http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2014; 37(4): 410–414 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.870193
RESEARCH ARTICLE
Effects of bentazone on lipid peroxidation and antioxidant systems in human erythrocytes in vitro
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M. Abudayyak, S. Ozden, B. Alpertunga, and G. O¨zhan Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazıt 34116, Istanbul, Turkey
Abstract
Keywords
Bentazone, a benzothiadiazole herbicide, is widely used for a variety of crops including cereals, maize, peas, rice and soy beans. The concern for human health is stil very high because bentazone is continuously monitored in environment and several studies to evaluate its potential carcinogenic effects when chronic and high doses were administered to animals. We aimed to investigate the possible effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes in human erythrocytes in vitro. For that, erythrocyte were incubated with bentazone in different concentrations (0–50 nM) at 37 C for 1 hr. Bentazone showed significant increase in the levels of malondialdehyde (MDA) at the highest concentration in erythrocytes as an index of lipid peroxidation. Besides, alterations in the levels of reduced glutathione (GSH) and activities of glutathione peroxidase (GSH-Px) were observed while the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GSH-Rd) were unchanged. In conclusion, findings from this study indicate that in vitro toxicity of bentazone may be associated with oxidative stress and this work warrants further in vivo investigations.
Antioxidant enzymes, bentazone, human erythrocytes, lipid peroxidation
Introduction Pesticides are used indiscriminately in large amounts, causing environmental pollution and risk to human and animal health. Bentazone (3-(1-methylethyl)-1-H-2,1,3-benzothiadiazin4(3H)-one 2,2-dioxide) is a benzothiadiazole herbicide widely used in the agrochemical field and acts as an inhibitor of photosynthesis in plants. Residual amounts of bentazone have been monitored in the water, soil, honey, plants, fruits and vegetables (Worthing, 1991). Moreover, the first clear report on the fatal poisoning of a human with bentazone has been reported (Mu¨ller et al., 2003). Based on concentration measured in suicidal case involving 59-year-old women who ingested 686–1371 mg/kg bentazone, they reported lethal dose of bentazone as 625 mg/kg body weight. Despite extensive use of bentazone, very limited studies on the mechanisms of its action exist. Long-term studies conducted in rats and mice have not indicated a carcinogenic potential, and a variety of in vitro and in vivo assays have indicated that bentazone is not genotoxic (EPA, 2000; Spencer, 1998). Pesticides or their degradation metabolites may exert toxicity related to oxidative stress and can cause oxidative damage in mammalian cells (Bukowska et al., 2006; Bukowska et al., 2008; Bozena et al., 2011; Cereser et al., ¨ zhan, Istanbul University, Faculty Address for correspondence: Dr. Gu¨l O of Pharmacy, Department of Pharmaceutical Toxicology, 34116 Beyazit, Istanbul, Turkey. Tel: +902124400000. Fax: +902124400252. E-mail: [email protected].
History Received 3 August 2013 Revised 12 November 2013 Accepted 25 November 2013 Published online 23 December 2013
2001; Chiapella et al., 2013; Cicchetti and Argentin, 2003; Moore et al., 2010). Several studies report bentazone has physiological and biochemical effects in higher plants and induces oxidative stress by generation of reactive oxygen species (ROS) in heterocystous cyanobacteria (Bagchi et al., 2012; Galhano et al., 2010; Galhano et al., 2011; Macedo et al., 2008). As reports on the oxidative damage of bentazone in mammalian cells are scanty, we aimed to investigate the dose-dependent effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes (SOD, CAT, GSH-Px and GSH-Rd).
Materials and methods Reagents Bentazone, technical purity 99.2% was obtained from Riedel de Hae¨n (Seelze, Germany). All chemicals were analytical reagent grade and chemicals required for all biochemical assays were obtained from Sigma-Aldrich Chemicals Co (St. Louis, MO) and Merck (Darmstadt, Germany). Erythrocytes preparation Human erythrocytes were obtained from whole blood from healthy donors (Bukowska et al., 2003). The whole blood was centrifuged at 1500 rpm for 10 min. The clear plasma and buffy coat were discarded. Erythrocytes were washed three times with phosphate-buffered saline (PBS), pH 7.4. Erythrocytes were kept up to seven days at 4 C.
Bentazone and oxidative stress
DOI: 10.3109/01480545.2013.870193
Experimental protocol Stock solutions of bentazone were prepared in DMSO at 50 nM concentration. Erythrocytes at 5% (20% for GSH) hematocrit in PBS were incubated with bentazone at the concentrations of 6.25; 12.5; 25; 50 nM for 1 h at 37 C. Samples of erythrocytes in PBS and DMSO without bentazone were used as control. After incubation, the mixtures were haemolysed in 20 C. The mixtures were thawed the following day and then centrifuged at 3600 rpm for 15 min. All assays were performed in these supernatants. The concentration of haemoglobin (Hb) was determined using the method of Drabkin (1946).
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Lipid peroxidation assay Quantitative measurement of lipid peroxidation was performed in erythrocyte hemolysate (5%) according to the method of Stocks & Dormandy (1971) based on the formation of thiobabituric acid reactive substances (TBARS) and expressed as the extent of malondialdehyde (MDA) production. The TBARS levels were calculated using 1,1,3,3-tetraethoxypropane as the standard and expressed as nmol MDA per g of Hb. GSH assay GSH levels were determined in erythrocyte hemolysate (20%) according to the method of Beutler (1975) with modification of the method of Ellman and Lysko (1979) by using 5,50 dithiobis-2-nitrobenzoic acid (DTNB) reagent. DTNB was reduced by free -SH groups of GSH to form 5-mercapto-2nitrobenzoate and its absorbance were measured by spectrophotometric means at 412 nm. Results were expressed as mmol GSH per g of Hb using standard calibration curve. SOD assay Activity of SOD was determined according to the method described by Sun et al. (1988). The principle of the method based on the inhibition of nitroblue tetrazolium (NBT) reduction by using the xanthine-xanthine oxidase system as a superoxide generator. Activity was assessed in the supernatants of 10 000 g of ethanol/chloroform (5/3, v/v) extracts of erythrocyte hemolysate (5%). One unit of SOD was defined as the enzyme amount causes 50% inhibition in NBT reduction rate. Specific activity was expressed as unit SOD per g of Hb using standard calibration curve. CAT assay Activity of CAT was assayed in erythrocyte hemolysate (5%) by the decomposition of hydrogen peroxide (H2O2) according to the method of Aebi (1984). This reaction follows a firstorder kinetic given by equation k ¼ (2.3/t) (log A0/A1). Specific activity was expressed as k per g of Hb using standard calibration curve. GSH-Px assay Activity GSH-Px was measured in erythrocyte hemolysate (5%) according to the method described by Paglia & Valentine (1967). The principle of the method based on the decrease in absorbance of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. GSHPx catalyzes the oxidation of glutathione by hydrogen
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peroxide. In the presence of glutathione reductase and NADPH, oxidized glutathione (GSSG) is immediately converted to the reduced form with a concominant oxidation of NADPH to NADPþ. Specific activity was expressed as unit GSH-Px per g of Hb using standard calibration curve. One unit of GSH-Px is defined as the amount of enzyme that oxidizes 1 mmol of NADPH per minute. GSH-Rd assay Activity of GSH-Rd was performed in erythrocyte hemolysate (5%) by monitoring the oxidation of NADPH in the presence of oxidized glutathione according to the method of Beutler (1969). Specific activity was expressed as milliunit GSH-Rd per g of Hb using standard calibration curve. One unit of GSH-Rd is defined as the amount of enzyme that oxidizes 1 mmol of NADPH per minute. Statistical analysis All data are expressed as mean standard deviation (SD). Data were analyzed by ANOVA test using ‘‘SPSS version 17.0 for Windows’’ (SPSS Inc., Chicago, IL) statistical program and the significance of differences between control and bentazone treated erythrocytes was calculated by Dunnett t-tests. p Values of less than 0.05 and 0.001 were selected as the levels of significance.
Results and discussion In the present study, the changes on oxidative status in human erythrocytes under the influence of bentazone were studied. Erythrocytes are used in studies on the toxicity evaluation of numerous pesticides (Altuntas et al., 2003; Bors et al., 2011; Bukowska et al., 2003). Besides, several investigations have shown that pesticides can damage the balance between prooxidants and antioxidants in body and lipid membrane as a result of lipid peroxidation (Mohammad et al., 2004). However, up to now, there are no studies concerning the effect of bentazone on lipid peroxidation and antioxidant systems in human erythrocytes in vitro. Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity of many xenobiotics (Gutteridge & Halliwell, 2000). The most widely used marker of lipid peroxidation is MDA formation, often assayed with the thiobarbituric acid assay. In the present study, as shown in Figure 1, content of MDA in the erythrocytes treated with bentazone was significantly increased 2.34-fold (p50.05) at the highest concentration (50 nM) when compared to the control group. This is the first study that investigated effects of bentazone on the oxidative stress biomarkes in vitro. Our results are consistent with the previous reports on effects of various pesticides such as chlorpyrifos-ethyl (Gultekin et al., 2000), 2,4-D, MCPA, 2,4,5-T (Duchnowicz et al., 2002), glyphosate (Pieniaz_ ek et al., 2004), trichlorfon (Catalgol et al., 2007), malathion (Durak et al., 2009) and 2,4-dichlorophenol (Bors et al., 2011) in erythrocytes. GSH, one of the most potent reducing biological molecules, affects scavenging of free radical reactions in the erythrocytes (El-Sharkawy et al., 1994). In the present study, GSH content in control erythrocytes averaged
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Drug Chem Toxicol, 2014; 37(4): 410–414
Figure 1. Effects of 1 h bentazone treatment on lipid peroxidation, glutathione level and antioxidant enzyme activitioes in erythrocytes. Units: MDA contents - nmol/g Hb; GSH levels - mmol/g Hb; SOD activities - U/g Hb; CAT activities - k/g Hb; GSH-Px activities - U/g Hb and GSH-Rd activities mU/g Hb. Values are expressed as mean SD; n ¼ 6 for each treatment group. * and ** mean are significantly different from control group at p50.05 and p50.001, respectively (One way ANOVA-Dunnett t-tests).
1.37 0.12 mmol/g Hb. Bentazone caused significant concentration-dependent increases in GSH levels in erythrocytes. The increase in GSH levels were 204%; 269%; 278% and 400% as compared to controls in the erythrocytes exposed to 6.25; 12.5; 25; 50 nM bentazone, respectively (Figure 1). Our results indicated that there was an alteration of GSH metabolic pathway in erythrocytes exposed to bentazone. We demonstrated that GSH is rapidly synthetized in response to bentazone injury. Conversly, Bors et al. (2011) showed that 2,4-dichlorophenol at 250 mg/mL concentration decreased GSH levels in human erythrocytes; Bukowska et al. (2003) also indicated the lower levels of GSH after 2,4-D and 2,4dichlorophenol treatment in human erythrocytes. Antioxidant enzymes, mainly SOD, CAT and GSH-Px are the first line of defense against free radical induced oxidative stress. SOD is responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals to hydrogen
peroxide (McCord & Fridovich, 1988). CAT is responsible for the catalytic decomposition of hydrogen peroxide to molecular oxygen and water (Aebi, 1984). GSH-Px, responsible for enzymatic defense against hydrogen peroxide, is strictly linked with the concentration of GSH because it catalyses the reaction between glutathione and hydrogen peroxide, resulting in the formation of glutathione disulphide (Paglia & Valentine, 1967). Our results show that activity of SOD was remained unchanged while the activity of CAT was nonsignificantly changed in the erythrocytes treated with bentazone (Figure 1). Catalgol et al. (2007) indicated that activity of CAT remained unchanged while the activity of SOD increased after 1 h exposure to trichlorfon in human eryhrocytes. As presented in Figure 1, the mean activity of GSH-Px 233.69 27.8 U/g Hb in control erythrocytes. Activity of GSH-Px non-significantly increased at the 6.25 nM bentazone treatment, while activity of GSH-Px
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DOI: 10.3109/01480545.2013.870193
decreased by 64.94%; 56.12% and 50.08% at the 12.5; 25; 50 nM bentazone treatments, respectively (Figure 1). These findings are in accordance with Catagol et al. (2007) who reported that 1 h treatment of trichlorfon caused a decrease in the activity of SOD in human eryhrocytes. In other study, Durak et al. (2009) reported that malathion treatment caused an decrease in the activity of SOD in human eryhrocytes. As shown in Figure 1, the activity of GSH-Rd in the bentazone treated erythrocytes remained unchanged when compared to control erythrocytes. In presesent study, bentazone caused some disturbances in the activities of antioxidant enzymes in erythrocytes, supporting that reactive oxygen species may be involved in the toxic effects of bentazone. A health-based value of 300 mg/L can be calculated on the basis of an acceptable daily intake (ADI) of 0.1 mg/kg of body weight established by JMPR (Joint FAO/WHO Meeting on Pesticide Residues) and a 10% allocation of the ADI to drinking-water (FAO, 1991). The acute oral lethal dose (LD50) for rats is 500 mg/kg (PPDB, 2009). In conclusion, it was demonstrated that in vitro treatment of bentazone results in induction of lipid peroxidation and alterations the activities of antioxidant enzymes at high doses in human erythrocytes. We suggest that further in vivo studies which include different exposure time points and dosages of bentazone treatment should be investigated to better understand the role of oxidative stress on bentazoneinduced toxicity.
Declaration of interest The authors declare that there are no conflicts of interest. This work was supported by the Research Fund of Istanbul University (BYP-29656, BYP-28594).
References Aebi H. (1984). Catalase in vitro. Meth Enzymol 105:121–126. Altuntas I, Delibas N, Doguc DK, et al. (2003). Role of reactive oxygen species in organophosphate insecticide phosalone toxicity in erythrocytes in vitro. Toxicol in Vitro 17:153–157. Bagchi SN, Das PK, Banerjee S, et al. (2012). A bentazone-resistant mutant of cyanobacterium, Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stres. Physiol Mol Biol Plants 18:115–123. Beutler E. (1969). Glutathione reductase: Stimulation in normal subjects by riboflavin supplementation. Science 165:613–615. Beutler E. (1975). Red cell metabolism. A manual of bichemical methods, second ed. New York: Grune & Stratton, 71–73. Bors M, Bukowska B, Pilarski R, et al. (2011). Protective activity of the Uncaria tomentosa extracts on human erythrocytes in oxidative stress induced by 2,4-dichlorophenol (2,4-DCP) and catechol. Food Chem Toxicol 49:2202–2211. Bozena S, Bozena B, Huras B. (2011). The effect of bromfenvinphos and its impurities on human erythrocyte. Food Chem Toxicol 49:502–507. Bukowska B, Goszcynska K, Duda W. (2003). Effect of 4-chloro-2methylphenoxyacetic acid and 2,4-dimethylphenol on human erythrocytes. Pestic Biochem Phys 77:92–98. Bukowska B, Kopka A, Michałowicz J, Duda W. (2006). Comparison of the effect of Aminopielik D pesticide and its active components on human erythrocytes. Environ Toxicol Pharmacol 22:189–193. Bukowska B, Rychlik B, Krokosz A, Michałowicz J. (2008). Phenoxyherbicides induce production of free radicals in human erythrocytes: Oxidation of dichlorodihydrofluorescine and
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dihydrorhodamine 123 by 2,4-D-Na and MCPA-Na. Food Chem Toxicol 46:359–367. Catalgol BK, Ozden S, Alpertunga B. (2007). Effects of trichlorfon on malondialdehyde and antioxidant system in human erythrocytes. Toxicol in Vitro 21:1538–1544. Cereser C, Boget S, Parvaz P, Revol A. (2001). Thiram-induced cytotoxicity is accompanied by a rapid and drastic oxidation of reduced glutathione with consecutive lipid peroxidation and cell death. Toxicology 163:153–162. Chiapella G, Flores-Martı´n J, Ridano ME, et al. (2013). The organophosphate chlorpyrifos disturbs redox balance and triggers antioxidant defense mechanisms in JEG-3 cells. Placenta 34:792–798. Cicchetti R, Argentin G. (2003). The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts. Mutagenesis 18:127–132. Drabkin DL. (1946). The crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. J Biol Chem 164:703–723. Duchnowicz P, Koter M, Duda W. (2002). Damage of erythrocyte by phenoxyacetic herbicides and their metabolites. Pestic Biochem Phys 74:1–7. Durak D, Uzun FG, Kalender S, et al. (2009). Malathion-induced oxidative stress in human erythrocytes and the protective effect of vitamins C and E in vitro. Environ Toxicol 24:235–242. Ellman G, Lysko H. (1979). A precise method for the determination of whole blood and plasma sulfhydryl groups. Anal Biochem 93: 98–102. El-Sharkawy AM, Abdel-Rahman SZ, Hassan AA, et al. (1994). Biochemical effects of some insecticides on the metabolic enzymes regulating glutathione metabolism. Bull Environ Contam Toxicol 52: 505–510. Environmental Protection Agency (EPA). (2000). Bentazon (Basagran) pesticide tolerance 2/00. Final rule. Washington, DC, USA. Food and Agriculture Organization (FAO). (1991). Pesticide residues in food-1991 evaluations. Part I- Residues. Rome, Food and Agriculture Organization of the United Nations, Joint FAO/WHO Meeting on Pesticide Residues (FAO Plant Production and Protection Paper 113/1). Galhano V, Peixoto F, Gomes-Laranjo J. (2010). Bentazon triggers the promotion of oxidative damage in the Portuguese ricefield cyanobacterium Anabaena cylindrica: response of the antioxidant system. Environ Toxicol 25:517–526. Galhano V, Santos H, Oliveira MM, et al. (2011). Changes in fatty acid profile and antioxidant systems in a Nostoc muscorum strain exposed to the herbicide bentazon. Process Biochem 46:2152–2162. Gultekin F, Ozturk M, Akdogan M. (2000). The effect of organophosphate insecticide chlorpyrifos-ethyl on lipid peroxidation and antioxidant enzymes (in vitro). Arch Toxicol 74:533–538. Gutteridge JM, Halliwell B. (2000). Free radicals and antioxidants in the year 2000. A historical look to the future. Ann N Y Acad Sci 899: 136–147. Macedo RS, Lombardi AT, Omachi CY, Rorig LR. (2008). Effects of the herbicide bentazon on growth and photosystem II maximum quantum yield of the marine diatom Skeletonema costatum. Toxicol in Vitro 22:716–722. McCord JM, Fridovich I. (1988). Superoxide dismutase: the first twenty years (1968–1988). Free Radic Biol Med 5:363–369. Mohammad A, Arkam R, Shahin S, et al. (2004). Pesticides and oxidative stress: A review. Med Sci Monit 10:141–147. Moore PD, Yedjou CG, Tchounwou PB. (2010). Malathion-induced oxidative stress, cytotoxicity and genotoxicity in human liver carcinoma (HepG2) cells. Environ Toxicol 25:221–226. Mu¨ller I.B, Willads Petersen H, Johansen S.S, Theilade P. (2003). Fatal overdose of the herbicide bentazone. Forensic Sci Int 135: 235–236. Paglia DE, Valentine WN. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70:158–169. Pieniaz_ ek D, Bukowska B, Duda W. (2004). Comparison of the effect of Roundup Ultra 360 SL pesticide and its active compound glyphosate on human erythrocytes. Pestic Biochem Phys 79:58–63.
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PPDB (The Pesticide Properties Database), University of Hertfordshire, http://sitem.herts.ac.uk/aeru/footprint/index.htm (last information retrieval June 2009). Spencer, HW. (1998). Toxicological review of Bentazon. Technical report. Washington, DC, USA: Environmental Protection Agency.
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Stocks J, Dormandy TL. (1971). The autoxidation of human red cell lipids induced by hydrogen peroxide. Brit J Haematol 20:95–111. Sun Y, Oberley LW, Li Y. (1988). A simple method for clinical assay of superoxide dismutase. Clin Chem 34:497–500. Worthing CR, ed. (1991). The pesticide manual, 9th ed. Farnham: British Crop Protection Council.
RESEARCH ARTICLE
Effects of bentazone on lipid peroxidation and antioxidant systems in human erythrocytes in vitro
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Universitat de Girona on 12/12/14 For personal use only.
M. Abudayyak, S. Ozden, B. Alpertunga, and G. O¨zhan Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazıt 34116, Istanbul, Turkey
Abstract
Keywords
Bentazone, a benzothiadiazole herbicide, is widely used for a variety of crops including cereals, maize, peas, rice and soy beans. The concern for human health is stil very high because bentazone is continuously monitored in environment and several studies to evaluate its potential carcinogenic effects when chronic and high doses were administered to animals. We aimed to investigate the possible effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes in human erythrocytes in vitro. For that, erythrocyte were incubated with bentazone in different concentrations (0–50 nM) at 37 C for 1 hr. Bentazone showed significant increase in the levels of malondialdehyde (MDA) at the highest concentration in erythrocytes as an index of lipid peroxidation. Besides, alterations in the levels of reduced glutathione (GSH) and activities of glutathione peroxidase (GSH-Px) were observed while the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GSH-Rd) were unchanged. In conclusion, findings from this study indicate that in vitro toxicity of bentazone may be associated with oxidative stress and this work warrants further in vivo investigations.
Antioxidant enzymes, bentazone, human erythrocytes, lipid peroxidation
Introduction Pesticides are used indiscriminately in large amounts, causing environmental pollution and risk to human and animal health. Bentazone (3-(1-methylethyl)-1-H-2,1,3-benzothiadiazin4(3H)-one 2,2-dioxide) is a benzothiadiazole herbicide widely used in the agrochemical field and acts as an inhibitor of photosynthesis in plants. Residual amounts of bentazone have been monitored in the water, soil, honey, plants, fruits and vegetables (Worthing, 1991). Moreover, the first clear report on the fatal poisoning of a human with bentazone has been reported (Mu¨ller et al., 2003). Based on concentration measured in suicidal case involving 59-year-old women who ingested 686–1371 mg/kg bentazone, they reported lethal dose of bentazone as 625 mg/kg body weight. Despite extensive use of bentazone, very limited studies on the mechanisms of its action exist. Long-term studies conducted in rats and mice have not indicated a carcinogenic potential, and a variety of in vitro and in vivo assays have indicated that bentazone is not genotoxic (EPA, 2000; Spencer, 1998). Pesticides or their degradation metabolites may exert toxicity related to oxidative stress and can cause oxidative damage in mammalian cells (Bukowska et al., 2006; Bukowska et al., 2008; Bozena et al., 2011; Cereser et al., ¨ zhan, Istanbul University, Faculty Address for correspondence: Dr. Gu¨l O of Pharmacy, Department of Pharmaceutical Toxicology, 34116 Beyazit, Istanbul, Turkey. Tel: +902124400000. Fax: +902124400252. E-mail: [email protected].
History Received 3 August 2013 Revised 12 November 2013 Accepted 25 November 2013 Published online 23 December 2013
2001; Chiapella et al., 2013; Cicchetti and Argentin, 2003; Moore et al., 2010). Several studies report bentazone has physiological and biochemical effects in higher plants and induces oxidative stress by generation of reactive oxygen species (ROS) in heterocystous cyanobacteria (Bagchi et al., 2012; Galhano et al., 2010; Galhano et al., 2011; Macedo et al., 2008). As reports on the oxidative damage of bentazone in mammalian cells are scanty, we aimed to investigate the dose-dependent effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes (SOD, CAT, GSH-Px and GSH-Rd).
Materials and methods Reagents Bentazone, technical purity 99.2% was obtained from Riedel de Hae¨n (Seelze, Germany). All chemicals were analytical reagent grade and chemicals required for all biochemical assays were obtained from Sigma-Aldrich Chemicals Co (St. Louis, MO) and Merck (Darmstadt, Germany). Erythrocytes preparation Human erythrocytes were obtained from whole blood from healthy donors (Bukowska et al., 2003). The whole blood was centrifuged at 1500 rpm for 10 min. The clear plasma and buffy coat were discarded. Erythrocytes were washed three times with phosphate-buffered saline (PBS), pH 7.4. Erythrocytes were kept up to seven days at 4 C.
Bentazone and oxidative stress
DOI: 10.3109/01480545.2013.870193
Experimental protocol Stock solutions of bentazone were prepared in DMSO at 50 nM concentration. Erythrocytes at 5% (20% for GSH) hematocrit in PBS were incubated with bentazone at the concentrations of 6.25; 12.5; 25; 50 nM for 1 h at 37 C. Samples of erythrocytes in PBS and DMSO without bentazone were used as control. After incubation, the mixtures were haemolysed in 20 C. The mixtures were thawed the following day and then centrifuged at 3600 rpm for 15 min. All assays were performed in these supernatants. The concentration of haemoglobin (Hb) was determined using the method of Drabkin (1946).
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Lipid peroxidation assay Quantitative measurement of lipid peroxidation was performed in erythrocyte hemolysate (5%) according to the method of Stocks & Dormandy (1971) based on the formation of thiobabituric acid reactive substances (TBARS) and expressed as the extent of malondialdehyde (MDA) production. The TBARS levels were calculated using 1,1,3,3-tetraethoxypropane as the standard and expressed as nmol MDA per g of Hb. GSH assay GSH levels were determined in erythrocyte hemolysate (20%) according to the method of Beutler (1975) with modification of the method of Ellman and Lysko (1979) by using 5,50 dithiobis-2-nitrobenzoic acid (DTNB) reagent. DTNB was reduced by free -SH groups of GSH to form 5-mercapto-2nitrobenzoate and its absorbance were measured by spectrophotometric means at 412 nm. Results were expressed as mmol GSH per g of Hb using standard calibration curve. SOD assay Activity of SOD was determined according to the method described by Sun et al. (1988). The principle of the method based on the inhibition of nitroblue tetrazolium (NBT) reduction by using the xanthine-xanthine oxidase system as a superoxide generator. Activity was assessed in the supernatants of 10 000 g of ethanol/chloroform (5/3, v/v) extracts of erythrocyte hemolysate (5%). One unit of SOD was defined as the enzyme amount causes 50% inhibition in NBT reduction rate. Specific activity was expressed as unit SOD per g of Hb using standard calibration curve. CAT assay Activity of CAT was assayed in erythrocyte hemolysate (5%) by the decomposition of hydrogen peroxide (H2O2) according to the method of Aebi (1984). This reaction follows a firstorder kinetic given by equation k ¼ (2.3/t) (log A0/A1). Specific activity was expressed as k per g of Hb using standard calibration curve. GSH-Px assay Activity GSH-Px was measured in erythrocyte hemolysate (5%) according to the method described by Paglia & Valentine (1967). The principle of the method based on the decrease in absorbance of reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. GSHPx catalyzes the oxidation of glutathione by hydrogen
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peroxide. In the presence of glutathione reductase and NADPH, oxidized glutathione (GSSG) is immediately converted to the reduced form with a concominant oxidation of NADPH to NADPþ. Specific activity was expressed as unit GSH-Px per g of Hb using standard calibration curve. One unit of GSH-Px is defined as the amount of enzyme that oxidizes 1 mmol of NADPH per minute. GSH-Rd assay Activity of GSH-Rd was performed in erythrocyte hemolysate (5%) by monitoring the oxidation of NADPH in the presence of oxidized glutathione according to the method of Beutler (1969). Specific activity was expressed as milliunit GSH-Rd per g of Hb using standard calibration curve. One unit of GSH-Rd is defined as the amount of enzyme that oxidizes 1 mmol of NADPH per minute. Statistical analysis All data are expressed as mean standard deviation (SD). Data were analyzed by ANOVA test using ‘‘SPSS version 17.0 for Windows’’ (SPSS Inc., Chicago, IL) statistical program and the significance of differences between control and bentazone treated erythrocytes was calculated by Dunnett t-tests. p Values of less than 0.05 and 0.001 were selected as the levels of significance.
Results and discussion In the present study, the changes on oxidative status in human erythrocytes under the influence of bentazone were studied. Erythrocytes are used in studies on the toxicity evaluation of numerous pesticides (Altuntas et al., 2003; Bors et al., 2011; Bukowska et al., 2003). Besides, several investigations have shown that pesticides can damage the balance between prooxidants and antioxidants in body and lipid membrane as a result of lipid peroxidation (Mohammad et al., 2004). However, up to now, there are no studies concerning the effect of bentazone on lipid peroxidation and antioxidant systems in human erythrocytes in vitro. Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity of many xenobiotics (Gutteridge & Halliwell, 2000). The most widely used marker of lipid peroxidation is MDA formation, often assayed with the thiobarbituric acid assay. In the present study, as shown in Figure 1, content of MDA in the erythrocytes treated with bentazone was significantly increased 2.34-fold (p50.05) at the highest concentration (50 nM) when compared to the control group. This is the first study that investigated effects of bentazone on the oxidative stress biomarkes in vitro. Our results are consistent with the previous reports on effects of various pesticides such as chlorpyrifos-ethyl (Gultekin et al., 2000), 2,4-D, MCPA, 2,4,5-T (Duchnowicz et al., 2002), glyphosate (Pieniaz_ ek et al., 2004), trichlorfon (Catalgol et al., 2007), malathion (Durak et al., 2009) and 2,4-dichlorophenol (Bors et al., 2011) in erythrocytes. GSH, one of the most potent reducing biological molecules, affects scavenging of free radical reactions in the erythrocytes (El-Sharkawy et al., 1994). In the present study, GSH content in control erythrocytes averaged
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Figure 1. Effects of 1 h bentazone treatment on lipid peroxidation, glutathione level and antioxidant enzyme activitioes in erythrocytes. Units: MDA contents - nmol/g Hb; GSH levels - mmol/g Hb; SOD activities - U/g Hb; CAT activities - k/g Hb; GSH-Px activities - U/g Hb and GSH-Rd activities mU/g Hb. Values are expressed as mean SD; n ¼ 6 for each treatment group. * and ** mean are significantly different from control group at p50.05 and p50.001, respectively (One way ANOVA-Dunnett t-tests).
1.37 0.12 mmol/g Hb. Bentazone caused significant concentration-dependent increases in GSH levels in erythrocytes. The increase in GSH levels were 204%; 269%; 278% and 400% as compared to controls in the erythrocytes exposed to 6.25; 12.5; 25; 50 nM bentazone, respectively (Figure 1). Our results indicated that there was an alteration of GSH metabolic pathway in erythrocytes exposed to bentazone. We demonstrated that GSH is rapidly synthetized in response to bentazone injury. Conversly, Bors et al. (2011) showed that 2,4-dichlorophenol at 250 mg/mL concentration decreased GSH levels in human erythrocytes; Bukowska et al. (2003) also indicated the lower levels of GSH after 2,4-D and 2,4dichlorophenol treatment in human erythrocytes. Antioxidant enzymes, mainly SOD, CAT and GSH-Px are the first line of defense against free radical induced oxidative stress. SOD is responsible for catalytic dismutation of highly reactive and potentially toxic superoxide radicals to hydrogen
peroxide (McCord & Fridovich, 1988). CAT is responsible for the catalytic decomposition of hydrogen peroxide to molecular oxygen and water (Aebi, 1984). GSH-Px, responsible for enzymatic defense against hydrogen peroxide, is strictly linked with the concentration of GSH because it catalyses the reaction between glutathione and hydrogen peroxide, resulting in the formation of glutathione disulphide (Paglia & Valentine, 1967). Our results show that activity of SOD was remained unchanged while the activity of CAT was nonsignificantly changed in the erythrocytes treated with bentazone (Figure 1). Catalgol et al. (2007) indicated that activity of CAT remained unchanged while the activity of SOD increased after 1 h exposure to trichlorfon in human eryhrocytes. As presented in Figure 1, the mean activity of GSH-Px 233.69 27.8 U/g Hb in control erythrocytes. Activity of GSH-Px non-significantly increased at the 6.25 nM bentazone treatment, while activity of GSH-Px
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DOI: 10.3109/01480545.2013.870193
decreased by 64.94%; 56.12% and 50.08% at the 12.5; 25; 50 nM bentazone treatments, respectively (Figure 1). These findings are in accordance with Catagol et al. (2007) who reported that 1 h treatment of trichlorfon caused a decrease in the activity of SOD in human eryhrocytes. In other study, Durak et al. (2009) reported that malathion treatment caused an decrease in the activity of SOD in human eryhrocytes. As shown in Figure 1, the activity of GSH-Rd in the bentazone treated erythrocytes remained unchanged when compared to control erythrocytes. In presesent study, bentazone caused some disturbances in the activities of antioxidant enzymes in erythrocytes, supporting that reactive oxygen species may be involved in the toxic effects of bentazone. A health-based value of 300 mg/L can be calculated on the basis of an acceptable daily intake (ADI) of 0.1 mg/kg of body weight established by JMPR (Joint FAO/WHO Meeting on Pesticide Residues) and a 10% allocation of the ADI to drinking-water (FAO, 1991). The acute oral lethal dose (LD50) for rats is 500 mg/kg (PPDB, 2009). In conclusion, it was demonstrated that in vitro treatment of bentazone results in induction of lipid peroxidation and alterations the activities of antioxidant enzymes at high doses in human erythrocytes. We suggest that further in vivo studies which include different exposure time points and dosages of bentazone treatment should be investigated to better understand the role of oxidative stress on bentazoneinduced toxicity.
Declaration of interest The authors declare that there are no conflicts of interest. This work was supported by the Research Fund of Istanbul University (BYP-29656, BYP-28594).
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