Arch Toxicol (2000) 74: 533±538 DOI 10.1007/s002040000167
MOLECULAR TOXICOLOGY
F. Gultekin á M. Ozturk á M. Akdogan
The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro)
Received: 11 February 2000 / Accepted: 18 August 2000 / Published online: 11 October 2000 Ó Springer-Verlag 2000
Abstract Organophosphates are known primarily as neurotoxins. However, reactive oxygen species (ROS) caused by organophosphates may be involved in the toxicity of various pesticides. Therefore, in this study we aimed to examine how an organophosphate insecticide, chlorpyrifos-ethyl (CE) [0,0-diethyl 0 (3,5,6-trichloro-2pyridyl) phosphorothioate], aects lipid peroxidation and the antioxidant defense system in vitro. For this purpose, four experiments were carried out. In experiment 1, erythrocyte packets obtained from six (three male, three female) volunteers were divided into six portions, and to each was added CE in both a high concentration range (0, 0.4, 2, 10, 50, 100 g/l) and a low concentration range (0, 0.01, 0.1 g/l). Additionally, each concentration group was divided into ®ve tubes, and incubated at +4 °C for 0, 30, 60, 120, and 240 min. After incubation, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were determined in the erythrocytes in all tubes. In experiment 2, to examine the eect of CE (or its main metabolites) on the activity of puri®ed, commercially available enzymes, CE at concentrations of 0, 0.01, 0.1, 0.4, and 10 g/l was incubated with puri®ed SOD, GSHPx and CAT at the concentrations observed in control group at the 0 CE concentration level in experiment 1
for 1 h at room temperature (25 °C). In experiment 3, the xanthine-xanthine oxidase system was used to determine whether the activities of SOD, GSH-Px and CAT were inactivated other than by CE, for example by superoxide radicals inducing lipid peroxidation in erythrocytes. Samples with xanthine and xanthine oxidase were mixed and incubated for 1 h at room temperature (25 °C). In experiment 4, to determine whether enzyme activities were still inhibited if lipid peroxidation was prevented by exogenous antioxidants, experiment 1 was repeated with the CE concentrations of 0.01, 0.1, 0.4, and 10 g/l by adding butylated hydroxytoluene and vitamin E to the medium. The MDA levels were determined spectrophotometrically. Enzymatic methods were used for the determination of SOD, GSH-Px, and CAT activities. The Friedman test and Wilcoxon's Signed Ranks test were used to compare paired groups. MDA values and GSH-Px activities increased with increasing CE concentration and incubation period (P < 0.05), but SOD and CAT activities decreased with increasing CE concentration and incubation period (P < 0.01). From these results, it can be concluded that in vitro administration of CE resulted in the induction of erythrocyte lipid peroxidation and signi®cant changes in antioxidant enzyme activities, suggesting that ROS and/or free radicals may be involved in the toxic eects of CE.
Presented as a poster at The Sixth Meeting of the Balkan Clinical Laboratory Federation in Antalya, Turkey (1999). We declare that the experiments reported here comply with the current laws and regulations of the Turkish Republic.
Key words Lipid peroxidation á Antioxidant enzymes á Chlorpyrifos-ethyl
F. Gultekin (&) á M. Akdogan Department of Biochemistry and Clinical Biochemistry, Suleyman Demirel University, School of Medicine, 32040 Isparta, Turkey e-mail:
[email protected] Fax: +90-246-2329422
Introduction
M. Ozturk Department of Public Health, Suleyman Demirel University, School of Medicine, Isparta, Turkey
Organophosphorus (OPS) pesticides are generally considered to be neurotoxic and are to considered to act as inhibitors of neuronal acetylcholinesterase (AchE) activity. It has been previously suggested that several pesticides also exert their biological eects mainly through electrophilic attack of cellular constituents with simultaneous generation of reactive oxygen species
534
(ROS). ROS may, therefore, be involved in the toxicity of various pesticides (Dwivedi et al. 1998). Malondialdehyde (MDA) is a marker of membrane lipid peroxidation (LP) resulting from the interaction of ROS and the cellular membrane (Aslan et al. 1997). The ®nal membrane damage can lead to a loss of cellular homeostasis by changing the membrane characteristics (Swann et al. 1991). ROS are produced by the univalent reduction of dioxygen to superoxide anion (áO±2), which in turn disproportionates to H2O2 and O2 spontaneously or through a reaction catalyzed by superoxide dismutase (SOD). Endogenous H2O2 may be converted to H2O either by catalase (CAT) or glutathione peroxidase (GSH-Px). Otherwise, it may generate a highly reactive free hydroxyl radical (áOH) via a Fenton reaction, which is strongly believed to be responsible for oxidative damage. GSH-Px converts H2O2 or other lipid peroxides to water or hydroxy lipids, and during this process glutathione (GSH) is converted to oxidized glutathione (GSSG). To recycle GSSG, the cell utilizes the enzyme NADPH-dependent GSH reductase, the NADPH being supplied to the reaction by glucose-6-phosphate dehydrogenase (Bachowski et al. 1997). Chlorpyrifos-ethyl (CE) [0,0-diethyl 0 (3,5,6-trichloro-2-pyridyl) phosphorothioate] is one of the OPS insecticides commonly used in Isparta by farmers. We could not ®nd any investigations on the peroxidative eects of CE on human erythrocytes in vitro. Therefore, the present research had the following objectives: 1. To determine the eects of various doses of CE on the antioxidant enzymes SOD, GSH-Px and CAT, and LP in erythrocytes. 2. To determine the eects of CE on commercially available puri®ed enzymes SOD, GSH-Px and CAT without using erythrocytes. 3. To determine the eects of superoxide radical which induces LP on the activities of SOD, GSH-Px and CAT in erythrocytes in the absence of CE. 4. To determine the eects of CE on the antioxidant enzymes SOD, GSH-Px and CAT in the presence of exogenous antioxidants, such as butylated hydroxytoluene (BHT) and vitamin E in erythrocytes.
Materials and methods Four experiments were carried out. In experiment 1, the eects of various doses of CE (Durspan 25) on SOD, GSH-Px and CAT and LP in erythrocytes were studied. In this experiment, a high dose range (0, 0.4, 2, 10, 50, and 100 g/l) and a low dose range (0, 0.01, and 0.1 g/l) of CE were used to dierentiate between the eects of various doses of CE. Each CE dose was incubated with a previously prepared erythrocyte sample at +4 °C for 0, 30, 60, 120, or 240 min. In experiment 2, in order to examine the eect of CE (or its main metabolites) on the activity of puri®ed, commercially available enzymes, CE at 0.01, 0.1, 0.4, and 10 g/l was incubated with SOD (Randox, UK), GSH-Px (G 6137, Sigma) and CAT (C30, Sigma) at the approximate concentrations found for the control group at CE dose level 0 in experiment 1 for 1 h at room temper-
ature (25 °C). Three measurements were made for each sample to determine the enzyme activities. In experiment 3, to test the inactivation of SOD, GSH-Px and CAT by means other than CE, for example by superoxide radicals inducing LP in erythrocytes, the xanthine-xanthine oxidase system was used to induce LP. Sample (0.5 ml), xanthine (10 mM, 0.1 ml) and xanthine oxidase (0.1 ml) were mixed and incubated for 1 h at room temperature (25 °C). MDA and antioxidant enzymes were measured before and after incubation. Xanthine oxidase was prepared by diluting xanthine oxidase (2.9 mg protein/ml, 1.6 U/mg protein; X-4500, Sigma) 40 times in a 2 mol/l solution of (NH4)2SO4. The ®nal amount of xanthine oxidase in the assay medium was 0.012 U. In experiment 4, to determine whether enzyme activities were still inhibited if LP was prevented by exogenous antioxidants, experiment 1 was repeated with the CE doses of 0.01, 0.1, 0.4, and 10 g/l by adding BHT and vitamin E to the medium. BHT and vitamin E were added to the samples to give concentrations of 0.88 mg/ml and 0.2 mg/ml, respectively, in the whole medium. A 20-ml venous blood sample was obtained in EDTA from each of six healthy volunteers (three male, three female). Plasma was separated. Erythrocyte packets were prepared by washing the erythrocytes three times with cold isotonic saline. Washed erythrocytes were diluted twice with isotonic saline, and the hemoglobin concentration was determined using a hemocounter (Coulter STKS, USA). Each erythrocyte packet was divided into six portions, and mixed with CE. In the zero concentration group (control group), saline was used instead of CE. Additionally, each erythrocyte/CE mixture (including the control) was divided into enough tubes for the various incubation times. After incubation the mixtures were stored at )20 °C. The mixtures were thawed 1 day later and the erythrocyte MDA levels, and SOD, GSH-Px, and CAT activities were determined. MDA was estimated by the double heating method of Draper and Hadley (1990). The principle of the method is the spectrophotometric measurement of the color generated by the reaction of thiobarbituric acid (TBA) with MDA. For this purpose, 2.5 ml of 100 g/l trichloroacetic acid solution was added to 0.5 ml erythrocyte/CE mixture in each centrifuge tube and the tubes placed in a boiling waterbath for 15 min. After cooling in tapwater, the tubes were 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 the tube placed in a boiling waterbath for 15 min. The solution was then cooled in tapwater and its absorbance was measured using a Shimadzu UV-1601 spectrophotometer (Japan) at 532 nm. The concentrations of MDA were calculated by the absorbance coef®cient of the MDA-TBA complex (1.56 ´ 105 cm±1 á M±1) and are expressed in nanomoles per gram hemoglobin. Erythrocyte SOD and GSH-Px activities were estimated using commercial kits (Randox-Ransod, UK) and an Olympus AU640 autoanalyser (Japan). 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 GSH by cumene hydroperoxide. In the presence of glutathione reductase and NADPH, the GSSH is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in absorbance of NADPH is measured at 340 nm. The determination of SOD was based on the reaction of xanthine with xanthine oxidase to generate superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. The SOD activity is then determined as the degree of inhibition of this reaction. CAT activity was measured according to the method of Aebi (1984). The assay is based on the determination of the rate constant (s±1, k) of hydrogen peroxide decomposition. The rate constant was calculated using the formula k
2:3=Dt
a=b log
A1 =A2 , where A1 and A2 are the absorbance values of hydrogen peroxide at times t1 (0th second) and t2 (15th second), a is a dilution factor, and b is the hemoglobin content of the erythrocytes. For statistical analyses, ®rst the degree of normality was investigated, and it was found that some of the values of the
535 parameters did not ®t a normal distribution. Therefore, as recommended by Dawson-Saunders and Trapp (1994), considering the small number of cases, the Friedman and Wilcoxon's Signed Ranks nonparametric tests were used to compare paired groups.
Results The mean ages of the male and female volunteers were 25 years (range 21±28 years) and 27 years (range 23± 29 years), respectively. All of them were healthy and taking no medication, and none of them was a farmer or an agricultural worker. The results and comparison of the groups in experiment 1 with the high CE dose range are shown in Table 1 and with the low CE dose range in Table 2. MDA formation increased with increasing CE dose and incubation period. Similarly, GSH-Px activity increased with increasing CE dose and incubation period. However, SOD and CAT activities decreased with increasing CE dose and incubation period. From the Table 2, there were no signi®cant changes in the activities of SOD, GSH-Px and CAT, but MDA increased with increasing CE dose and incubation period. The eects of CE on puri®ed enzymes can be seen in Table 3 (experiment 2). CAT activity decreased and GSH-Px increased with increasing CE dose while SOD was not aected by CE. The eects of superoxide radicals without CE on LP in erythrocytes are shown in Table 4 (experiment 3). GSH-Px activity was inhibited
by CE while MDA increased. The results of experiment 4 determining whether enzyme activities were still inhibited by CE if LP was prevented by exogenous antioxidants are given in Table 5. GSH-Px increased and CAT decreased with increasing CE dose while SOD was not aected.
Discussion Insecticides are known to interfere with a number of biochemical processes such as mitochondrial respiration, drug metabolism, carbohydrate metabolism and protein biosynthesis (Gupta et al. 1992). The frequent and widespread use of OPI has resulted in their distribution in the environment and they have been shown to exert deleterious eects on biological systems. Organophosphorus and organochlorine compounds have been shown to interact with membrane events including nerve conductance, and plasma membrane and organelle enzyme activities (Binder et al. 1976; Rosenstock et al. 1990; Schneider 1975). Recent investigations have provided further evidence that OPS insecticides, besides their typical action as inhibitors of AchE, interfere with the allosteric behavior of the enzyme through interaction with the membrane lipids (Domenech et al. 1977). Bagchi et al. (1995) have shown that dierent classes of pesticides induce production of ROS and oxidative
Table 1 Concentrations of MDA and activity of SOD, GSH-Px and CAT following incubation of erythrocytes for various times with CE in the high concentration range. Values are means (SD, n=6) (ns not signi®cant) Incubation time (min) MDA (nmol/gHb)
SOD (U/gHb)
GSH-Px (U/gHb)
CAT (k/gHb)
a b
0
0.4
43.27 (5.12)
0
30 60 120 240 P-valueb
P-valuea
CE concentration (g/l)
ns
44.11 44.25 44.50 45.97
(7.12) (6.31) (4.25) (5.44)
43.27 (5.12) 66.70 (9.22) 68.00 (7.21) 69.07 (6.35) 73.01 (8.64)
