ANTIOXIDANT EFFECT OF SELENIUM ON HEPATOTOXICITY INDUCED BY CHLORPYRIFOS IN MALE RATS

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1 Acdemic Sciences Interntionl Journl of Phrmcy nd Phrmceuticl Sciences ISSN Vol 4, Suppl 4, 2012 Reserch Article ANTIOXIDANT EFFECT OF SELENIUM ON HEPATOTOXICITY INDUCED BY CHLORPYRIFOS IN MALE RATS TAREK M. HEIKAL 1*, MAHMOUD EL-SHERBINY 2, SOHAIR A. HASSAN 2, AZZA ARAFA 2, HASSAN Z. GHANEM 2 1* Environmentl Toxicology Reserch Unit (ETRU), Pesticide Chmistry Deprtment, 2 Therpeutic Chemistry Deprtment, Ntionl Reserch Centre, Ciro, Egypt. Emil: trekhl@yhoo.com ABSTRACT Received: 10 Jun 2012, Revised nd Accepted: 15 July 2012 The present study ws undertken to evlute the protective effect of selenium ginst chlorpyrifos-induced heptotoxicity in experimentl rts. The wy of ppliction selected for the study ws orl gvge for 28 consecutive dys. Wister dult mle rts were rndomly divided into four groups. The first group ws served s control, wheres the remining groups were respectively treted with sodium selenite (3 mg/kg.wt.), chlorpyrifos (13.5 mg/kg.wt., 1/10 LD50) nd comintion of chlorpyrifos nd sodium selenite. The exposure of rts to chlorpyrifos promoted oxidtive stress resulted in n increse nd decrese of liver mlondildehyde (MDA) nd reduced glutthione levels compred to control, respectively. Also, decreses in liver glutthione peroxidse (GPx), superoxide dismutse (SOD), ctlse (CAT) nd lctte dehydrogense (LDH) ctivities were oserved. In ddition, plsm trnsminses (ALT&AST), lkline phosphtse (ALP) nd lctte dehydrogense (LDH) ctivities were incresed. A significnt decrese in ody weight nd n increse in solute nd reltive liver weights were oserved in chlorpyrifos-treted rts compred to the corresponding controls. The co-dministrtion of ttenuted the iochemicl prmeters cited ove s well s the chnges in ody nd liver weights. Liver histologicl studies hve confirmed the chnges oserved in iochemicl prmeters nd proved the eneficil role of lenium. In Conclusion, the use of selenium ppered to e eneficil to rts, to gret extent in ttenuting nd restoring the oxidtive dmge sustined y insecticide exposure. Keywords: Antioxidnt enzymes, Chlorpyrifos, Histopthology, Lipid peroxidtion, Oxidtive stress, lenium. INTRODUCTION For centuries, pesticides hve een used in griculture to enhnce food production y erdicting unwnted insects nd controlling disese vectors 1. The use of pesticides cuses severe environmentl nd helth hzrds to orgnisms 2, 3, 4. Due to their high insecticidl ctivity, low environmentl persistence nd moderte toxicity, the orgnophosphorus (OP) compounds re the most fvored insecticides. They re widely used in griculture nd medicine. However, the unregulted use nd its eril ppliction over lrge griculturl nd urn res hve cused severe environmentl pollution 5. Exposure to OP is ssocited with toxic effects on humns nd nimls 2, 3, 6, 7. One such orgnophosphte which hs spurred interest is Chlorpyrifos (). is rod-spectrum orgnophosphorus insecticide utilized extensively in griculture 7. is thought to e primrily metolized in the liver involving the intervention of multiple, specific cytochrome P450 s through severl rection pthwys 8. elicits numer of dditionl effects, including heptic dysfunction, hemtologicl nd immunologicl normlities, emryotoxicity, genotoxicity, nd neuroehviorl chnges 4, 6, 9, 10. Pesticides re known to increse the production of rective oxygen species (ROS), which in turn prompt oxidtive stress in different tissues 3, 10, 11. Mny studies hve implicted oxidtive dmge s the centrl mechnism of toxicity 12. Oxidtive dmge primrily occurs through production of rective oxygen species (ROS), including hydroxyl rdicls nd hydrogen peroxide tht re generted during the rection nd rect with iologicl molecules, eventully dmging memrnes nd other tissues 13. Mny insecticides re hydrophoic molecules tht ind extensively to iologicl memrnes, especilly phospholipids ilyers 14, nd they my dmge memrnes y inducing lipid peroxidtion 3, 15, 16. Free rdicl genertion is expected to induce heptotoxicity. Therefore, supplementtion of ntioxidnts cn e considered s the lterntive method for chelting therpy. In fct, severl studies demonstrted tht the cellulr ntioxidnt ctivity is reinforced y the presence of dietry ntioxidnts 17, 18. Accordingly, interest hs recently grown in the role of nturl ntioxidnts used s strtegy to prevent oxidtive dmge s fctor in the pthophysiology of vrious helth disorders 3, 16, 18. Among ntioxidnts, selenium () used s nutritionl supplements, is the essentil elements in lmost ll iologicl systems. is n essentil element for humns, which improves the ctivity of the seleno-enzyme. It is present in the ctive center of glutthione peroxidse (GPx), n ntioxidnt enzyme, which protects lipid memrnes nd mcromolecules from oxidtive dmge produced y peroxides 19, 20. Furthermore, other uthors (Yun nd Tng; Akhtr et l.) reported tht hs the ility to counterct free rdicls nd protect the structure nd function of proteins, DNA nd chromosomes ginst oxidtion injury 21, 22. Since, the potentil hzrd of exposure to on the liver is well documented, therefore the present study imed to elucidte the heptoprotective effect of when co-dministered orlly to dult mle rt using iochemicl ltertions nd histopthologicl findings s criteri. MATERIALS AND METHODS Animls Helthy mle Wister rts weighing 150 ± 5 g, were otined from the Animl Breeding House of the Ntionl Reserch Centre (NRC), Dokki, Ciro, Egypt, nd mintined in clen plstic cges in the lortory niml room (23 ± 2 C). On stndrd pellet diet, tp wter d liitum, nd dily drk/light cycle (12/12 hrs.), the rts were cclimtized for 1 week prior to the strt of experiments. The experimentl work on rts ws performed with the pprovl of the Animl Cre & Experimentl Committee, Ntionl Reserch Centre, Ciro, Egypt, nd interntionl guidelines for cre nd use of lortory nimls. Chemicls nd Regents Chlorpyrifos (M. Wt ; 99% purity) ws otined from Dow AgroSciences (Indinpolis, Indin, USA) nd Sodium selenite (N2O3) ws purchsed from Sigm (St. Louis, MO, USA). The ssy kits used for iochemicl mesurements of mlondildehyde (MDA), ALT, AST, ALP, LDH, GSH, CAT, SOD nd GPx were ll purchsed from Biodignostic Compny, 29 Thrir Str., Dokki, Giz, Egypt. All other chemicls were of regent grdes nd were otined from the locl scientific distriutors in Egypt. Experimentl Design The nimls were rndomly divided into 4 groups of six nimls ech. The route of dministrtion selected for the study ws orl

2 Heikl et l. Int J Phrm Phrm Sci, Vol 4, Suppl 4, dily gvge for 28 consecutive dys. Animls in Group 1 were served s control nd given only stndrd pellet diet nd corn oil (0.5 ml/rt). Animls in Group 2 were given dily N2O3 t dose of 3 mg/kg.wt./dy. Animls in Group 3 were given dily chlorpyrifos lone t dose of 13.5 mg/kg.wt. (1/10 LD50, Tomlin, 2004) dissolved in corn oil (0.5 ml/rt) 23. Animls in Group 4 were simultneously given of (13.5 mg/kg.wt.) nd N2O3 (3 mg/kg.wt./dy). This dose of which corresponded to 1/10 of LD50 ws selected on the sis of previous studies 6, 24, wheres, N2O3 dose ws selected sed on the clinicl ppliction nd on results from previous studies on humn nd experimentl nimls 25, 26. During the experimentl durtion, ody weights were weekly recorded nd the doses were modulted ccordingly. After completion of tretment period, lood smples were withdrwn from the nimls under light ether nesthesi y puncturing the retro-oritl venous plexus of the nimls with fine sterilized glss cpillry tue. Blood smples were collected, sujected to plsm seprtion nd stored t - 20 ⁰C for iochemicl nlysis within one week. Rts were then killed y decpittion. Then, livers were dissected out, clened nd weighed. Some smples were rinsed nd homogenized (10%, w/v) in n pproprite uffer (ph, 7.4) nd centrifuged. The resulting superntnts were used for iochemicl ssys. Other smples were immeditely removed, clened nd fixed in 10% formlin solution nd emedded in prffin for histologicl exmintion. Biochemicl Anlysis The plsm nd liver superntnts otined from different tretments were sujected to certin iochemicl nlyses spectrophotometriclly y using Shimdzu UV- VIS Recording 2401 PC (Jpn). Protein content Liver protein contents were mesured ccording to the method of Lowry et l. using ovine serum lumin s stndrd 27. Liver function tests Plsm trnsminses (ALT & AST) ctivities were determined y colorimetric method ccording to Reitmn nd Frnkel 28. Plsm lkline phosphtse (ALP) ctivity ws determined y enzymtic colorimetric method ccording to Young et l. 29. Indictors of liver cell necrosis Liver nd plsm lctte dehydrogense (LDH) ctivity s indictor of necrotic cell deth ws determined using kinetic method ccording to Vssult et l. 30. Oxidtive stress prmeters The liver mlondildehyde (MDA) concentrtions, lipid peroxidtion index, were determined spectrophotometriclly ccording to Drper nd Hdley 31. Briefly, n liquot of liver extrct superntnt ws mixed with 1ml of 5% trichlorocetic cid nd centrifuged t 3000g for 10 min. An mount of 1ml of thiorituric cid regent (0.67%) ws dded to 500 ml of superntnt nd heted t 90 ⁰C for 15 min. The mixture ws then cooled nd mesured for sornce t 532 nm. The mlondildehyde vlues were clculted using 1, 1, 3, 3-tetrethoxypropne s stndrd nd expressed s nno moles of mlondildehyde/g of liver. Liver glutthione (GSH) levels in the liver ws determined y the method of Ellmn 32 modified y Jollow et l. 33. The method is sed on the development of yellow color when 5, 5-dithiotis-2 nitroenzoic cid (DTNB) ws dded to compounds contining sulfhydryl groups. Five hundred microlitres of tissue homogente in phosphte uffer were dded to 3ml of 4% sulfoslicylic cid. The mixture ws centrifuged t 1800g for 15min. Five hundred milliliters of superntnts were tken nd dded to Ellmn s regent. The sornce ws mesured t 412 nm fter 10min. Totl GSH content ws expressed s mg/mg of protein in the liver. Antioxidnt enzymes Ctlse (CAT) ctivity ws ssyed y the method of Aei 34. Enzymtic rection ws initited y dding n liquot of 20 ml of the homogenized tissue nd the sustrte (H2O2) to concentrtion of 0.5 M in medium contining 100 mm phosphte uffer (ph 7.4). Chnges in sornce were recorded t 240 nm. CAT ctivity ws clculted in terms of mmole H2O2 consumed/min/mg of protein. Superoxide dismutse (SOD) ctivity ws estimted ccording to Beuchmp nd Fridovich 35. The rection mixture contined 50 mm of tissue homogentes in potssium phosphte uffer (ph 7.8), 0.1 mm EDTA, 13 mm l-methionine, 2 mm rioflvin nd 75 mm nitro lue tetrzolium (NBT). The developed lue color in the rection ws mesured t 560 nm. Units of SOD ctivity were expressed s the mount of enzyme required to inhiit the reduction of NBT y 50% nd the ctivity ws expressed s units/mg of protein. Glutthione peroxidse (GPx) ctivity ws mesured ccording to Flohe nd Gunzler 36. The enzyme ctivity ws expressed s nmole of GSH oxidized/ min/mg protein. Histopthologicl Exmintion For light microscopic investigtions, specimens from liver were fixed in 10% phosphte uffer formlin, dehydrted in lcohols nd emedded in prffin. Five micron tissue sections were stined with hemtoxylin nd eosin stin (H&E) for generl histopthologicl exmintion. Scoring of histopthologicl chnges ws done s follow: (-) sent; (+) mild; (++) moderte; (+++) severe, nd (++++) extremely severe 37. Sttisticl Anlysis The results were expressed s mens ± SD. All dt were done with the Sttisticl Pckge for Socil Sciences (SPSS 11.0 for windows). The results were nlyzed using one wy nlysis of vrince (ANOVA) followed y Duncn s test for comprison etween different tretment groups. Sttisticl significnce ws set t p < RESULTS To ddress the hypothesis whether would prevent or ttenute -induced oxidtive stress nd heptotoxicity confirmed y iochemicl perturtions nd the histopthologicl findings, the principl nd design of the experiment were conducted. Tle 1: Chnges in food intke, ody weight, nd solute nd reltive liver weights of control nd treted rts. Prmeters Tretment groups Control Initil ody weight (g) 152.4± ± ± ±4.28 Finl ody weight (g) 213.6± ± ±11.77 c 201.3±13.37 Body weight gin (%) 28.5± ± ± ±5.53 Asolute liver weights (g) 7.24± ± ± ±0.57 Reltive liver weights (g/100g w) 3.41± ± ± ±0.35 Food intke (g/dy/rt) 29.7± ± ± ±5.75 Ech vlue is men of 6 rts ± SD;,, c vlues re not shring superscripts letters (,, c) differ significntly t p < 0.05; % of ody weight gin = [(finl.w t. initil.wt.)/ finl.wt.] X 100. : Chlorpyrifos; : lenium. 604

3 [ Control Heikl et l. Int J Phrm Phrm Sci, Vol 4, Suppl 4, The Effects of on Generl Rt Helth Generlly, deth ws not oserved in ny of the experimentl groups during the tretment period (28 dys). However, in -treted group, few clinicl signs such s huddling, reduced ctivity, incresing wekness, slight dirrhe nd hir loss were oserved. The oserved signs were relted to the cholinergic crisis; is consistent sign in orgnophosphte poisoning. However, except of huddling, no other significnt clinicl mnifesttion ws oserved following supplementtion. Also, in -treted rts, food intke ws reduced y 25.6%. However, supplementtion of ttenuted the reduction in food intke to 10.8% (Tle 1). Evlution of Body, Asolute nd Reltive Liver Weights At the end of the experimentl course, there ws no significnt difference in ody, solute nd reltive liver weights etween nd untreted rts. However significnt (p< 0.05) loss of ody weight gin ccompnied y significnt increse in the solute nd reltive liver weights were recorded in rts treted with compred to the control. The co-dministrtion of selenium with group restored these prmeters to non significnt difference compred to the control (Tle 1). Enzymtic Antioxidnt Sttus in Liver In the liver homogentes of -treted rts, ctlse (CAT), superoxide dismutse (SOD) nd glutthione peroxidse (GPx) ctivities decresed significntly (p < 0.05) y 40.8%, 43.4% nd 43.3% in mle rts, respectively, when compred to the corresponding controls (Tle 2). However, supplementtion of regenerted SOD nd GPx ctivities nd prtilly meliorted CAT ctivity when compred to -group. Tle 2: Liver ntioxidnt enzyme ctivities of control nd treted rts. Prmeters / Tretments Control Ctlse (CAT) 23.13± ± ± ±3.39 c (µmoles H2O2 degrded/min/mg protein) Superoxide dismutse (SOD 17.13± ± ± ±2.89 (units/mg protein) Glutthione peroxidse (GPx) (nmoles of GSH/min/mg protein) 90.3± ± ±10.78 c 79.3±12.86 Ech vlue is men of 6 rts ± SD;,, c vlues re not shring superscripts letters (,, c) differ significntly t p < 0.05; : Chlorpyrifos; : : lenium. Oxidtive Stress Biomrkers (LPO nd GSH) In Liver nmoles of MDA / g tissue A) Mlonldildehyde (MDA) B) Glutthione (GSH) GSH µg / g tissue c Control Fig. 1: Effects of selenium () on liver MDA (Fig. 1A) nd GSH (Fig. 1B) fter tretment. Dt represented s mens of 6 rts ± SD;,, c columns re not shring ove letters (,, c) differ significntly t p < 0.05; : Chlorpyrifos; : lenium; MDA: Mlonldildehyde; GSH: Glutthione reduced. Administrtion of led to significnt increment (p<0.05) in lipid peroxidtion (LPO) s evidenced y the increse in liver MDA levels y +57.3% s compred to the control group. However, co-dministrtion of to -treted rts mitigted the ugmenttion in liver MDA levels (Fig. 1A). Reduced glutthione (GSH), nturl ntioxidnt in liver tissue ws significntly reduced in treted group ( 48.7%), when compred to the control group. Supplementtion of selenium to treted group restored GSH level (Fig. 1B). Tle 3: Plsm ctivities of lnine minotrnsferse (ALT), sprtte minotrnsferse (AST) nd lkline phosphtse (ALP) enzymes of control nd treted rts. Prmeters / Tretments Control ALT (IU/L) 32.3± ± ± ±5.39 AST (IU/L) 65.4± ± ± ±11.14 ALP (IU/L) 113.0± ± ± ±16.25 Ech vlue is men of 6 rts ± SD;,, c vlues re not shring superscripts letters (,, c) differ significntly t p < 0.05; : Chlorpyrifos; : : lenium. 605

4 Control Heikl et l. Int J Phrm Phrm Sci, Vol 4, Suppl 4, Effects of on Biochemicl Mrkers In Plsm nd Liver The results of enzymes ctivities of mle rts re shown in Tle 3. Mle rts exposed to (13.5 mg/kg/dy) showed significnt increse (p<0.05) in the plsm enzyme ctivities of ALT, AST nd ALP levels comprle to control. -treted group did not show ny significnt chnges s compred to the control. The tretment of ( +) decresed the enzymes ctivity s compred to treted group. The LDH ctivity mesured s n indictor of necrotic cell deth were incresed y 89.3% in the plsm (Fig. 2A), ut were decresed y 34.8% in the liver (Fig. 2B). Supplementtion of to the -treted group restored ll the prmeters cited ove. Histopthologicl Exmintion The histopthologicl exmintion of the liver tissue is shown in Fig. 3 nd the semiquntittive histologicl scoring of liver dmge is presented in Tle 4. Liver sections from the control rts showed norml heptic loules formed of heptocytes rditing from centrl vein to the periphery of the loules (Fig. 3A1 nd A2). However, only dministrtion of showed norml ppernce of heptocytes (Fig. 3D). In contrst, the exposure of rts to induced degenertive chnges in the liver orgn. cused inflmmtory cellulr infiltrtion in etween degenerted heptocytes, kupffer cells prolifertion, ftty infiltrtion nd degenertion in heptocytes (Fig. 3B1, B2, B3 nd B4). Codministrtion of improved the histopthologicl fetures (Fig. 3C). The liver of +-treted rts showed mrked improvement in their histologicl structure compred to treted group nd represented y nil to mild degree in inflmmtory cellulr infiltrtion in etween degenerted heptocytes, kupffer cells prolifertion, ftty infiltrtion nd dilttion nd congestion of portl nd centrl vein (Tle 4). Plsm LDH ctivity (IU/L) A) Plsm Lctte dehydrogense (LDH) Liver LDH ctivity (IU/ g tissue B) Liver Lctte dehydrogense (LDH) Control Fig. 2: Lctte dehydrogense (LDH) levels in plsm (Fig. 2A) nd in liver (Fig. 2B) of control nd treted rts. Dt represented s mens of 6 rts ± SD;, columns re not shring ove letters (, ) differ significntly t p < 0.05; : Chlorpyrifos; : lenium; LDH: Lctte dehydrogense. Tle 4: miquntittive scoring of rchitecturl dmge on histopthologicl exmintion of the rt livers in the different tretment groups. Tretments Control Degenertion of heptocytes Ftty chnge in heptocytes ++ Inflmmtory cells infiltrtion Diffuse kupffer cells prolifertion in etween heptocytes Dilttion nd congestion of centrl vein ++ + : Chlorpyrifos; : selenium. ( ) indictes norml, (+) indictes mild, (++) indictes moderte, (+++) indictes severe, nd (++++) indictes extremely severe. DISCUSSION In toxicologicl studies, orgn nd reltive orgn weights re importnt criteri for evlution of orgn toxicity 3, 38. In our study, rts exposed to during 28 dys showed decrese in their ody nd orgn weights. The reduction of dily food consumption in -treted rts supported these findings. On the other hnd, the reduction in ody weight my e due to the overll incresed degrdtion of lipids nd proteins s result of the direct effects of s n orgnophosphte compound 6. Moreover, the increse in liver weight could e ttriuted to the reltionship etween liver weight increse nd vrious toxicologicl effects or to the reduction in ody weight gin of experimentl nimls 3, 39, 40, 41, 42. These results re consistent with mny previous investigtors with nd other OP compounds 2, 43, 44. Co-dministrtion of selenium to the -treted group improved ody nd liver weights, which could e ttriuted to n increse in dily food consumption. Indeed, previous studies of Nvrro-Alrcon nd Crer-Vique showed n ctivted growth nd development fter intke of selenium 45. Due to the role of liver in detoxifiction of environmentl xenoiotics, it is t gret risk of injury nd induces heptotoxicity. The results of the present study indicted tht excertion of oxidtive injury in liver of -treted rts ws more thn control group s evidenced y elevted MDA levels nd reduced GSH content. Indeed, the involvement of oxidtive stress following exposure to OP hs een reported 3, 43, 44. Different mechnisms hve een postulted to explin -induced liver injury, such s lipid peroxidtion nd interction with memrne components. In fct, lipid peroxidtion represents one of the most frequent rections resulting from free rdicls ttck on iologicl structures 46. GSH is n importnt nturlly occurring ntioxidnt, which prevents free rdicl dmge nd helps detoxifiction y conjugting with chemicls. In ddition, GSH is centrl to the cellulr ntioxidnt defenses nd cts s n essentil cofctor for ntioxidnt enzymes including GPx nd GST 47, 48. Under oxidtive stress, GSH is consumed y GSH relted enzymes to detoxify the peroxides produced due to incresed lipid peroxidtion 49. In this respect, severl studies oserved depletion of GSH in -treted nimls 6, 9. It ws found 606

5 Heikl et l. Int J Phrm Phrm Sci, Vol 4, Suppl 4, tht the co-dministrtion of in - induced toxicity hs protected liver from lipid peroxidtion nd from ny chnges in GSH nd ntioxidnt enzymes 50, 51. This finding could e explined ccording to Ognjnovic et l. 52 y the importnt role of in preventing hydroxyl rdicls formtion nd in protecting the integrity nd the functions of tissues 53. Fig. 3: Photomicrogrph of H&E stined sections of liver from control rt showing norml histologicl structure of the centrl vein (CV), portl re (p) surrounding heptocytes (h) (Fig. 3A1-80x & A2-60x ). Chlorpyrifos-treted rt liver showing inflmmtory cells infiltrtion (white rrow) in the portl re (p) with degenertive chnges (d) (Fig. 3B1-80x), degenertion (d) in heptocytes (Fig. 3B2-80x),, congestion ws oserved in the portl vein (pv) ssocited with dilttion in the ile duct (d), inflmmtory cells infiltrtion (m) nd collgen prolifertion (c) in the portl re (Fig. 3B3-80x), ftty chnges (f) with diffuse Kupffer cells prolifertion (lck rrow) in etween the degenerted heptocytes (d) (Fig. 3B4-80 ). co-dministrted with treted rt liver showing inucleted heptocytes (white rrow), diffuse Kupffer cells prolifertion (lck rrow) ssocited with degenerted heptocytes (d) (Fig. 3C-80x). Sodium selenite () treted rt liver showing norml ppernce of heptocytes s well s centrl vein (CV) (Fig. 3D-80x). 607

6 Heikl et l. Int J Phrm Phrm Sci, Vol 4, Suppl 4, The cellulr ntioxidnt sttus determines the susceptiility to oxidtive dmge nd is usully ltered in response to oxidtive stress. In the current study, induced oxidtive dmge y producing rective oxygen species nd decresing the iologicl ctivities of some liver ntioxidnt enzymes, like SOD, CAT nd GPx. Our results were in line with previous studies which hve shown tht exposure to genertes lipid peroxidtion nd lters the ntioxidnt sttus of severl tissues in rts 24, 41. On the other hnd, the effect of on the enhnced GPx ctivity my e ttriuted to the increse in the iovilility of following co-tretment with sodium selenite 54. Wheres, CAT ecme significntly higher thn in control nimls, reflecting, most proly, n dptive response towrds free rdicl dmge in the liver, s reported y El Heni et l. 55. Another iochemicl mrker used to evlute liver function ws lctte dehydrogense (LDH) ctivity. Its ctivity decresed in liver tissue nd incresed in plsm in the -treted group. This my e ttriuted to generlized increse in memrne permeility, s reported y 56. Moreover, elevtion of LDH ctivity indictes cell lysis nd deth s well s the switching over of neroic glycolysis to eroic respirtion. LDH cn e used s n indictor for cellulr dmge nd cytotoxicity of toxic gents 57. Besides, the decresed levels of liver LDH ctivity result from superoxide nions nd hydroxyl rdicls which cuse oxidtive dmge to the cell memrne 58. The co-dministrtion of selenium with -treted rts improved LDH ctivity due proly to its ntirdicl/ntioxidnt efficcy. In ddition, ALT, AST nd ALP re the most sensitive iomrkers directly implicted in the extent of heptic dmge nd toxicity 59, 60. In our finding, we demonstrted tht dministrted to rts provoked mrked elevtion in plsm AST, ALT nd ALP ctivities which indicting heptocellulr dmge s previously reported y El-Demerdsh nd Klender et l. 60, 61. This elevtion could potentilly e ttriuted to the relese of these enzymes from the cytoplsm into the lood circultion 62, indicting necrosis nd inflmmtory rections 63. lenium tended to llevite plsm trnsminses nd ALP s demonstrted y us nd y other studies 60, 64. The heptic function tests corroorted the histopthologicl lesions oserved in the present study. Degenertion nd necrosis in the heptocytes, inflmmtory cells infiltrtion, nd Kupffer cells prolifertion were frequently oserved in -treted group. These oservtions indicted mrker chnges in the overll historchitecture of liver in response to, which could e due to its toxic effects primrily y the genertion of rective oxygen species cusing dmge to the vrious memrne components of the cell. Our results re supported y other studies conducted on nd other OP insecticides 3, 4, 41, 42. The co-tretment of improved the histologicl ltertions induced y, which could e ttriuted to the ntirdicl/ntioxidnt nd metl-chelting efficcy of this metl. Moreover, these results re in good ccordnce with those otined y other studies which hve postulted the eneficil role of on histopthologicl nd enzymtic chnges of rts 50, 51, 65. In conclusion, the results of present study showed tht induced genertion of free rdicls tht cused oxidtive dmge to mcromolecules nd cell memrne ccompnied y histopthologicl ltertions. Also, the results of the present study implicted the cpility of to induce heptotoxicity. In contrst reduces oxidtive stress y virtue of its ntioxidnt properties thus improving the structurl integrity of cell memrne nd eventully llevites the histopthologicl chnges s well s the iochemicl perturtions. Bsed on our present oservtions, we propose tht my provide cushion for prolonged therpeutic option ginst toxins-induced heptotoxicity without hrmful side effects. REFERENCES 1. Prksm A, thupthy S, Llith S. Plsm nd RBCs ntioxidnt sttus in occuptionl mle pesticide spryers. Clin. Chim. Act. 2001; 310: Heikl TM, Solimn MS. 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