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Combined effects of 2,4-D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus
Comparative Biochemistry and Physiology Part C 127 (2000) 291 – 296
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Combined effects of 2,4-D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus Elif O8 zcan Oruc¸ *, Nevin U8 ner Department of Biology, Faculty of Arts and Sciences, Uni6ersity of C ¸ ukuro6a, 01330 Balcali, Adana, Turkey Received 1 January 2000; received in revised form 3 August 2000; accepted 8 August 2000
Abstract This study aims to investigate the effects of the herbicide 2,4-D and the insecticide azinphosmethyl on hepatic antioxidant enzyme activities and lipid peroxidation in tilapia. Fish were exposed to 27 ppm 2,4-D, 0.03 ppm azinphosmethyl and to a mixture of both for 24, 48, 72 and 96 h. Activities of catalase (EC 1.11.1.6), glutathione-S-transferase (GST, EC 2.5.1.18) and the level of malondialdehyde (MDA) in the liver of Oreochromis niloticus exposed to 2,4-D and azinphosmethyl, both individually and in combination, were not affected by the pesticide exposures. However, glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and glutathione reductase (GR, EC 1.6.4.2) activities in individual and combined treatments, increased significantly compared to controls. Furthermore, glutathione peroxidase (GPx, EC 1.11.1.9) activity increased in individual treatment, while the same enzyme activity decreased in combination. 2,4-D did not affect the activity of superoxide dismutase (SOD, EC 1.15.1.1), but the activity of this enzyme in azinphosmethyl treatment decreased, while its activity increased in combination. Combined treatment of the pesticides exerted synergistic effects in the activity of SOD, while antagonistic effects were found in the activities of G6PD, GPx, GR. The results indicate that O. niloticus resisted oxidative stress by antioxidant mechanisms and prevented increases in lipid peroxidation. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Oreochromis niloticus; 2,4-D; Azinphosmethyl; Insecticide; Herbicide; Oxidative stress; Antioxidant enzyme; Lipid peroxidation
1. Introduction Aerobic organisms generate superoxide anion radical, (O− 2 ) hydrogen peroxide (H2O2) and hydroxyl radical (OH) as a result of oxidative metabolism. OH can initiate lipid peroxidation in tissues (Halliwell and Gutteridge, 1984). The sensitivity of the cell to oxidants is attenuated by antioxidant enzymes such as superoxide dismutase * Corresponding author. Fax: + 90-322-3386070. E-mail address: [email protected] (E.O8 . Oruc¸).
(SOD), glutathione peroxidase (GPx), catalase, glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). The antioxidant enzymes maintain a relatively low rate of the reactive and harmful OH. Oxidative stress occurs as a result of the effect of xenobiotics causing the disturbances in the antioxidant enzyme systems. Glutathione-S-transferase (GST) is a group of multifunctional enzyme involved in biotransformation and detoxification of xenobiotics (Smith and Litwack, 1980). Highly reactive electrophilic components can be removed before they cova-
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lently bind to tissue nucleophilic compounds which would lead to toxic effects. The liver was chosen due to its important role in degradation and bioactivation of pesticides. 2,4-D and azinphosmethyl are pesticides that have been used extensively in South Anatolia to control a wide variety of pests. However, these pesticides cause several metabolic alterations and tissue necrosis in non-target organisms including important members of the food chain such as fish (Marinovich et al., 1994; Singh and Bhati, 1994; Palmeira et al., 1997). Earlier studies revealed that these pesticides alter protein and carbohydrate metabolism and related enzyme activity levels (Oruc¸ and U8 ner, 1998, 1999). 2,4-D produces hepatotoxicity and initiates the process of cell death by decreasing cellular reduced glutathione (GSH), ATP and NADH (Palmeira et al., 1994). However, little is known about its toxic mechanism in fish, in spite of its widespread application in agriculture. Although GST is an important enzyme in the detoxification of 2,4-D in Oncorhynchus mykiss, in vitro studies indicate that the enzyme activity decreases (Dierickx, 1985). Nevertheless, it is argued that the results of in vivo studies may be different. For example, Ictalurus punctatus exposed to picloram, 2,4-D and their mixture for 10 days did not exhibit an alteration in the activities of GST and catalase (Gallagher and Di Giulio, 1991). The effects of individual treatments of 2,4-D and azinphosmethyl are not explored sufficiently in fish. Also, no results are recorded related to biochemical and physiological effects of their combined treatments in fish. Therefore, it is believed that the evaluation of the effects of the individual and combined treatments with 2,4-D and azinphosmethyl on the antioxidant enzyme activities and lipid peroxidation in liver of Oreochromis niloticus, would contribute significantly to the understanding of pesticide effects.
2. Materials and methods Male O. niloticus (Perciformes: Cichlidae) (131.40 930.91 g, 21.229 2.82 cm) were taken from unpolluted fish culture pools at the University of C ¸ ukurova and transferred to the laboratory where the temperature was kept at 209 2°C (12:12 L:D). Tap water was used throughout, at a pH of 7.60, an alkalinity of 326 ppm CaCO3, and
an oxygen concentration of 7.02 mg/l. The fish were allowed to acclimatise to these conditions for 2 weeks and were fed with commercial fish food (Pinar, Turkey). Commercial 2,4-D (2,4-dichlorophenoxy acetic acid dimethyl amine salt, Bayer, 500 g/l) and azinphosmethyl {S-(3,4-dihydro-4-oxobenzo [d] [1,2,3] triazin-3-ylmethyl) O-dimethyl phosphorodithioate, Bayer, Guthion EC 20, 230 g/l} were used in aquaria containing 160 l test solution. LC50 values for 96 h of 2,4-D and azinphosmethyl to O. niloticus were found to be 80 and 0.09 ppm, respectively. The fish were exposed to 27 ppm 2,4-D, 0.03 ppm azinphosmethyl, 27 ppm 2,4D+ 0.03 ppm azinphosmethyl for 24, 48, 72, and 96 h. A control group was maintained in tap water. Six fish were put into each aquarium and the water was refreshed every 2 days. At the end of the experiments, fish were killed by decapitation. Liver tissues were dissected and put in petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples were taken and kept at − 85°C until analysis. The tissues were homogenized for 5 min in 1.15% KCl solution (1:10 w/v) using a glass-teflon homogenizer and then centrifuged at 9500× g for 30 min. All processes were carried out at 4°C. Supernatants were used to determine the antioxidant enzyme activities and the malondialdehyde (MDA) levels. GR (EC 1.6.4.2) activity was assayed by following at 37°C and 340 nm the oxidation of NADPH by oxidised glutathione (GSSG) (Beutler, 1984). G6PD (EC 1.1.1.49) activity was determined at 37°C and 340 nm in the presence of 1 M Tris– HCl, 5 mM EDTA (pH 8.0), 0.1 M MgCl2, 2 mM NADP, water and 6 mM G6P (Beutler, 1984). When superoxide radicals, which occur as a result of the reactions between xanthine and xanthine oxidase, and 2-[4-iodophenyl]-3-[4-nitrophenyl]-5phenyltetrazolium chloride (INT) react, a red coloured formazon is formed which is used to determine the SOD (EC 1.15.1.1) activity. In the presence of SOD in the medium, superoxide radicals are removed and the formation of formazon is therefore inhibited. SOD activity is measured at 505 nm and 37°C and calculated with inhibition percent of formazon formation (McCord and Fridovich, 1969). To determine GPx (EC 1.11.1.9) activity, t-butyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GPx and NADPH. The activity of GPx was as-
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sayed at 37°C and 340 nm by calculating the difference in absorbance values during the oxidation of NADPH to NADP+ (Beutler, 1984). One M Tris–HCl, 5mM EDTA (pH 8.0), 10 mM H2O2 and H2O were mixed and the rate of H2O2 consumption at 230 nm and 37°C was used for quantitative determination of CAT (EC 1.11.1.6) activity (Beutler, 1984). An extinction coefficient for H2O2 at 230 nm was used to calculate the activity of the enzyme. GST (EC 2.5.1.18) was assayed at 25°C spectrophotometrically by following the conjugation of glutathione with 1-chloro2,4-dinitrobenzene (CDNB) at 340 nm as described by Mannervik and Guthenberg (1981). MDA occurs in lipid peroxidation and was measured after incubation at 95°C with thiobarbituric acid in aerobic conditions (pH 3.4). The pink colour produced by these reactions was measured spectrophotometrically at 532 nm to measure MDA levels (Ohkawa et al., 1979). Specific activity is defined as units of activity per mg of protein. Protein was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. Oneway analysis of variance (ANOVA) was used to determine the treatment toxic effects, and Duncan’s Significant Difference Test was used for mean separation (Duncan, 1955).
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Fig. 2. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glutathione reductase (GR) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9S.D. (n =6). * PB 0.01.
The specific activity of G6PD after 2,4-D and the combination exposures for 24 h was higher than the control and azinphosmethyl treatment groups (Fig. 1). Azinphosmethyl exposure for 24 h did not alter the G6PD activity. However, 2,4-D exposure for 96 h did not affect the enzyme activity while azinphosmethyl and 2,4-D+azin-
phosmethyl exposures caused an elevation (PB 0.01). Also, an antagonistic effect was found in mixture exposure of pesticide. The specific activity of GR increased following exposure to pesticides for 24, 72 and 96 h (PB 0.01). For 48 h, the activity did not change when 2,4-D and azinphosmethyl were applied individually, but it increased when they were applied in combination (Fig. 2). An antagonistic effect was borderline after combined treatment for 48 h. The specific activity of GPx did not change after the individual and combined treatment for 24 h (Fig. 3). Individual treatment with pesticides for 48 and 72 h did not change the GPx specific activity while the combined treatment caused a decline. After an individual treatment for 96 h, GPx activity was significantly higher than in the other groups. 2,4-D+ azinphosmethyl exposure for 96 h did not affect the GPx activity. The results indicate that combined exposure of pesticides produced antagonistic effect in the GPx specific activity. The activity of SOD remained at control levels after individual treatment with of 2,4-D or azinphosmethyl for 24, 48 and 72 h, while the com-
Fig. 1. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glucose-6-phosphate dehydrogenase (G-6PD) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9 S.D. (n=6). * PB 0.01.
Fig. 3. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glutathione peroxidase (GPx) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9S.D. (n =6). * PB 0.01.
3. Results
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Fig. 4. Effects of 2,4-D, azinphosmethyl and their combined treatment on superoxide dismutase (SOD) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means9S.D. (n =6). * PB 0.01.
bined treatments with pesticides increased the SOD specific activity (P B 0.01) (Fig. 4). After 96 h exposure, 2,4-D did not affect the enzyme activity, while azinphosmethyl decreased the enzyme activity. Also, an elevation was observed in the activity after the combined treatment for 96 h. Combined treatment for 24, 48, 72 h caused a slightly additive effect, while combined treatment for 96 h had a synergistic effect on the SOD specific activity. Twenty seven ppm 2,4-D, 0.03 ppm azinphosmethyl and the mixture of both neither affect of the specific activities of catalase and GST nor the level of MDA (P\ 0.05). Control values are given in Table 1.
4. Discussion The SOD activity in the liver of O. niloticus after 2,4-D + azinphosmethyl exposure was higher than in the control, or 2,4-D and azinphosmethyl exposed groups. The same enzyme activity in fish exposed to azinphosmethyl for 96 h is less than in the other treatment groups. O− 2 is dismutated by SOD to H2O2. Induction of SOD could occur during high production of superoxide anion radi-
cal. Therefore, an increase in the SOD activity indicates an increase of O− 2 production. Similar to the authors’ results, the SOD isomers in the liver of Leuciscus cephalus increased as a result of pollution (Lenartova et al., 1997). In contrast, the superoxide radicals by themselves or after their transformation to H2O2 cause an oxidation of the cysteine in the enzyme and decrease SOD activity (Dimitrova et al., 1994). Decreases in SOD activity level were found in erythrocytes of Cyprinus carpio exposed to MS 222 (Bartowiak et al., 1981). The sensitivity of a cell to oxidants is decreased by antioxidant enzymes (Amstad et al., 1991). Nevertheless, the physiological role of a single antioxidant enzyme in the cell is poorly understood because of complex interactions and interrelationships among individual components. SOD, GPx and catalase enzymes are related in the terms of their functions (Amstad et al., 1994). However, in this study, no parallel alteration was found in the activities of SOD-catalase and SOD-GPx in the liver of O. niloticus after treatment with 2,4-D, azinphosmethyl or their mixture treatments. Catalase removes H2O2. Therefore, the SODcatalase system provides the first defense against oxygen toxicity. It is reported that SOD and catalase activities increased at the combined effect of zinc and lead for 24 h and 10 days in C. carpio (Dimitrova et al., 1994). However, in the current study, 2,4-D and azinphosmethyl and combined treatments did not affect the activity of catalase in the liver of O. niloticus. Similar to the present observation, combined effect of 2,4-D and picloram in fish did not alter the catalase activity (Gallagher and Di Giulio, 1991). Lindane (60 mg/kg b.w.) (Jungueira et al., 1988) and 10 − 4 M thiram (Babo and Vasseur, 1992) caused an increase in catalase activity, while a decline in its activity was reported at 10 − 6 and 10 − 8 M thiram doses (Babo and Vasseur, 1992).
Table 1 The levels of catalase specific activity (U/mg protein), glutathione S-transferase (GST) specific activity (U/mg protein) and the content of malondialdehyde (MDA) (nmol/mg protein) in the liver of Oreochromis niloticus a Exposure duration
Catalase GST MDA a
24 h
48 h
72 h
96 h
100.9693.66 820.579 252.17 5.229 0.56
106.4792.70 825.489 218.41 4.919 0.69
95.19 929.72 832.17 9 146.86 5.80 90.25
105.42 9 4.49 120.63 978.10 5.06 9 2.21
Values are means 9 S.D. (n= 6).
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An increase in G6PD activity was observed in the liver tissue of O. niloticus as a consequence of pesticide toxicity. This alteration may indicate the operation of hexose monophosphate pathway, since the increased G6PD activity facilitates increased production of NADPH for detoxification processes. Several studies show increased G6PD activity in response to pesticide toxicity (Rao and Rao, 1987; Reddy and Yellama, 1991). Diplodus annularis living in polluted sea water displayed an increase of the G6PD activity, while GPx activity was reduced (Bagnasco et al., 1991). Similarly, the current study showed that G6PD and GR activities were increased after pesticide exposure, while GPx activity was reduced after the combined effect of 2,4-D and azinphosmethyl for 48 and 72 h. The contrast, GPx activity was elevated after 2,4D and azinphosmethyl treatments for 96 h. GPx activity was reduced in 90 days of lindane treatment and catalase activity decreased in 60 and 90 days treatments, while the content of thiobarbiturate increased. Despite these changes, lindane treatment did not cause liver injury. Therefore, the antioxidant mechanisms seem to operate at an adequate level to deal with the mediators of cell injury (Bainy et al., 1993). The decreased activity of GPx may be the result of O− production 2 (Bagnasco et al., 1991) or a direct action of pesticides on the synthesis of the enzyme (Bainy et al., 1993). Oxidative stress causes an elevation in the GPx activity. This probably could reflect an adaptation to oxidative conditions to which fish had been exposed (Lenartova et al., 1997). MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation (Freeman and Crapo, 1981). MDA content did not vary significantly after 2,4-D, azinphosmethyl and their combined treatments, indicating lipid peroxidation did not increase in O. niloticus exposed to pesticide. In this study, 2,4-D, azinphosmethyl and their mixtures treatments did not affect the activity of GST. Similarly, 4-chloro-2-methyl phenoxy acetic acid (MCPA) exposure for 3 days did not affect the activity of GST in liver and kidney tissues of C. carpio (Riviere et al., 1990). I. punctatus exposed to picloram, 2,4-D and their mixtures for 10 days did not show any alteration in GST activity (Gallagher and Di Giulio, 1991). However, a decreased GST activity was found in D. annularis (Dimitrova et al., 1994) and D. labrax
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(Lemaire et al., 1996). It was also shown that the activity of GST in the liver of O. mykiss was stimulated after atrazin exposure for 14 days (Egaas et al., 1993). Findings of these studies suggest that GST activity is dependent on interspecies differences, as well as on xenobiotic type and concentration and exposure period. It was concluded that the metabolism of pesticide-exposed O. niloticus resisted the oxidative stress using the antioxidant mechanism and prevented the increase of lipid peroxidation. Acknowledgements We thank Professor Dr Levent Kayrin for his valuable help. We are also greatful to Dr Nazmi Tekelioglu for providing fish. This work was supported by grant from The University Grant Commission (The University of C ¸ ukurova, No. FBE. 96-166). References Amstad, P., Peskin, A., Shah, A.G., Mirault, M.E., Moret, R., Zbinden, I., Cerutti, P., 1991. The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress. Biochemist 30, 9305 – 9313. Amstad, P., Moret, R., Cerutti, P., 1994. Glutathione peroxidase compensates for the hypersensitivity of Cu, Zn-superoxide dismutase overproducers to oxidant stress. J. Biol. Chem. 269 (3), 1606 – 1609. Babo, S., Vasseur, P., 1992. In vitro effects of thiram on liver antioxidant enzyme activities of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 22, 6168. Bagnasco, M., Camoirano, A., De Flora, S., Melodia, F., Arillo, A., 1991. Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. Mutat. Res. 262, 129 – 137. Bainy, A.C.D., Arisi, A.C.M., Azzalis, L.A., Simizu, K., Barios, S.B.M., Videla, L.A., Jungueira, V.B.C., 1993. Differential effects of short-term lindane administration on parameters related to oxidative stress in rat liver and erythrocytes. J. Biochem. Toxicol. 8 (4), 187 – 194. Bartowiak, A., Grzelinska, E., Varga, I.S., Leyko, W., 1981. Studies on superoxide dismutase from cod (Gadus morhua) liver. Int. J. Biochem. 13, 1039 – 1042. Beutler, E., 1984. Red Cell Metabolism: A Manual of Biochemical Methods, 2nd edition. Grune and Starton, New York.
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Dierickx, P.J., 1985. Hepatic glutathione-S-transferases in rainbow trout and their interaction with 2,4dichlorophenoxyacetic acid and 1,4-benzoquinon. Comp. Biochem. Physiol. 82C (21), 495–500. Dimitrova, M.S.T., Tsinova, V., Velcheva, V., 1994. Combined effect of zinc and lead on the hepatic superoxide dismutase-catalase system in carp (Cyprinus carpio). Comp. Biochem. Physiol. 108C (1), 43–46. Duncan, D.B., 1955. Multiple range and multiple f tests. Biometrics 11, 1–42. Egaas, E., Skaare, J.U., Swendsen, N.O., Sandvik, M., Falls, J.G., Dauterman, W.C., Collier, T.K., Netland, J., 1993. A comparative study of effects of atrazine on xenobiotic metabolizing enzymes in fish and insect, and of the in vitro phase II atrazine metabolism in some fish, insects, mammals and one plant species. Comp. Biochem. Physiol. 160C (1), 141–149. Freeman, B.A., Crapo, J.D., 1981. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256, 10986–10992. Gallagher, E.P., Di Giulio, R.T., 1991. Effects of 2,4dichlorophenoxy-acetic acid and picloram on biotransformation, peroxisomal and serum enzyme activities in channel catfish (Ictalurus punctatus). Toxicol. Lett. 57, 65–72. Halliwell, B., Gutteridge, J.M.C., 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1–14. Jungueira, V.B.C., Simizu, K., Halsema, L.V., Koch, O.R., Barios, S.B.M., Videla, A.L., 1988. Lindaneinduced oxidative stress. I. Time course of changes in hepatic microsomal parameters, antioxidant enzymes, lipid peroxidative indices and morphological characteristics. Xenobiotica 18 (11), 1297–1304. Lemaire, P., Forlin, L., Livingstone, D.R., 1996. Responses of hepatic biotransformation and antioxidant enzymes to cyp1a-inducers (3-methylcholanthrene, b-naphthoflavone) in sea bass (Dicentrarchus labrax), dab (Limanda limanda) and rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 36, 141–160. Lenartova, V., Holovska, K., Pedrajas, J.R., MartinezLara, E., Peinado, J., Lopez-Barea, J., Rosival, I., Kosuth, P., 1997. Antioxidant and detoxifying fish enzymes as biomarkers of river pollution. Biomarkers 2, 247–252. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193, 265–275.
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Mannervik, B., Guthenberg, C., 1981. Glutathione-Stransferase (Human placenta). Methods Enzymol. 77, 231 – 235. Marinovich, M., Guizette, M., Galli, L.C., 1994. Mixtures of benomyl, pirimiphos-methyl, dimethoate, diazinon and azinphos-methyl affect protein synthesis in HL-60 cells differently. Toxicology 94, 173 – 185. 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 – 362. Oruc¸, E.O8 ., U8 ner, N., 1998. Effects of azinphosmethyl on some biochemical parameters in blood, muscle, and liver tissues of Cyprinus carpio (L.). Pestic. Biochem. Physiol. 62, 65 – 71. Oruc¸, E.O8 ., U8 ner, N., 1999. Effects of 2,4-D on some biochemical parameters of protein and carbohydrate metabolisms in the serum, muscle, and liver tissues of Cyprinus carpio (L.). Environ. Pollut. 105, 267 – 272. Palmeira, C.M., Moreno, A.J., Madeira, V.M.C., 1994. Metabolic alterations in hepatocytes promoted by the herbicides paraquat, dinoseb and 2,4-D. Arch. Toxicol. 68, 24 – 31. Palmeira, C.M., Moreno, A.J., Bairos, V., Madeira, V.M.C., 1997. Structural alterations in isolated hepatocytes induced by the herbicides paraquat, dinoseb and 2,4-D. Med. Sci. Res. 25, 339 – 342. Rao, K.S.P., Rao, K.V.R., 1987. The possible role of glucose-6-phosphate dehydrogenase in the detoxification of methyl parathion. Toxicol. Lett. 39, 211 – 214. Reddy, Y., Yellama, K., 1991. Perturbations in carbohydrate metabolism during cypermethrin toxicity in fish, Oreochromis mossambica. Biochem. Int. 23 (4), 633 – 638. Riviere, J.L., Devaux, A., Gonin, O., Monod, G., 1990. Effects of b-naphtoflavone and MCPA on liver and kidney drug-metabolizing enzymes from the carp, Cyprinus carpio. Ecotoxicol. Environ. Safe. 19, 276 – 284. Singh, S., Bhati, D.P.S., 1994. Evaluation of liver protein due to stress under 2,4-D intoxication in Channa punctatus (Bloch.). Bull. Environ. Contam. Toxicol. 53, 149 – 152. Smith, G.J., Litwack, G., 1980. Roles of ligandin and the glutathione S-transferases in binding steroid metabolites, carcinogens and other compounds. Rev. Biochem. Toxicol. 2, 1 – 47.
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Combined effects of 2,4-D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus Elif O8 zcan Oruc¸ *, Nevin U8 ner Department of Biology, Faculty of Arts and Sciences, Uni6ersity of C ¸ ukuro6a, 01330 Balcali, Adana, Turkey Received 1 January 2000; received in revised form 3 August 2000; accepted 8 August 2000
Abstract This study aims to investigate the effects of the herbicide 2,4-D and the insecticide azinphosmethyl on hepatic antioxidant enzyme activities and lipid peroxidation in tilapia. Fish were exposed to 27 ppm 2,4-D, 0.03 ppm azinphosmethyl and to a mixture of both for 24, 48, 72 and 96 h. Activities of catalase (EC 1.11.1.6), glutathione-S-transferase (GST, EC 2.5.1.18) and the level of malondialdehyde (MDA) in the liver of Oreochromis niloticus exposed to 2,4-D and azinphosmethyl, both individually and in combination, were not affected by the pesticide exposures. However, glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and glutathione reductase (GR, EC 1.6.4.2) activities in individual and combined treatments, increased significantly compared to controls. Furthermore, glutathione peroxidase (GPx, EC 1.11.1.9) activity increased in individual treatment, while the same enzyme activity decreased in combination. 2,4-D did not affect the activity of superoxide dismutase (SOD, EC 1.15.1.1), but the activity of this enzyme in azinphosmethyl treatment decreased, while its activity increased in combination. Combined treatment of the pesticides exerted synergistic effects in the activity of SOD, while antagonistic effects were found in the activities of G6PD, GPx, GR. The results indicate that O. niloticus resisted oxidative stress by antioxidant mechanisms and prevented increases in lipid peroxidation. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Oreochromis niloticus; 2,4-D; Azinphosmethyl; Insecticide; Herbicide; Oxidative stress; Antioxidant enzyme; Lipid peroxidation
1. Introduction Aerobic organisms generate superoxide anion radical, (O− 2 ) hydrogen peroxide (H2O2) and hydroxyl radical (OH) as a result of oxidative metabolism. OH can initiate lipid peroxidation in tissues (Halliwell and Gutteridge, 1984). The sensitivity of the cell to oxidants is attenuated by antioxidant enzymes such as superoxide dismutase * Corresponding author. Fax: + 90-322-3386070. E-mail address: [email protected] (E.O8 . Oruc¸).
(SOD), glutathione peroxidase (GPx), catalase, glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). The antioxidant enzymes maintain a relatively low rate of the reactive and harmful OH. Oxidative stress occurs as a result of the effect of xenobiotics causing the disturbances in the antioxidant enzyme systems. Glutathione-S-transferase (GST) is a group of multifunctional enzyme involved in biotransformation and detoxification of xenobiotics (Smith and Litwack, 1980). Highly reactive electrophilic components can be removed before they cova-
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lently bind to tissue nucleophilic compounds which would lead to toxic effects. The liver was chosen due to its important role in degradation and bioactivation of pesticides. 2,4-D and azinphosmethyl are pesticides that have been used extensively in South Anatolia to control a wide variety of pests. However, these pesticides cause several metabolic alterations and tissue necrosis in non-target organisms including important members of the food chain such as fish (Marinovich et al., 1994; Singh and Bhati, 1994; Palmeira et al., 1997). Earlier studies revealed that these pesticides alter protein and carbohydrate metabolism and related enzyme activity levels (Oruc¸ and U8 ner, 1998, 1999). 2,4-D produces hepatotoxicity and initiates the process of cell death by decreasing cellular reduced glutathione (GSH), ATP and NADH (Palmeira et al., 1994). However, little is known about its toxic mechanism in fish, in spite of its widespread application in agriculture. Although GST is an important enzyme in the detoxification of 2,4-D in Oncorhynchus mykiss, in vitro studies indicate that the enzyme activity decreases (Dierickx, 1985). Nevertheless, it is argued that the results of in vivo studies may be different. For example, Ictalurus punctatus exposed to picloram, 2,4-D and their mixture for 10 days did not exhibit an alteration in the activities of GST and catalase (Gallagher and Di Giulio, 1991). The effects of individual treatments of 2,4-D and azinphosmethyl are not explored sufficiently in fish. Also, no results are recorded related to biochemical and physiological effects of their combined treatments in fish. Therefore, it is believed that the evaluation of the effects of the individual and combined treatments with 2,4-D and azinphosmethyl on the antioxidant enzyme activities and lipid peroxidation in liver of Oreochromis niloticus, would contribute significantly to the understanding of pesticide effects.
2. Materials and methods Male O. niloticus (Perciformes: Cichlidae) (131.40 930.91 g, 21.229 2.82 cm) were taken from unpolluted fish culture pools at the University of C ¸ ukurova and transferred to the laboratory where the temperature was kept at 209 2°C (12:12 L:D). Tap water was used throughout, at a pH of 7.60, an alkalinity of 326 ppm CaCO3, and
an oxygen concentration of 7.02 mg/l. The fish were allowed to acclimatise to these conditions for 2 weeks and were fed with commercial fish food (Pinar, Turkey). Commercial 2,4-D (2,4-dichlorophenoxy acetic acid dimethyl amine salt, Bayer, 500 g/l) and azinphosmethyl {S-(3,4-dihydro-4-oxobenzo [d] [1,2,3] triazin-3-ylmethyl) O-dimethyl phosphorodithioate, Bayer, Guthion EC 20, 230 g/l} were used in aquaria containing 160 l test solution. LC50 values for 96 h of 2,4-D and azinphosmethyl to O. niloticus were found to be 80 and 0.09 ppm, respectively. The fish were exposed to 27 ppm 2,4-D, 0.03 ppm azinphosmethyl, 27 ppm 2,4D+ 0.03 ppm azinphosmethyl for 24, 48, 72, and 96 h. A control group was maintained in tap water. Six fish were put into each aquarium and the water was refreshed every 2 days. At the end of the experiments, fish were killed by decapitation. Liver tissues were dissected and put in petri dishes. After washing the tissues with physiological saline (0.9% NaCl), samples were taken and kept at − 85°C until analysis. The tissues were homogenized for 5 min in 1.15% KCl solution (1:10 w/v) using a glass-teflon homogenizer and then centrifuged at 9500× g for 30 min. All processes were carried out at 4°C. Supernatants were used to determine the antioxidant enzyme activities and the malondialdehyde (MDA) levels. GR (EC 1.6.4.2) activity was assayed by following at 37°C and 340 nm the oxidation of NADPH by oxidised glutathione (GSSG) (Beutler, 1984). G6PD (EC 1.1.1.49) activity was determined at 37°C and 340 nm in the presence of 1 M Tris– HCl, 5 mM EDTA (pH 8.0), 0.1 M MgCl2, 2 mM NADP, water and 6 mM G6P (Beutler, 1984). When superoxide radicals, which occur as a result of the reactions between xanthine and xanthine oxidase, and 2-[4-iodophenyl]-3-[4-nitrophenyl]-5phenyltetrazolium chloride (INT) react, a red coloured formazon is formed which is used to determine the SOD (EC 1.15.1.1) activity. In the presence of SOD in the medium, superoxide radicals are removed and the formation of formazon is therefore inhibited. SOD activity is measured at 505 nm and 37°C and calculated with inhibition percent of formazon formation (McCord and Fridovich, 1969). To determine GPx (EC 1.11.1.9) activity, t-butyl hydroperoxide was used. The GSSG in the medium was reduced to GSH by GPx and NADPH. The activity of GPx was as-
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sayed at 37°C and 340 nm by calculating the difference in absorbance values during the oxidation of NADPH to NADP+ (Beutler, 1984). One M Tris–HCl, 5mM EDTA (pH 8.0), 10 mM H2O2 and H2O were mixed and the rate of H2O2 consumption at 230 nm and 37°C was used for quantitative determination of CAT (EC 1.11.1.6) activity (Beutler, 1984). An extinction coefficient for H2O2 at 230 nm was used to calculate the activity of the enzyme. GST (EC 2.5.1.18) was assayed at 25°C spectrophotometrically by following the conjugation of glutathione with 1-chloro2,4-dinitrobenzene (CDNB) at 340 nm as described by Mannervik and Guthenberg (1981). MDA occurs in lipid peroxidation and was measured after incubation at 95°C with thiobarbituric acid in aerobic conditions (pH 3.4). The pink colour produced by these reactions was measured spectrophotometrically at 532 nm to measure MDA levels (Ohkawa et al., 1979). Specific activity is defined as units of activity per mg of protein. Protein was determined by the Lowry method (Lowry et al., 1951) using bovine serum albumin as a standard. Oneway analysis of variance (ANOVA) was used to determine the treatment toxic effects, and Duncan’s Significant Difference Test was used for mean separation (Duncan, 1955).
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Fig. 2. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glutathione reductase (GR) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9S.D. (n =6). * PB 0.01.
The specific activity of G6PD after 2,4-D and the combination exposures for 24 h was higher than the control and azinphosmethyl treatment groups (Fig. 1). Azinphosmethyl exposure for 24 h did not alter the G6PD activity. However, 2,4-D exposure for 96 h did not affect the enzyme activity while azinphosmethyl and 2,4-D+azin-
phosmethyl exposures caused an elevation (PB 0.01). Also, an antagonistic effect was found in mixture exposure of pesticide. The specific activity of GR increased following exposure to pesticides for 24, 72 and 96 h (PB 0.01). For 48 h, the activity did not change when 2,4-D and azinphosmethyl were applied individually, but it increased when they were applied in combination (Fig. 2). An antagonistic effect was borderline after combined treatment for 48 h. The specific activity of GPx did not change after the individual and combined treatment for 24 h (Fig. 3). Individual treatment with pesticides for 48 and 72 h did not change the GPx specific activity while the combined treatment caused a decline. After an individual treatment for 96 h, GPx activity was significantly higher than in the other groups. 2,4-D+ azinphosmethyl exposure for 96 h did not affect the GPx activity. The results indicate that combined exposure of pesticides produced antagonistic effect in the GPx specific activity. The activity of SOD remained at control levels after individual treatment with of 2,4-D or azinphosmethyl for 24, 48 and 72 h, while the com-
Fig. 1. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glucose-6-phosphate dehydrogenase (G-6PD) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9 S.D. (n=6). * PB 0.01.
Fig. 3. Effects of 2,4-D, azinphosmethyl and their combined treatment on the glutathione peroxidase (GPx) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means 9S.D. (n =6). * PB 0.01.
3. Results
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Fig. 4. Effects of 2,4-D, azinphosmethyl and their combined treatment on superoxide dismutase (SOD) specific activity (U/mg protein) in the liver of Oreochromis niloticus. Values are means9S.D. (n =6). * PB 0.01.
bined treatments with pesticides increased the SOD specific activity (P B 0.01) (Fig. 4). After 96 h exposure, 2,4-D did not affect the enzyme activity, while azinphosmethyl decreased the enzyme activity. Also, an elevation was observed in the activity after the combined treatment for 96 h. Combined treatment for 24, 48, 72 h caused a slightly additive effect, while combined treatment for 96 h had a synergistic effect on the SOD specific activity. Twenty seven ppm 2,4-D, 0.03 ppm azinphosmethyl and the mixture of both neither affect of the specific activities of catalase and GST nor the level of MDA (P\ 0.05). Control values are given in Table 1.
4. Discussion The SOD activity in the liver of O. niloticus after 2,4-D + azinphosmethyl exposure was higher than in the control, or 2,4-D and azinphosmethyl exposed groups. The same enzyme activity in fish exposed to azinphosmethyl for 96 h is less than in the other treatment groups. O− 2 is dismutated by SOD to H2O2. Induction of SOD could occur during high production of superoxide anion radi-
cal. Therefore, an increase in the SOD activity indicates an increase of O− 2 production. Similar to the authors’ results, the SOD isomers in the liver of Leuciscus cephalus increased as a result of pollution (Lenartova et al., 1997). In contrast, the superoxide radicals by themselves or after their transformation to H2O2 cause an oxidation of the cysteine in the enzyme and decrease SOD activity (Dimitrova et al., 1994). Decreases in SOD activity level were found in erythrocytes of Cyprinus carpio exposed to MS 222 (Bartowiak et al., 1981). The sensitivity of a cell to oxidants is decreased by antioxidant enzymes (Amstad et al., 1991). Nevertheless, the physiological role of a single antioxidant enzyme in the cell is poorly understood because of complex interactions and interrelationships among individual components. SOD, GPx and catalase enzymes are related in the terms of their functions (Amstad et al., 1994). However, in this study, no parallel alteration was found in the activities of SOD-catalase and SOD-GPx in the liver of O. niloticus after treatment with 2,4-D, azinphosmethyl or their mixture treatments. Catalase removes H2O2. Therefore, the SODcatalase system provides the first defense against oxygen toxicity. It is reported that SOD and catalase activities increased at the combined effect of zinc and lead for 24 h and 10 days in C. carpio (Dimitrova et al., 1994). However, in the current study, 2,4-D and azinphosmethyl and combined treatments did not affect the activity of catalase in the liver of O. niloticus. Similar to the present observation, combined effect of 2,4-D and picloram in fish did not alter the catalase activity (Gallagher and Di Giulio, 1991). Lindane (60 mg/kg b.w.) (Jungueira et al., 1988) and 10 − 4 M thiram (Babo and Vasseur, 1992) caused an increase in catalase activity, while a decline in its activity was reported at 10 − 6 and 10 − 8 M thiram doses (Babo and Vasseur, 1992).
Table 1 The levels of catalase specific activity (U/mg protein), glutathione S-transferase (GST) specific activity (U/mg protein) and the content of malondialdehyde (MDA) (nmol/mg protein) in the liver of Oreochromis niloticus a Exposure duration
Catalase GST MDA a
24 h
48 h
72 h
96 h
100.9693.66 820.579 252.17 5.229 0.56
106.4792.70 825.489 218.41 4.919 0.69
95.19 929.72 832.17 9 146.86 5.80 90.25
105.42 9 4.49 120.63 978.10 5.06 9 2.21
Values are means 9 S.D. (n= 6).
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An increase in G6PD activity was observed in the liver tissue of O. niloticus as a consequence of pesticide toxicity. This alteration may indicate the operation of hexose monophosphate pathway, since the increased G6PD activity facilitates increased production of NADPH for detoxification processes. Several studies show increased G6PD activity in response to pesticide toxicity (Rao and Rao, 1987; Reddy and Yellama, 1991). Diplodus annularis living in polluted sea water displayed an increase of the G6PD activity, while GPx activity was reduced (Bagnasco et al., 1991). Similarly, the current study showed that G6PD and GR activities were increased after pesticide exposure, while GPx activity was reduced after the combined effect of 2,4-D and azinphosmethyl for 48 and 72 h. The contrast, GPx activity was elevated after 2,4D and azinphosmethyl treatments for 96 h. GPx activity was reduced in 90 days of lindane treatment and catalase activity decreased in 60 and 90 days treatments, while the content of thiobarbiturate increased. Despite these changes, lindane treatment did not cause liver injury. Therefore, the antioxidant mechanisms seem to operate at an adequate level to deal with the mediators of cell injury (Bainy et al., 1993). The decreased activity of GPx may be the result of O− production 2 (Bagnasco et al., 1991) or a direct action of pesticides on the synthesis of the enzyme (Bainy et al., 1993). Oxidative stress causes an elevation in the GPx activity. This probably could reflect an adaptation to oxidative conditions to which fish had been exposed (Lenartova et al., 1997). MDA is a major oxidation product of peroxidized polyunsaturated fatty acids and increased MDA content is an important indicator of lipid peroxidation (Freeman and Crapo, 1981). MDA content did not vary significantly after 2,4-D, azinphosmethyl and their combined treatments, indicating lipid peroxidation did not increase in O. niloticus exposed to pesticide. In this study, 2,4-D, azinphosmethyl and their mixtures treatments did not affect the activity of GST. Similarly, 4-chloro-2-methyl phenoxy acetic acid (MCPA) exposure for 3 days did not affect the activity of GST in liver and kidney tissues of C. carpio (Riviere et al., 1990). I. punctatus exposed to picloram, 2,4-D and their mixtures for 10 days did not show any alteration in GST activity (Gallagher and Di Giulio, 1991). However, a decreased GST activity was found in D. annularis (Dimitrova et al., 1994) and D. labrax
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(Lemaire et al., 1996). It was also shown that the activity of GST in the liver of O. mykiss was stimulated after atrazin exposure for 14 days (Egaas et al., 1993). Findings of these studies suggest that GST activity is dependent on interspecies differences, as well as on xenobiotic type and concentration and exposure period. It was concluded that the metabolism of pesticide-exposed O. niloticus resisted the oxidative stress using the antioxidant mechanism and prevented the increase of lipid peroxidation. Acknowledgements We thank Professor Dr Levent Kayrin for his valuable help. We are also greatful to Dr Nazmi Tekelioglu for providing fish. This work was supported by grant from The University Grant Commission (The University of C ¸ ukurova, No. FBE. 96-166). References Amstad, P., Peskin, A., Shah, A.G., Mirault, M.E., Moret, R., Zbinden, I., Cerutti, P., 1991. The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress. Biochemist 30, 9305 – 9313. Amstad, P., Moret, R., Cerutti, P., 1994. Glutathione peroxidase compensates for the hypersensitivity of Cu, Zn-superoxide dismutase overproducers to oxidant stress. J. Biol. Chem. 269 (3), 1606 – 1609. Babo, S., Vasseur, P., 1992. In vitro effects of thiram on liver antioxidant enzyme activities of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 22, 6168. Bagnasco, M., Camoirano, A., De Flora, S., Melodia, F., Arillo, A., 1991. Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. Mutat. Res. 262, 129 – 137. Bainy, A.C.D., Arisi, A.C.M., Azzalis, L.A., Simizu, K., Barios, S.B.M., Videla, L.A., Jungueira, V.B.C., 1993. Differential effects of short-term lindane administration on parameters related to oxidative stress in rat liver and erythrocytes. J. Biochem. Toxicol. 8 (4), 187 – 194. Bartowiak, A., Grzelinska, E., Varga, I.S., Leyko, W., 1981. Studies on superoxide dismutase from cod (Gadus morhua) liver. Int. J. Biochem. 13, 1039 – 1042. Beutler, E., 1984. Red Cell Metabolism: A Manual of Biochemical Methods, 2nd edition. Grune and Starton, New York.
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