The effects of diazinon on lipid peroxidation and antioxidant enzymes ...

Toxicology and Industrial Health 2007; 23: 13–17 http://tih.sagepub.com

The effects of diazinon on lipid peroxidation and antioxidant enzymes in rat erythrocytes: role of vitamins E and C . Recep Sutcua, Irfan Altuntasa, Bora Buyukvanlib, Onur Akturka, Ozlem Ozturka, Halis Koylub, Namik Delibasa aDepartment bDepartment

of Biochemistry and Clinical Biochemistry, Süleyman Demirel University, School of Medicine, Isparta, Turkey of Physiology, SÜleyman Demirel University, School of Medicine, Isparta, Turkey

Reactive oxygen species caused by organophosphates may be involved in the toxicity of various pesticides. Therefore, in this study, we aimed to investigate the effects of acute exposure to organophosphate insecticide diazinon (DI) and possible ameliorating role of vitamins E and C, with the following parameters: lipid peroxidation (LPO) and the activity of the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in rat erythrocytes. The experimental groups were arranged as control group, DI-treated group (DI) and DI ⫹ vitamin E ⫹ vitamin C–treated group (DI ⫹ Vit). DI ⫹ Vit groups were treated orally with a single dose of 335 mg/kg DI body weight. Vitamins E and C were injected at doses of 150 mg/kg body weight intramuscular (in) and 200 mg/kg body weight intraperitoneal (ip), respectively, 30 min after the treatment of DI in DI ⫹ Vit group. Blood samples were taken 24 h after the DI. The results showed that DI administration caused to increase in LPO and the activities of SOD and GSH-Px enzymes in erythrocytes. Also, the combination of vitamins E and C decreased LPO and the activities of GSH-Px and SOD compared with the DI group. In conclusion, although treating rats with single dose DI increases LPO and antioxidant enzyme activities in erythrocytes, vitamins C and E combination can reduce LPO caused by DI. Toxicology and Industrial Health 2007; 23: 13–17. Key words: antioxidant enzymes; diazinon; lipid peroxidation; vitamin C; vitamin E

Introduction Organophosphorus insecticides (OPIs), widely used in agriculture, show several interesting features for environmental safety, such as limited persistence and selective toxicity to insects with respect to mammals. However, in spite of their selectivity of action, they are often highly toxic to humans and are responsible for

Address all correspondence to: Dr. Recep Sutcu, Department of Biochemistry and Clinical Biochemistry, S¨uleyman Demirel University, School of Medicine, 32200 Isparta, Turkey Fax: ⫹90 246 2371651; E-mail: [email protected]

© 2007 SAGE Publications

most accidental intoxications in agriculture and the pesticide industry (Vittozzi et al., 2001). In blood, normal erythrocyte function depends on the intactness of erythrocyte membrane, which is target for many toxic factors, including the OPIs. Available in vivo and in vitro studies showed that OPIs increased lipid peroxidation (LPO) and altered the activities of antioxidant enzymes in erythrocytes. In these studies, LPO has been suggested as one of the molecular mechanisms involved in OPI-induced toxicity (Datta et al., 1992; Ahmed et al., 2000; Gultekin et al., 2001; John et al., 2001). Diazinon (DI: O, O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothionate), as an OPI with a wide spectrum 10.1177/0748233707076758

The effects of diazinon on oxidative stress R Sutcu et al.

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of activity, has been used for several decades in agriculture (Baily et al., 2000). DI is also one of the widely used OPIs in the region of Isparta, Turkey (Agriculture Ministry Office of Isparta province). Previous study from our laboratory investigated the effects of DI on LPO and antioxidant enzymes in erythrocytes in vitro. In the present study, the rats were administered orally DI to determine its effect on LPO and antioxidant enzymes in rat erythrocytes. Additionally, a combination of vitamins E and C after the treatment of DI as administered to rats evaluates their protective effects on DI-induced toxicity.

Materials and methods Animals Twenty Wistar albino rats weighing between 200 and 290 g were divided into three experimental groups: control group (n ⫽ 8), DI-treated group (DI, n ⫽ 6) and DI ⫹ vitamin E ⫹ vitamin C-treated group (DI ⫹ Vit, n ⫽ 6). DI and DI ⫹ Vit groups were treated orally with a single dose of 335 mg/kg body weight of DI (0.25 LD50) (Basudin; Syngenta, Turkey) in corn oil at 0 h. Only corn oil was given in the same way to the control group. Vitamin E, as ␣-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 im (Appenroth et al., 1997; Gultekin et al., 2001; Altuntas et al., 2002a) and 200 mg/kg body weight ip (Appenroth et al., 1997; Gultekin et al., 2001; Altuntas and Delibas, 2002; Altuntas et al., 2002a,) respectively, 30 min after the treatment of DI in DI ⫹ Vit group. Equal amounts of physiologic saline instead of vitamins were given to the rats of control and DI groups. The rats were caged individually and fed ad libitum without water restriction. The animals starved overnight for 12 h before the blood was collected. Rats were anaesthetized with ether and venous blood samples were collected by direct right ventricle heart puncture at 24 h. Blood samples were centrifuged and serum was discarded.1 1We

hereby declare that the experiments reported here comply with the current laws and regulations of the Turkish Republic on the care and handling of experimental animals.

Biochemical parameters Blood samples were centrifuged and plasma was discarded. Erythrocyte packets were prepared by washing erythrocytes three times with cold isotonic saline. Hemoglobin concentration was determined by the cyanmethemoglobin method from the washed erythrocytes (Van Kampen and Zijlstra, 1965). The erythrocytes were then stored at ⫺20°C and all measurements were made within a week. The erythrocytes were thawed and the levels of malondialdehyde (MDA) and the activities of superoxide dimutose (SOD) and gluthione peroxidase (GSH-Px) were assessed. MDA, as a marker for LPO, was determined by the double heating method of Draper and Hadley. The principle of the method was spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 mL erythrocytes in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000 g for 10 min, and 2 mL of the supernatant was added to 1 mL of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a Shimadzu UV-1601 spectrophotometer (Japan) at 532 nm. The concentration of MDA was calculated by the absorbance coefficient of MDA-TBA complex 1.56 ⫻ 105 cm⫺1 M⫺1 and expressed in nmol/g Hb. The measurement of SOD was based on the principle in which xanthine reacts with xanthine oxidase to generate superoxide radicals that react with 2-(4iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazon dye. The 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 nicotinamide adenine dinucleotide phosphate reduced form with a concomitant oxidation of (NADPH) to NADP⫹. The decrease in absorbance of NADPH was measured at 340 nm. An autoanalyser, Abbott Aeroset (IL, USA), was used to determine the activities of SOD and GSH-Px,


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and the spectrophotometer, Shimadzu UV-1601 (Japan), was used to estimate the levels of MDA. Statistical evaluation For statistical analyses, normality was first investigated, and it was shown that some values of the parameters did not fit to the normal distribution. Therefore, as stated by Dawson-Saunders and Trapp (1994) in considering the small number of cases, non-parametric the Kruskal–Wallis test and Mann–Whitney U-test were used to compare groups.

Results Severe fasciculations were observed after DI administration in rats. These findings continued approximately 1–1.5 h, after that fatigue symptoms were observed. No significant symptoms were observed after 24 h in rats. The in vivo results are shown Table 1 and Figure 1. The levels of MDA increased significantly in DI group Table 1. The levels of MDA and the activities of SOD and GSH-Px in control, DI and DI⫹Vit groups in erythrocytes

MDA (nmol/g Hb) SOD (U/g Hb) GSH-Px (U/g Hb)

Control

DI

DI ⫹ Vit

88 ⫾ 5 1846 ⫾ 290 150 ⫾ 20

120 ⫾ 21a 2463 ⫾ 251a 193 ⫾ 23a

88 ⫾ 6b 1969 ⫾ 190b 157 ⫾ 19b

Values are expressed as means ⫾ SD for groups. aP ⬍ 0.05, as control group is compared with the DI group. bP ⬍ 0.05, as DI group is compared with the DI ⫹ Vit group.

compared with control group (P ⬍ 0.05) and decreased significantly in DI ⫹ Vit group compared with DI group (P ⬍ 0.05). There was no statistical difference in MDA levels between DI ⫹ Vit and control groups. The activities of SOD and GSH-Px increased significantly in DI group compared with control group (P ⬍ 0.05) and decreased significantly in DI ⫹ Vit group compared with DI group (P ⬍ 0.05).

Discussion The most important feature DI and OPIs toxicity is related to their irreversible blood ChE inhibition, which at high doses could lead to animal death (Neishabouri et al., 2004). However, some studies indicate that oxidative stress could be an important component to the mechanism of toxicity of OPIs. OPIs may induce oxidative stress, leading to generation of free radicals and alterations in antioxidants or reactive oxygen species (ROS) scavenging enzymes (Baghci et al., 1995; Ahmed et al., 2000; Gultekin et al., 2000;). Some studies showed that LPO has been suggested as one of the molecular mechanisms involved in OPIs-induced toxicity (Yamano and Morita, 1992; Baghci et al., 1995). The cell has several mechanisms to deal with the effects of oxidative stress, either by repairing the damage (damaged nucleotides and LPO by-products) or by directly diminishing the occurrence of oxidative damage by means of enzymatic and nonenzymatic antioxidants. Enzymatic and nonenzymatic antioxidants have also been shown to scavenge free radicals and

Figure 1. The levels of MDA and the activities of SOD and GSH-Px in erythrocytes. Mann–Whitney U-test). The values of SOD were given as 1/10.

a,bIndicate

significant differences between groups (P ⬍ 0.05,


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ROS. The enzymatic antioxidants in erythrocytes may counteract oxidative stress (Gultekin et al., 2001). In our previous in vitro and in vivo studies, MDA formation increased with the administration of MD, chlorpyrifos-ethyl, fenthion, phosalone and DI (Gultekin et al., 2001; Altuntas et al., 2002c; Altuntas et al., 2003; Altuntas et al., 2004) in a similar manner to the present study. The increase in MDA formation may be modulated by DI itself inducing LPO or by a possible increase in ROS induced by DI. It has been reported that chlorpyrifos-ethyl, malathion, methidathion, fenthion and phosalone caused a decrease in the SOD activity (Yarsan et al., 1999; Gultekin et al., 2000; Gultekin et al., 2001; Altuntas and Delibas, 2002; Altuntas et al., 2002a, 2002b, 2002c; Altuntas et al., 2003; Altuntas et al., 2004) However, dimethoat, malathion and phosphomidon caused an increase in SOD activity (Datta et al., 1992; Banerjee et al., 1999; Yarsan et al., 1999; John et al., 2001). In the present study, DI caused an increase in the SOD activity in erythrocytes. In our previous studies (in vitro), the activity of SOD respectively decreased with increasing DI concentrations above the 0.033 mM DI concentrations. The highest SOD activity was seen at the DI concentration of 0.033 mM (Altuntas et al., 2004). The activation of SOD is not directly mediated only by DI. The increase in SOD activity in erythrocytes after DI intoxication appears to be due to increased generation of ROS. It has been reported that malathion, methidathion and phosalone caused a decrease, (Yarsan et al., 1999; Altuntas et al., 2002a, 2002b; Altuntas et al., 2003) and chlorpyrifos-ethyl, malathion and phosphomidon caused an increase in the GSH-Px activity (Datta et al., 1992; Banerjee et al., 1999; Ahmed et al., 2000; Gultekin et al., 2000). In the present study, DI caused an increase in GSH-Px activity in erythrocytes. This suggests that the activation of GSH-Px is directly mediated by DI. Nonenzymatic antioxidants such as vitamins E and C can also act to overcome the oxidative stress, being a part of total antioxidant system. Vitamin E, a constituent of a 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. Vitamin C is hydrophilic and a most 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, vitamin E and vitamin C can have interactive effects (Stoyanovsky et al., 1995). Altuntas et al. (2002a) showed that the treatment with a combination of vitamins E and C 30 min after the administration of methidathion led to a significant decrease in LPO; in addition, it caused a significant increase in GSH-Px activity. But the activity of SOD was not affected by the combination of vitamins E and C. The results of the present study showed that the treatment with the combination of vitamin E and C 30 min after the administration of DI led to a significant decrease in LPO; in addition, it caused a significant decrease in SOD and GSH-Px activity. Conflicting results concerning antioxidant enzymes may be due to the timing and dosing of vitamins E and C administration. In conclusion, these findings demonstrate that treating rats with DI results in the induction of erythrocyte LPO and increases the activities of antioxidant enzymes and suggesting that ROS may be involved in the toxic effects of pesticidal use of DI. Furthermore, single dose treatment with the combination of vitamins E and C 30 min after the administration of DI can reduce LPO caused by DI.

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