Vitamin C and Chitosan Alleviate Toxic Effects of Paraquat on Some Biochemical Parameters in Hepatocytes of Common Carp

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1 Irnin Journl Toxiology Vol 10, No 1, Jn-Fe 2016 Originl Artile Vitmin C n Chitosn Allevite Toxi Effets Prqut on Some Biohemil Prmeters in Heptoytes Common Crp Zein Shrifins 1, Mhi Bnee*1, Mohmm Mohiseni 1, Ahm Noori 2 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Reeive: Aepte: ABSTRACT Bkgroun: Prqut is nonseletive ipyriyl heriie tht inues heptotoxiity through oxitive stress. Vitmin C n hitosn hve ntioxint s well s ril svenger properties n show protetive effets ginst retive oxygen speies (ROS). In the present stuy, heptoprotetive effets hitosn n vitmin C were evlute in ommon rp expose to prqut. Methos: While expose to 0.02 mg. L-1prqut for 21 ys, ommon rp were fe hitosn (1000 mg. kg-1 fee), vitmin C (1000 mg. kg-1 fee), n vitmin C omine with hitosn. At the en the experiment, tivities hepti enzymes n oxitive stress iomrkers were evlute. Results: Prqut inues hnges in the tivity lnine minotrnsferse, sprtte minotrnsferse, gmm-glutmyl trnsferse, lkline phosphtse n ltte ehyrogense in liver tissue fish. However, these enzymes were restore to norml levels in fish fe with vitmin C n vitmin C omine with hitosn following exposure to prqut. Inrese levels mlonilehye were oserve in liver fter exposure to prqut, while gluose-6-phosphte ehyrogense n tlse tivities n the totl ntioxint levels erese. Aministrtion vitmin C omine with hitosn signifintly reue mlonilehye levels n inrese the totl ntioxint pity, gluose-6-phosphte ehyrogense n tlse tivities. Conlusion: Aministrtion vitmin C is effetive in reuing liver toxiity prqut. However, ministrting oth vitmin C n hitosn is more effetive. In other wors, hitosn n vitmin C hve synergi effet. They oul e use s heptoprotetive gents ginst prqut-inue heptotoxiity in fish. Keywors: Biohemil prmeters, Chitosn, Oxitive stress, Prqut, Vitmin C. IJT 2016; INTRODUCTION Prqut (1, 1 -imethyl-4 4 -ipyriinium ihlorie) is one the most importnt ipyriylium heriies with unusul toxiologil properties. This heriie is one the most ommon tive grohemils use in Irnin griulture. Contmintion the quti environment y prqut is minly vi runf from frmln. However, only smll frtion the prqut pplie to the griulturl fiels my ultimtely enter surfe wters. Prqut exerts its effet s n eletron onor n trnsforms moleulr oxygen to tive rils, whih re responsile for lipi peroxition. Prqut n its metolites n ffet some physiologil n iohemil proesses in fish y influening the tivities severl enzymes [1]. Fishes hve evolve omplex ntioxint system ompose y oth non-enzymti n enzymti omponents to prevent the hrmful effets ROS. In reent yers, however, the emphsis hs een shifte to stuying gents tht n hve either protetive or meliortive effets on iohemil prmeters fish expose to vrious xenoiotis [2, 3]. Reue iovilility prqut in wter or erese iologil effet prqut my e effetive in mintining the helth fish living in pollute wters. Chitosn, eetylte erivtive hitin, is nturlly ourring polyshrie 1. Deprtment Aquulture, Behhn Khtm Alni University Tehnology, Irn. 2. Deprtment Aquulture, Hormozgn University, Bnr As, Irn. *Corresponing Author: E-mil: nee@ktu..ir

2 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Irnin Journl Toxiology foun unntly in mrine rustens, insets n fungi. Chitosn n e use in the tretment wter ontminte with pestiies [4-6]. The potentil hitosn to remove prqut from pollute wter hs een proven [7]. Therefore, hitosn n reue the iovilility prqut in wter. Moreover, hitosn n its erivtives re use in elivery rugs, hormones, vines n vitmins [8, 9]. Chitosn my lso e effetive in heling tissue mge use y toxiity pestiies. Vitmin C (sori i) is nturlly ourring ntioxint. It is n essentil nutrient for the iosynthesis ollgen, L-rnitine, n the onversion opmine to norepinephrine [10]. Vitmin C neutrlizes retive oxygen speies (ROSs) y reuing their mount. Thus, y inresing the iovilility vitmin C in the oy orgnisms, the iologil effets this vitmin woul e ugmente [11, 12]. Chitosn nnoprtiles re ple rrying Vitmin C in iologil systems [11]. Therefore, hitosn n inrese the iovilility vitmin C [12]. Heptoprotetive effets relte to the ntioxint tivity hitosn on prqutinue hepti injury hve not previously een stuie. However, the potentil ntioxint effets vitmin C ginst heptotoxiity n oxitive stress use y other pestiies were reporte y some uthors [13]. Therefore, in this stuy we ime to fin out the effiy vitmin C, hitosn n the omintion them ginst toxiity sulethl onentrtions prqut in ommon rp. MATERIALS AND METHODS Chemil Mterils Low weight hitosn (80% eetylte) purhse from Alrih Chemil Compny In., USA. Prqut (Grmoxone) t 20% onentrtion ws otine s ommeril preprtion (Jingsu Hi Bng Compny, Chin importe y Irn). Vitmin C (Asori i) ws ought from Rooyn Drou Compny, Irn. All iohemil kits were purhse from Prs Azmoun Co, Irn. Other hemil mterils were otine from Merk Chemil Compny. Germny. Fishes Tretment One hunre n eighty juvenile Common rp, Cyprinus rpio, weighing (38.50 ± 5.80 g) Zein Shrifins et l were use oring to the Ntionl Ethil Frmework for Animl Reserh in Irn [14]. The fishes were purhse from ommeril frm in Behhn, Khuzestn Provine, Irn, n were trnsferre to the Aquulture Lortory Aquulture Deprtment, Nturl Resoures n Environment Fulty, Behhn Khtm Alni University Tehnology. The speimens were rnomly istriute in 18 plsti tnks (80 l) n limtize in erte freshwter (24 ± 2 C; ph 7.4 ± 0.2; 16 L/8D; 40% wter exhnge rte/y) for two weeks efore use. During the limtiztion perio, the fishes were fe two times per y with ommeril iet from Beyz Fee Mill, Shirz, Irn. The fishes were rnomly ssigne to six groups. Group I: the speimens were fe norml iet for 21 ys n were onsiere the ontrol group; Group II: the speimens were expose to 0.02 mg.l-1 prqut; Group III: the fishes were fe iet enrihe with 1000 mg hitosn per 1 kg fee for 21 ys; Group IV: the speimens were expose to 0.02 mg.l-1 prqut n were fe 1000 mg hitosn per 1 kg fee for 21 ys; Group V: the speimens were expose to 0.02 mg.l-1 prqut n were fe iet enrihe with 100 mg vitmin C per 1 kg fee for 21 ys; Group VI: the speimens were expose to 0.02 mg.l-1prqut n were fe iet enrihe with 100 mg vitmin C omine with 1000 mg hitosnper 1 kg fee for 21 ys. Eh tretment ws pplie to triplite groups in ompletely rnomize esign. To prevent the uil-up metoli wstes n to keep onentrtions prqut ner the nominl level, the wter ws hnge ily. On 21 ys the stuy, the speimens were euthnize y epittion n their livers were refully remove, wshe repetely in ie-ol physiologil sline n urtely weighe. Tissue smples were homogenize in ie ol phosphte uffer for two min (ph 7.4; 1:10, w/v) using glss homogenizer n then entrifuge for 15 min t g t 4 C in refrigerte entrifuge. The superntnts were immeitely use to mesure iohemil prmeters vi spetrophotometri ssys. Biohemil Prmeters Anlysis Ltte ehyrogense (LDH) tivity ws mesure se on the onversion pyruvte to L-ltte y monitoring the oxition NADH. Asprtte minotrnsferse (AST) ws ssye 32 Vol 10, No 1, Jn-Fe 2016;

3 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Vitmin C n Chitosn Allevite Toxi Effets in ouple retion with mlte ehyrogense in the presene NADH. In lnine minotrnsferse (ALT) ssy, the enzyme rets with lnine n α-ketoglutrte to form glutmte n pyruvte. Ltte ehyrogense onverts pyruvte to ltte n NAD+. All these tivities were monitore y mesuring hnges in sorne t 340 nm. Alkline phosphtse (ALP) ssy ws se on the enzyme-meite onversion p-nitrophenol phosphte to nitrophenol in n lkline uffer t 405 nm. Gmm-glutmyl trnsferse (GGT) tivity ws etermine y ouple enzyme ssy, in whih the GGT trnsfers the g-glutmyl group from the sustrte L-g-Glutmylp-nitronilie, n liertes hromogen p-nitronilie proportionl to the GGT present. GGT tivity ws reore uring 3 min t 418 nm [15]. In G6PDH ssy, this enzyme tlyzes the oxition gluose6-phosphte (G6P) to 6-phospho-D-gluonte, long with the onomitnt reution NADP+ to NADPH. The rte NADPH formtion is proportionl to G6PDH tivity n is mesure using spetrophotometry s inrese in sorne t 340 nm [16]. Protein levels in tissues were etermine y stnr proeures use in linil iohemistry lortories oring to theiohemil kits user mnuls (Prs Azmoun Co, Irn) [17]. CAT tivity ws etermine oring to Góth [18], with some moifitions. Ctlse tivity ws mesure y hyrogen peroxise ssy se on formtion its stle omplex with mmonium molyte. 200 µl the superntnt ws inute in working solution inluing 1000 µl hyrogen peroxie n 500 µl phosphte uffer (ph: 7.4) t 25 C for 60 S. Then 1000 µl 32.4 mmol.l-1 mmonium molyte ws e to the retion solution n the onentrtion the yellow omplex molyte n hyrogen peroxie ws mesure t 405 nm wvelengths. Blnk 1 ontine 1.0 ml sustrte, 1.0 ml molyte n 0.2 ml istille wter; lnk 2 ontine 1.0 ml sustrte, 1.0 ml molyte n 0.2 ml uffer; lnk 3 ontine 1.0 ml uffer, 1.0 ml molyte n 0.2 ml uffer. Totl ntioxint pity ws estimte oring to the ferri reuing ility plsm Irnin Journl Toxiology (FRAP). Briefly, the FRAP regent ontine 5 ml (10 mmol/l) TPTZ (2,4,6- tripyriyl- strizine) solution in 40 mmol/l HCL plus 5 ml FeCl3 (20 mmol/l) n 50 ml ette uffer (0.3 mol/l, ph=3.6) n ws prepre freshly. Aliquots (100 µl) the superntnt were mixe with 3 ml FRAP regent. The onversion rte ferri tripyriyl-s-trizine (Fe3+-TPTZ) omplex to ferrous tripyriyl-strizine (Fe2+-TPTZ) t ph 3.6 n 25 C is iretly proportionl to the onentrtion totl ntioxint in the smple. Fe2+-TPTZ hs n intense lue olor tht n e monitore for up to 5 min t 593 nm y UV/VIS spetrophotometer. The lultions were performe using lirtion urve FeSO4 7H2O (100 to 1000 µm/l) [19]. Mlonilehye (MDA) ontent ws ssesse y moifie thiorituri i ssy n ws expresse s µmol/g tissue [20]. Briefly, 500 µl the superntnt ws trnsferre to Pyrex tue n mixe with 2500 µl trihloroeti i (20%) n 1000 µml thiorituri i (67%). The tues were then ple in oiling wter (100 C) for 15 min. After ooling, the hromogeni sustrte ws extrte into the orgni phse with 1000 µl istille wter n 5000 µl n-utnol: pyriine (15: 1). The mixture ws then entrifuge t 2000 g for 15 min t 4 C. The pink olor proue y these retions ws mesure y spetrophotometry t 532 nm to mesure MDA levels. MDA onentrtion ws lulte using MDA stnr. Tetrethoxypropne n solute ethnol were use to prepre the MDA stnrs. Conentrtions MDA in whole oy smples re expresse in µm per g protein. All iohemil prmeters were mesure y UV/VIS spetrophotometer (moel UNICCO 2100). Sttistil Anlysis All t were exmine for normlity (Shpiro-Wilk test). Sttistil tests were performe with SPSS (IBM, Relese 19) stwre y mens one wy nlysis vrine, followe y Dunn multiple omprison test (P<0.01). Dt re presente s men ± SD in eh experimentl group. Signifint ifferenes etween vlues were hrterize y lphetil symols (P<0.05) Vol 10, No 1, Jn-Fe 2016

4 Irnin Journl Toxiology Zein Shrifins et l to prqut (P<0.05), wheres CAT tivity ws elevte in fishes fe iet enrihe with vitmin C n vitmin C omine with hitosn s ompre to the group expose to prqut lone. Chitosn ministrtion i not signifintly moify the tivities the CAT in liver tissue fishes expose to prqut (Figure 6). Ativity G6PDH signifintly erese in liver tissue fish expose to prqut s ompre to ontrol group. However, its tivity in fish trete with prqut long with vitmin C n the group whih were trete with prqut long with hitosn, ws signifintly (P<0.05) lower thn tht ontrol group (Figure 7). Levels mlonilehye (MDA) in liver signifintly inrese (P<0.05) in the group expose to 0.02 mg.l-1 prqut when ompre with the non-expose group on y 21. The MDA levels in heptoytes reue signifintly in groups, whih sori i n sori i omine with hitosn, were use. However, MDA levels in heptoytes fishes expose to prqut n trete with hitosn were reue ompre with nontrete group expose to prqut. The MDA level in liver fishes expose to prqut n trete with hitosn ws greter thn tht ontrol group (Figure 8). No mortlity ws oserve uring the experiment. Altertions in the iohemil prmeters liver tissue re presente in Figure 1-9. Asprtte minotrnsferse (AST) tivity signifintly erese in liver tissue fishes expose to prqut s ompre to ontrol group. However, ministrtion vitmin C n vitmin C omine with hitosn showe n inrese in the tivity AST s ompre to the group expose to prqut lone (Figure 1). Prqut erese lnine minotrnsferse (ALT) tivity. Comine tretment vitmin C n hitosn resulte in signifint improvement in ALT tivity in liver tissue prqut-expose fishes (Figure 2). Exposure to prqut erese lkline phosphtse (ALP) n gmm-glutmyl trnsferse (GGT) tivities in liver tissue. Aministrtion vitmin C n hitosn i not signifintly ffet ALP n GGT tivities in liver tissue fishes expose to prqut (Figure 3 & 4). Ltte ehyrogense (LDH) tivity signifintly inrese in fishes expose to prqut lone. Simultneous ministrtion hitosn n vitmin C resulte in mrke normliztion LDH tivity when ompre to the prqut trete groups (Figure 5). There ws signifint erese in tlse tivity in liver fishes expose 3.50 AST tivity (U. g-1 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 RESULTS Chitosn Prqut Vitmin Chitosn Vitmin C C & Chitosn Tretment Figure 1. Ameliortive effet hitosn n vitmin C on the AST tivity in the liver tissue ommon rp expose to prqut. 34 Vol 10, No 1, Jn-Fe 2016;

5 Irnin Journl Toxiology Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment GGT tivity (U. g-1 Figure 2. Ameliortive effet hitosn n vitmin C on the ALT tivity in the liver tissue ommon rp expose to prqut Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment ALP tivity (U. g-1 Figure 3. Ameliortive effet hitosn n vitmin C on the GGT tivity in the liver tissue ommon rp expose to prqut Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment Figure 4. Ameliortive effet hitosn n vitmin C on the ALP tivity in the liver tissue ommon rp expose to prqut. LDH tivity (U. g -1 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 ALT tivity (U. g-1 prrotein) Vitmin C n Chitosn Allevite Toxi Effets Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment Figure 5. Ameliortive effet hitosn n vitmin C on the LDH tivity in the liver tissue ommon rp expose to prqut Vol 10, No 1, Jn-Fe 2016

6 Irnin Journl Toxiology CAT tivity (U. g-1 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Zein Shrifins et l Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment G6PDH tivity (U. g-1 Figure 6. Ameliortive effet hitosn n vitmin C on the CAT tivity in the liver tissue ommon rp expose to prqut Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment Figure 7. Ameliortive effet hitosn n vitmin C on the G6PDH tivity in the liver tissue ommon rp expose to prqut. MDA Levels (µm.g-1 tissue) Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment TAO levels (µmol.g-1 Figure 8. Ameliortive effet hitosn n vitmin C on the MDA levels in the liver tissue ommon rp expose to prqut Chitosn Prqut Vitmin C Chitosn Vitmin C & Chitosn Tretment Figure 9. Ameliortive effet hitosn n vitmin C on the TAO levels in the liver tissue ommon rp expose to prqut. 36 Vol 10, No 1, Jn-Fe 2016;

7 Vitmin C n Chitosn Allevite Toxi Effets Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Totl ntioxint levels reue signifintly in liver fishes expose to prqut with no supplementry feeing. However, sori i n sori i omine with hitosn tretment inrese totl ntioxint levels in liver tissue prqutexpose fishes. Chitosn tretment lso improve totl ntioxint levels (Figure 9). DISCUSSION In the present stuy, the protetive effets sori i (vitmin C) n hitosn ginst prqut-inue toxiity in the liver ommon rp n the resulting hepti oxitive mge were investigte. Biohemil prmeters the trete fishes were ompre to those untrete ontrols (Tle 1). Mussi n Clterr suggeste lipi peroxition s n importnt prmeter the toxiity prqut [21]. Our results inite signifint erese in the totl ntioxint levels n n inrese in MDA levels in the fishes expose to prqut s ompre to ontrols (P<0.05). Therefore, the inrese in MDA levels in the present stuy oul e relte to n inrese in the genertion ROS in the liver the fishes expose to prqut. An inrese in MDA levels ws reporte in ifferent tissues fish expose to fenpropthrin [22], Deltmethrin [23], methyl prthion [24], hlorpyrifos [24], rmzepine [25], n trzine [26]. This les to the loss ellulr memrne integrity resulting in pores in the memrnes. Due to ell memrne lipi peroxition unsturte ftty is, short hin ftty is ontining R-COOH, R-OOH, R-CHO, n R-OH re rete whih seriously ffet the ellulr memrne funtions suh s the tivity hormone reeptors n neurl meitors, ion trnsport hnnels n the tivity memrne enzymes n trnsporttion speifi moleules. On the other hn, the formtion mlonilehye (MAD) uring peroxition ftty is with oule ons n rete ovlent ons n polymerize ellulr memrne omponents. Totl ntioxint levels reue signifintly in liver fish expose to prqut with no supplementry feeing. The ellulr totl ntioxint pity plys ruil role in intrellulr protetion ginst the retive oxygen speies [27]. The overproution Irnin Journl Toxiology retive oxygen speies uring pestiie etoxifition my e ssoite with erese in the hepti totl ntioxint pity [28, 29]. Impirment in the synthesis enzymti n non-enzymti ntioxints my e the most importnt ftor in reuing levels ellulr totl ntioxint pity. Therefore, eline in the ellulr ntioxint pity mkes the ells more vulnerle to oxitive stress mge. Conomitnt supplementtion with vitmin C n hitosn tretment elevte the level totl ntioxint in heptoytes. This enhne ellulr totl ntioxint pity seems to e essentil in mintining the reox stte n to ope with the oxitive stress in the fish expose to prqut. Signifint erese in the tivity tlse ws oserve in liver tissue fish fter exposure to prqut (P<0.05). Ctlse plys n importnt role in the elimintion hyrogen peroxie in ells [29]. Ctlse ts on H2O2 y eomposing it, n protets the ell ginst hyrogen peroxie meite lipi peroxition. Therefore, signifint erese in CAT tivity in liver tissue fish expose to prqut might e iohemil response to overproution H2O2 in liver ells. Hene, reue CAT tivity in heptoytes my inite efet in the ntioxint efense system. Inrese tlse tivity in liver tissue fish fe iet supplemente with vitmin C or Vitmin C omine with hitosn my reflet the effets vitmin C on reuing levels retive oxygen speies. Our result is supporte y stuy Mehrpk et l. [13], where vitmin C n hitosn ws emonstrte to llevite mium-inue oxitive stress in ommon rp. Gluose-6-phosphte ehyrogense (G6PDH) ws signifintly inhiite y prqut. Derese G6PDH le to reue iosynthesis niotinmie-enine inuleotie phosphte (NADPH) n ellulr reue glutthione (GSH) level [30]. In ition, NADPH is require for the genertion reue glutthione, whih is importnt for protetive effet ginst oxitive mge. Therefore, the results this stuy show tht erese G6PD tivity is suffiient to inrese oxitive stress. However, orl ministrtion hitosn or vitmin C supplementtion signifintly inrese G6PDH tivity s 37 Vol 10, No 1, Jn-Fe 2016

8 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Irnin Journl Toxiology ompre to fishes expose to prqut. Aministrting omintion vitmin C n hitosn h more preventive effet on the erese G6PDH use y prqut n improve the iminishe tivity G6PDH in liver tissue fishes expose to prqut. Aminotrnsferse enzymes suh s AST, ALT, n GGT re importnt in ellulr nitrogen metolism, oxition mino is, n liver gluoneogenesis [31]. In stress situtions, inrese tivity liver enzymes hs stimultory effets on gluoneogeni mehnism [31, 32]. After 21 ys, the results showe tht prqut tretment signifintly erese AST, ALT n GGT tivities in liver tissue. Thus, ltertions in minotrnsferse tivities my inite interferene with the ellulr energy supply for fish expose to prqut. Similr hnges were oserve in Leo rohit n A. mossulensis fter exposure to fenvlerte n fenpropthrin, respetively [22, 33]. However, orl ministrtion vitmin C omine with hitosn to fish expose to prqut reverses the tivity AST, n ALT to norml levels. Similr finings were reporte y Merhpk et l. [13] who oserve tht vitmin C n hitosn reues mium heptotoxiity in fish. The inrese LDH tivity use y prqut oul e expline y hnges in the ellulr metolism proess uring the tretment. Prqut signifintly inrese LDH tivity in the liver ommon rp, whih inite serious liver mge. This suggests impirment in the eroi pity the liver tissue. The oserve inrese LDH tivity n e ttriute to the onversion umulte pyruvte into ltte, trnsporte through musles to liver n regenerte gluose n glyogen to supply energy for fish expose to insetiies. Bnee et l [27] emonstrte the verse effets mlthion on liver n resulting inrese in LDH, AST n ALT tivities. However, LDH tivity returne to norml levels in fish fe iet supplemente with sori i n sori i omine with hitosn. The present results gree with those otine y Mehrpk et l. [13] who revele tht vitmin C n hitosn re ple lmost ompletely lleviting liver toxiity in fish expose to mium hlorie. ALP plys signifint role in phosphte hyrolysis n in memrne trnsport n ts Zein Shrifins et l s goo io-initor stress in iologil systems. The erese in ALP tivity y prqut proly inites ltere trnsport phosphte n inhiitory effet on the ell growth n prolifertion. In ontrst, inrese ALP tivity in the whole oy the freshwter fish Alurnus mossulensis in exposure to fenpropthrin hs een reporte [22]. Moreover, o-tretment vitmin C n hitosn i not inue ny signifint hnges in ALP tivity in heptoyte fish expose to prqut. CONCLUSION Prqut toxiity in liver oul e ttriute to the oxitive stress on the hepti ells, whih les to erese in ellulr totl ntioxint levels. The inrese in MDA n CAT tivity is initive retive oxygen speies formtion in the heptoytes fish expose to prqut. Aministrtion sori i n sori i omine with hitosn llowe reovering some enzymti tivities, hnge y prqut tretment. Nevertheless, hitosn supplementtion ws not suffiiently effetive in regulting liver enzymes tivities in fish expose to prqut. Nonetheless, sori i n sori i omine with hitosn oul e useful in eresing prqut toxiity y inhiiting the prqut-inue oxitive stress. ACKNOWLEDGMENT This stuy ws supporte y grnt from Behhn Khtm Alni University Tehnology. In ition, the uthors re grteful to Mrym Bnee, our English eitor, for proreing the mnusript. The uthors elre tht there is no onflit interests. REFERENCES 1. Prvez S, Risuin S. Effets prqut on the freshwter fish Chnn puntt (Bloh): nonenzymti ntioxints s iomrkers exposure. Arh Environ Contm Toxiol 2006; 50: Yn Y, Wnshun L, Boqin H, Bing H, Chenwei F. Protetive effets hitosn oligoshrie n its erivtives ginst ron tetrhlorieinue liver mge in mie. Heptol Res 2006; 35: Li R, Zhou Y, Ji J, Wng L. Oxitive mges y mium n the protetive effets lowmoleulr-weight hitosn in the freshwter r 38 Vol 10, No 1, Jn-Fe 2016;

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10 Downloe from ijt.rkmu..ir t 6: on Tuesy Septemer 25th 2018 Irnin Journl Toxiology 27. Bnee M, Sure A, Shhf S, Fzilt N. Protetive effets silymrin extrt on mlthion-inue zer ihli (Cihlsom nigrsitum) heptotoxiity. Irn J Toxiol 2015; 9(28): Monteiro DA, e Almei JA, Rntin FT, Klinin AL. Oxitive stress iomrkers in the freshwter hri fish, Bryon ephlus, expose to orgnophosphorus insetiie Folisuper 600 (methyl prthion). Compr Biohem Physiol Prt C 2006; 143: Bnee M, Sure A, Mirvghefi AR, Ahmi K. Biohemil n histologil hnges in the liver tissue Rinow trout (Onorhynhus mykiss) expose to su-lethl onentrtions izinon. Fish Physiol Biohem 2013; 39: Singh S, Ann A, Srivstv PK. Regultion n properties gluose-6-phosphte Zein Shrifins et l ehyrogense: A review. Int J Plnt Physiol Biohem 2012; 4(1): Bnee M. Physiologil ysfuntion in fish fter insetiies exposure: Insetiies ten Unesire ut still so Importnt, Eite y Stnislv Trn, Pulishe y InTeh. 2013; Chpter 4, p Bnee M. Averse effet insetiies on vrious spets fish s iology n physiology: Insetiies Bsi n Other Applitions Book, Eite y Soni Soloneski n Mrelo Lrrmeny, Pulishe y InTeh. 2012; Chpter 6, p Prusty A.K, Kohli MPS, Shu NP, Pl AK, Shrn N, Mohptr S, Gupt SK (2011) Effet short term exposure fenvlerte on iohemil n hemtologil responses in Leo rohit (Hmilton) fingerlings. Pestiie Biohem Physiol 100: Vol 10, No 1, Jn-Fe 2016;