Nanoparticles of zinc oxide defeat chlorpyrifos-induced immunotoxic effects and histopathological alterations

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1 Veterinry World, EISSN: RESEARCH ARTICLE Open Aess Nnoprtiles of zin oxide defet hlorpyrifos-indued immunotoxi effets nd histopthologil ltertions Sr S. Ess 1, Eimn M. El-Sied 2, Osm S. El-Twil 2, Ins M. Gml 1 nd Shr S. Ad El-Rhmn 3 1. Immune Setion, Reserh Institute for Animl Reprodution, Ciro, Egypt; 2. Deprtment of Toxiology, Forensi Mediine nd Veterinry Regultions, Fulty of Veterinry Mediine, Ciro University, Egypt; 3. Deprtment of Pthology, Fulty of Veterinry Mediine, Ciro University, Ciro, Egypt. Corresponding uthor: Shr S. Ad El-Rhmn, e-mil: shrsmirmh@u.edu.eg Co-uthors: SSE: sr_yn79@yhoo.om, EME: emn_moustf84@yhoo.om, OSE: osmeltwil@u.edu.eg, IMG: insgml@yhoo.om Reeived: , Aepted: , Pulished online: doi: /vetworld How to ite this rtile: Ess SS, El-Sied EM, El-Twil OS, Gml IM, Ad El- Rhmn SS (2019) Nnoprtiles of zin oxide defet hlorpyrifos-indued immunotoxi effets nd histopthologil ltertions, Veterinry World, 12(3): Astrt Bkground nd Aim: Chlorpyrifos (CPF) is widely used orgnophosphte insetiide. Nnoprtiles of zin oxide (ZnO NPs) physilly showed effetive dsoring property for some insetiides. The study ws onduted to estimte the potentil effet of ZnO NPs ginst CPF toxiity. Mterils nd Methods: Four groups of mle rts were used; ontrol group nd three groups reeived drinking wter ontined 75 mg/l CPF, omined 75 mg/l CPF nd 200 mg/l ZnO NPs, nd 200 mg/l ZnO NPs, respetively. Results: CPF signifintly deresed mrophge tivity, serum lysozyme tivity, nd levels of interleukin-2 (IL-2) nd IL-6; inresed the perentge of DNA degenertion on omet ssy of lymphoytes nd signifintly elevted hepti nd spleni mlondildehyde ontents; nd deresed their glutthione ontents. The liver nd spleen showed mrked histologil ltertions fter exposure to CPF with deresed expression of etylholinesterse. The odministrtion of ZnO NPs meliorted most of the undesirle effets of CPF, through elevtion of mrophge nd serum lysozyme tivities, inresed the levels of IL-2 nd IL-6, orreted the oxidtive stress mrkers, nd llevited most of the dverse effet exerted y CPF in liver nd spleen tissues. Conlusion: The ddition of ZnO NPs to CPF-ontminted drinking wter my e useful s powerful ntioxidnt gent ginst toxi dmge indued y CPF prtiulrly in individuls who re on dily ouptionl exposure to low doses of CPF. Keywords: etylholinesterse, hlorpyrifos, immune system, pthology, zin oxide nnoprtiles. Introdution Commeril insetiides used in griulturl nd non-griulturl purposes hve the potentil to use signifint humn nd niml illnesses through diret or indiret exposure during pplition. Due to the extensive use of insetiides, they persist in soil, surfe wters, ir, nd griulturl produts [1]. Chlorpyrifos (CPF) is well-known orgnophosphorothiote insetiide tht is used for griulturl nd non-griulturl res. It is rod-spetrum insetiide used to kill wide rnge of insets [2,3]. CPF inhiits etylholinesterse (ACHE) enzyme of the nervous system neessry for proper funtion of the nervous system. Symptoms ssoited with the CPF poisoning inluding; nuse, vomiting, dirrhe, hedhe, onvulsions, om, nd deth in severe onditions. Long period exposure to CPF results in Copyright: Ess, et l. Open Aess. This rtile is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pulidomin/zero/1.0/) pplies to the dt mde ville in this rtile, unless otherwise stted. serious hrm effets to the nervous system, respirtory trt, nd rdiovsulr systems. CPF metolites persist in the environment for long period; therefore, it eomes puli onern. CPF n e oxidized y vrious oxidizing gents, whih give CPF-oxon fter the replement of sulfur y oxygen in the thiophosphoryl ond [4]. CPF-oxon is more toxi ompred to its prent ompound [5]. Nnotehnology offers fst nd effetive solutions for environmentl lenup. It hs ttrted onsiderle interest of oth sientifi nd industril ommunities euse it is often desried s n emerging tehnology ple of revolutionizing pprohes to ommon prolems [6]. Nnostrutured memrnes with size-seletive pores my provide effiient wys of seprting solutes from wter [7]. Besides filtrtion, whih is generlly energy intensive, the removl of ontminnts y sequestrtion (dsorptive remedition) or degrdtion to less toxi produts (retive remedition) my represent n effetive lterntive. Nnomterils possess very lrge surfe-to-volume rtio tht fvors intertion with their environment. For exmple, nnomterils hve the potentil to effetively dsor moleules or tlyze hemil retions t their interfe [8]. Shhrm et l. [9] Veterinry World, EISSN:

2 showed tht nnoprtile (NPs) form of zin oxide (ZnO) ws effetive dsoring gent for permethrin insetiide in wter system, nd the mount of redution is relted to permethrin onentrtion. For this reson, ZnO in the NP form ould e n eminent ndidte for preventing CPF dverse effets. As per our knowledge, no work hs een reported onerning the role of ZnO, in the NP form ginst CPF toxiity in in vivo system. Hene, this work imed to investigte the potentil effets of this formultion to relieve the toxi effet of CPF on immune system prospeting its pplition industrilly nd medilly to remove this insetiide from niml s wter to derese its toxiity. In ddition, this work ws employed to onfirm tht ZnO NPs hve no hrmful effets when it is dded to the drinking wter of the nimls. Mterils nd Methods Ethil pprovl The Institutionl Animl Cre nd Use Committee (IACUC) of Ciro University pproved the design of the experiment (IACUC protool numer: CU-II-S-50-17). Chemils CPF ws provided from Centrl Agriulturl Pestiide Lortory, Ntionl Center for Agriulturl Reserh, Ministry of Agriulture, Giz, Egypt. ZnO NPs (100 nm) were purhsed from Nno- Teh, Dremlnd, 6 th Otoer, Giz, Egypt. Other hemils were purhsed from Sigm-Aldrih Chemils Co., St. Louis, MO, USA, nd Cusio Bioteh Co. Ltd. Animls Sixty mture mle Sprgue Dwley rts weighing (180±10 g) were used in this investigtion. They were otined from n niml house elonging to the Deprtment of Veterinry Hygiene nd Mngement, Fulty of Veterinry Mediine, Ciro University. Animls were mintined in plsti ges, fed on stndrd ommeril pelleted feed nd wter tht supplied d liitum. They were oserved for helth sttus nd were limted to the lortory environment for 2 weeks efore use. Experimentl protool Rts were rndomly divided into four equl groups. The first group served s ontrol, the seond group reeived drinking wter ontined 75 mg/l CPF, the third group reeived drinking wter ontined 75 mg/l CPF nd 200 mg/l ZnO NPs, while the fourth group reeived drinking wter ontined 200 mg/l ZnO NPs. The experiment ws extended for 9 weeks. The seleted onentrtion of CPF ws nerly equivlent to 1 / 20 of the LD 50 [10], while the seleted onentrtion of ZnO NPs ws nerly equivlent to 30 mg/kg ody weight whih onsidered of no oservle dverse effets [11]. Blood smples were olleted every 3 weeks s will e mentioned, while t the end of the experimentl period, nimls of ll groups were srified under nesthesi using ketmine nd xylzine (40 mg/kg nd 5 mg/kg, respetively) intrperitonelly, then liver nd spleen were refully disseted out, lotted free of lood nd eh orgn ws divided into two prts, one prt ws kept in 10% uffered neutrl formlin for histopthologil exmintion nd the other ws kept t 20 C for further ssessment of ntioxidnt mrkers. Immunologil ssessment Every 3 weeks, ten nimls from eh group were rndomly seleted for lood smple olletion from the retro-oritl venous plexus under nesthesi using the mix of ketmine nd xylzine (40 mg/kg nd 5 mg/kg, respetively) IP. Blood smples were used for the ssessment of mrophge tivity fter 1 nd 2 h nd omet ssy on lymphoytes. In ddition, serum smples were used to estimte the tivity of serum lysozyme nd the titer of interleukin-2 (IL-2) nd IL-2. Determintion of mrophge tivity The mount of nitrite produed from isolted nd ultivted mrophges during exposure to infetion ws determined y mixing the superntnt with the olorless Griess regent forming purple omplex. The degree of the developed olor ws mesured spetrophotometrilly fter 1 nd 2 h using ELISA reder t 570 nm [12]. Comet ssy of lymphoytes DNA single-strnd reks (frnk strnd reks nd inomplete exision repir sites) were investigted t the single ell level using single-ell gel eletrophoresis s previously mentioned y Tie et l. [13]. Determintion of serum lysozyme tivity Lysozyme is n enzyme tht is known to kill the orgnism through hydrolysis of their ell wlls. Lysozyme enzyme ws llowed to diffuse through the grose gel ontining suspension of Miroous lysodeiktius s the sustrte produing ler zone ring of lysis on the grose gel. At the end of the inution period, the dimeters of the ler zone rings were mesured to the nerest 0.1 mm with n enlrger viewer (Klesttd Lortories., In., nd Austin, TX). For eh lysoplte, the lysozyme onentrtion in the smples ws determined from plotted stndrd urve ginst the orresponding ler zone ring dimeter on the liner xis [14]. Determintion of serum IL-2 nd IL-6 The quntittive sndwih enzyme immunossy tehnique ws employed for the determintion of IL-2 ording to Goldsmith nd Greene [15] nd IL-6 ording to Hirno [16]. Antiodies speifi for IL-2 or IL-6 hve een preoted onto miropltes. Stndrds nd smples were pipetted into the wells nd, if the smples ontin ny IL-2 or IL-6, it will e ound y the immoilized ntiody. Any Veterinry World, EISSN:

3 unound sustne ws removed, nd then, iotin-onjugted horserdish peroxidse ws dded to the wells. Any unound vidin-enzyme regent ws wshed followed y the ddition of sustrte solution to the wells. The developed olor will e in proportion to the mount of IL-2 or IL-6 ound in the initil step. The olor development ws stopped, nd the intensity of the olor ws mesured. Determintion of the ntioxidnt mrkers in liver nd spleen tissues homogentes Briefly, homogeniztion of liver nd spleen tissue speimens ws performed in 5-10 ml old uffer (50 mm) of potssium phosphte (ph 7.5) nd 1 mm EDTA or 1 mm EDTA per grm tissue for the determintion of the lipid peroxidtion mrker; mlondildehyde (MDA) nd glutthione (GSH) levels, respetively. Following entrifugtion of the otined homogentes t 5000 rpm for 20 min t 4 C, the superntnts were spirted nd trnsferred into Eppendorf Tues nd then preserved t 80 C until used. Both MDA nd GSH ontents were ssessed in the hepti nd spleni tissue homogentes ording to Ellmn [17] nd Ruiz-Lrre et l. [18], respetively. Histopthologil nd immunohistohemil evlution After fixtion of liver nd spleen speimens in 10% uffered neutrl formlin for 72 h, the speimens were wshed, dehydrted in grded series of lohol nd lered in xylol, nd then emedded in prffin. Seril setions of 4-5-µm thikness were otined nd stined with hemtoxylin nd eosin [19]. Eletri light mirosope Olympus BH2 (Tokyo, Jpn) ws used for the histopthologil exmintion of hemtoxylin nd eosin setions. Expression of ACHE in prffin setions of hepti tissue of ll groups ws demonstrted immunohistohemilly ording to the method desried y Hsu et l. [20] using vidin-iotin-peroxidse (3,3-diminoenzidine tetrhydrohloride [DAB], Sigm Chemil Co.). After inution of liver setions with monolonl ntiody for ACHE (Dko Corp, Crpinteri, CA) nd regents of vidin-iotin-peroxidse method (Vetstin ABC Peroxidse Kit, Vetor Lortories), ntigen-ntiody omplex ws deteted. The immunoretive ells were visulized using hromgen DAB (Sigm Chemil Co.). Following exmintion, the intensity of immunohistohemil stining of ACHE ws quntified in rndom five high mirosopi fields s n optil density using imge nlysis softwre (Imge J, 1.46, NIH, USA). Sttistil nlysis The results were expressed s men±stndrd error of the men. Dt were nlyzed using one-wy nlysis of vrine followed y Dunn s multiple rnge test nd GrphPd softwre CoStt. p<0.05 ws onsidered sttistilly signifint. Results Mrophge phgoyti tivity Mrophges tivity of rts whih reeived wter ontined 75 mg/l CPF fter 1 nd 2 h from its ultivtion showed signifint (p<0.05) derese ompred to the ontrol group long the three periods (fter 3, 6, nd 9 weeks) of ssessments, while mrophges tivity of those rts whih reeived wter ontined CPF nd 200 mg/l ZnO NPs ws signifintly higher thn tht of CPF-intoxited group long the experimentl period. The tivity of mrophges of ZnO NP-dministrted group showed non-signifint differenes ompred with tht of ontrol sets in the three periods ssessments (Figure-1 nd ). Comet ssy of lymphoytes DNA degenertion perentge in ontrol nd the other treted groups is presented in Figure-2. The n ulei of the lymphoyte ells of the ontrol group in the lst week of the experiment (9 th week) hd no til reveling intt DNA (Figure-2). While those of rts reeived CPF, their DNA showed signifint (p<0.05) high degenertion perentge ompred with the ontrol group nd mny nulei ppered with hed nd til (Figure-2). On the other hnd, rts whih drnk wter ontined CPF nd ZnO NPs (Figure-2d) reveled signifint (p<0.05) lower DNA degenertion perentge ompred with tht oserved in the CPF-intoxited group. ZnO NP-dministrted group reveled non-signifint differene from the ontrol group. Serum lysozyme tivity Serum lysozyme tivity of CPF-intoxited group showed signifint (p<0.05) derese when it ompred with tht of ontrols t eh time point. The dministrtion of ZnO NPs to CPF-intoxited rts signifintly (p<0.05) inresed the lysozyme tivity Figure-1: Men vlues±stndrd error of the mrophge tivity (µm/ml) fter 1 h () nd 2 h () from its ultivtion of ontrol nd experimentl rts whih reeived wter ontined 75 mg/l hlorpyrifos nd/or 200 mg/l zin oxide nnoprtiles. Columns hve different sripts t the sme time re signifintly different t p 0.05 (n=10). Veterinry World, EISSN:

4 Figure-2: Comet ssy of lymphoytes. () The men vlues±stndrd error of the of DNA degenertion perentge of lymphoyte in ll groups. (-d) Nulei of the lymphoytes of; () ontrol rt hve no til (rrow) reveling intt DNA, () rts whih reeived wter ontined hlorpyrifos (CPF) hve hed nd til (rrow) reveling mny DNA reks, (d) rt whih reeived wter ontined omined CPF zin oxide nnoprtiles reveled mny intt DNA tht hs no til (rrow) nd few DNA reks whih hve hed nd til (dshed rrow). Columns hve different sripts t the sme time re signifintly different t p 0.05 (n=10). d ompred with tht of CPF-intoxited group prtiulrly t the 6 th nd 9 th weeks of ssessment. Lysozyme tivity of rts whih reeived wter ontined ZnO NPs showed no differene from tht of the ontrols (Figure-3). Serum IL-2 A grdul inrese in serum IL-2 titer ws reorded in the serum of ontrol group long the periods of ssessment (3 rd, 6 th, nd 9 th weeks). However, serum IL-2 titer of rts intoxited with CPF (75 mg/l) showed signifint (p<0.05) derese ompred with tht of ontrol nd the other experimentl groups. Rts whih reeived wter ontined omintion of CPF nd ZnO NPs showed signifint inrese in their IL-2 titer ompred to tht of CPF intoxited rts, while serum titer of IL-2 of rts whih dministrted ZnO NPs in their drinking wter showed no differene from the ontrol group long the experimentl period (Figure-3). Serum IL-6 Serum IL-6 titer of rts whih dministrted CPF showed signifint (p<0.05) derese ompred to tht of ontrols t eh time point. The titer of the group intoxited with CPF nd treted with ZnO NPs showed signifint (p<0.05) inrese ompred with tht of CPF-intoxited group exept t the 3 rd week of the experiment. Serum IL-6 titer for ZnO NPs-treted group showed non-signifint differene from tht of the ontrol group long the whole period of the experiment (Figure-3). Oxidtive stress mrkers Signifint (p<0.05) elevtion of oth hepti nd spleni MDA levels (Figure-4) ompnied with signifint (p<0.05) redution in their GSH ontent (Figure-4) ws oserved in CPF-intoxited rts ompred with ontrols nd those reeived ZnO NPs in their drinking wter. The odministrtion of ZnO NPs with CPF in the drinking wter of rts exhiited signifint (p<0.05) reupertion of tht ltered oxidtive stress mrkers y signifint (p<0.05) derese in MDA level nd signifint inrese in GSH ontent. Histopthologil nd immunohistohemil findings Mirosopi exmintion of the liver nd spleen of norml ontrol (Figure-5 nd ) rts nd those dministrted ZnO NPs reveled norml histologil struture (Figure-5 nd d). Liver setions of CPFdministrted rts reveled ongestion of the entrl veins nd sinusoids (Figure-5e). Hepti sinusoids were dilted nd showed leukoytosis. Widespred heptoellulr vuolr degenertion nd nerosis were evident prtiulrly in the entriloulr re (Figure-5f). The neroti ells ppered with pyknoti or kryorrheti nulei or without ny nuler struture. Livers of rts reeived wter ontined omined CPF nd ZnO NPs showed good protetion of the hepti prenhyml ells ginst the hrmful effet of CPF with the only pperne of miniml degenertive hnges (Figure-6). The spleen of rts whih reeived CPF in their drinking wter showed mrked lymphoyti depletion of the white pulp lymphoid folliles with inresed numer of the tingile ody mrophges nd lymphoyte poptosis (Figure-6). Mrked loss of lymphoytes in the germinl enter with the pperne of the underlying retiulr msh ws oserved (Figure-6). The entrl rteriole showed thikened wll nd fol res of hyliniztion. However, the spleens of omined Veterinry World, EISSN:

5 CPF nd ZnO NPs dministrted rts showed miniml lymphoyti degenertion nd nerosis (Figure-6d). ACHE showed mrked immunopositivity in the livers of ontrol (Figure-7) s well s ZnO NP (Figure-7) dministrted rts, while the livers of rts whih reeived CPF in their drinking wter showed signifint (p<0.05) deresed expression of hepti ACHE (Figure-7). The ltter derese ws signifintly (p<0.05) restored on the omined use of ZnO NPs with CPF (Figure-7d) s deteted y the quntittive nlysis of ACHE immune expression in vrious groups (Figure-7e). Disussion CPF is rod-spetrum ntiholinesterse insetiide, utilized extensively in griulture nd residentil pest ontrol throughout the world [21]. Figure-3: Men vlues±stndrd error of () serum lysozyme tivity (u/ml), () interleukin-2 (IL-2) titer (pg/ ml), nd () IL-2 (pg/ml) in ontrol nd experimentl groups whih reeived wter ontined 75 mg/l hlorpyrifos nd/ or 200 mg/l zin oxide nnoprtiles. Columns hving different sripts t the sme time re signifintly different t p 0.05 (n=10). Monitoring studies onduted in different ountries reveled the presene of residues of this insetiide in different vrieties of food ommodities [22,23]. Moreover, CPF ws lso ytotoxi nd immunotoxi even t lower onentrtion [24]. ZnO NPs physilly showed effetive dsoring property for some insetiides [9]. The present study ws onduted to follow up the protetive effiy of ZnO NPs (100 nm) ginst CPF immunotoxiity in mle rts when they left to gin ess to CPFontminted drinking wter. In the urrent study, ellulr immune response of mle rts ws signifintly redued s result of drinking wter ontminted with 75 mg/l CPF. The reorded immunotoxi effets of CPF ould e ttriuted to its diret toxi effet on lood ells s reorded y signifint low perentges of phgoyti tivity of mrophges fter 1 nd 2 h from its ultivtion nd serum lysozyme tivity or indiretly on the hemtopoieti orgns s oserved y the histopthologil nd immunohistohemil ltertions in liver nd spleen. Despite CPF is ACHE inhiitor, its dverse effets re not only onfined to holinergi system ut lso to other ody systems. Mny studies eluidted tht CPF exerted severl dverse effets inluding hemotoxiity, immunologil normlities s well s hepti nd renl dysfuntions [25-27]. Other studies reveled leukopeni pprently due to lymphopeni, neutropeni, nd monoytopeni in the CPFdministrted nimls [25,28] nd oxidtive stress s well [29]. In the present investigtion, immunotoxi mnifesttions indued y CPF my e ssoited with the enhned prodution of retive oxygen speies (ROS), whih use dmge to vrious memrne omponents of the ells of the immune system. Mnsour et l. [27] identified ROS s use of toxi effets exerted y orgnophosphorus pestiides. These ROS re responsile for induing oxidtive stress in the tissues nd hroni permnent dmge. Comet ssy of the lymphoytes in the present study showed generl trend of inresed DNA degenertion in the CPF-intoxited rts when it ompred with the ontrol nd the other experimentl groups. ROS produed due to CPF toxiity [21] ould e Figure-4: Men vlues±stndrd error of hepti nd spleni ontents of () mlondildehyde nd () glutthione in ontrol nd experimentl groups whih reeived wter ontined 75 mg/l hlorpyrifos nd/or 200 mg/l nnoprtiles of zin oxide. Columns hving different sripts t the sme time re signifintly different t p 0.05 (n=10). Veterinry World, EISSN:

6 e d d f Figure-5: The liver nd spleen of norml ontrol ( nd ) nd nnoprtiles of zin oxide-dministrted rts ( nd d). (e nd f) The liver of hlorpyrifos-dministrted rts showing; (e) ongestion of entrl vein (CO), dilttion of hepti sinusoids (dshed rrow) nd (e) nd (f) entriloulr (CL) heptoellulr degenertion nd nerosis. d Figure-6: () The liver of hlorpyrifos (CPF) nd nnoprtiles of zin oxide (ZnO NPs) dministrted rt showing miniml degenertive hnges in the hepti prenhyml ells. ( nd ) Spleen of rt whih reeived CPF showing lymphoyti depletion with mny tingile ody mrophges (rrow) nd lymphoytes poptosis in the folliles, loss of the lymphoytes in the germinl enter with pperne of the underlying retiulr msh. (d) The spleen of omined CPF nd ZnO NP-dministrted rt showing miniml lymphoyti nerosis. eliited detrimentl DNA dmge. Mny reports hve identified two potentil ellulr trgets for CPF, ell signling sdes from one side nd the expression nd funtion of gene trnsription ftors from the other side [30]. Oxidtive stress is known to e key ftor in severl diseses. In ft, one of the moleulr mehnisms of some pestiides toxiity seems to e lipid peroxidtion; s onsequene, these ompounds n distur the iohemil nd physiologil funtions of the immune system ells s well s the liver nd kidney [31]. The urrent results illustrted tht CPF hd strong inhiitory effet on oth IL-2 nd IL-6; these ytokines stimulte T-lymphoyte prolifertion nd stimulte the tivities of T-helper ells nd ytotoxi T-ells. They lso indue the Veterinry World, EISSN: e Figure-7: Aetylholinesterse (ACHE) immune expression in the liver of ontrol () nd experimentl groups whih reeived wter ontined 200 mg/l nnoprtiles of zin oxide (ZnO NPs) (), 75 mg/l hlorpyrifos (), nd omined CPE nd ZnO NPs (d). (e) The quntittive imge nlysis of ACHE immune expression in vrious groups expressed s optil density in 5 mirosopi fields. : signifintly different from ontrol group t p<0.05, : signifintly different from hlorpyrifos group t p<0.05. differentition of Th1 ells nd inhiit the differentition of Th2 ells to regulte immune response [32]. Similr studies showed tht CPF hd n inhiitory effet on oth IL-2 nd interferon-γ prodution on rts [28] nd lso CPF suppressed T-lymphoyte prolifertion nd redued serum IgG nd IgM levels in rts [26,33]. As demonstrted y the mirosopi exmintion, CPF exerted mrked histologil ltertions in the exmined liver nd spleen tissues whih ould e ttriuted to the formtion of ROS y CPE exposure. It hd een reported tht iologilly tive sustnes suh s pestiides oost the formtion of ROS whih is responsile for the tuting oxidtive stress in the tissues nd susequently their dmge [34,35]. In ddition, some studies identified ROS s use of toxi effets exerted y CPF s it inresed oxidtive stress in different tissues nd orgns [36,37] whih supported with our results of inresed hepti nd spleni ontents of MDA nd deresed their GSH ontents. However, the ddition of ZnO NPs to the drinking wter ontined CPF nd mrkedly relieved the tissue ltertions exerted y CPF. In this regrd, severl studies hve demonstrted tht zin possesses ntioxidnt properties nd onsequently n protet the ody ells from oxidtive dmge indued y ertin xenoiotis [38]. In ddition, zin plys n essentil role in ellulr GSH regultion whih is vitl proess to ellulr ntioxidnt defense, thus 445

7 proteting the ell ginst ROS indued y OP pestiides [31]. The protetive effet of zin ould e hieved through its intertion with ell memrnes, thus stilizing them ginst vrious dmging effets, inluding those used y oxidtive injuries. ACHE expression ws signifintly deresed in the liver of CPF-dministrted rts. CHE is known to e synthesized minly in heptoytes nd then sereted into the loodstrem [39]. As result of liver dysfuntion, CHE synthesis nd tivity will e delined in omprison with the other serum hepti funtion enzymes tht relted to the linil ssessment of liver funtion whose levels inresed due to their inresed relese from their ellulr soures following dmge of ell memrne [40]. Like other orgnophosphorus insetiides, CPF is n ACHE enzyme inhiitor whih leds to the umultion of etylholine nd results in exessive stimultion of postsynpti reeptors nd onsequent signs of toxiity [41]. CPF does not diretly inhiit ACHE enzyme; it is first metolized to the orresponding oxygen nlog (CPF-oxon), more potent inhiitor of ACHE enzyme. The tivtion of CPF into CPF-oxon is medited y ytohrome P 450 mixed funtion oxidses, primrily within the liver [42]; however, extrhepti metolism hs een reported in other tissues inluding the rin [21]. Short-term exposure of CPF in rts used signifint inhiition of ACHE enzyme tivity in different tissues inluding the liver, kidney, nd spleen [27]. The urrent results demonstrted tht ll the reorded CPF dverse effets on the immune system of mle rts were signifintly relieved y ddition of ZnO NPs (200 mg/l) to the ontminted wter. These findings suggested tht ZnO NPs diretly llevited these effets y dsoring CPF nd onsequently preventing it from induing its toxi effet on the immune system. Tht dsoring proess is hiefly ourred s result of the eletrostti ttrtion etween negtively hrged CPF nions nd positively hrged surfe of the sorent [43]. Another mehnism of ZnO NPs for lleviting the toxi effets of CPF in this reserh ould e ttriuted to the ntioxidnt effet of ZnO NPs. As one of the essentil nutrients, zin n protet ginst oxidtive dmge used y ertin xenoiotis nd thus my hve ntioxidnt properties. It ts s oftor for importnt enzymes involved in the proper funtioning of the ntioxidnt defense system [38,44]. Exposure to ZnO-NPs in this study does not ffet the explortory ehviors of mle rts. A similr finding ws reorded y Soheili et l. [45] nd Amr et l. [46]. Conlusion The present results onluded tht the odministrtion of ZnO in the NP form to CPFontminted drinking wter restored the viility nd funtion of the immune ells, the oxidtive stress mrkers, the histopthologil ltertions s well s the immune-expression of ACHE. These results give light on the enefiil use of ZnO NPs in CPFontminted wter nd in individuls who re ouptionlly on dily exposure to low onentrtion of suh insetiide. Authors Contriutions EME nd OSE designed the study nd nlyzed the dt. IMG nd SSAE ontriuted to the regents/ mterils/nlysis tools nd nlyzed the dt. SSE ontriuted to the regents/mterils/nlysis tools, olleted the mteril, nd nlyzed the dt. All uthors red nd pproved the finl mnusript. Aknowledgments We thnk the Immune setion t the Reserh Institute for Animl Reprodution, Ciro, Egypt s well s Fulty of Veterinry Mediine, Ciro University, Egypt for providing ll filities to ondut this study. This reserh did not reeive ny speifi grnt from funding genies in the puli, ommeril, or not-for-profit setors. Competing Interests The uthors delre tht they hve no ompeting interests. Pulisher s Note Veterinry World remins neutrl with regrd to jurisditionl lims in pulished institutionl ffilition. Referenes 1. Crvlho, F.P. (2017) Pestiides, environment, nd food sfety. Food Energy Seur., 6(2): LKind, J.S., Sous, J.R., Goodmn, M., Brr, D. B., Furst, P., Alertini, R.J., Arukle, T.E., Shoeters, G., Tn, Y.M., Teegurden, J., Tornero-Velez, R. nd Weisel, C.P. (2014) A proposl for ssessing study qulity: Biomonitoring, environmentl epidemiology, nd shortlived hemils (BEES-C) instrument. Environ. Int., 73(12): Antonio, V., Sergio, H., Mrth, R. nd Irmene, O. (2014) Detetion of residul orgnohlorine nd orgnophosphorus pestiides in griulturl soil in Rio Verde region of Sn Luis Potosi, Mexio. J. Environ. Si. Helth B., 49(7): Duirk, S.E., Desetto, L.M. nd Dvis, G.M. (2009) Trnsformtion of orgnophosphorus pestiides in the presene of queous hlorine: Kinetis, pthwys, nd struture-tivity reltionships. Environ. Si. Tehnol., 43(7): Lee, I., Eriksson, P., Fredriksson, A., Burtovi, S. nd Vierg, H. (2015) Developmentl neurotoxi effets of two pestiides: Behvior nd iomoleulr studies on hlorpyrifos nd rryl. Toxiol. Appl. Phrmol., 288(3): Shnnon, M.A. (2008) Siene nd tehnology for wter purifition in the oming dedes. Nture, 452(7185): Yng, H.Y. (2013) Cron nnotue memrnes with ultrhigh speifi dsorption pity for wter deslintion nd purifition. Nt. Commun., 4(8): Yun, J. (2008) Super wetting nnowire memrnes for Veterinry World, EISSN:

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9 Biol. Tre Elem. Res., 176(2): Soheili, S., Seed, M., Attollh, S. nd Msoud, G. (2013) Histopthologil effets of ZnO nnoprtiles on liver nd hert tissues in Wistr rts. Adv. Biores., 4(2): ******** 46. Amr, S., Ben-Slm, I., Mrd, I., Rihne, N. nd Jeljeli, M. (2014) Aute exposure to zin oxide nnoprtiles does not ffet the ognitive pity nd neurotrnsmitters levels in dult rts. Nnotoxiology, 8(Suppl 1): Veterinry World, EISSN: