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Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl
Comparative Biochemistry and Physiology Part C 137 (2004) 43–51
Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl E. Ozcan Oruc*, Y. Sevgiler, N. Uner Faculty of Arts and Science, Department of Biology, Cukurova University, Balcali, 01330 Adana, Turkey Received 17 July 2003; received in revised form 11 November 2003; accepted 12 November 2003
Abstract Species- and tissue-specific defenses against the possibility of oxidative stress and lipid peroxidation were compared in adult fish, Oreochromis niloticus and Cyprinus carpio, exposed to 2,4-dichlorophenoxyacetic acid (2,4-D), azinphosmethyl and their combination for 96 h. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities were monitored in kidney, brain and gill. In all exposure groups there was a marked increase in SOD activity in gill tissues in both fish species, while it was at the control level in other tissues. The highest elevation of SOD activity by combined treatment was observed in C. carpio. Individual and combined treatments caused an elevation in catalase and GPx activities in kidney of C. carpio. Catalase activity was unaffected in brain of O. niloticus, while GPx activity was decreased after all treatments. Glutathione S-transferase (GST) activity was higher than the control levels in kidney of both fish exposed to pesticides. No significant changes were observed in malondialdehyde level in kidney and brain of C. carpio. Our results indicate that the toxicities of azinphosmethyl and 2,4-D may be related to oxidative stress. Also, the results show that SOD activity in gill and GST activity in kidney may be used as biomarkers for pollution monitoring and indicate that the activities of certain biomarkers in C. carpio are more sensitive to pesticides than those in O. niloticus. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Fish; 2,4-D; Azinphosmethyl; Oxidative stress; Antioxidant enzyme; Lipid peroxidation; Enzymes; Pesticides
1. Introduction Chemical pollution in the environment by pesticides has been increasing due to their extensive usage in agriculture. Alterations in the chemical composition of natural aquatic environments can affect the freshwater fauna, particularly fish. Many of these compounds or their metabolites have shown toxic effects related to oxidative stress (Winston and Di Giulio, 1991). In recent years *Corresponding author. Tel.: q90-322-338-6084x2485; fax: q90-322-338-6070. E-mail address: [email protected] (E. Ozcan Oruc).
increasing emphasis has been placed on the use of biomarkers as a tool for monitoring both environmental quality and adaptation of organisms (Stegeman et al., 1991). The antioxidant defence system has been increasingly studied because of the potential usefulness of oxyradical-mediated responses to provide biochemical biomarkers (Di Giulio et al., 1989; Winston and Di Giulio, 1991). Key components are enzymes that can be induced by oxidative stress. These enzymes include superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT EC 1.11.1.16) and glutathione peroxidase (GPx; EC 1.11.1.9) and their reactions with oxyradicals have been studied in fish (Di
1532-0456/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2003.11.006
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E. Ozcan Oruc et al. / Comparative Biochemistry and Physiology Part C 137 (2004) 43–51
Giulio et al., 1989). It is shown that the antioxidants of fish may be useful biomarkers of exposure to aquatic pollutants (Ahmad et al., 2000). Knowledge of the major qualitative and quantitative similarities and differences in antioxidant defence systems among different species is necessary for the development of biomarkers (Livingstone, 1998). Pesticides may cause oxidative stress leading to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems (Almeida et al., 1997). Lipid peroxidation has been suggested as one of the molecular mechanisms involved in pesticideinduced toxicity (Khrer, 1993). 2,4-Dichlorophenoxyacetic acid (2,4-D) and azinphosmethyl are pesticides that have been used commonly in Cukurova, the most important agricultural region in our country. Chlorophenoxy herbicides such as 2,4-D are chemical compounds with physiological properties similar to natural plant hormones and have been used as common weed killers (Seiler, 1978). Phenoxyacid herbicides are structurally related to the hypolipidemic drug ethyl 2-(4-chlorophenoxy)-2-methylpropionate, which is considered the representative of hepatocarcinogenic agents that combine induction of peroxisome proliferation with changes in a number of enzymatic activities in the liver of some mammalian species (Rao and Reddy, 1987). Rowe and Hymas (1954) considered 2,4-D to be only moderately toxic to animals. Although wS-(3,4-dihydro-4-oxobenzoazinphosmethyl w1,2,3-dxtriazin-3-ylmethyl) O-dimethyl phosphorodithioatex is an organophosphate insecticide used for pest control on a wide variety of crops (Sine, 1992), little is known about the biochemical or physiological effects in aquatic organisms. Most of these studies show the results concerning AChE inhibition in fish, resistance mechanism in insects and stabilityysolubility in water (Carlini et al., 1995; Gruber and Munn, 1998; Sabik and Jeannot, 2000). Several studies dealing with the responses of antioxidant enzymes to pesticides, which enhance reactive oxygen species production, have been reported. However, they were inconclusive and show wide individual differences. With respect to the studies of the toxic effects of environmental pollutants, biomarkers were defined to assess the health status of organisms and to obtain earlywarning responses of environmental risk (Payne et
al., 1984). Antioxidant enzyme activities, glutathione redox status, the level of lipid peroxidation product and the specific induction of new glutathione S-transferase (GST; 2.5.1.18) isozymes are most frequently used as biomarkers in toxicological evaluation. The toxicity of chemicals may be different in exposed animals (James et al., 1988). It is therefore useful to evaluate the response of potential test species to xenobiotics. Investigations on the effects of pesticides on fish have a diagnostic significance in evaluating the adverse effects of pesticides to human health (Begum and Vijayaraghavan, 1996). Selection of fish is also adequate since they were proven to be useful in biomonitoring of exposure to aquatic pollutants (Pandey et al., 2001). Unfortunately, studies on oxidative stress in freshwater fish exposed to different pesticides are less extensive than those carried out on marine fish. The mechanism of toxic action of environmental chemicals is not usually thought to be restricted to one specific process but rather multiple sites and mechanisms are involved (Fatima et al., 2000). The primary aim of this study was to compare the inducing effects of pesticides on antioxidant enzyme activities of kidney, brain and gill tissues of commercially valuable freshwater fish Cyprinus carpio and Oreochromis niloticus. The gill was chosen as the major respiratory tissue and major site of uptake of xenobiotic chemicals, whereas kidney was selected as the major route for elimination and rapid clearance of these compounds. Brain was the target of oxidative damage of environmental pollutants (Hai et al., 1997). There is a high content of polyunsaturated fatty acids in brain membrane and low level of antioxidant enzymes. The pollutants attack non-target organisms simultaneously, and quite often the effect of a single compound is different when it is present together with other chemicals at comparable concentrations (Gill et al., 1991). Therefore, speciesand tissue-specific defences against individual and combined treatments of 2,4-D and azinphosmethyl were examined in fish, O. niloticus and C. carpio. Particular attention was given to malondialdehyde (MDA) content, which is known as an end-product of lipid peroxidation.
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2. Materials and methods Adult male specimens of Nile tilapia (O. niloticus) and common carp (C. carpio) were obtained from the culture pools. Randomly selected fish were allowed to acclimatise for at least 2 weeks at 22 8C in glass aquaria supplied with dechlorinated tap water, pH 7.6, alkalinity, 326 ppm CaCO3 and oxygen concentration 7.02 mgyl. The stocks and experimental fish were fed 1% body mass daily, with a commercial fish food (Pinar, Turkey) and submitted to a 12-h light cycle. The water was replaced every 2 days. Renewal assay for static test was used at a rate of 2 or 3 l of solution per fish. Commercial preparations of 2,4-D (2,4-dichlorophenoxy acetic acid dimethyl amine salt, Bayer, 500 gyl, ˙Istanbul, Turkey) and azinphosmethyl (wS-(3,4-dihydro-4-oxobenzo-w1,2,3-dxtriazin-3-ylmethyl) O-dimethyl phosphorodithioatex, Bayer, Guthion 20 EC, 230 gyl, ˙Istanbul, Turkey) were used in the experiments. Experiments were conducted in aquaria containing 160-l test solution. LC50 values for 96 h of 2,4-D and azinphosmethyl to C. carpio were 264 ppm (Feei, 1987) and 0.695 ppm (Pimentel, 1971), respectively. The experiments were designed identically: 24 fish were assigned non-systematically to four groups with six fish per group. The fish were exposed to 87ppm 2,4-D and 0.23-ppm azinphosmethyl, sublethal concentrations, and to their combination for 96 h. For the study of combined effect, one third of 96 h LC50 values of each compound were mixed at 1:1 ratio. No mortality was observed during the 96-h time courses of these experiments. A control group was maintained in tap water. At the end of the fourth day, fish were decapitated. The gill, kidney and brain tissues were dissected, washed in physiological saline solution (0.9% NaCl) and frozen at y85 8C until required for use. The tissues were homogenized by glass– Teflon homogenizer (Heidolph S01 10R2RO) in 1:10 wyv cold 1.15% KCl solution and then centrifuged at 9500=g for 30 min in a Sorvall RC2B centrifuge at 4 8C. Supernatants were used to determine the antioxidant enzyme activities and MDA levels. SOD activity (EC 1.15.1.1) was determined according to the method of McCord and Fridovich (1969) based on the measurement of inhibition percentage of formazan formation at 505 nm and 37 8C.
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GPx activity (EC 1.11.1.9) was determined according to the method of Beutler (1984). GPx catalyzes the oxidation of GSH to GSSG by H2O2. The rate of GSSG formation was then measured by following a decrease in absorbance of the reaction mixture containing NADPH and glutathione reductase at 37 8C and 340 nm as NADPH is converted to NADP. t-Butyl hydroperoxide was used as a substrate. Catalase activity (EC 1.11.1.16) was determined according to the method of Beutler (1984). 1 M Tris–HCl, 5 mM EDTA (pH 8.0), 10 mM H2O2 and H2O were mixed and the rate of H2O2 consumption at 230 nm and 37 8C was used for quantitative determination of CAT activity. An extinction coefficient for H2O2 at 230 nm was used to calculate the activity of the enzyme. GST activity (EC 2.5.1.18) was assayed according to the method of Mannervik and Guthenberg (1981). The enzyme activity was measured by following the change in absorbance at 340 nm of the substrate, CDNB, conjugated with GSH. MDA content was measured after incubation at 95 8C with thiobarbituric acid in aerobic conditions (pH 3.4). The pink colour produced by these reactions was measured spectrophotometrically at 532 nm (Ohkawa et al., 1979). Specific activity was defined as the unit of activity per milligram protein. Protein content was determined according to the method of Lowry et al. (1951) using bovine serum albumin as the standard. The data are expressed as mean"standard error. For differences between several mean values of the control and exposed fish, analysis of variance (ANOVA) was performed. Student Newman Keul’s test was used to determine which groups were significantly different from the control. P0.05 accepted as statistically significant. Statistical analysis was performed using the Statistical Analysis System (SPSS). 3. Results The activities of antioxidant enzymes and MDA content in the kidney, brain and gill tissues of control, C. carpio and O. niloticus, were determined and recorded in Tables 1–5. The results demonstrated that SOD and CAT activities in the kidney of O. niloticus were higher than C. carpio (Tables 1 and 2). Oppositely, GPx activity in O. niloticus was lower than C. carpio (Table 3). This study also showed that GST
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Table 1 Effects of 2,4-D, azinphosmethyl and their combination on SOD activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus SOD activity (Uymg protein) Kidney
Brain y3
Gill y2
C. carpio
Control 2,4-D Azinphosmethyl Combination
0.160"2.60=10 0.145"4.70=10y2 0.155"3.25=10y2 0.150"6.31=10y2
a a a a
0.160"1.15=10 0.170"6.67=10y2 0.180"1.00=10y2 0.170"2.08=10y2
a a a a
0.038"0.009 0.860"0.031 2.027"0.100 4.033"0.284
a b c d
O. niloticus
Control 2,4-D Azinphosmethyl Combination
2.53"0.17 2.096"0.05 2.53"0.03 2.19"0.07
a a a a
0.241"5.36=10y2 0.247"3.28=10y2 0.237"2.67=10y2 0.250"3.06=10y2
a a a a
0.030"1.45=10y3 0.098"3.28=10y3 0.095"5.24=10y3 0.081"4.631=10y3
a b b b
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b, c and d show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
Table 2 Effects of 2,4-D, azinphosmethyl and their combination on CAT activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus CAT activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
5.90"0.22 9.78"0.06 8.32"0.30 8.60"0.88
a b b b
1.55"0.19 2.17"0.32 1.47"0.06 1.41"0.15
a a a a
4.23"1.14 4.08"0.22 3.61"0.33 4.33"0.80
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
135.33"14.62 154.67"19.10 169.67"10.98 169.00"17.35
a a a a
2.30"0.38 2.16"0.31 2.13"0.07 1.98"0.20
a a a a
7.00"0.09 6.32"0.52 6.43"0.18 6.46"0.29
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
Table 3 Effects of 2,4-D, azinphosmethyl and their combination on GPx activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus GPx activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
0.24"0.020 0.36"0.012 0.35"0.017 0.31"0.035
a b b b
0.150"0.027 0.147"0.033 0.133"0.033 0.107"0.012
a a a a
0.333"0.024 0.327"0.035 0.383"0.003 0.357"0.017
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
0.053"0.014 0.076"0.023 0.070"0.069 0.083"0.010
a b b b
0.090"0.012 0.053"0.003 0.057"0.003 0.055"0.001
a b b b
0.016"0.002 0.015"0.001 0.015"0.002 0.017"0.001
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
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Table 4 Effects of 2,4-D, azinphosmethyl and their combination on GST activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus GST activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
86.82"09.77 203.83"26.22 212.81"17.26 138.25"05.17
a b b b
168.56"18.71 199.40"08.03 183.97"02.23 151.11"35.89
a a a a
73.68"11.71 76.91"03.56 92.05"06.99 99.59"01.83
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
98.98"01.97 201.86"14.48 201.10"12.04 212.72"01.99
a b b b
71.70"08.99 81.35"04.22 67.03"04.32 75.14"04.72
a a a a
61.50"01.42 60.92"01.82 62.40"03.60 63.21"03.54
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
activity in the kidney is similar in O. niloticus and C. carpio. In the brain tissue, activities of SOD and CAT in O. niloticus were higher than that in C. carpio. Nevertheless, the activities of GST (Table 4) and GPx in O. niloticus were lower than that found in C. carpio. In the gill tissues, activity of SOD and GST did not show any significant difference between species. Although CAT activity in the gill of O. niloticus was higher than that in C. carpio, GPx activity in the gill of O. niloticus was lower than that found in C. carpio. In all tissues, levels of MDA were similar in both species (Table 5). This study also demonstrates significant differences in the effects of 2,4-D and azinphosmethyl in individual and in combined treatments on the antioxidant enzyme activity and MDA content,
with respect to the tissue distribution of those effects. The most affected tissues were kidney and gill. The results in Table 1 demonstrate that an elevation in SOD activity was observed in gill tissue. For both species, the activity in gill was higher in 2,4-D, azinphosmethyl and their combined treatments than that in control fish. Azinphosmethyl treatment caused more significant elevation (53.3-fold) than 2,4-D treatment (22.6fold) in the gill tissue of C. carpio. The combination of pesticides result in an additive effect on the SOD activity in the gill of C. carpio (106fold). In O. niloticus, SOD activity was increased to more than 2.5-fold of control activity following exposure to pesticides individually and combined treatment.
Table 5 Effects of 2,4-D, azinphosmethyl and their combination on MDA levels (nmolymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus MDA level (nmolymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
2.42"0.61 3.51"0.15 3.06"0.09 3.18"0.38
a a a a
2.61"0.27 3.45"0.59 2.72"0.88 3.64"0.46
a a a a
5.33"0.54 6.54"0.07 5.55"0.81 5.90"1.03
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
3.98"0.14 3.05"0.33 4.50"0.71 3.81"0.53
a a a a
2.91"0.23 2.08"0.05 2.97"0.67 2.53"0.86
a a a a
4.89"0.26 3.88"0.32 3.55"0.44 4.24"0.57
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. No difference was found among exposure groups (P-0.05).
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CAT and GPx activities are shown in Tables 2 and 3, respectively, for control and exposed fish. CAT activity was unaffected in the tissues of O. niloticus. GPx activity was decreased only in brain of O. niloticus by these compounds, while it was increased in kidney. In C. carpio, CAT and GPx activities in kidney increased, while it was at the control level in gill and brain tissues. We found that 2,4-D and azinphosmethyl caused an increase in GST activity (twofold) in kidney tissues, while no response was found in brain and gill tissues (Table 4). No marked species differences were found in the induction of renal GST activities in individual treatments of 2,4-D and azinphosmethyl. In contrast with these results, combined treatment of pesticides caused an elevation more than 2.15-fold in O. niloticus, while 1.59-fold elevation was observed in C. carpio. In contrast with the results found in antioxidant enzyme activities, pesticides had no statistically significant effect on MDA content in either fish species (Table 5). 4. Discussion Pesticides may induce oxidative stress leading to the generation of free radicals and cause lipid peroxidation as molecular mechanisms involved in pesticide-induced toxicity (Agrawal et al., 1991; Khrer, 1993; Almeida et al., 1997). Increased lipid peroxidation and oxidative stress can affect the activities of protective enzymatic antioxidants that have been shown to be sensitive indicators of increased oxidative stress. The present study evaluated the effects of different pesticides and their combined treatments on lipid peroxidation and antioxidant enzyme activities in different tissues of fish, C. carpio and O. niloticus. Although there were some differences in responses between the fish species, adaptive responses have been observed and a common mechanism of adaptive response is the induction of antioxidant enzymes. It was also found that antioxidant responses vary among tissues. Similar results were reported in various fish species (Radi and Matkovics, 1988; Winston and Di Giulio, 1991). Both oxidative responses and the antioxidant potential of fish differ depending on species habitat and feeding behaviour (Ahmad et al., 2000).
Various GSTs were seen to be at relatively high constitutive levels, especially in metabolically active tissues such as liver and kidney (Hayes and Pulford, 1995). GST-mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products, which have a number of adverse biological effects when present in high amounts. Induced GST activity indicates the role of this enzyme in protection against the toxicity of xenobiotic-induced lipid peroxidation (Leaver and George, 1998). 2,4-D and azinphosmethyl treatments caused significant elevations in kidney GST activity. Elevated GST activity may reflect the possibility of better protection against pesticide toxicity. Recent in vitro studies demonstrated that a,bunsaturated aldehydes, produced as a result of cell membrane lipid peroxidation, increase GST mRNA and protein (Tjalkens et al., 1998). Bassi et al. (2000) showed the alterations in response to oxidative stress to be decreased lipid peroxidation and increased xenobiotic metabolism enzymes such as GST. In our study, GPx activity was increased in kidney after pesticide exposure. Ahmad et al. (2000) reported that increased GPx activity may be due to increased production and enzyme induction by H2O2 derived from Oy 2 . Hai et al. (1997) showed that GPx activation is enhanced in C. carpio and Ictalurus (now Ameiurus) nebulosus exposed to dichlorvos. Matkovics et al. (1984) have demonstrated the inductive effect of paraquat in C. carpio and Hypophthalmichthys molitrix. In endosulfan exposed fish, Clarias punctatus, elevated levels of GPx were found (Pandey et al., 2001). Decreased GPx activity in brain tissue of O. niloticus was observed. Brain is the target for oxidative damage of xenobiotics (Hai et al., 1997). The main factors that contribute to the vulnerability of brain include high content of polyunsaturated fatty acids in brain membrane and level of enzymatic and non-enzymatic antioxidants (PerezCampo et al., 1993). The cause of tissue differences could be due to different rates of free radical generation. Moreover, even though the brain is very susceptible to oxidative damage, it is not particularly enriched with antioxidant enzymes (Giuffrida Stella and Lajtha, 1987). Fish being more susceptible to oxidative damage generally have a high GPx activity (Aksnes and Njaa, 1981).
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GPx activity may be increased due to increased production and enzyme-inducing effect of H2O2 derived from Oy 2 . Fatima et al. (2000) stated that low activity of GPx in different tissues of exposed fish demonstrates inefficiency of these organs in neutralizing the impact of peroxides. The brain tissues of fish might be strongly protected against paraquat toxicity since the blood–brain barrier reduces the entry of paraquat from the plasma to the brain tissue in mammals (Rose and Smith, 1977). As a result of exposure to pesticides, an increased CAT activity was observed in kidney tissue of C. carpio. CAT activity in the kidney of O. niloticus was 23-fold higher than that in carp, and together with the high SOD activity can exert an abundant protective effect. A pro-oxidant condition elicited by the presence of pesticides could be triggering an increase in the activity of this antioxidant enzyme, as an adaptive response (Alves et al., 2002). The higher CAT activity might be in response to the increased oxygen consumption giving a great potential for H2O2 production (Ritola et al., 2002). Catalase is an inducible enzyme that protects the biological system against reactive oxygen species (Romeo et al., 2000). Different responses of CAT were found in fish after xenobiotic exposure. Increased activity of this enzyme was also observed in studies with C. carpio and Ictalurus punctatus after dichlorvos exposure (Hai et al., 1997). The treatment of Ameiurus nebulosus with menadione led to decreased CAT activity (Pandey et al., 2001). The gill showed the highest changes in SOD activity among the tissues. The highest SOD activity stimulated by pesticides was measured in C. carpio. Vig and Nemcsok (1989) stated that as a result of paraquat treatment, the changes in SOD activity were highest in the gill, considerable in the liver, but hardly perceptible in the brain. Since the gill is in direct contact with the medium, the damaging effect of paraquat in this tissue is extreme, whereas its effects are limited in liver and brain. The reduction of superoxide radicals to H2O2 is catalyzed by SOD. The increase in SOD activity may be due to increased generation of reactive oxygen species. Palace et al. (1996) have stated that SOD is the most responsive indicator of exposure to contaminants eliciting oxidative stress.
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Lipid peroxidation may be the first step of cellular membrane damage by pesticides. However, in this study, 2,4-D, azinphosmethyl and their combined treatments were unable to stimulate the lipid peroxidation process in the exposed fish, statistically. However, very slight stimulation of lipid peroxidation by pesticides cannot be excluded in the kidney and brain of C. carpio. Low levels or lack of lipid peroxidation in the tissues reflects the protective effects of oxidative enzymes. In conclusion, it is possible to restore susceptibility and to adapt to oxidative stress by increasing SOD and GST activities. The present study revealed that fish exposed to pesticides develop tissue-specific adaptive responses to protect cells against oxidative stress. C. carpio is found to be more sensitive than O. niloticus. Moreover, according to our results, the elevations in gill SOD activity and kidney GST activity serve as biomarkers of oxidative stress and may be helpful in assessing the risk of environmental contaminants. Acknowledgments We wish to thank The Cukurova University Grant Commission for their financial support. References Agrawal, D., Sultana, P., Gupta, G.S.D., 1991. Oxidative damage and changes in the glutathione redox system in erythrocytes from rats treated with hexachlorocyclohexane. Food Chem. Toxicol. 29, 459–462. Ahmad, I., Hamid, T., Fatima, M., et al., 2000. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus) is a biomarker of paper mill effluent exposure. Biochim. Biophys. Acta 1519, 37–48. Aksnes, A., Njaa, L.R., 1981. Catalase, glutathione peroxidase and superoxide dismutase in different fish species. Comp. Biochem. Physiol. B 69, 893–896. Almeida, M.G., Fanini, F., Davino, S.C., Aznar, A.E., Koch, O.R., Barros, S.B.M., 1997. Pro- and anti-oxidant parameters in rat liver after short-term exposure to hexachlorobenzene. Hum. Exp. Toxicol. 16, 257–261. Alves, S.R.C., Severino, P.C., Ibbotson, D.P., et al., 2002. Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae. Mar. Environ. Res. 54, 1–5. Bassi, A.M., Ledda, S., Penco, S., et al., 2000. Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation. Free Rad. Biol. Med. 29, 1186–1196. Begum, G., Vijayaraghavan, S., 1996. Alterations in protein metabolism of muscle tissue in the fish Clarias batrachus
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(Linn) by commercial grade dimethoate. Bull. Environ. Contam. Toxicol. 57, 223–228. Beutler, E., 1984. Red Cell Metabolism: A Manual of Biochemical Methods. Grune and Stratton, New York second reprint. Carlini, E.J., McPheron, B.A., Felland, C.M., Hull, L.A., 1995. Biochemical mechanisms of azinphosmethyl resistance in the tufted apple bud moth Platynota idaeusalis. Pestic. Biochem. Physiol. 51, 38–47. Di Giulio, R.T., Washburn, P.C., Wennig, R.J., Winston, G.W., Jewell, C.S., 1989. Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environ. Toxicol. Chem. 8, 1103–1123. Fatima, M., Ahmad, I., Sayeed, I., Athar, M., Raisuddin, S., 2000. Pollutant-induced over-activation of phagocytes is concomitantly associated with peroxidative damage in fish tissues. Aquat. Toxicol. 49, 243–250. Feei, S., 1987. Evaluating acute toxicity of pesticides to aquatic organisms carp, mosquito fish and daphnids. Plant Protect. Bull. 29, 385–396. Gill, T.S., Pande, J., Tevari, H., 1991. Individual and combined toxicity of common pesticides to teleost Puntius conchonius Hamilton. Indian J. Exp. Biol. 29, 145–148. Giuffrida Stella, A.M., Lajtha, A., 1987. Macromolecular turnover in brain during aging. Gerontology 33, 136–148. Gruber, S.J., Munn, M.D., 1998. Organophosphate and carbamate insecticides in agricultural waters and cholinesterase (ChE) inhibition in common carp (Cyprinus carpio). Arch. Environ. Contam. Tox. 35, 391–396. Hai, D.Q., Varga, Sz.I., Matkovics, B., 1997. Organophosphate effects on antioxidant system on carp (Cyprinus carpio) and catfish (Ictalurus nebulosus). Comp. Biochem. Physiol. C 117, 83–88. Hayes, J.D., Pulford, D.J., 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30, 445–600. James, M.O., Heard, C.S., Hawkins, W.E., 1988. Effects of 3methylcholanthrene on monooxygenase, epoxide hydrolase, and glutathione S-transferase activities in small estuarine and freshwater fish. Aquat. Toxicol. 12, 1–15. Khrer, J.P., 1993. Free radicals as mediator of tissue injury and disease. Crit. Rev. Toxicol. 23, 21–48. Leaver, M.J., George, S.G., 1998. A piscidine glutathione Stransferase which efficiently conjugates the end-products of lipid peroxidation. Mar. Environ. Res. 46, 71–74. Livingstone, D.R., 1998. The fate of organic xenobiotics in aquatic ecosystems: quantitative and qualitative differences in biotransformation by invertebrates and fish. Comp. Biochem. Physiol. A 120, 43–49. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin-phenol reagent. J. Biol. Chem. 193, 265–275. Mannervik, B., Guthenberg, C., 1981. Glutathione S-transferase (human placenta). Method. Enzymol. 77, 231–235. Matkovics, B., Szabo, L., Varga, S.z.I., Barabas, K., Berencsi, G., Nemcsok, J., 1984. Effects of a herbicide on the peroxide metabolism enzymes and lipid peroxidation in carp fish (Hypophthalmichthys molitrix). Acta Biol. Hung. 35, 91–96.
McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049–6053. Ohkawa, H., Ohishi, N., Tagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Chem. 95, 351–358. Palace, V.P., Dick, T.A., Brown, S.B., Baron, C.L., Klaverkamp, J.F., 1996. Oxidative stress in Lake Sturgeon (Acipenser fulvescens) orally exposed to 2,3,7,8-tetrachlorodibenzofuran. Aquat. Toxicol. 35, 79–92. Pandey, S., Ahmad, I., Parvez, S., Bin-Hafeez, R., Naque, R., Raisuddin, S., 2001. Effect of endosulfan on antioxidants of freshwater fish Channa punctatus Bloch: 1. Protection against lipid peroxidation in liver by copper pre-exposure. Arch. Environ. Contam. Toxicol. 41, 345–352. Payne, J.F., Bauld, C., Dey, A.C., Kiceniuk, J.W., Williams, U., 1984. Selectivity of mixed-function oxygenase enzyme induction in flounder (Pseudopleuronectes americanus) collected at the site of the Baie Verte. Newfoundland oil spill. Comp. Biochem. Physiol. C 79, 15–19. Perez-Campo, R., Lopez-Torres, M., Rojas, C., Cadenas, S., Barja, G.A., 1993. Comparative study of free radicals in vertebrates I. Antioxidant enzymes. Comp. Biochem. Physiol. B 105, 749–755. Pimentel, D., 1971. Ecological Effects of Pesticides on NonTarget Organisms. US Gov. Printing Office, Washington DC, pp. 659. Radi, A.A.R., Matkovics, B., 1988. Effects of metal ions on the antioxidant enzyme activities, protein contents and lipid peroxidation of carp tissues. Comp. Biochem. Physiol. C 90, 69–72. Rao, M., Reddy, J., 1987. Peroxisome proliferation and hepatocarcinogenesis. Carcinogenesis 8, 631–636. Ritola, O., Livingstone, D.R., Peters, L.D., Lindstrom-Seppa, P., 2002. Antioxidant processes are affected in juvenile rainbow trout (Oncorhynchus mykiss) exposed to ozone and oxygen-supersaturated water. Aquaculture 210, 1–19. Romeo, M., Bennani, N., Gnassia-Barelli, M., La Faurie, M., Girard, J.P., 2000. Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax. Aquat. Toxicol. 48, 185–194. Rose, M.S., Smith, L., 1977. The relevance of paraquat accumulation by tissue. In: Autor, A.P. (Ed.), Biochemical Mechanisms of Paraquat Toxicity. Academic Press, New York, pp. 71–91. Rowe, V.R., Hymas, T.H., 1954. Summary of toxicological information on 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. Am. J. Vet. Res. 15, 622–629. Sabik, H., Jeannot, R., 2000. Stability of organophosphorus insecticides on graphitized carbon black extraction cartridges used for large volumes of surface water. J. Chromatography A 879, 73–82. Seiler, J.P., 1978. The genetic toxicology of phenoxy acids other than 2,4,5-T. Mut. Res. 55, 197–226. Sine, C., 1992. Farm Chemicals Handbook. Meister, Willoughby, OH, pp. C34–C35. Stegeman, J.J., Smolowitz, R.M., Hahn, M.E., 1991. Immunohistochemical localization of environmentally induced cytochrome P450IA1 in multiple organs of the marine
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Vig, E., Nemcsok, J., 1989. The effects of hypoxia and paraquat on the superoxide dismutase activity in different organs of carp, Cyprinus carpio L. J. Fish Biol. 35, 23–25. Winston, G.W., Di Giulio, R.T., 1991. Pro-oxidant and antioxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19, 137–161.
Tissue-specific oxidative stress responses in fish exposed to 2,4-D and azinphosmethyl E. Ozcan Oruc*, Y. Sevgiler, N. Uner Faculty of Arts and Science, Department of Biology, Cukurova University, Balcali, 01330 Adana, Turkey Received 17 July 2003; received in revised form 11 November 2003; accepted 12 November 2003
Abstract Species- and tissue-specific defenses against the possibility of oxidative stress and lipid peroxidation were compared in adult fish, Oreochromis niloticus and Cyprinus carpio, exposed to 2,4-dichlorophenoxyacetic acid (2,4-D), azinphosmethyl and their combination for 96 h. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activities were monitored in kidney, brain and gill. In all exposure groups there was a marked increase in SOD activity in gill tissues in both fish species, while it was at the control level in other tissues. The highest elevation of SOD activity by combined treatment was observed in C. carpio. Individual and combined treatments caused an elevation in catalase and GPx activities in kidney of C. carpio. Catalase activity was unaffected in brain of O. niloticus, while GPx activity was decreased after all treatments. Glutathione S-transferase (GST) activity was higher than the control levels in kidney of both fish exposed to pesticides. No significant changes were observed in malondialdehyde level in kidney and brain of C. carpio. Our results indicate that the toxicities of azinphosmethyl and 2,4-D may be related to oxidative stress. Also, the results show that SOD activity in gill and GST activity in kidney may be used as biomarkers for pollution monitoring and indicate that the activities of certain biomarkers in C. carpio are more sensitive to pesticides than those in O. niloticus. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Fish; 2,4-D; Azinphosmethyl; Oxidative stress; Antioxidant enzyme; Lipid peroxidation; Enzymes; Pesticides
1. Introduction Chemical pollution in the environment by pesticides has been increasing due to their extensive usage in agriculture. Alterations in the chemical composition of natural aquatic environments can affect the freshwater fauna, particularly fish. Many of these compounds or their metabolites have shown toxic effects related to oxidative stress (Winston and Di Giulio, 1991). In recent years *Corresponding author. Tel.: q90-322-338-6084x2485; fax: q90-322-338-6070. E-mail address: [email protected] (E. Ozcan Oruc).
increasing emphasis has been placed on the use of biomarkers as a tool for monitoring both environmental quality and adaptation of organisms (Stegeman et al., 1991). The antioxidant defence system has been increasingly studied because of the potential usefulness of oxyradical-mediated responses to provide biochemical biomarkers (Di Giulio et al., 1989; Winston and Di Giulio, 1991). Key components are enzymes that can be induced by oxidative stress. These enzymes include superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT EC 1.11.1.16) and glutathione peroxidase (GPx; EC 1.11.1.9) and their reactions with oxyradicals have been studied in fish (Di
1532-0456/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cca.2003.11.006
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Giulio et al., 1989). It is shown that the antioxidants of fish may be useful biomarkers of exposure to aquatic pollutants (Ahmad et al., 2000). Knowledge of the major qualitative and quantitative similarities and differences in antioxidant defence systems among different species is necessary for the development of biomarkers (Livingstone, 1998). Pesticides may cause oxidative stress leading to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems (Almeida et al., 1997). Lipid peroxidation has been suggested as one of the molecular mechanisms involved in pesticideinduced toxicity (Khrer, 1993). 2,4-Dichlorophenoxyacetic acid (2,4-D) and azinphosmethyl are pesticides that have been used commonly in Cukurova, the most important agricultural region in our country. Chlorophenoxy herbicides such as 2,4-D are chemical compounds with physiological properties similar to natural plant hormones and have been used as common weed killers (Seiler, 1978). Phenoxyacid herbicides are structurally related to the hypolipidemic drug ethyl 2-(4-chlorophenoxy)-2-methylpropionate, which is considered the representative of hepatocarcinogenic agents that combine induction of peroxisome proliferation with changes in a number of enzymatic activities in the liver of some mammalian species (Rao and Reddy, 1987). Rowe and Hymas (1954) considered 2,4-D to be only moderately toxic to animals. Although wS-(3,4-dihydro-4-oxobenzoazinphosmethyl w1,2,3-dxtriazin-3-ylmethyl) O-dimethyl phosphorodithioatex is an organophosphate insecticide used for pest control on a wide variety of crops (Sine, 1992), little is known about the biochemical or physiological effects in aquatic organisms. Most of these studies show the results concerning AChE inhibition in fish, resistance mechanism in insects and stabilityysolubility in water (Carlini et al., 1995; Gruber and Munn, 1998; Sabik and Jeannot, 2000). Several studies dealing with the responses of antioxidant enzymes to pesticides, which enhance reactive oxygen species production, have been reported. However, they were inconclusive and show wide individual differences. With respect to the studies of the toxic effects of environmental pollutants, biomarkers were defined to assess the health status of organisms and to obtain earlywarning responses of environmental risk (Payne et
al., 1984). Antioxidant enzyme activities, glutathione redox status, the level of lipid peroxidation product and the specific induction of new glutathione S-transferase (GST; 2.5.1.18) isozymes are most frequently used as biomarkers in toxicological evaluation. The toxicity of chemicals may be different in exposed animals (James et al., 1988). It is therefore useful to evaluate the response of potential test species to xenobiotics. Investigations on the effects of pesticides on fish have a diagnostic significance in evaluating the adverse effects of pesticides to human health (Begum and Vijayaraghavan, 1996). Selection of fish is also adequate since they were proven to be useful in biomonitoring of exposure to aquatic pollutants (Pandey et al., 2001). Unfortunately, studies on oxidative stress in freshwater fish exposed to different pesticides are less extensive than those carried out on marine fish. The mechanism of toxic action of environmental chemicals is not usually thought to be restricted to one specific process but rather multiple sites and mechanisms are involved (Fatima et al., 2000). The primary aim of this study was to compare the inducing effects of pesticides on antioxidant enzyme activities of kidney, brain and gill tissues of commercially valuable freshwater fish Cyprinus carpio and Oreochromis niloticus. The gill was chosen as the major respiratory tissue and major site of uptake of xenobiotic chemicals, whereas kidney was selected as the major route for elimination and rapid clearance of these compounds. Brain was the target of oxidative damage of environmental pollutants (Hai et al., 1997). There is a high content of polyunsaturated fatty acids in brain membrane and low level of antioxidant enzymes. The pollutants attack non-target organisms simultaneously, and quite often the effect of a single compound is different when it is present together with other chemicals at comparable concentrations (Gill et al., 1991). Therefore, speciesand tissue-specific defences against individual and combined treatments of 2,4-D and azinphosmethyl were examined in fish, O. niloticus and C. carpio. Particular attention was given to malondialdehyde (MDA) content, which is known as an end-product of lipid peroxidation.
E. Ozcan Oruc et al. / Comparative Biochemistry and Physiology Part C 137 (2004) 43–51
2. Materials and methods Adult male specimens of Nile tilapia (O. niloticus) and common carp (C. carpio) were obtained from the culture pools. Randomly selected fish were allowed to acclimatise for at least 2 weeks at 22 8C in glass aquaria supplied with dechlorinated tap water, pH 7.6, alkalinity, 326 ppm CaCO3 and oxygen concentration 7.02 mgyl. The stocks and experimental fish were fed 1% body mass daily, with a commercial fish food (Pinar, Turkey) and submitted to a 12-h light cycle. The water was replaced every 2 days. Renewal assay for static test was used at a rate of 2 or 3 l of solution per fish. Commercial preparations of 2,4-D (2,4-dichlorophenoxy acetic acid dimethyl amine salt, Bayer, 500 gyl, ˙Istanbul, Turkey) and azinphosmethyl (wS-(3,4-dihydro-4-oxobenzo-w1,2,3-dxtriazin-3-ylmethyl) O-dimethyl phosphorodithioatex, Bayer, Guthion 20 EC, 230 gyl, ˙Istanbul, Turkey) were used in the experiments. Experiments were conducted in aquaria containing 160-l test solution. LC50 values for 96 h of 2,4-D and azinphosmethyl to C. carpio were 264 ppm (Feei, 1987) and 0.695 ppm (Pimentel, 1971), respectively. The experiments were designed identically: 24 fish were assigned non-systematically to four groups with six fish per group. The fish were exposed to 87ppm 2,4-D and 0.23-ppm azinphosmethyl, sublethal concentrations, and to their combination for 96 h. For the study of combined effect, one third of 96 h LC50 values of each compound were mixed at 1:1 ratio. No mortality was observed during the 96-h time courses of these experiments. A control group was maintained in tap water. At the end of the fourth day, fish were decapitated. The gill, kidney and brain tissues were dissected, washed in physiological saline solution (0.9% NaCl) and frozen at y85 8C until required for use. The tissues were homogenized by glass– Teflon homogenizer (Heidolph S01 10R2RO) in 1:10 wyv cold 1.15% KCl solution and then centrifuged at 9500=g for 30 min in a Sorvall RC2B centrifuge at 4 8C. Supernatants were used to determine the antioxidant enzyme activities and MDA levels. SOD activity (EC 1.15.1.1) was determined according to the method of McCord and Fridovich (1969) based on the measurement of inhibition percentage of formazan formation at 505 nm and 37 8C.
45
GPx activity (EC 1.11.1.9) was determined according to the method of Beutler (1984). GPx catalyzes the oxidation of GSH to GSSG by H2O2. The rate of GSSG formation was then measured by following a decrease in absorbance of the reaction mixture containing NADPH and glutathione reductase at 37 8C and 340 nm as NADPH is converted to NADP. t-Butyl hydroperoxide was used as a substrate. Catalase activity (EC 1.11.1.16) was determined according to the method of Beutler (1984). 1 M Tris–HCl, 5 mM EDTA (pH 8.0), 10 mM H2O2 and H2O were mixed and the rate of H2O2 consumption at 230 nm and 37 8C was used for quantitative determination of CAT activity. An extinction coefficient for H2O2 at 230 nm was used to calculate the activity of the enzyme. GST activity (EC 2.5.1.18) was assayed according to the method of Mannervik and Guthenberg (1981). The enzyme activity was measured by following the change in absorbance at 340 nm of the substrate, CDNB, conjugated with GSH. MDA content was measured after incubation at 95 8C with thiobarbituric acid in aerobic conditions (pH 3.4). The pink colour produced by these reactions was measured spectrophotometrically at 532 nm (Ohkawa et al., 1979). Specific activity was defined as the unit of activity per milligram protein. Protein content was determined according to the method of Lowry et al. (1951) using bovine serum albumin as the standard. The data are expressed as mean"standard error. For differences between several mean values of the control and exposed fish, analysis of variance (ANOVA) was performed. Student Newman Keul’s test was used to determine which groups were significantly different from the control. P0.05 accepted as statistically significant. Statistical analysis was performed using the Statistical Analysis System (SPSS). 3. Results The activities of antioxidant enzymes and MDA content in the kidney, brain and gill tissues of control, C. carpio and O. niloticus, were determined and recorded in Tables 1–5. The results demonstrated that SOD and CAT activities in the kidney of O. niloticus were higher than C. carpio (Tables 1 and 2). Oppositely, GPx activity in O. niloticus was lower than C. carpio (Table 3). This study also showed that GST
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Table 1 Effects of 2,4-D, azinphosmethyl and their combination on SOD activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus SOD activity (Uymg protein) Kidney
Brain y3
Gill y2
C. carpio
Control 2,4-D Azinphosmethyl Combination
0.160"2.60=10 0.145"4.70=10y2 0.155"3.25=10y2 0.150"6.31=10y2
a a a a
0.160"1.15=10 0.170"6.67=10y2 0.180"1.00=10y2 0.170"2.08=10y2
a a a a
0.038"0.009 0.860"0.031 2.027"0.100 4.033"0.284
a b c d
O. niloticus
Control 2,4-D Azinphosmethyl Combination
2.53"0.17 2.096"0.05 2.53"0.03 2.19"0.07
a a a a
0.241"5.36=10y2 0.247"3.28=10y2 0.237"2.67=10y2 0.250"3.06=10y2
a a a a
0.030"1.45=10y3 0.098"3.28=10y3 0.095"5.24=10y3 0.081"4.631=10y3
a b b b
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b, c and d show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
Table 2 Effects of 2,4-D, azinphosmethyl and their combination on CAT activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus CAT activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
5.90"0.22 9.78"0.06 8.32"0.30 8.60"0.88
a b b b
1.55"0.19 2.17"0.32 1.47"0.06 1.41"0.15
a a a a
4.23"1.14 4.08"0.22 3.61"0.33 4.33"0.80
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
135.33"14.62 154.67"19.10 169.67"10.98 169.00"17.35
a a a a
2.30"0.38 2.16"0.31 2.13"0.07 1.98"0.20
a a a a
7.00"0.09 6.32"0.52 6.43"0.18 6.46"0.29
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
Table 3 Effects of 2,4-D, azinphosmethyl and their combination on GPx activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus GPx activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
0.24"0.020 0.36"0.012 0.35"0.017 0.31"0.035
a b b b
0.150"0.027 0.147"0.033 0.133"0.033 0.107"0.012
a a a a
0.333"0.024 0.327"0.035 0.383"0.003 0.357"0.017
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
0.053"0.014 0.076"0.023 0.070"0.069 0.083"0.010
a b b b
0.090"0.012 0.053"0.003 0.057"0.003 0.055"0.001
a b b b
0.016"0.002 0.015"0.001 0.015"0.002 0.017"0.001
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
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47
Table 4 Effects of 2,4-D, azinphosmethyl and their combination on GST activity (Uymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus GST activity (Uymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
86.82"09.77 203.83"26.22 212.81"17.26 138.25"05.17
a b b b
168.56"18.71 199.40"08.03 183.97"02.23 151.11"35.89
a a a a
73.68"11.71 76.91"03.56 92.05"06.99 99.59"01.83
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
98.98"01.97 201.86"14.48 201.10"12.04 212.72"01.99
a b b b
71.70"08.99 81.35"04.22 67.03"04.32 75.14"04.72
a a a a
61.50"01.42 60.92"01.82 62.40"03.60 63.21"03.54
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. Letters a, b show the differences among exposure groups. Data shown with different letters are significantly different at the P-0.05 level.
activity in the kidney is similar in O. niloticus and C. carpio. In the brain tissue, activities of SOD and CAT in O. niloticus were higher than that in C. carpio. Nevertheless, the activities of GST (Table 4) and GPx in O. niloticus were lower than that found in C. carpio. In the gill tissues, activity of SOD and GST did not show any significant difference between species. Although CAT activity in the gill of O. niloticus was higher than that in C. carpio, GPx activity in the gill of O. niloticus was lower than that found in C. carpio. In all tissues, levels of MDA were similar in both species (Table 5). This study also demonstrates significant differences in the effects of 2,4-D and azinphosmethyl in individual and in combined treatments on the antioxidant enzyme activity and MDA content,
with respect to the tissue distribution of those effects. The most affected tissues were kidney and gill. The results in Table 1 demonstrate that an elevation in SOD activity was observed in gill tissue. For both species, the activity in gill was higher in 2,4-D, azinphosmethyl and their combined treatments than that in control fish. Azinphosmethyl treatment caused more significant elevation (53.3-fold) than 2,4-D treatment (22.6fold) in the gill tissue of C. carpio. The combination of pesticides result in an additive effect on the SOD activity in the gill of C. carpio (106fold). In O. niloticus, SOD activity was increased to more than 2.5-fold of control activity following exposure to pesticides individually and combined treatment.
Table 5 Effects of 2,4-D, azinphosmethyl and their combination on MDA levels (nmolymg protein) in kidney, brain and gill tissues of C. carpio and O. niloticus MDA level (nmolymg protein) Kidney
Brain
Gill
C. carpio
Control 2,4-D Azinphosmethyl Combination
2.42"0.61 3.51"0.15 3.06"0.09 3.18"0.38
a a a a
2.61"0.27 3.45"0.59 2.72"0.88 3.64"0.46
a a a a
5.33"0.54 6.54"0.07 5.55"0.81 5.90"1.03
a a a a
O. niloticus
Control 2,4-D Azinphosmethyl Combination
3.98"0.14 3.05"0.33 4.50"0.71 3.81"0.53
a a a a
2.91"0.23 2.08"0.05 2.97"0.67 2.53"0.86
a a a a
4.89"0.26 3.88"0.32 3.55"0.44 4.24"0.57
a a a a
Values are means"S.E.M. (ns6). Statistical comparisons were done between control and exposure data from the same species. No difference was found among exposure groups (P-0.05).
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CAT and GPx activities are shown in Tables 2 and 3, respectively, for control and exposed fish. CAT activity was unaffected in the tissues of O. niloticus. GPx activity was decreased only in brain of O. niloticus by these compounds, while it was increased in kidney. In C. carpio, CAT and GPx activities in kidney increased, while it was at the control level in gill and brain tissues. We found that 2,4-D and azinphosmethyl caused an increase in GST activity (twofold) in kidney tissues, while no response was found in brain and gill tissues (Table 4). No marked species differences were found in the induction of renal GST activities in individual treatments of 2,4-D and azinphosmethyl. In contrast with these results, combined treatment of pesticides caused an elevation more than 2.15-fold in O. niloticus, while 1.59-fold elevation was observed in C. carpio. In contrast with the results found in antioxidant enzyme activities, pesticides had no statistically significant effect on MDA content in either fish species (Table 5). 4. Discussion Pesticides may induce oxidative stress leading to the generation of free radicals and cause lipid peroxidation as molecular mechanisms involved in pesticide-induced toxicity (Agrawal et al., 1991; Khrer, 1993; Almeida et al., 1997). Increased lipid peroxidation and oxidative stress can affect the activities of protective enzymatic antioxidants that have been shown to be sensitive indicators of increased oxidative stress. The present study evaluated the effects of different pesticides and their combined treatments on lipid peroxidation and antioxidant enzyme activities in different tissues of fish, C. carpio and O. niloticus. Although there were some differences in responses between the fish species, adaptive responses have been observed and a common mechanism of adaptive response is the induction of antioxidant enzymes. It was also found that antioxidant responses vary among tissues. Similar results were reported in various fish species (Radi and Matkovics, 1988; Winston and Di Giulio, 1991). Both oxidative responses and the antioxidant potential of fish differ depending on species habitat and feeding behaviour (Ahmad et al., 2000).
Various GSTs were seen to be at relatively high constitutive levels, especially in metabolically active tissues such as liver and kidney (Hayes and Pulford, 1995). GST-mediated conjugation may be an important mechanism for detoxifying peroxidised lipid breakdown products, which have a number of adverse biological effects when present in high amounts. Induced GST activity indicates the role of this enzyme in protection against the toxicity of xenobiotic-induced lipid peroxidation (Leaver and George, 1998). 2,4-D and azinphosmethyl treatments caused significant elevations in kidney GST activity. Elevated GST activity may reflect the possibility of better protection against pesticide toxicity. Recent in vitro studies demonstrated that a,bunsaturated aldehydes, produced as a result of cell membrane lipid peroxidation, increase GST mRNA and protein (Tjalkens et al., 1998). Bassi et al. (2000) showed the alterations in response to oxidative stress to be decreased lipid peroxidation and increased xenobiotic metabolism enzymes such as GST. In our study, GPx activity was increased in kidney after pesticide exposure. Ahmad et al. (2000) reported that increased GPx activity may be due to increased production and enzyme induction by H2O2 derived from Oy 2 . Hai et al. (1997) showed that GPx activation is enhanced in C. carpio and Ictalurus (now Ameiurus) nebulosus exposed to dichlorvos. Matkovics et al. (1984) have demonstrated the inductive effect of paraquat in C. carpio and Hypophthalmichthys molitrix. In endosulfan exposed fish, Clarias punctatus, elevated levels of GPx were found (Pandey et al., 2001). Decreased GPx activity in brain tissue of O. niloticus was observed. Brain is the target for oxidative damage of xenobiotics (Hai et al., 1997). The main factors that contribute to the vulnerability of brain include high content of polyunsaturated fatty acids in brain membrane and level of enzymatic and non-enzymatic antioxidants (PerezCampo et al., 1993). The cause of tissue differences could be due to different rates of free radical generation. Moreover, even though the brain is very susceptible to oxidative damage, it is not particularly enriched with antioxidant enzymes (Giuffrida Stella and Lajtha, 1987). Fish being more susceptible to oxidative damage generally have a high GPx activity (Aksnes and Njaa, 1981).
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GPx activity may be increased due to increased production and enzyme-inducing effect of H2O2 derived from Oy 2 . Fatima et al. (2000) stated that low activity of GPx in different tissues of exposed fish demonstrates inefficiency of these organs in neutralizing the impact of peroxides. The brain tissues of fish might be strongly protected against paraquat toxicity since the blood–brain barrier reduces the entry of paraquat from the plasma to the brain tissue in mammals (Rose and Smith, 1977). As a result of exposure to pesticides, an increased CAT activity was observed in kidney tissue of C. carpio. CAT activity in the kidney of O. niloticus was 23-fold higher than that in carp, and together with the high SOD activity can exert an abundant protective effect. A pro-oxidant condition elicited by the presence of pesticides could be triggering an increase in the activity of this antioxidant enzyme, as an adaptive response (Alves et al., 2002). The higher CAT activity might be in response to the increased oxygen consumption giving a great potential for H2O2 production (Ritola et al., 2002). Catalase is an inducible enzyme that protects the biological system against reactive oxygen species (Romeo et al., 2000). Different responses of CAT were found in fish after xenobiotic exposure. Increased activity of this enzyme was also observed in studies with C. carpio and Ictalurus punctatus after dichlorvos exposure (Hai et al., 1997). The treatment of Ameiurus nebulosus with menadione led to decreased CAT activity (Pandey et al., 2001). The gill showed the highest changes in SOD activity among the tissues. The highest SOD activity stimulated by pesticides was measured in C. carpio. Vig and Nemcsok (1989) stated that as a result of paraquat treatment, the changes in SOD activity were highest in the gill, considerable in the liver, but hardly perceptible in the brain. Since the gill is in direct contact with the medium, the damaging effect of paraquat in this tissue is extreme, whereas its effects are limited in liver and brain. The reduction of superoxide radicals to H2O2 is catalyzed by SOD. The increase in SOD activity may be due to increased generation of reactive oxygen species. Palace et al. (1996) have stated that SOD is the most responsive indicator of exposure to contaminants eliciting oxidative stress.
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Lipid peroxidation may be the first step of cellular membrane damage by pesticides. However, in this study, 2,4-D, azinphosmethyl and their combined treatments were unable to stimulate the lipid peroxidation process in the exposed fish, statistically. However, very slight stimulation of lipid peroxidation by pesticides cannot be excluded in the kidney and brain of C. carpio. Low levels or lack of lipid peroxidation in the tissues reflects the protective effects of oxidative enzymes. In conclusion, it is possible to restore susceptibility and to adapt to oxidative stress by increasing SOD and GST activities. The present study revealed that fish exposed to pesticides develop tissue-specific adaptive responses to protect cells against oxidative stress. C. carpio is found to be more sensitive than O. niloticus. Moreover, according to our results, the elevations in gill SOD activity and kidney GST activity serve as biomarkers of oxidative stress and may be helpful in assessing the risk of environmental contaminants. Acknowledgments We wish to thank The Cukurova University Grant Commission for their financial support. References Agrawal, D., Sultana, P., Gupta, G.S.D., 1991. Oxidative damage and changes in the glutathione redox system in erythrocytes from rats treated with hexachlorocyclohexane. Food Chem. Toxicol. 29, 459–462. Ahmad, I., Hamid, T., Fatima, M., et al., 2000. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus) is a biomarker of paper mill effluent exposure. Biochim. Biophys. Acta 1519, 37–48. Aksnes, A., Njaa, L.R., 1981. Catalase, glutathione peroxidase and superoxide dismutase in different fish species. Comp. Biochem. Physiol. B 69, 893–896. Almeida, M.G., Fanini, F., Davino, S.C., Aznar, A.E., Koch, O.R., Barros, S.B.M., 1997. Pro- and anti-oxidant parameters in rat liver after short-term exposure to hexachlorobenzene. Hum. Exp. Toxicol. 16, 257–261. Alves, S.R.C., Severino, P.C., Ibbotson, D.P., et al., 2002. Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae. Mar. Environ. Res. 54, 1–5. Bassi, A.M., Ledda, S., Penco, S., et al., 2000. Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation. Free Rad. Biol. Med. 29, 1186–1196. Begum, G., Vijayaraghavan, S., 1996. Alterations in protein metabolism of muscle tissue in the fish Clarias batrachus
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