The modulatory effect of deltamethrin on antioxidants in mice

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The modulatory effect of deltamethrin on antioxidants in mice
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  The modulatory effect of deltamethrin on antioxidants in mice Hasibur Rehman, Mehboob Ali, Fahim Atif, Manpreet Kaur,Kanchan Bhatia, Sheikh Raisuddin*  Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India Received 9 September 2005; received in revised form 7 January 2006; accepted 9 January 2006Available online 24 February 2006 Abstract  Background:  Deltamethrin is a  a -cyano pyrethroid insecticide used extensively in pest control. Although initially thought to be least toxic, anumber of recent reports showed its toxicity in mammalian and non-mammalian laboratory and wildlife animal species. In fish, it is a potent oxidative stress-inducing agent. We studied the oxidative stress-inducing effect of deltamethrin in mice.  Methods:  Male Swiss albino mice were orally administered 2 doses of deltamethrin viz., 5.6 and 18mg/kg body weight (bw), for 15 days.  Results:  Both the doses of deltamethrin significantly induced lipid peroxidation (LPO) in liver and kidney. Along with the induction of LPO,activities of vital antioxidant enzymes such as glutathione peroxidase (GPx), glutathione  S  -transferase (GST) and catalase (CAT) were alsosuppressed in both the tissues. Glutathione (GSH) level was also decreased. GSH decrease was more pronounced in kidney than the liver. Conclusion:  Toxicity of many chlorinated and organophosphate insecticides is mediated by the reactive oxygen species (ROS). Findings of the present investigation also suggest a role for ROS in deltamethrin toxicity. An increased LPO indicates that these ROS might have causeddegradation of biomembrane in deltamethrin-exposed animals. D  2006 Elsevier B.V. All rights reserved.  Keywords:  Deltamethrin; Oxidative stress; Antioxidant enzymes; Lipid peroxidation; DNA damage 1. Introduction The use of pyrethroids as insecticidal and anti-parasiticformulations has markedly increased in last 2 decades[1,2]. The main advantages of their use are their  photostability, high efficacy at low concentrations, easydisintegration and low toxicity to birds and mammals[3,4]. The selective neurotoxicity of deltamethrin isattributed to their effect on voltage sensitive sodiumchannels (VSSCs) [5]. Deltamethrin is globally used incrop protection and control of malaria and other vector- borne diseases [1,6]. It has a potent insecticidal activity with an appreciable safety margin [2]. However, anumber of studies have demonstrated genotoxic, immu-notoxic and tumorogenic effects of deltamethrin inmammalian and non-mammalian species [7–10]. Since intended use of deltamethrin involves spraying in thecrop fields to control insect pests and impregnation of  bednets to ward off the mosquitoes, concern has beenexpressed about aquatic ecotoxicological implications of its use. Recently, we have reported on oxidative stress-inducing effect of deltamethrin in a freshwater fish Channa punctata  Bloch [11].For many pesticides, induction of oxidative stress is oneof the main mechanisms of their action [12,13]. The damage to membrane lipids, protein and DNA is the endpoint  biomarker of oxidative stress-inducing effects of pesticides[12–14]. In our previous study on deltamethrin in fish, weobserved that various adaptive and compensatory responsesare also induced as a result of exposure to deltamethrin [11].In the present investigation we report effect of 2 sub-lethaldoses of deltamethrin on redox cycle enzymes andglutathione (GSH) in order to understand mammalianresponse to deltamethrin exposure. 0009-8981/$ - see front matter   D  2006 Elsevier B.V. All rights reserved.doi:10.1016/j.cca.2006.01.010* Corresponding author. Tel.: +91 11 26059688x5568; fax: +91 1126059663.  E-mail address:  sraisuddin@jamiahamdard.ac.in (S. Raisuddin).Clinica Chimica Acta 369 (2006) 61 – 65www.elsevier.com/locate/clinchim  2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene (BHT), 1-chloro-2,4-dinitro- benzene (CDNB), EDTA disodium salt, and sulfosalicylicacid were procured from Ameresco (Solon, OH). Dithio-bis-2-nitrobenzoic acid (DTNB), Folin reagent, glutathionereduced (GSH), glutathione reductase (GR), and NADPreduced (NADPH) were from Sigma Chemical Co. (St.Louis, MO).  o -phosphoric acid (OPA) was from CDHChemicals (Mumbai, India). 2-Thiobarbituric acid (TBA)was from Hi-Media Lab (Mumbai) and deltamethrin(99.9%) was from Hoechst Schering Agro Evid Limited(Ankleshwar, India). 2.2. Animals and dose schedule Swiss albino male mice (30 T 2g) were provided by theCentral Animal House Facility of Jamia Hamdard. Theanimals were bred and maintained under standard labora-tory conditions. Commercial pellet diet and water weregiven ad libitum. The study was approved by theInstitutional Animal Ethical Committee (IAEC) of theuniversity. Animals were divided into three groups. Eachgroup comprised of at least 6 animals. Control animals(group I) were administered vehicle (0.2ml corn oil) orally.Treatment group animals (group II and III) were orallyadministered 2 doses of deltamethrin: 5.6 and 18mg/kg bw, suspended in corn oil. All the doses were given dailyonce for 15 days. The dose schedule is based on the preliminary investigation involving a range of doses and previous reports of deltamethrin in mice [6,8,10,15]. 2.3. Biochemical investigations After the termination of treatment, animals weresacrificed by cervical dislocation under mild anesthesiaand their kidney and liver were removed and used for themeasurement of GSH, lipid peroxidation (LPO) andactivities of catalase (CAT), glutathione peroxidase(GPx), and glutathione  S  -transferase (GST). Tissues werehomogenized in chilled phosphate buffer (0.1mol/l, pH7.4) containing KCl (1.17%), using a Potter-Elvehjemhomogenizer and the supernatant was centrifuged at 10,500   g   for 30min at 4 - C to obtain post-mitochondrialsupernatant (PMS). 2.3.1. Estimation of GSH  GSH was measured by the method of Haque et al. [16].Briefly, PMS (1ml) was precipitated with 1ml of sulfosa-licylic acid (4.0%). The samples were incubated at 4 - C for 1h and then centrifuged at 1200   g   for 15min at 4 - C. Theassay mixture contained 0.2ml of filtered aliquot, 2.6ml of sodium phosphate buffer (0.1mol/l sodium phosphate buffer, pH 7.4) and 0.2ml DTNB (stock 100mmol/l in0.1mol/l sodium phosphate buffer, pH 7.4) in a totalvolume of 3ml. The optical density (OD) of reaction product was measured immediately at 412nm usingspectrophotometer. The GSH content is expressed as nmolGSH/g tissue. 2.3.2. Antioxidant enzyme measurements Activities of antioxidant enzymes viz., GST, GPx, andCAT were measured by the method Haque et al. [16]. For  GSTactivity measurement, the reaction mixture consisted of 1.675ml sodium phosphate buffer (0.1mol/l, pH 7.4), 0.2mlGSH (1mmol/l), 0.025ml of CDNB (1mmol/l) and 0.1mlPMS (10%) in a total assay volume of 2ml. The change inabsorbance was recorded at 340nm and the enzyme activitywas calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6  10 3 l/mol cm. For GPx activity measurement, the assaymixture consisted of 1.44ml sodium phosphate buffer, 0.1mlEDTA (1 mmol/l), 0.1ml sodium azide (1mmol/l), 0.05ml of GR (1IU/ml), 0.1ml GSH (1mmol/l), 0.1ml NADPH(0.02mmol/l), 0.01ml H 2 O 2  (0.25mmol/l) and 0.1ml PMS(10%) in a total volume of 2ml. Oxidation of NADPH wasrecorded spectrophotometrically at 340nm at room temper-ature. The enzyme activity was calculated as nmol NADPHoxidized/min/mg of protein, using a molar extinctioncoefficient of 6.223  10 3 l/mol cm. For CAT activitymeasurement, the assay mixture consisted of 1.95ml phos- phate buffer (0.1M, pH 7.4), 1ml H 2 O 2  (0.09mol/l) and0.05ml 10% PMS in final volume of 3ml. Change inabsorbance was recorded at 240nm. Catalase activity wascalculated in terms of nmol H 2 O 2  consumed/min/mg protein. 2.3.3. LPO measurement  LPO was measured using the procedure of Uchiyama andMihara [17]. The tissues were homogenized in chilled 0.1mol/l potassium chloride solution. The mixture consistedof 10mmol/l BHT, 0.67% TBA, 1% chilled OPA and tissuehomogenate (10%). The mixture was incubated at 90 - C for 45min. The absorbance of supernatant was measured at 535nm. The rate of LPO was determined as nmol of TBA-reactive substances (TBARS) formed/h/g of tissue using amolar extinction coefficient of 1.56  10 5 l/mol cm. 2.3.4. Protein estimation Protein content in various samples was measured by themethod of Lowry et al. [18]. 2.3.5. Statistical analysis Data (means T SE) were compared using one-way anal-ysis of variance (ANOVA). Data significant at   p <0.05 inANOVA were further analyzed by post hoc Bonferroni’smultiple-comparison test to determine statistical differences between the groups.  P   value<0.05 was considered signif-icant. GraphPad Prism 3 software (GraphPad Software, Inc.San Diego, CA) was used for statistical analysis.  H. Rehman et al. / Clinica Chimica Acta 369 (2006) 61–65 62  3. Results 3.1. Effect of deltamethrin on antioxidant enzymes in liver of  mice A significant decrease in the activities of GPx, GST andCAT was observed in the liver of deltamethrin-treatedanimals when compared with vehicle-treated (control)animals (Table 1). Doses of 5.6 and 18mg/kg bw of deltamethrin resulted in 30.27% (  p <0.001) and 44%(  p <0.001) decrease in GPx activity, respectively, whencompared with control animals. The GST activity of 5.6and 18mg/kg body weight treatment groups decreased by4.7% (  p <0.05) and 18.5% (  p <0.001), respectively,whereas CAT activity recorded a decrease of 54.3%(  p <0.001) and 64.86% (  p <0.001). At a higher dose of deltamethrin (18mg/kg body weight), significant decreasein the activities of GST (  p <0.01) and CAT (  p <0.001)was noticed when compared with lower dose group(5.6mg/kg body weight). There was no significant differ-ence in GPx activities between both the deltamethrin-treated groups. Also, there was no noteworthy difference between data of vehicle-treated and normal saline animals(data not reported). 3.2. Effect of deltamethrin on antioxidant enzymes in kidneyof mice GPx, GST and CAT activities in kidney of deltamethrin-treated animals were significantly decreased when com- pared with vehicle-treated animals (Table 2). GPx activityrecorded a decrease of 8.06% (  p <0.05) and 20.6%(  p <0.001) in animals treated with 5.6 and 18mg/kgdeltamethrin, respectively. Doses of 5.6 and 18mg/kg bwof deltamethrin resulted in 55.40% (  p <0.001) and 64%(  p <0.001) decrease in GST activity, respectively, whencompared with controls. The CATactivity also decreased by63.27% (  p <0.001) and 69.19% (  p <0.001) in treatment groups. At higher dose (18mg/kg), significant (  p <0.05– 0.001) inhibition was observed in activities of all theenzymes when compared with lower dose (5.6mg/kg) groupdata. 3.3. Effect of deltamethrin on reduced glutathione in liver and kidney of mice GSH content in liver was significantly decreased by43.55% (  p <0.001) to 63.67% (  p <0.001), as a result toexposure of 5.6 and 18mg/kg deltamethrin, respectively,when compared with vehicle-treated animals (Fig. 1). GSH in kidney also decreased significantly by 93.18%(  p <0.001) and 89.23% (  p <0.001) at 5.6 and 18mg/kgdose, respectively (Fig. 1). Comparison between data of   both the treatment groups showed no significant differencein GSH content in either liver or kidney. 3.4. Effect of deltamethrin on LPO in liver and kidney of  mice LPO in liver was significantly increased by 95.83%(  p <0.001) and 130% (  p <0.001) at 5.6 and 18mg/kg dosesof deltamethrin, respectively, when compared with vehicle-treated animals (Fig. 2). The percent increase in LPO in kidney was 67.63% (  p <0.001) and 83.9% (  p <0.001) at 5.6 Table 1Effect of deltamethrin on antioxidant enzymes in the liver of miceGroup Antioxidant enzyme activityGPx GST CATI (vehicle) 87.19 T 3.32 143.0 T 5.03 135.2 T 0.82II (5.6mg/kg) 60.79 T 2.25** 136.33 T 1.56* 61.74 T 1.12**III (18mg/kg) 48.82 T 4.23** 116.42 T 1.11** ,a  47.47 T 1.92** ,b Values are means T SE ( n =6). GPx is expressed as nmol NADPH oxidized/ min/mg/protein, GST as nmol CDNB conjugate formed/min/mg protein,catalase as nmol H 2 O 2  consumed/min/mg protein. Significant differencesare indicated by *  p <0.05 and **  p <0.001 when compared with vehicle-treated group animals and  a   p <0.01 and  b  p <0.001 when compared with5.6mg/kg body weight of deltamethrin.Table 2Effect of deltamethrin on antioxidant enzymes in the kidney of miceGroup Antioxidant enzyme activityGPx GST CATI (vehicle) 141.5 T 4.71 248.80 T 3.02 168.3 T 2.01II (5.6mg/kg) 130.1 T 2.55* 110.94 T 0.88** 61.81 T 1.63**III (18mg/kg) 112.3 T 2.52** ,a  89.72 T 1.85** ,c 51.85 T 1.40** ,b Values are means T SE ( n =6). GPx is expressed as nmol NADPH oxidized/ min/mg/protein, GST as nmol CDNB conjugate formed/min/mg protein,catalase as nmol H 2 O 2  consumed/min/mg protein. Significant differencesare indicated by *  p <0.05 and **  p <0.001 when compared with vehicle-treated group animals and  a   p <0.05,  b  p <0.01 and  c  p <0.001 whencompared with 5.6mg/kg body weight of deltamethrin. 00.10.20.30.40.50.6Vehicle 5.6mg/kg 18mg/kg Treatment   n  m  o   l   G   S   H   /  g   t   i  s  s  u  e Liver Kidney **** Fig. 1. Effect of deltamethrin on reduced glutathione in liver and kidney.Values are expressed as means T SE nmol GSH/g tissue ( n =6). Significant differences is indicated by *  p <0.001 when compared with vehicle-treatedanimals.  H. Rehman et al. / Clinica Chimica Acta 369 (2006) 61–65  63  and 18mg/kg, respectively, over control values (Fig. 2). The LPO was also found to be significantly greater at 18mg/kgdose as compared to 5.6mg/kg dose for kidney (  p <0.05) aswell as liver (  p <0.001). 4. Discussion The orally administered deltamethrin-induced LPO anddecreased various vital antioxidants in liver and kidney of mice. Both the organs showed almost a similar response. Inmost of the in vivo toxicity studies in mammals oral route of administration of deltamethrin has been employed, asgastrointestinal tract is the main site of its absorption. Themain site of metabolism of deltamethrin is liver  [1,19]. The disruption of antioxidant balance in liver may indirectlyaffect its activation balance and disposition of deltamethrin.Deltamethrin induced discernible oxidative stress re-sponse in mice as measured by increased LPO. Oxidativedamage has been recognized as one of the primary causes of subcellular toxicity of pesticides [12]. Studies on pyrethroid insecticides have also suggested a putative role for freeradicals in LPO and other oxidative stress-mediated injuries[11,20]. In the present study liver and kidney showed almost a similar pattern of induction of LPO with greater degree of LPO at the higher dose. LPO is caused by the action of ROS. ROS also cause damage to DNA and proteinsresulting in various harmful consequences [12,13]. El- Gohary et al. [21] reported deltamethrin-induced LPO andnitric oxide (NO) production in plasma of rats and its role intesticular apoptosis. Recently, Imamura et al. [22] havereported deltamethrin-induced expression of activity-depen-dent gene expression of brain-derived neurotrophic factor (BDNF) in SD rats. Li et al. [23] advocated a role for oxidative stress in deltamethrin-induced neurotoxicity. Thefindings of present investigation on liver and kidney warrant further investigation on induction of redox cycling bydeltamethrin in brain and its correlation with BDNF.LPO induction was found to be dose-dependant in boththe tissues. Both the liver and kidney showed an equal levelof susceptibility to deltamethrin exposure. However, whenthe relative responses of LPO and GSH were compared,effect of deltamethrin was more pronounced in case of decrease in GSH than the LPO induction in both the tissue.Manna et al. [24] showed that deltamethrin exposure(15mg/kg for 30 days p.o.) led to 13-fold increase in LPOin rat liver. On the pattern of our observations, they alsofound that the activities of hepatic CAT and GSH decreasedsignificantly in deltamethrin-treated animals. Yarsan et al.[25] have also reported on LPO inducing effect of deltamethrin in plasma and decrease in activities of Cu– Zn–SOD and GPx in erythrocytes in mice.In the present study decrease in GSH content was foundto be more pronounced in kidney than the liver. This showsthat kidney is relatively more susceptible to deltamethrintoxicity than the liver. There is no report in support of thisobservation. Decrease in the GSH level and disruption of activities of antioxidant enzymes as reported here might contribute to histological changes. Tos-Luty et al. [15]reported on degenerative changes with inflammatory foci inmouse liver exposed to 5mg/kg of deltamethrin with more pronounced at higher dose (25mg/kg). Inflammatorychanges are a clear indication of involvement of freeradicals in deltamethrin-induced toxicity. Findings of the present study also suggest a role for free radicals indeltamethrin-induced heptotoxicity and nephrotoxicity. Fur-thermore, role of oxygen free radical-mediated oxidativestress is strengthened by the findings of Jayasree et al. [26]who have shown that administration of vitamin E (anantioxidant) was helpful in reversing the toxic effects of deltamethrin as measured by various oxidative stress biomarkers in broiler chicks. Similar studies in mammalsmay provide means of abrogation of deltamethrin-inducedoxidative stress and its deleterious consequences. Acknowledgements Financial support from the University Grants Commis-sion (UGC), Government of India is acknowledged. We alsoacknowledge the support offered by the Dr. Ehsan A. Khan,Head of the Department for our research work. References [1] IPCS (International Programme on Chemical Safety). EnvironmentalHealth Criteria 97. Deltamethrin. Geneva ’  World Health Organization;1990. 050100150200250300350Vehicle 5.6mg/kg 18mg/kg Treatment   n  m  o   l  e   T   B   A   R   S   f  o  r  m  e   d   /   h   /  g   t   i  s  s  u  e Liver Kidney * ** a  * b Fig. 2. Effect of deltamethrin on lipid peroxidation in liver and kidney.Values are expressed as means T SE ( n =6). LPO was measured as nmolTBARS formed/h/g tissue. Significant differences are indicated by*  p <0.001 when compared with vehicle-treated animals,  a   p <0.001 and  b  p <0.05 when compared with 5.6mg/kg treatment group in liver andkidney, respectively.  H. Rehman et al. / Clinica Chimica Acta 369 (2006) 61–65 64  [2] Mestres R, Mestres G. 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Toxicol Appl Pharmacol Sep 14 2005, doi:10.1016/j.taap.2005.07.021 [Electronic publication ahead of print].[20] Kale M, Rathore N, John S, Bhatnagar D. Lipid peroxidative damageon pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species.Toxicol Lett 1999;105:197–205.[21] El-Gohary M, Awara WM, Nassar S, Hawas S. Deltamethrin-inducedtesticular apoptosis in rats: the protective effect of nitric oxidesynthase inhibitor. Toxicol 1999;132:1–8.[22] Imamura L, Yasuda M, Kuramitsu K, Hara D, Tabuchi A, Tsuda M.Deltamethrin, a pyrethroid insecticide, is a potent inducer for theactivity-dependent gene expression of brain-derived neurotrophicfactor in neurons. J Pharmacol Exp Ther 2006;316:136–43.[23] Li HY, Shi N, Chen D, et al. Oxidative stress of deltamethrin on rat nervous system. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi2005;23:97–101.[24] Manna S, Bhattacharyya D, Mandal TK, Das S. 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