Cell Biol Toxicol 2006; 22: 455–461. DOI: 10.1007/s10565-006-0138-5
C Springer 2006
The effects of diazinon on lipid peroxidation and antioxidant enzymes in rat heart and ameliorating role of vitamin E and vitamin C O. Akturk1 , H. Demirin1 , R. Sutcu1 , N. Yilmaz1 , H. Koylu2 and I. Altuntas1 1 Department of Biochemistry and Clinical Biochemistry, 2 Department of Physiology, School of Medicine, Suleyman Demirel University, Isparta, Turkey Received 2 June 2006; accepted 17 August 2006; Published online 11 September 2006
Keywords: diazinon, catalase, glutathione peroxidase, lipid peroxidation, superoxide dismutase Abstract Diazinon is one of the most widely used organophosphate insecticides (OPIs) in agriculture and public health programs. Reactive oxygen species (ROS) caused by OPIs may be involved in the toxicity of various pesticides. The aim of this study was to investigate how diazinon affects lipid peroxidation (LPO) and the antioxidant defense system in vivo and the possible ameliorating role of vitamins E and C. For this purpose, experiments were done to study the effects of DI on LPO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in adult rat heart. Experimental groups were: (1) control group, (2) diazinon treated (DI) group, (3) DI+vitamins E and C-treated (DI+Vit) group. The levels of malondialdehyde (MDA) and the activities of SOD and CAT increased significantly in the DI group compared with the control group. The activity of SOD and the levels of MDA decreased significantly in the DI+Vit group compared with the DI group. The differences between the DI+Vit and control groups according to the MDA levels and the activities of both SOD and CAT were statistically significant. These results suggest that treating rats with a single dose of diazinon increases LPO and some antioxidant enzyme activities in the rat myocardium and, in addition, that single-dose treatment with a combination of vitamins E and C after the administration of diazinon can reduce LPO caused by diazinon, though this treatment was not sufficiently effective to reduce the values to those in control group. Abbreviations: CAT, catalase; DI, diazinon; GSH-Px, glutathione peroxidase; LPO, lipid peroxidation; MDA, malondialdehyde; OPI, organophosphate insecticides; ROS, reactive oxygen species; SOD, superoxide dismutase (SOD) Introduction Pesticide poisoning is an important cause of morbidity and mortality in developing countries, although only one-quarter of the total world consumption of pesticides is attributed these regions (Banerjee et al., 1999). Every year there are three million cases of severe poisoning and 220 000
deaths; the majority of these poisonings and 99% of the resulting deaths occur in the Third World (Tinoco and Halperin, 1998). Since the mechanism of action of organophosphate insecticides (OPIs) has not been fully elucidated, further information on the factors regulating the toxicity of these compounds should allow a better assessment of their environmental
456 impact (Keizer et al., 1995). The toxic effect of some OPIs (e.g., paraoxon, sarin) is not limited to inhibition of cholinesterase: following the cholinergic crisis changes in noncholinergic neurotoxic parameters, such as specific damage to cell membranes, are observed (Tonkopii, 2003). Bagchi et al. (1995) found that different classes of pesticides, including OPIs, may induce in vitro and in vivo generation of reactive oxygen species (ROS), such as hydrogen peroxide (H2 O2 ), su− peroxide (O− 2 ) and the hydroxyl radical (HO ). The enzymes that provide the first line of defense against O− 2 and H2 O2 include superoxide dismutase (SOD), catalase (CAT), and gluthathione peroxidase (GPx). The extent of lipid peroxidation is determined by the balance between the production of oxidants and the removal and scavenging of those oxidants by antioxidants. It has been reported that OP pesticides exert their toxic effects via lipid peroxidation (Hai et al., 1997; Hazarika et al., 2003). In the region of Isparta, Turkey, the commonly used OPIs in agricultural production systems in recent years are reported to be chlorpyrifos-ethyl, methidathion, fenthion, phosalone, diazinon (DI), parathion-methyl, malathion, and oxydemetonmethyl (Agricultural Ministry Office of Isparta Province). We therefore, have considerable interest in evaluating the toxicity of some of these OPIs by series of in vitro and in vivo studies in our laboratory. Previous studies from our laboratory focused on in vitro and in vivo effects of OPIs, such as chlorpyrifos-ethyl, diazinon, methidathion, fenthion, and phosalone, on LPO and antioxidant enzymes status. The results indicate that these OPIs can affect the antioxidant enzyme status at different levels, and generally cause the levels of LPO to increase. It was also shown that these OPIs significantly altered the activities of the main antioxidant enzymes such as SOD, GSH-Px, and CAT. In these studies, LPO has been suggested as one of the molecular mechanisms involved in OPIinduced toxicity (Gultekin et al., 2001; Altuntas et al., 2002a,b,c).
Diazinon (O,O-diethyl-O-[2-isopropyl-6-methylpyrimidin-4-yl] phosphorothionate) attracted our interest because of its effect on LPO and the activities of some antioxidant enzymes in vitro or in vivo, since no experimental results have yet been reported on these effects of diazinon on LPO of rat heart tissue. It is possible that the heart is particularly sensitive to this peroxidative damage of some OPIs due to its limited capacity in antioxidant defense systems that can enzymatically detoxify drug-related hydroxyl radicals (Goodman and Hochstein, 1977; Doroshow et al., 1980). The effects of methidathion on LPO in rat heart tissue was studied in one of our previous studies (Yavuz et al., 2003). Povoa et al. (1997) have reported that acute toxicity of malathion induced myocardial necrosis and elevated levels of cardiac enzymes. However, there are no current data available concerning biochemical changes caused by acute heart exposure to diazinon. It is well known that vitamins E and C are very potent cellular scavengers of reactive oxygen species (ROS). We showed in our previous studies that a combination of vitamins E and C decreased the levels of LPO and restored the antioxidant enzyme activities (Gultekin et al., 2001; Altuntas et al., 2002a,c; Yavuz et al., 2004). Therefore, the present study was planned to determine the effects of diazinon on LPO and the activities of SOD, GSH-Px, and CAT in rat heart, and whether administration of vitamins C and E after single dose diazinon treatment can alleviate these effects.
Materials and methods Animals and treatment Twenty Wistar albino rats weighing between 200 and 290 g were divided into three experimental groups: (1) control group (C, n = 8), (2)
457 diazinon-treated group (DI, n = 6), (3) DI vitamin E + vitamin C-treated group (DI+Vit, n = 6). The DI and DI+Vit groups were treated orally with a single dose of 335 mg/kg body weight of diazinon (Basudin; Syngenta, Turkey), in corn oil at 0 h. This diazinon dose was adjusted due to 25% of LD50 of diazinon (Office of Prevention, Pesticides and Toxic Substances, 1999). Attempts at suicide using diazinon are frequently encountered in Turkey. We therefore aimed to use a high dose (25% LD50 ) and to investigate possible beneficial effects of vitamins E and C combination immediately after the exposure. Only corn oil was given orally to the control group. Vitamin E as α-tocopherol acetate (Evigen; Aksu Farma, Istanbul, Turkey) and vitamin C as sodium L-ascorbate (Redoxon; Roche, Basel, Switzerland) were injected at doses of 150 mg/kg body weight intramuscularly and 200 mg/kg body weight intraperitoneally (Appenroth et al., 1997; Gultekin et al., 2001), respectively, to DI+Vit group 30 min after the treatment with the OPI. On the assumption that patients exposed to acute toxicity would be taken to an emergency unit approximately within 30 min and that vitamin therapy would only be given after this. We chose the doses according to studies we had previously carried out in our laboratory to investigate the ameliorative effects of the same vitamins against OPI administration. Equal volumes of physiological saline instead of vitamin solutions were given to the rats of control and DI groups. After all the rats received the above treatments, they were fed ad libitum for 24 h. Rats were sacrificed by decapitation under ether anesthesia. The heart tissues were removed and suspended in 3 ml of Tris-HCl buffer pH 7.3 that contained 0.25 mol/L sucrose, and then stored at −80◦ C until biochemical analysis. These experiments complied with the current laws and regulations of The Turkish Republic on the care and handling of experimental animals.
Tissue homogenates For biochemical analyses, the hearts of rats were washed with physiological saline and then homogenized for 3 min (Ultra-Turrax T25, Janke & Kunkel GmbH & Co. KG, IKAR -Labortechnik, Staufen, Germany) in cold phosphate buffer in order to provide a 10% homogenate. The homogenates were centrifuged at 6000 g for 10 min to obtain supernatants. The levels of protein, malondialdehyde (MDA), SOD, GSH-Px, and CAT were determined in the supernatants. Protein content of homogenates was determined by the Lowry method (Lowry et al., 1951). MDA, as a marker for LPO, was determined by an HPLC method improved by Mateos et al. (2005). The principle of the method is the chromatographic separation of the pyrazole and hydrazone derivatives after derivatization of MDA with 2,4-dinitrophenylhydrazine (DNPH). For this, 0.5 g tissue is added to 2.5 ml 0.25 mol/L Trizma base buffer, and 250 μl of 500 ppm butylated hydroxytoluene (BHT) is added to 0.5 g tissue sample, after which it is mixed well. Homogenization precedes centrifugation for 30 min at 10 000g at 4◦ C and then 250 μl of supernatant is collected and placed in a 1.5 ml Eppendorf tube. Sodium hydroxide (50 μl 6 mol/L) is added to the Eppendorf and incubated in a 60◦ C water bath for 30 min (alkaline hydrolysis of protein-bound MDA). After hydrolysis, 125 μl of 35% (v/v) perchloric acid is added for precipitation of proteins and the mixture is centrifuged at 2800g for 10 min. Supernatant (50 μl) is put into an Eppendorf and 25 μl of DNPH solution is added for derivatization, after which the mixture is vortexed and left in dark for 30 min at room temperature for incubation. Sample (50 μl) is injected to the HPLC system using a reversed-phase column and an Acetonitrile (ACN)–H2 O–acetate (38:62:0.2, by vol) mobile phase at a flow rate of 0.6 ml. For straightforward spectrophotometric determination of the MDA derivative, a wavelength of 310 nm is used. The HPLC instrument used was a Thermo Finnigan SpectraSystem with diode
458 array detector. The concentration of MDA was expressed in nmol/ml (Mateos et al., 2005). The measurement of SOD was based on the principle that xanthine reacts with xanthine oxidase to generate superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol) -5-phenyltetrazolium chloride (INT) to form a red formazan dye. SOD activity is then measured by the degree of inhibition of this reaction (Woolliams et al., 1983). The determination of GSH-Px activity was based on the method of Paglia and Valentine (1967). The principle of the method is as follows: GSH-Px catalyses the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+ . The decrease in absorbance of NADPH was measured at 340 nm wavelength. CAT activity was measured according to the method of Aebi (1984). The principle of the assay is based on the determination of the rate constant (k, s−1 ) of decomposition of hydrogen peroxide by the enzyme catalase. The rate constant was calculated from the formula: k = (2.3/t)(a/b) log(A1 /A2 ), where A1 and A2 are the absorbance values of hydrogen peroxide at t1 (0 second) and t2 (15 seconds), a is the dilution factor, and b is the protein level of tissue homogenate. An autoanalyser (Aeroset, Abbott, Abbott Park, IL, USA), was used to determine the activities of SOD and GSH-Px, and a Shimadzu UV-1601
spectrophotometer (Shimadzu, Kyoto, Japan) was used to estimate the activities of the enzyme CAT. Statistical evaluation Data are presented as means ± SD. A computer program (SPSS 9.0 for Windows, SPSS Inc. Chicago, IL, USA) was used for statistical analyses. Normality was first investigated using the Kolmogorow–Smirnov test, and it was shown that some values of the parameters did not fit the normal distribution. Therefore, as recommended by Dawson-Saunders and Trapp in considering small numbers of cases, the nonparametric Kruskal– Wallis test and the Mann–Whitney U-test were used to compare groups (Dawson-Saunders and Trapp, 1994). Results Clinical findings Moderate involuntary movements were observed 10 min after diazinon administration in the rats. These movements continued for approximately 30 min, after which symptoms of fatigue were observed. No significant symptoms were grossly observed after these findings during the rest of experiment. Biochemical findings The results are shown in Table 1. The levels of MDA increased significantly in the DI group
Table 1. The levels of MDA and the activities of SOD, GSH-Px, and CAT in rat heart in control, DI, and DI+Vit groups (values are mean ± SD) Experimental group Parameter MDA (nmol/mg protein) SOD (U/mg protein) GSH-Px (U/mg protein) CAT (k/mg protein)
DI
DI+Vit
0.637 ± 0.069 14.720 ± 1.094 0.255 ± 0.009 0.115 ± 0.018
0.794 ± 0.028a 17.554 ± 0.537a 0.261 ± 0.011 0.302 ± 0.020a
0.730 ± 0.021a,b 16.469 ± 0.586a,b 0.266 ± 0.014 0.278 ± 0.054a
< 0.05, the DI or the DI+Vit groups compared with the control group. p < 0.05, the DI+Vit group compared with the DI group.
ap b
Control
459 compared with the control group ( p < 0.05), and decreased significantly in the DI+Vit group compared with the DI group ( p < 0.05). The activities of both SOD and CAT increased significantly in the DI group compared with the control group ( p < 0.05). The activity of SOD decreased significantly in the DI+Vit group compared with the DI group ( p < 0.05), whereas the activity of CAT remained unchanged in the DI+Vit group compared with the DI group. The differences between the DI+Vit and control groups in terms of the MDA levels and the activities of both SOD and CAT were significant ( p < 0.05). There were no statistical differences in GSH-Px activities among all groups.
Discussion Recent findings indicate that toxic manifestations induced by OPIs may be associated with an enhanced production of reactive oxygen species (ROS) (Bagchi et al., 1995; Gultekin et al., 2000, 2001). Among ROS, superoxide anions, hydroxyl radicals, and hydrogen peroxide enhance the oxidative process and induce lipid peroxidative damage in cell membranes. The heart tissue may be susceptible to oxidative damage due to the presence of polyunsaturated fatty acids (PUFAs), and oxygen, which may produce oxidative changes in myocytes (Kale et al., 1999). The cell has several ways to alleviate the effects of oxidative stress, either by repairing the damage (damaged nucleotides and LPO byproducts) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and nonenzymatic antioxidants. These antioxidants have also been shown to scavenge free radicals and ROS; for instance SOD catalyzes the conversion of superoxide radical to hydrogen peroxide while CAT or GSH-Px converts hydrogen peroxide to water. These antioxidant enzymes can, therefore, alleviate the toxic effects of ROS (Gultekin et al., 2001; Altuntas et al., 2002a).
There have been several studies of the peroxidative effects of different OPIs on LPO and antioxidant enzymes. OPIs such as chlorpyrifos-ethyl, dimethoate, and malathion have been reported to increase LPO both in vivo and in vitro in erythrocytes (Datta et al., 1992; Banerjee et al., 1999; Yarsan et al., 1999; Ahmed et al., 2000; John et al., 2001). MDA, the end product of LPO, has also been measured to indicate the presence of free radicals and LPO-induced cardiotoxicity. In a previous study from our laboratory, Gultekin et al. (2001) have shown that chlorpyrifos-ethyl caused in vitro and in vivo increased LPO in erythrocytes. In addition, Altuntas et al. (2002a) have also shown that single-dose treatment with methidathion increased LPO in erythrocytes. Diazinon caused increase of LPO levels in rat erythrocytes and pancreas (Altuntas et al., 2004; Gokalp et al., 2005). In the present study, diazinon caused increase in LPO level. LPO is an autocatalytic process that is caused by free radicals. We thought that the significant increase ( p < 0.01) in MDA might be modulated by diazinon itself inducing LPO or by a possible increase in ROS induced by diazinon. Nonenzymatic antioxidants such as vitamins E and C can also act to overcome oxidative stress, being a part of the antioxidant system. Vitamin E, a constituent of plasma membrane, is an effective antioxidant and, as it is present at the site of free radical generation, it may neutralize the toxic effects of ROS (John et al., 2001). Vitamin C is hydrophilic and is a very important free-radical scavenger in extracellular fluids, trapping radicals in the aqueous phase and protecting biomembranes from peroxidative damage (Harapanhalli et al., 1996). In addition to its antioxidant effects, vitamin C is involved in the regeneration of tocopherol from tocopheroxyl radicals in the membrane. Thus, vitamins E and C can have interactive effects (Stoyanovsky et al., 1995). The results suggest that treating rats with diazinon increases LPO significantly in the rat heart tissue. These results support the hypothesis whereby LPO has been suggested as one of the molecular
460 mechanisms involved in OPI-induced toxicity. Furthermore, the treatment with a combination of vitamins E and C 30 min after the administration of diazinon led to a significant ( p < 0.05) decrease in LPO, but not enough to bring the values of the DI+Vit group close to those in controls ( p < 0.05). There are differing data concerning the effect of OPIs on antioxidant enzymes. Gultekin et al. (2000) showed that CAT activity was inhibited by the direct action of chlorpyrifos-ethyl and the superoxide radicals caused an inhibition of GSH-Px activity. Other researchers found several OPI to cause an increase in the activities of SOD, GSH-Px, and CAT (Datta et al., 1992; Ahmed et al., 2000). In the present study, diazinon caused significant increases in the activities of SOD and CAT ( p < 0.05), and the increase in GSH-Px remained unchanged in the heart tissue of rat. Ahmed et al. (2000) reported that malathion caused highly significant increases in SOD and CAT activities, and dietary ginger—said to have antioxidant effects—caused significant decreases in these enzyme activities in erythrocytes of malathion-treated rats. Also, in a previous in vitro study by us (Altuntas et al., 2004), diazinon caused an increase in SOD activity in erythrocytes. The increased activities of SOD and CAT reflect an activation of the compensatory mechanism through the effects of pesticides on cells, and its extent depends on the magnitude of the oxidative stress and hence on the dose of stressor. The elevated activity of CAT is due to the adaptive response to the generated free radicals, indicating the failure of the total antioxidant defense mechanism to protect the tissues from mechanical damage caused by pesticides, as evidenced by lipid peroxidation. Thus, the superoxide ion generated is dealt with by the enhanced SOD and is converted to H2 O2 by CAT or GSH-Px. In the present study, the administration of vitamins E and C was somewhat effective in restoring the activities of SOD and CAT; only SOD differed significantly and the effect did not normalize the
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Address for correspondence: Onur Akturk, Suleyman Demirel University, School of Medicine, Department of Biochemistry and Clinical Biochemistry, 32260 Isparta, Turkey. E-mail: onur
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