYP-induced ROS are not totally scavenged by the antioxidant enzymes. This was aggravated by the decrease in GSH-Px activities and GSH levels in these tissues. Lipid peroxidation has been shown to increase in plasma and some tissues in CYP and other insecticides toxicities (Parker et al., 1984; Akhtar et al., 1994; Gupta et al., 1999; ¨ ner et al. (2001) demonAldana et al., 2001). In addition, U strated that MDA increased in fish liver and kidney following CYP exposure. On the other hand, in a study carried out in Channa punctatus, Sayeed et al. 2003 reported that deltamethrin exposure increased MDA levels in fish liver, kidney, and gills. In our study, the increased level of MDA could also be attributed to free radicals generated by CYP exposure, and these increases in MDA may be due to the possible relationship between CYP toxicity and lipid peroxidation. Carotenoids are well known as highly efficient scavengers of singlet-oxygen (1O2) and other excited oxygen species. During 1O2 quenching, energy is transferred from 1O2 to the lycopene molecule, converting it to the energy-rich triplet state. In contrast, trapping of other ROS, like OH2, NO22 or peroxynitrite, leads to oxidative breakdown of the lycopene molecule. Thus, lycopene may protect in vivo against oxidation of lipids, proteins and DNA
Environmental Toxicology DOI 10.1002/tox
(Stahl and Sies, 2003; Velmurugan et al., 2004; Wertz et al., 2004). In this investigation, the significantly increased MDA level was returned to a level close to that of the control levels by lycopene, due to the free radical scavenging properties of the lycopene. Antioxidant enzymes such as SOD and CAT are considered to be a primary defense that prevents biological macromolecules from oxidative damage. SOD is a group of metalloenzymes that play a crucial role as antioxidants and constitute the primary defense system against the toxic effects of superoxide radicals (O2 2) in aerobic organisms (Kappus, 1985; Kohen and Nyska, 2002). CAT is an enzyme located in peroxisomes and facilitates the removal of hidrojen peroxide (H2O2), which is metabolized to molecular oxygen and water (Aebi, 1983; van der Oost et al., 2003; Yılmaz et al., 2006). SOD and CAT activities in tissues were increased in CYP treated fish probably to dismutate O2 2 and to decompose H2O2. The increase in these enzymes was probably a response toward increased ROS generation in CYP toxicity. Our results are in agreement ¨ ner et al., 2001), who demonstrated sigwith the study of (U nificant increases in SOD and CAT enzyme activities in fish liver and kidney following CYP exposure. The coadministration of lycopene showed decrease in tissues SOD and CAT activities, as lycopene scavenges ROS and lowers oxidative stress. These results indicated that lycopene might have a beneficial role in lowering pyrethroids toxicity probably due to its radical scavenging property. GSH redox cycles serve as a crucial component of cellular antioxidant defenses and are essential for the tissues to protect themselves against the ROS damage. They participate in the elimination of ROS, acting both as a nonenzymatic oxygen radical scavenger and as a substrate for various enzymes such as GSH-Px (Tsukamoto et al., 2002). GSH-Px is one of the enzymes protecting tissues from oxidative damage by reducing H2O2 and a wide range of organic hydroperoxides that form an important group of toxic compounds produced by oxygen metabolism (Helmy et al., 2000). The lower enzymatic activity of GSH-Px in our study could indicate facilitation of increased LPO due to the lack of the protective effect of this antioxidant enzyme. The decreased activity of GSH-Px in blood and tissues observed after CYP exposure may be the result of O2 2 production (Bagnasco et al., 1991) or a direct action of pesticides on the synthesis of the enzyme (Bainy et al., 1993). It is well known that GSH-Px prevents lipid peroxidation in the membranes and acts as a ROS scavenger (Orbea et al., 2000). Our results showed that lycopene supplementation was able to maintain GSH-Px activity close to control values in tissues after CYP exposure. GSH is the major cytosolic low molecular weight sulfhydryl compound that acts as a cellular reducing and a protective reagent against numerous pollutants. GSH protects cells from oxidative stress and plays a critical role in detoxification reactions by acting both as a nucleophilic scavenger of various
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
undesired compounds and their toxic metabolites, and as a specific substrate for the enzyme GSH-Px and GST (Zhang et al., 2004). Large numbers of researches have demonstrated that a shortage of sulfhydryl groups brings about the cells/tissues at risk of oxidative damage (Boulares et al., 2002; Bizzozero et al., 2006; Rosa et al., 2007; Fico et al., 2008). GSH depletion decreases the reduced/oxidized glutathione ratio, which leads to the production of ROS, facilitating the production of lipid peroxidation (Nehru and Bansal, 1997; Sk and Bhattacharya, 2006). In the present study, a significant decrease in the GSH levels in all tissues was observed in fish exposed to CYP compared with the control group. The depletion of GSH content in tissues of fish treated with CYP could be explained either by high GSH utilization for conjugation and/or participation of GSH as an antioxidant in neutralizing free radicals. The results showed that lycopene enhanced antioxidant capacity, protecting blood and tissues against the CYP-induced damages, as shown by the maintenance of GSH-Px activity and GSH content. In conclusion, the results showed that CYP exposure induced oxidative stress in C. carpio. The coadministration of lycopene attenuated the toxic effect of CYP. Thus, lycopene may ameliorate CYP-induced oxidative stress by decreasing oxidative stress and by altering the antioxidant defense system in blood and tissues.
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Received 27 November 2010; revised 8 June 2011; accepted 20 June 2011 ABSTRACT: The aim of this study was to investigate the ameliorative properties of lycopene against the toxic effects of cypermethrin (CYP) by examining oxidative damage markers such as lipid peroxidation and the antioxidant defense system components in carp (Cyprinus carpio). The fish were divided into seven groups of 10 fish each and received the following treatments: group 1, no treatment; group 2, orally administered corn oil; group 3, oral lycopene (10 mg/kg body weight); group 4, exposure to 0.202 lg/L CYP; group 5, exposure to 0.202 lg/L CYP plus oral administration of 10 mg/kg lycopene; group 6, exposure to 0.404 lg/L CYP; and group 7, exposure to 0.404 lg/L CYP plus oral administration of 10 mg/kg lycopene. Treatment was continued for 28 days, and at the end of this period, blood and tissue (liver, kidney, and gill) samples were collected. Levels of malondialdehyde (MDA) and reduced glutathione (GSH) as well as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were determined in blood and tissues for measurement of oxidant-antioxidant status. MDA level, as an index of lipid peroxidation, increased in blood and tissues. Antioxidant enzyme activities in blood and tissues were modified in CYP groups compared with controls. Administration of lycopene ameliorated these parameters. The present results suggest that administration of lycopene might alleviate CYP-induced oxidative stress. # 2011 Wiley Periodicals, Inc. Environ Toxicol 21: 000–000, 2011. Keywords: pesticides; pyrethroid insecticide; cypermethrin; lycopene; oxidative stress; antioxidant enzymes; fish
INTRODUCTION Pesticides have become an increasingly serious source of chemical pollution of the environment due to their extensive usage in agriculture. In natural aquatic environments, alterations in the chemical composition, such as those caused by pesticide contamination, can affect the freshwater fauna, particularly fish. Indeed, fish have been extensively used as bioindicators for environmental pollutants in evaluations of the water quality of aquatic systems. Recent studies indicate that pesticide toxicity in fish may be related Correspondence to: M.E. Yonar; e-mail: [email protected] Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20757
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to an increased production of reactive oxygen species (ROS), leading to oxidative damage. ROS are products of electron transport chains, enzymes, and redox cycling and their production may be enhanced by exposure to xenobiotics. Oxidative stress occurs when ROS overwhelm the cellular defenses, causing damage to proteins, membranes, and DNA and is defined as a disruption of the pro-antioxidant balance, which leads to potential damage (Winston and Di Giulio, 1991; Kelly et al., 1998; Adams and Greeley, 2000). Fish, like many other vertebrates, try to reduce the damage from oxidative stress by using an antioxidant defense system; the first line of defense consists of antioxidant molecules, such as glutathione (GSH), vitamin C and E, carotenoids (Alvarez et al., 2005). Antioxidant enzymes comprise another defense
2011 Wiley Periodicals, Inc.
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mechanism, including the following radical-scavenging enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione S-transferase (GST) (Storey, 1996; Droge, 2002; Valavanidis et al., 2006). Synthetic pyrethroid insecticides are extensively used in place of organochlorine, organophosphorus insecticides and carbamates to control pests. These insecticides are more likely to be toxic to fish and other aquatic organisms (Casida et al., 1983; Moore and Waring, 2001; Polat et al., 2002; Smith and Stratton, 1986). Cypermethrin (CYP) is a synthetic pyrethroid insecticide used to control many pests, such as moth pests attacking cotton, fruit and vegetable crops, including structural pest control, or landscape maintenance. This has resulted in its discharge into the aquatic environment and consequently several laboratory studies have been performed, which have shown that CYP is extremely toxic to fish and aquatic invertebrates at very low concentrations. Fish sensitivity to pyrethroids may be explained by their relatively slow metabolism and elimination of these compounds (David et al., 2003). Carotenoids are a family of fat-soluble pigments that are prevalent in numerous fruits and vegetables, and many studies have investigated their potential at ameliorating oxidative stress (Cohen, 2002; Visioli et al., 2003; Tapiero et al., 2004). Lycopene, a naturally occurring carotenoid in tomatoes and tomato products, has attracted considerable attention as a potential chemopreventive agent. Recently, lycopene has become the focus of much interest because of its highly efficient antioxidant scavenging activity against singlet-oxygen and free radicals (Cohen, 2002; Heber and Lu, 2002; Jonker et al., 2003; Michael and Bausch, 2003; Stahl and Sies, 2003; Tapiero et al., 2004; Velmurugan et al., 2004; Wertz et al., 2004). This antioxidant activity is a potential mechanism by which lycopene may contribute to the prevention of a range of oxidative damage, toxicity, and disease. Among the carotenoids, lycopene is one of the more effective antioxidants against biological ROS and may contribute to the prevention or amelioration of oxidative damage to cells and tissues both in vivo and in vitro (Matos et al., 2000; Velmurugan et al., 2002; Gupta et al., 2003; Reifen et al., 2004). In view of the known antioxidant effects of lycopene, the aim of this study was to evaluate the possible protective or ameliorative effects of lycopene on CYP-induced oxidative stress in Cyprinus carpio.
MATERIALS AND METHODS Chemicals The chemicals used in this study were obtained from SigmaAldrich (St. Louis, MO). CYP, (R,S)-alphacyano-3-phenoxybenzyl (1RS)-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcy
Environmental Toxicology DOI 10.1002/tox
clopropanecarboxylate, was obtained from Novartis, in form of Polytrin 200 EC, (purity 20%, dissolved in 80% acetone).
Fish Cyprinus carpio (72.59 6 13.30 g) were obtained from fish culture pools of the State Hydraulic Works, Elazig, Turkey. The fish were transported to the Fish Diseases Laboratory in the Fisheries Faculty and acclimatized in stock tanks (540 L capacity; 80 3 75 3 90 cm3) to laboratory conditions for 2 weeks. During this period, the fish were fed ad libitum with pellet feedstuff twice a day. The use of fish and the experimental protocol were approved by the Animal Experimentation Ethics Committee of the Firat University (FUAEEC) (Elazig, Turkey).
Water Composition Water composition was as follows: temperature 5 228C 6 28C; total hardness 5 161.3 6 15 mg/L CaCO3; pH 5 7.08 6 0.04; dissolved oxygen 5 7.62 6 0.5 mg/L; alkalinity 5 124 6 16 mg/L. Water used was aerated dechlorinated tap water; moreover, temperature, dissolved oxygen and pH were analyzed daily and a 12:12 h photoperiod was used.
Feed Preparation A commercial basal diet (Ecobio, Elazig/Turkey; including 45% crude protein, 20% crude fat, 11% ash, 3% crude fiber, 8.5% moisture, 12.5% nitrogen free extract, and 5124 kcal/ kg gross energy) was crushed and divided into two parts. The first part was mixed with 10 mg lycopene per kg fish weight; the second part was mixed with corn oil. The diets were thoroughly mixed, and water was added with a commercial food mixer; the diets were then repelleted with a mincer and subsequently spread to dry in a current of air at room temperature for 48 h. After that, the feeds were broken up and sieved into convenient pellet sizes for the fish and stored at 148C for the feeding experiment. Remade pellets were given to the fish manually at a rate of~2% fish body weight per day.
Experimental Setup and Exposure After 2 weeks of acclimation, the fish were randomly divided into seven groups that each contained 10 fish. The first group was held in tap water as a control group. Fish in group 2 received corn oil orally for 28 days. Fish in group 3 received lycopene orally for 28 days. Fish in group 4 were exposed to 0.202 lg/L CYP in their environment for 28 days. Fish in group 5 were exposed to 0.202 lg/L CYP, while lycopene was simultaneously administered for 28 days. Fish in group 6 were exposed to 0.404 lg/L CYP for 28 days. Fish in group 7 were exposed to 0.404 lg/L CYP while lycopene was simultaneously administered for 28
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
days. Control and experimental units were prepared with three replicates. Sublethal concentrations were selected based on 96-h LC50 value for C. carpio. According to Aydın et al. (2005), the 96-h LC50 of CYP for C. carpio was 0.809 lg/L. Fish were exposed to 0.202 lg/L (~1/4 of 96-h LC50) and 0.404 lg/L (~1/2 of 96-h LC50) of CYP for 28 days. Experimental aquaria were aerated and test media were replaced every day. No fish mortality occurred during these exposures.
Sample Collection and Preparation At the end of the experiments, blood was collected with heparinized syringes from the caudal vein of individual fish after anesthetization with benzocaine (25 mg/L). Tissue samples (liver, kidney, and gill) were collected from the individual fish after the blood samples. The levels of malondialdehyde (MDA) and reduced glutathione (GSH) and the catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities were determined in blood, liver, kidney, and gill tissues for oxidant-antioxidant status measurements. Blood samples were centrifuged at 1500 3 g for 10 min at 48C for the separation of plasma. The washing and hemolysis of erythrocytes were performed in accordance with the method described by Winterbourn et al. (1975). The washed erythrocytes were hemolyzed by ice cold distilled water and the hemolysate was used for the biochemical measurements. Immediately after the collection of blood samples from the fish, the liver, kidney, and gills were removed. Tissues were homogenized with a Teflon-glass homogenizer, in a buffer containing 1.15% KCl, to obtain 1:10 (w/v) whole homogenate. The homogenates were centrifuged at 18,000 3 g for 30 min at 48C, then used for determination of malondialdehyde (MDA) and reduced glutathione (GSH) concentrations, and catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activities.
Biochemical Analysis MDA Concentration in Plasma and Tissues Concentrations of MDA, as indices of lipid peroxidation in the plasma and tissue samples, were measured using the thiobarbituric acid reaction (Placer et al., 1966). The quantification of the thiobarbituric acid reactive substances was determined by comparing the absorption with the standard curve of malondialdehyde equivalents, generated by the acid-catalyzed hydrolysis of 1,1,3,3-tetramethoxypropane.
SOD Activity in Blood and Tissues SOD activity was determined according to the method described by Sun et al. (1988), based on the principle
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wherein xanthine reacts with xanthine oxidase to generate superoxide radicals that react with nitrobluetetrazolium to form a colored formazan dye. For this purpose, 600 lL SOD in a reaction mixture containing 0.1 mM xanthine, 0.1 mM EDTA, 50 mg of bovine serum albumin, and 25 lmol of nitrobluetetrazolium per liter, was added to 125 lL of the supernatant or 125 lL SOD standard solution. A 25 lL volume of 9.9 nM xanthine oxidase solution was added to each tube at 30 s intervals. Each tube was incubated for 20 min at 258C and the reaction was terminated by adding 0.5 mL of 0.8 mM CuCl2 solution per tube every 30 s. The amount of formazan product was determined by measurement of absorbance at 560 nm.
CAT Activity in Blood and Tissues CAT activity was determined according to the method of Aebi (1983), and the principle of the assay was based on the determination of the rate constant of hydrogen peroxide decomposition by the CAT enzyme. In the experiment, 2 mL of the sample was added to 1 mL of 40 mM H2O2 in phosphate buffer (50 mM, pH 7.0 prepared by mixing the solutions 0.681 g KH2PO4 in 100 mL and 1.335 g Na2HPO4.2H2O in 150 mL) and the decrease in H2O2 was measured spectrophotometrically at 240 nm for 2 min.
GSH-Px Activity in Blood and Tissues GSH-Px activity was determined using the method of Beutler (1975), which records the disappearance of NADPH. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide (NaN3), 0.2 mM NADPH, 1 EU/mL GSH-Px, 1 mM GSH, and 0.25 mM H2O2. An enzyme source (0.1 mL) was added to 0.8 mL of this mixture, and this mixture was incubated at 258C for 5 min before the initiation of the reaction by the addition of 0.1 mL peroxide solution. The absorbance at 340 nm was recorded for 5 min. The activity was then calculated from the slope of the lines as micromoles of NADPH oxidized per minute.
GSH Concentration in Blood and Tissues GSH concentration was measured by an assay using the dithionitrobenzoic acid recycling method described by Ellman (1959). In this method, the chromophoric product resulting from the reaction of the reagent DTNB and erythrocyte-free sulfhydryl groups possessed a molar absorption at 412 nm. One milliliter samples were deproteinated by addition of solution containing 1.67 g metaphosphoric acid, 0.2 g Na2EDTA, and 30 g NaCl, in distilled water. Na2HPO4 of 2.4 mL, and 0.3 mL DTNB were added to the supernatants and cleared by centrifugation (10 min, 3000 3 g/min). The formation of 5-thio-2-
Environmental Toxicology DOI 10.1002/tox
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TABLE I. Plasma MDA level, blood SOD, CAT, GSH-Px activities, and blood GSH concentration in the control and experimental groups Groupsa 1 2 3 4 5 6 7
MDA (nmol/ml)
SOD (U/mg Hb)
CAT (k/g Hb)
GSH-Px (U/g Hb)
GSH (lmol/g Hb)
3.11 6 0.39b 3.20 6 0.61b 3.06 6 0.40b 9.17 6 1.15c 4.89 6 0.74b 12.07 6 0.46d 5.91 6 0.82b
1.96 6 0.18c 2.02 6 0.24c 1.99 6 0.31c 3.11 6 0.83d 2.18 6 0.37c,d 3.59 6 0.62d 2.38 6 0.55c,d
58.42 6 10.51b 57.21 6 9.76b 63.63 6 10.37c 66.14 6 8.64c 55.47 6 11.45b 71.23 6 12.71d 62.27 6 10.40c
35.20 6 6.64d 34.19 6 9.33d 37.08 6 10.44d 28.11 6 8.22c,d 33.47 6 9.10d 24.36 6 8.29c 31.52 6 10.41c,d
4.31 6 0.46d 4.16 6 0.30d 4.29 6 0.52d 3.32 6 0.29c,d 4.01 6 0.63d 2.71 6 0.55c 3.88 6 0.38c,d
k, the first-order rate constant; Hb, Haemoglobin. a Group 1, control; Group 2, corn oil; Group 3, lycopene (10 mg/kg fish/day); Group 4, CYP (0.202 lg/L); Group 5, CYP (0.202 lg/L) plus lycopene (10 mg/kg fish/day); Group 6, CYP (0.404 lg/L); Group 7, CYP (0.404 lg/L) plus lycopene (10 mg/kg fish/day). b,c,d The groups in the same column with different letters are statistically significant (p \ 0.05).
TABLE II. Liver MDA level, SOD, CAT, GSH-Px activities, and GSH concentration in the control and experimental groups Groups
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
42.38 6 7.49 40.27 6 9.22a 39.02 6 6.61a 71.14 6 12.43c 54.29 6 9.85d 68.47 6 9.50c 49.03 6 11.09a,d
2.76 6 0.61 2.88 6 0.47a 2.91 6 0.55a 4.65 6 0.89d 3.29 6 0.72a,d 5.38 6 0.78c 3.47 6 0.68a,d
166.29 6 34.09 161.88 6 27.51b 163.62 6 37.75b 197.34 6 42.32d 180.20 6 50.16a 212.06 6 46.41c 174.71 6 61.34a,b
56.29 6 7.42 54.37 6 9.11c 57.30 6 10.27c 43.18 6 8.89a,d 49.06 6 10.17d 37.29 6 10.91a 51.22 6 8.72d
3.58 6 0.66c 3.69 6 0.49c 3.86 6 0.53c 2.39 6 0.71a 3.02 6 0.82d 2.07 6 0.72a 2.94 6 0.90d
a
1 2 3 4 5 6 7
a
b
c
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
nitrobenzoic acid, which was proportional to GSH concentration, was monitored at 412 nm, at 258C, against reagent controls. Protein concentrations were measured according to Lowry et al. (1951). The concentration of hemoglobin (Hb) was determined using the method of Drabkin (1946).
Statistical Analysis The SPSS 10.1 package program was used in analyses. Study data were given as arithmetic means with standard deviations. The one way analysis of variance and Duncan’s test were used for the determination of the significance of differences among the groups. A value of p \ 0.05 was considered statistically significant.
RESULTS Fish Behavioral During the experiment, control and experimental carp showed normal feeding behavior. Furthermore, there were no signs of respiratory distress such as rapid ventilation, increased rate of gill cover movements, or floating at the
Environmental Toxicology DOI 10.1002/tox
surface of water. During the experiment, there were no mortalities in the groups.
MDA Concentration in Plasma and Tissues Statistically significant differences were observed in the plasma MDA levels of the treatment groups compared with the control (Table I). The MDA levels increased in the groups exposed to CYP alone and decreased in the groups administered lycopene in association with CYP exposure. The group that was administered lycopene alone showed no statistically significant difference from the control group (Table I). Significant increases in MDA were seen in the liver, kidney, and gill samples of the groups exposed to CYP alone. Cotreatment with lycopene resulted in a decrease in tissue MDA levels when compared with the CYP-treated groups. In the group that was administered lycopene alone, no statistically significant difference was seen in tissue MDA levels compared with the control group (Tables II–IV).
SOD Activity in Blood and Tissues Lycopene alone had no significant effect on blood SOD activity compared with the control. Exposure to CYP
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
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TABLE III. Kidney MDA level, SOD, CAT, GSH-Px activities, and GSH concentration in the control and experimental groups Groups 1 2 3 4 5 6 7
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
61.36 6 11.28a 64.59 6 10.37a 60.41 6 10.26a 87.13 6 14.59d 70.08 6 12.63b 96.62 6 16.44c 75.70 6 12.39b
2.38 6 0.34a 2.69 6 0.41c,a 2.32 6 0.25a 3.87 6 0.56c,d 2.96 6 0.75b 4.51 6 0.87c 3.12 6 0.62d
67.42 6 7.11b 65.09 6 9.34b 69.36 6 9.49b 81.11 6 12.63d 72.19 6 9.31b,d 88.57 6 12.76c 76.40 6 14.22d
34.39 6 2.44c 32.51 6 4.08c 33.14 6 3.57c 21.89 6 5.34b 29.27 6 5.79c,d 23.41 6 7.88d 27.00 6 8.11c,d
4.19 6 1.38c,d 4.02 6 0.63c,d 5.29 6 1.52c 3.40 6 0.76d 3.96 6 0.80c,d 3.08 6 1.41d 4.11 6 1.24c,d
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
TABLE IV. Gill MDA level, SOD, CAT and GSH-Px activities, and GSH concentration in the control and experimental groups Groups 1 2 3 4 5 6 7
MDA (nmol/g protein)
SOD (U/mg protein)
CAT (k/g protein)
GSH-Px (U/g protein)
GSH (lmol/g protein)
53.24 6 8.14a 52.71 6 9.89a 50.45 6 7.26a 104.92 6 12.43b 76.53 6 14.38d 117.33 6 20.11c 79.61 6 15.66d
1.65 6 0.26b 1.67 6 0.21b 1.64 6 0.39b 2.94 6 0.42c 1.97 6 0.53b,c 2.82 6 0.64c 1.77 6 0.60b
17.39 6 1.52b 15.60 6 1.89b 16.01 6 0.74b 24.52 6 0.53c 20.08 6 0.68b,c 26.41 6 1.25c 18.79 6 0.98b
19.51 6 3.34c 18.84 6 2.36c 18.22 6 3.51c 12.07 6 4.09b 14.63 6 3.95b,c 10.14 6 5.18b 14.26 6 4.63b,c
3.10 6 0.66c 3.39 6 0.73c 3.58 6 0.97c 2.77 6 0.51b,c 3.41 6 0.45c 2.29 6 0.83b 2.63 6 0.36b,c
The groups in the same column with different letters are statistically significant (p \ 0.05).
a,b,c,d
alone resulted in a statistically significant increase in the blood SOD activity compared with the control group. The CYP-exposed groups treated with lycopene had a lower SOD activity than the groups exposed to CYP alone, but a higher SOD activity than the control group (Table I). SOD activity was significantly increased in the liver, kidney, and gill of the groups exposed to CYP alone. Cotreatment with lycopene provided a marked normalization of tissue SOD activities when compared with the CYP groups. In the group that was administered lycopene alone, no statistically significant difference was found in tissue SOD activities when compared with the control group (Tables II–IV).
GSH-Px Activity in Blood and Tissues Blood GSH-Px activity was significantly decreased by CYP exposure. Cotreatment with lycopene caused a significant increase in erythrocyte GSH-Px activity when compared with the CYP groups (Table I). A statistically significant inhibition was also demonstrated in GSH-Px activities the tissue in the groups which were administered both doses of CYP. Treatment with lycopene provided a marked normalization of the tissue GSH-Px activities. Administration of lycopene alone had no significant effect when compared with the control group (Tables II–IV).
GSH Concentration in Blood and Tissues CAT Activity in Blood and Tissues Blood CAT activity was significantly increased by exposure to CYP compared with the control. However, the CAT activities of the groups treated with lycopene were closer to the control group and significantly lower than the groups exposed to CYP alone. Blood CAT activity was significantly increased by administration of lycopene alone compared with the control group (Table I). Tissue CAT activities showed a statistically significant increase in the groups that were exposed to CYP alone, compared with control group tissues. Tissues from fish cotreated with lycopene had lower CAT activities than the CYP groups, but higher activities than the control group (Table II–IV).
Administration of lycopene alone resulted in blood GSH values that were close to the values of the control group. Exposure to CYP alone resulted in a statistically significant decrease in GSH level compared with the control. Cotreatment with lycopene resulted in increased GSH level, but the values were closer to those of the control group and were significantly higher than those from blood from fish exposed to CYP alone (Table I). Compared with the control group, a statistically significant decrease in GSH was seen in tissues of the groups which were administered both doses of CYP. Treatment with lycopene provided a marked normalization of tissue GSH levels when compared with the CYP groups. No statistically significant difference was
Environmental Toxicology DOI 10.1002/tox
6
YONAR
determined between the groups that were administered lycopene alone and the control group, except for the kidney GSH level (Tables II–IV).
DISCUSSION Currently, synthetic pyrethroid insecticides pose a risk to humans especially those professionally involved in their production, those who use them in agriculture and the general population who consume contaminated food products. These compounds are more hydrophobic than other classes of insecticides (Michelangeli et al., 1990) and therefore their general site of action is biological membranes which cause deleterious effects. Pesticides may induce oxidative stress, leading to the generation of free radicals and causing lipid peroxidation, and may be the underlying molecular mechanism that gives rise to pesticide-induced toxicity (Agrawal et al., 1991; Khrer, 1993; Almeida et al., 1997; Ko¨pru¨cu¨ et al., 2008). Increased lipid peroxidation and oxidative stress can affect the activities of a number of protective enzymatic and nonenzymatic antioxidants that are known to be sensitive indicators of increased oxidative stress. In the present study, the protective effect of lycopene against CYP-induced oxidative damage has been evaluated in the blood and tissues of fish. In this study, the high levels of antioxidant enzymes (SOD and CAT) demonstrate an CYP-induced adaptive response in attempting to neutralize the generated ROS. However, the enhanced lipid peroxidation in blood and tissues of C.carpio shows that CYP-induced ROS are not totally scavenged by the antioxidant enzymes. This was aggravated by the decrease in GSH-Px activities and GSH levels in these tissues. Lipid peroxidation has been shown to increase in plasma and some tissues in CYP and other insecticides toxicities (Parker et al., 1984; Akhtar et al., 1994; Gupta et al., 1999; ¨ ner et al. (2001) demonAldana et al., 2001). In addition, U strated that MDA increased in fish liver and kidney following CYP exposure. On the other hand, in a study carried out in Channa punctatus, Sayeed et al. 2003 reported that deltamethrin exposure increased MDA levels in fish liver, kidney, and gills. In our study, the increased level of MDA could also be attributed to free radicals generated by CYP exposure, and these increases in MDA may be due to the possible relationship between CYP toxicity and lipid peroxidation. Carotenoids are well known as highly efficient scavengers of singlet-oxygen (1O2) and other excited oxygen species. During 1O2 quenching, energy is transferred from 1O2 to the lycopene molecule, converting it to the energy-rich triplet state. In contrast, trapping of other ROS, like OH2, NO22 or peroxynitrite, leads to oxidative breakdown of the lycopene molecule. Thus, lycopene may protect in vivo against oxidation of lipids, proteins and DNA
Environmental Toxicology DOI 10.1002/tox
(Stahl and Sies, 2003; Velmurugan et al., 2004; Wertz et al., 2004). In this investigation, the significantly increased MDA level was returned to a level close to that of the control levels by lycopene, due to the free radical scavenging properties of the lycopene. Antioxidant enzymes such as SOD and CAT are considered to be a primary defense that prevents biological macromolecules from oxidative damage. SOD is a group of metalloenzymes that play a crucial role as antioxidants and constitute the primary defense system against the toxic effects of superoxide radicals (O2 2) in aerobic organisms (Kappus, 1985; Kohen and Nyska, 2002). CAT is an enzyme located in peroxisomes and facilitates the removal of hidrojen peroxide (H2O2), which is metabolized to molecular oxygen and water (Aebi, 1983; van der Oost et al., 2003; Yılmaz et al., 2006). SOD and CAT activities in tissues were increased in CYP treated fish probably to dismutate O2 2 and to decompose H2O2. The increase in these enzymes was probably a response toward increased ROS generation in CYP toxicity. Our results are in agreement ¨ ner et al., 2001), who demonstrated sigwith the study of (U nificant increases in SOD and CAT enzyme activities in fish liver and kidney following CYP exposure. The coadministration of lycopene showed decrease in tissues SOD and CAT activities, as lycopene scavenges ROS and lowers oxidative stress. These results indicated that lycopene might have a beneficial role in lowering pyrethroids toxicity probably due to its radical scavenging property. GSH redox cycles serve as a crucial component of cellular antioxidant defenses and are essential for the tissues to protect themselves against the ROS damage. They participate in the elimination of ROS, acting both as a nonenzymatic oxygen radical scavenger and as a substrate for various enzymes such as GSH-Px (Tsukamoto et al., 2002). GSH-Px is one of the enzymes protecting tissues from oxidative damage by reducing H2O2 and a wide range of organic hydroperoxides that form an important group of toxic compounds produced by oxygen metabolism (Helmy et al., 2000). The lower enzymatic activity of GSH-Px in our study could indicate facilitation of increased LPO due to the lack of the protective effect of this antioxidant enzyme. The decreased activity of GSH-Px in blood and tissues observed after CYP exposure may be the result of O2 2 production (Bagnasco et al., 1991) or a direct action of pesticides on the synthesis of the enzyme (Bainy et al., 1993). It is well known that GSH-Px prevents lipid peroxidation in the membranes and acts as a ROS scavenger (Orbea et al., 2000). Our results showed that lycopene supplementation was able to maintain GSH-Px activity close to control values in tissues after CYP exposure. GSH is the major cytosolic low molecular weight sulfhydryl compound that acts as a cellular reducing and a protective reagent against numerous pollutants. GSH protects cells from oxidative stress and plays a critical role in detoxification reactions by acting both as a nucleophilic scavenger of various
PROTECTIVE EFFECT OF LYCOPENE AGAINST CYPERMETHRIN
undesired compounds and their toxic metabolites, and as a specific substrate for the enzyme GSH-Px and GST (Zhang et al., 2004). Large numbers of researches have demonstrated that a shortage of sulfhydryl groups brings about the cells/tissues at risk of oxidative damage (Boulares et al., 2002; Bizzozero et al., 2006; Rosa et al., 2007; Fico et al., 2008). GSH depletion decreases the reduced/oxidized glutathione ratio, which leads to the production of ROS, facilitating the production of lipid peroxidation (Nehru and Bansal, 1997; Sk and Bhattacharya, 2006). In the present study, a significant decrease in the GSH levels in all tissues was observed in fish exposed to CYP compared with the control group. The depletion of GSH content in tissues of fish treated with CYP could be explained either by high GSH utilization for conjugation and/or participation of GSH as an antioxidant in neutralizing free radicals. The results showed that lycopene enhanced antioxidant capacity, protecting blood and tissues against the CYP-induced damages, as shown by the maintenance of GSH-Px activity and GSH content. In conclusion, the results showed that CYP exposure induced oxidative stress in C. carpio. The coadministration of lycopene attenuated the toxic effect of CYP. Thus, lycopene may ameliorate CYP-induced oxidative stress by decreasing oxidative stress and by altering the antioxidant defense system in blood and tissues.
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