Glutamate ameliorates copper-induced oxidative injury by regulating antioxidant defences in fish intestine

Transcription

1 British Journl of Nutrition (2016), 116, The Authors 2016 oi: /s Glutmte meliortes opper-inue oxitive injury y regulting ntioxint efenes in fish intestine Jun Jing 1,2,3, Xio-Yun Wu 1,2, Xio-Qiu Zhou 2,3, Lin Feng 2,3, Yng Liu 2,3, Wei-Dn Jing 2,3, Pei Wu 2,3 n Ye Zho 1,2 * 1 College of Animl Siene n Tehnology, Sihun Agriulturl University, Chengu , People s Repuli of Chin 2 Animl Nutrition Institute, Sihun Agriulturl University, Chengu , People s Repuli of Chin 3 Fish Nutrition n Sfety Proution University Key Lortory of Sihun Provine, Sihun Agriulturl University, Chengu , People s Repuli of Chin (Sumitte 26 Septemer 2015 Finl revision reeive 8 Mrh 2016 Aepte 23 Mrh 2016) Astrt The ojetive of this stuy ws to etermine the protetive effet of glutmte (Glu) in Cu-inue oxitive injury in fish intestine in vivo n enteroytes in vitro. The results inite tht exposure to 6 mg/l Cu for 72 h inue the proution of retive oxygen speies, therey inresing protein oxition n lipi peroxition in enteroytes of grss rp in vitro. Cells expose to Cu lone resulte in signifint inrese in ltte ehyrogense relese, whih is ompnie y epletions of ntioxints, inluing totl superoxie ismutse (T-SOD), glutthione S-trnsferse (GST), glutthione reutse (GR), nti-superoxie nion (ASA), nti-hyroxy ril (AHR) tivities n GSH ontent. Pre-tretment with Glu remrkly prevente the toxi effets of Cu on the T-SOD, GST, GR, AHR, n ASA tivities n GSH ontent in enteroytes. However, Cu inue n ptive inrese in the tivities of tlse n glutthione peroxise (GPx). Glu supplementtion further inrese GPx tivity in enteroytes. Interestingly, the experiment in vivo showe tht Glu pre-supplementtion signifintly elevte SOD, GPx, GST, GR, ASA n AHR tivities, s well s GSH ontent. Further results showe tht pre-tretment with Glu oul llevite Cu-inue oxitive injury y elevting ntioxint enzyme tivities through regulting the expression of NF-E2-relte nuler ftor 2 (Nrf2) mrna. Together, these results inite tht Glu oul ttenute Cu-inue ellulr oxitive mge in fish intestine, likely meite through Nrf2 signlling pthwys regulting mrna expressions of ntioxint enzyme genes n synthesis of GSH. Key wors: Glutmte: Copper: Oxitive injury: Antioxints: NF-E2-relte nuler ftor 2: Intestine L-Glutmte (Glu) is non-essentil mino i with verstile funtions in niml physiology n metolism (1). Glu hs n importnt role in the mino i metolism through its onversion to α-ketoglutrte or other mino is, inluing lnine, sprtte, ornithine n proline in intestine (2,3). Thus, Glu n serve oth lolly insie enteroytes n through the proution of other mino is in n interorgn metoli perspetive (2). In ition, Glu is key trnsmintion prtner n is require for the synthesis of GSH, whih is n importnt omponent in the efene ginst oxitive stress (4). Our previous stuies emonstrte tht ietry Glu supplementtion inrese intestinl nti-superoxie nion (ASA), nti-hyroxy ril (AHR), glutthione reutse (GR), tlse (CAT) n totl superoxie ismutse (T-SOD) tivities, n GSH ontent in grss rp Ctenophryngoon iell (5), improve ntioxint pity n regulte ntioxint-relte signlling moleule expression of fish enteroytes (6). Sivkumr et l. (7) reporte tht Glu llevite isoproterenol-inue oxitive stress in rts y inresing ntioxint enzyme tivities n GSH ontent from exogenous Glu. These results suggeste tht Glu oul offer protetion uring intestinl oxitive stress. Cu is known for its essentility for living orgnisms, inluing fish, whih is require for mintining ellulr funtion n ting s oftor for numer of key metoli enzymes (8,9). Nevertheless, Cu is one of the most importnt pollution-using metls, euse it is ommonly relese into the environment through inustril wstes n use in the form of opper Arevitions: AHR, nti-hyroxy ril; AKP, lkline phosphtse; ASA, nti-superoxie nion; CAT, tlse; DMEM, Duleo s Moifie Egle s Meium; Glu, glutmte; GPx, glutthione peroxise; GR, glutthione reutse; GST, glutthione S-trnsferse; LDH, ltte ehyrogense; MDA, mlonilehye; MTS, 3-(4, 5-imethylthizol-2-yl)-5-(3-roxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrzolium; Nrf2, NF-E2-relte nuler ftor 2; OD, optil ensity; PC, protein ronyl; ROS, retive oxygen speies; SOD, superoxie ismutse. * Corresponing uthor: Y. Zho, fx , emil zhye3@foxmil.om Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

2 Glu meliortes Cu-inue oxitive injury 71 sulphte s lgeie, fungiie, teriie n heriie. In fish ulture systems, Cu is regulrly use in the form of opper sulphte (CuSO 4 ) to ontrol lgl looms n quti mrophyte infesttions (16). Reently, quti orgnisms my suffer from exposure to Cu onentrtions tht might e ten to fifty times higher thn the require onentrtions (10). The nger of Cu is ggrvte y their lmost inefinite persistene in wter euse they nnot e estroye iologilly ut re only trnsforme from one stte to nother (11). Also, high onentrtions of wterorne Cu n e toxi, euse perturtions in Cu homoeostsis result in oxitive stress n inrese free-ril proution (12 14). The intestine is very sensitive to wie rnge of stressors (15). The reent reports show tht the intestine is mjor trget for wterorne Cu toxiity in oth freshwter n sewter fish (16 18). Although Cu uptke is vi gills, Cu exposure highly elevtes fish intestinl Cu lo (17).In grss rp, umultion of Cu in intestine is higher thn tht in gill (19). Our previous stuy emonstrte tht Cu exposure oul inue oxitive stress in intestine n the enteroytes of juvenile Jin rp Cyprinus rpio vr. Jin (16). However, few stuies exmine how to effiiently protet the intestine ginst Cu-inue oxitive mge. Given the inresing relese of Cu into the environment n its potentilly hrmful effets on fish, it is importnt to expn our knowlege of how to protet fish ginst Cu toxiity. Some nutrients oul prevent Cuinue oxitive mge n hnge ntioxint pity in fish enteroytes (16). Pisine ntioxint pity n e ssesse y the ontent of non-enzymti ompouns (e.g. GSH) n tivities of ntioxint enzymes inluing superoxie ismutse (SOD), CAT n glutthione peroxise (GPx), glutthione S-trnsferse (GST) n GR (20). These ntioxint ompouns n enzymes hve key roles in eliminting the retive oxygen speies (ROS) in fish (21 23). ROS re generte uring norml ellulr funtion, ut high oses n/or inequte removl of ROS results in oxitive stress tht my use severe metoli mlfuntions n impir ell helth sttus (24). NF-E2-relte nuler ftor 2 (Nrf2) is n importnt trnsription ftor tht n in to the ntioxint responsive element (ARE) n inue trnsription of ntioxint enzyme genes (25). Kelh-like ECH-ssoite protein 1 (Kep1) ws ientifie s n Nrf2-ining protein, whih epresses Nrf2 trnslotion to the nuleus (26). Our previous report showe tht ntioxint enzyme tivities were regulte y the Nrf2 n Kep1 signlling moleules in fish (27). Chen et l. (28) reporte tht the up-regultion of Nrf2 expression oul elevte the ntioxint gene (inluing SOD, CAT, GPx, GR n GST) expression levels in the mouse liver. However, to te, no stuy hs resse the effet of Glu on Nrf2 signlling pthwy in fish. Our reent stuy showe tht Glu oul regulte Nrf2 n Kep1 gene expression, mye meiting the signl trnsution involve in inrese gene expressions of ntioxint enzymes in fish enteroytes (6). The present stuy ws esigne to investigte whether Glu oul ttenute Cu-inue ellulr oxitive mge, meiting through Nrf2 signlling pthwys regulting mrna expressions of ntioxint enzymes genes n synthesis of GSH in fish intestine. Methos In vivo experiments Animl olletion n limtion onitions. Animl Cre visory Committee of Sihun Agriulturl University speifilly pprove this stuy. Yong grss rp were otine from Tong Wei fisheries n limte for 4 weeks. Dissolve O 2 ws not <6 0 mg/l. Wter temperture n ph were 24 (SEM 3) C n 7 5 (SEM 0 5), respetively. Protetive effet of glutmte in opper-inue oxitive stress in the intestine. The formultions of the sl n experimentl iets (Tle 1) were similr s in our previous stuy (5). In rief, it ontine 280 g of rue protein/kg iet. The sl iet ws Glu unsupplemente ontrol (Ctrl). Glu ws e to the sl iet to provie 8 g Glu/kg iet, whih ws the require Glu onentrtion for optiml growth estlishe y our previous stuy (5). Proeures for iet preprtion n storge were the sme s those esrie y Shiu & Su (29).A totl of 180 fish with n verge initil weight of 247 (SEM 7 5) g from the limtistion tnk were rnomly ssigne into two groups of three replites eh. The groups were fe either the Ctrl iet or the Glu iet for 56. The experimentl onitions were the sme s in our previous stuy (5). At the en of the feeing tril, the fish in eh tnk were weighe n ollete for Cu exposure. Fish with similr oy weight from oth the Ctrl n Glu groups were expose to 0 7 mg Cu/l wter for 96 h, whih hs een prove to inue oxitive stress in grss rp oring to our previous stuy (30). Tle 1. Fee formultion n hemil omposition of iets Diets Ctrl group Glu group Ctrl group Glu group Formultion (g/kg iet) Essentil mino is Soyen mel Lys Rpesee mel Met Cotton mel Thr Whet flour Arg Soy oil Leu Monolium phosphte His Choline hlorie 6 6 Ile Vitmin premix* Phe Minerl premix Vl L-Glu 0 8 Non-essentil mino i Totl Al Asp Anlyse hemil Glu omposition (g/kg DM) Proximte nutrients Gly Crue protein Pro Crue lipi Ser Crue sh Tyr * Vitmin premix (g/kg): retinyl ette (150 g/kg), 0 80 g; holeliferol (12 5 g/kg), 0 48 g; DL-α-toopheryl ette (500 g/kg), 2000 g; menione (230 g/ kg), 0 22 g; thimine hyrohlorie (980 g/kg), 0 12 g; rioflvin (800 g/kg), 0 99 g; pyrioxine hyrohlorie (980 g/kg), 0 62 g; ynoolmin (10 g/kg), 0 10 g; niin (990g/kg), 2 58 g; D-iotin (20 g/kg), 5 00 g; meso-inositol (990 g/kg), g; foli i (960 g/kg), 0 52 g; soryl ette (930 g/kg), 7 16 g; lium- D-pntothente (900 g/kg), 2 78 g. All ingreients were ilute with mize strh to 1 kg. Minerl premix (g/kg): FeSO 4.H 2 O, g; CuSO 4.5H 2 O, 0 60 g; ZnSO 4.H 2 O, 4 35 g; MnSO 4.H 2 O, 2 04 g; KI, 1 10 g; NSeO 3, 2 50 g; MgSO 4.H 2 O, g. All ingreients were ilute with CCO 3 to 1 kg. Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

3 72 J. Jing et l. Control group Ctrl/Ctrl group Ctrl/Cu group Inutor (Shnghi Boxun Inustry & Commere Co. Lt) in n ir tmosphere. The ells were llowe to tth to pltes for 72 h. Glu group Growth tril for 56 Fig. 1. Overview of experiment esign in vivo. Glu/Cu group Cu exposure for 96 h In ition, the Ctrl/Ctrl tretment (fish from the Ctrl) ws performe y exposing the fish from the Glu unsupplemente group to Cu-free wter. Therefore, there were three ifferent pre-tretment/exposure groups, Ctrl/Ctrl, Ctrl/Cu n Glu/Cu, with three replites per group n twelve fish per replite (thirty-six fish for eh group) (Fig. 1). During the Cu exposure perio in replites, the experimentl onitions were the sme s those in the growth tril, ut no foo ws provie n the wter ws not renewe (16). At the en of the hllenge tril, ll of the living fish from eh tnk were nesthetise in enzoine th oring to Bsi et l. (31). The intestines of fish were quikly remove, frozen in liqui N 2 n store t 80 C for further nlysis. Beuse the fish were fste for 96 h uring Cu exposure, it ws not neessry to empty the intestinl lumen (32). Prevention of opper-inue oxitive stress y glutmte in fish enteroytes. To investigte the effet of Glu on Cu-inue oxitive stress in fish enteroytes, ells were pre-trete with ifferent onentrtions of Glu (0 12 mmol/l) for 72 h. Next, ells were expose to 6 mg Cu/l Glu-free meium for 24 h in 27 C inutor. The Cu exposure onentrtion ws hosen euse previous experiments showe tht 6 mg/l Cu of meium oul inue oxitive stress in rp enteroytes (16). Thus, there were eight groups (pre-tretment/ Cu exposure): Ctrl/Ctrl, Ctrl/Cu, 2mmol/l Glu/Cu, 4mmol/l Glu/ Cu, 6mmol/l Glu/Cu, 8mmol/l Glu/Cu, 10mmol/l Glu/Cu n 12mmol/l Glu/Cu. At the en of the exposure, the MTS ssy ws performe. Cytotoxiity ws etermine y mesuring ltte ehyrogense (LDH) tivity n mlonilehye (MDA) in the ulture mei superntnts. Cell lystes were ollete to etet protein ronyl (PC) n GSH ontents, lkline phosphtse (AKP), ASA, AHR, SOD, CAT, GPx, GST n GR tivities, n CAT, GPx, GST, GR, Nrf2 n Kep1 mrna expression. In vitro experiments Chemils. Copper sulphte penthyrte (CuSO 4.5H 2 O), Glu, insulin, ollgense, ispse, D-soritol, Triton X-100, trnsferrin, enzyl peniillin n streptomyin sulphte were purhse from Sigm. Duleo s Moifie Egle s Meium (DMEM), Hnk s lne slt solution (HBSS) n fetl ovine serum (FBS) were purhse from Hylone. Glu-free DMEM ws orere from Beijing Tsing Skywing Bio Teh Co. Lt. 3-(4, 5-imethylthizol-2-yl)-5-(3-roxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrzolium (MTS) ws purhse from Promeg Corportion. Primry enteroyte ulture. The isoltion n ulture of primry enteroytes from grss rp C. iell intestine were performe oring to the methos of Jing et l. (22) with minor moifitions. In rief, helthy grss rp with n verge weight of 48 5 (SEM 1 2) g were foo eprive for 24 h efore the experiment n kille y epittion. The intestines were rpily seprte from the rss, opene n rinse with HBSS ontining ntiiotis (100 U/ml peniillin n 100 μg/ml streptomyin). Enteroytes were isolte y ollgense n ispse igestion. Next, ells were suspene in DMEM (ontining 2 % D-soritol, S-DMEM) n wshe with S-DMEM five times to remove ny unigeste mteril n single ells oring to Booth & O She (33) with slight moifitions. Isolte enteroytes were seee in twenty-four-well ulture pltes (Flon) t the ensity of ells per well tht h een previously ote with ollgen I (Sigm), s previously esrie y us (34). The ells were ulture in DMEM supplemente with 5 % FBS, 0 02 mg trnsferrin/ml, 0 01 mg insulin/ml n ntiiotis (100 U/ml peniillin n 100 μg/ml streptomyin) t 26 (SEM 0 5) C uner Biohemil Anlysis n mesurement Cell viility n ifferentition ssys. Cell viility in vivo experiments were quntifie using the CellTiter 96 AQueous One Solution ell prolifertion ssy kit (Promeg). In rief, t the en of the experiment, 40 µl of MTS working solution ws e to eh well. After inution for 2 h, the mount of formzn ws etermine y mesuring the optil ensity (OD) t 490 nm on plte reer (Wellsn MK3; Lsystems). AKP tivity ws ssye oring to Kroghl et l. (35). Ltte ehyrogense, mlonilehye, protein ronyl n ntioxint prmeter nlysis. Cu-inue ytotoxiity ws quntifie y mesuring the mounts of LDH relese into the ulture meium from injure ell in vitro experiments (36,37). The mount of LDH relese ws mesure using the metho of Mulier et l. (38). MDA ontent ws nlyse s esrie y Zhng et l. (39) using the thiorituri i retion. The ontent of PC ws etermine oring to the metho esrie y Armenteros et l. (40) using 2, 4-initrophenylhyrzine regent. The tivities of SOD, CAT n GPx were etermine y the metho esrie y Chen et l. (28). GST n GR tivities were mesure y the metho esrie y Pney et l. (41) with minor moifition. GSH ontents were etermine y using metho esrie y Chen et l. (23) with minor moifition. The metho is se on the formtion of yellow olour when ithio nitroenzoi i rets with ompouns ontining sulfhyryl groups. The mount of GSH ws expresse s nmol of GSH per mg protein. ASA (nti-superoxie nion) n AHR (nti-hyroxy ril) tivities were ssye oring to the metho esrie y Jing et l. (42). ASA ws etermine using the Superoxie Anion Free Ril Detetion Kit (Nnjing Jinheng Bioengineer Institute). Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

4 Glu meliortes Cu-inue oxitive injury 73 Superoxie nion (O 2 ) were generte y the tion of xnthine n xnthine oxise. When the eletron eptor e, olortion retion is evelope using the nitro lue tetrzolium. The olortion egree is iretly proportionl to the quntity of superoxie nion in the retion. If the smple hs nti-superoxie nion tivity, the superoxie nion in the retion will erese, so the olortion will e wek; if the smple n promote the proution of superoxie nion, the olortion will e strong. The superoxie nion n e mesure y olorimetry, n then ASA of the homogente ws lulte y the following formul: ASA (U/mg protein) = (OD ontrol OD smple )/(OD stnr OD lnk ) stnr V C onentrtion (mmol/l)/protein ontin (mg). One unit is 1 mg of homogente svenge superoxie nion free ril, whih equls 1 mg vitmin C svenging in 40 min t 37 C. AHR ws etermine using the Hyroxyl Ril Detetion Kit (Nnjing Jinheng Bioengineer Institute). It ws on the sis of Fenton retion (Fe 2+ +H 2 O 2 Fe 3+ +OH + OH). Aoring to the priniple, hyroxyl rils re generte y Fenton retion. When the eletron eptor e, olortion retion is evelope using the nitro lue tetrzolium. The olortion egree is iretly proportionl to the quntity of hyroxyl rils in the retion. If the smple hs nti-hyroxy ril tivity, the hyroxyl rils in the retion will erese, so the olortion will e wek; if the smple n promote the proution of hyroxyl rils, the olortion will e strong. The hyroxyl rils n e mesure y olorimetry, n then AHR of the homogente ws lulte y the following formul: AHR (U/mg protein) = (OD Control OD Smple )/(OD stnr OD lnk ) stnr H 2 O 2 onentrtion (mmol/l)/protein ontin (mg). One unit is 1 mg homogente erese 1 mmol/l H 2 O 2 in 1 min t 37 C. The protein ontents were mesure using the metho of Brfor with ovine serum lumin stnrs (43). Rel-time quntittive PCR. Totl RNA ws isolte using TRIZOL regent (Invitrogen) oring to the mnufturer s instrutions. The RNA purity of eh smple ws etermine y lulting the 260:280 rtio. The RNA integrity ws ssesse y inspetion of the 28S n 18S riosoml RNA ns in 1 % grose gel. Susequently, the 2 μl totl RNA ws use to synthesise omplementry DNA (DNA) using the PrimeSript RT regent Kit with gdna Erser (Tkr Biotehnology Co. Lt). Rel-time quntittive PCR nlysis of CAT, GPx, GST, GR n house-keeping gene (β-tin) ws performe in CFX96 Rel-Time PCR Detetion System (Bio-R). The gene-speifi primers use in this stuy were liste in Tle 2. The PCR mixture onsiste of l μl of the first-strn DNA smple, 0 5 μl eh of forwr n reverse primers from 10 μm stoks, 3 μl of RNse-free H 2 O n 5 μl of2 Ssofst EvGreen Supermix (Bio-R). Cyling onitions were 98 C for 10 s, followe forty yles of 98 C for 5 s, nneling t ifferent temperture (Tle 2) for eh gene for 10 s n 72 C for 15 s. Trget gene mrna levels were normlise to the mrna levels of the referene gene β-tin. The mount of the trget gene ws se on the threshol yle numer (CT), n the CT for eh smple ws etermine using the CFX Mnger TM softwre. All of the primer mplifition effiienies were pproximtely 100 %. The gene expression results were nlyse using the 2 ΔΔCT metho oring to Jing et l. (44). Sttistil nlysis A t test ws use for omprisons etween two groups in the growth tril. The other t were nlyse y one-wy ANOVA using SPSS 13.0 (SPSS In.). Dunn s multiple-rnge test ws use to etermine signifint ifferenes. Dt re presente s mens with their stnr errors. P < 0 05 ws onsiere to e sttistilly signifint. Results Glutmte prevente opper-inue oxitive mge of the intestine in vivo Dietry supplementtion with Glu signifintly inrese the growth of grss rp when ompre with the Ctrl group finl weight: 614 (SEM 12 2) v. 560 (SEM 5 6) g (P < 0 05). The effets of Glu on MDA, PC, SOD, CAT, GPx, GST, GR, ASA, AHR n GSH ontents in intestine of grss rp uner Cu exposure re Tle 2. The primers n nneling tempertures use in rel-time quntittive PCR Nmes Sequene (5' 3') Anneling temperture ( C) GenBnk ID CAT QF-GAAGTTCTACACCGATGAGG 58 7 FJ QR-CCAGAAATCCCAAACCAT GR QF-GTGTCCAACTTCTCCTGTG 59 4 JX QR-ACTCTGGGGTCCAAAACG GPx QF-GGGCTGGTTATTCTGGGC 61 5 EU QR-AGGCGATGTCATTCCTGTTC GST QF-TCTCAAGGAACCCGTCTG 58 4 EU QR-CCAAGTATCCGTCCCACA Nrf2 QF-CTGGACGAGGAGACTGGA 62 5 KF QR-ATCTGTGGTAGGTGGAAC Kep1 QF-TTCCACGCCCTCCTCAA 63 0 KF QR-TGTACCCTCCCGCTATG β-atin QF-GGCTGTGCTGTCCCTGTA 61 4 M25013 QR-GGGCATAACCCTCGTAGAT CAT, tlse; GR, glutthione reutse; GPx, glutthione peroxise; GST, glutthione S-trnsferse; Nrf2, NF-E2-relte nuler ftor 2; Kep1, Kelh-like ECH-ssoite protein 1. Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

5 74 J. Jing et l. Tle 3. Mlonilehye (MDA), protein ronyl (PC) n GSH ontents (nmol/mg protein), nti-superoxie nion (ASA), nti-hyroxy ril (AHR), superoxie ismutse (SOD), tlse (CAT), glutthione peroxise (GPx), glutthione S-trnsferse (GST), glutthione reutse (GR) tivities (U/mg protein) in the intestine of grss rp-fe iets ontining ifferent glutmte levels for 56, followe y exposure to 0 7 mg/l Cu for 96 h (Men vlues with their stnr errors, n 6) MDA PC ASA AHR SOD CAT GPx GST GR GSH Groups Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Ctrl/Ctrl Ctrl/Cu g/kg Glu/Cu ,, Men vlues within olumn with unlike supersript letters were signifintly ifferent (P < 0 05). Tle 4. Effet of ifferent onentrtions of glutmte on ltte ehyrogense (LDH) tivity n mlonilehye (MDA) ontent in mei, 3-(4, 5-imethylthizol-2-yl)-5-(3-roxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrzolium optil ensity (MTS OD), lkline phosphtse (AKP) tivities n protein ronyl (PC) ontent in opper-expose grss rp enteroytes* (Men vlues with their stnr errors, n 6) MTS OD AKP (U/g protein) LDH (U/g protein) MDA (nmol/ml) PC (nmol/mg protein) Groups Men SEM Men SEM Men SEM Men SEM Men SEM Ctrl/Ctrl Ctrl/Cu mmol/l Glu/Cu 0 152, mmol/l Glu/Cu , mmol/l Glu/Cu mmol/l Glu/Cu , mmol/l Glu/Cu , mmol/l Glu/Cu ,,, Men vlues within olumn with unlike supersript letters were signifintly ifferent (P < 0 05). * The ells were pre-trete with ifferent onentrtions of Glu for 72 h, followe y exposure to 6 mg/l Cu for 24 h. isplye in Tle 3. Compre with the Ctrl/Ctrl group, Ctrl/Cu exposure use signifint inrese in MDA n PC ontent in the intestine (P < 0 05). However, ietry Glu presupplementtion (Glu/Cu) signifintly erese MDA n PC formtion (P < 0 05). The tivity of CAT in the intestine of grss rp ws signifintly inrese y Cu exposure (Ctrl/Cu) (P < 0 05), wheres ietry Glu pre-supplementtion (Glu/Cu) signifintly prevente the inrese in CAT tivity (P < 0 05). In ontrst, Ctrl/Cu exposure signifintly erese ASA, AHR, SOD, GPx, GST n GR tivities in the intestine (P < 0 05). Glu pre-supplementtion (Glu/Cu) signifintly prevente the verse effets of Cu on these enzyme tivities (P < 0 05). The intestinl GSH ontent ws the lowest in the Ctrl/Cu tretment, followe y the Glu/Cu tretment, n the GSH ontent ws the highest in the Ctrl/Ctrl group (P < 0 05). Glutmte reue opper-inue oxitive mge of enteroytes in vitro The present stuy hs investigte the protetive effets of Glu pre-tretment on Cu-inue enteroyte oxitive stress. The Cu exposure (Ctrl/Cu) signifintly inrese LDH relese, MDA ontent in the meium n PC ontent in enteroytes s ompre with the unexpose Ctrl group (Ctrl/Ctrl) (P < 0 05) (Tle 4). However, pre-tretment with 6 12 mmol/l efore exposure to Cu signifintly reue LDH relese, MDA genertion n PC formtion inue y Cu (P < 0 05) (Tle 4). Exposure to Cu ws shown to use signifint erese in MTS OD vlues n AKP tivity ompre with tht of the Ctrl (P < 0 05) (Tle 4). As expete, pre-tretment with Glu prtilly prevente the erese in ell viility n AKP tivity inue y Cu (P < 0 05) (Tle 4). ASA n AHR tivities of enteroytes re shown in Fig. 2. Cu exposure lone signifintly erese ASA n AHR tivities in enteroytes. When ells were pre-trete with inresing oses of Glu, efore Cu stress, ASA n AHR tivities were inrese in ose-epenent mnner (P < 0 05). The effets of Glu on ntioxint prmeters in enteroytes uner Cu exposure re isplye in Tle 5. The T-SOD, GST n GR tivities n GSH ontent were signifintly erese in ells expose to Cu s ompre with the unexpose Ctrl tretment (P < 0 05). However, pre-tretment with Glu prtilly prevente mrke reution in T-SOD, GST n GR tivities n GSH ontent inue y Cu (P < 0 05) (Tle 5). In ontrst, Cu exposure lone signifintly inrese the CAT n GPx tivities. However, pre-tretment with Glu prtilly prevente the inrese in CAT tivity. Interestingly, ells pretrete with Glu further inrese high GPx tivity (Tle 5). Effets of glutmte on ntioxint-relte n NF-E2- relte nuler ftor 2 signlling moleule gene expression in the intestine in vivo As shown in Fig. 3, the Ctrl/Cu tretment inrese the reltive mrna expression levels of CAT in the intestine of grss rp Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

6 Glu meliortes Cu-inue oxitive injury (A) (B) 1.3 ASA (U/g protein) 15 10, AHR (U/g protein) (Control) 0 2 mmol/l 4 mmol/l 6 mmol/l 8 mmol/l 10 mmol/l 12 mmol/l 0 (Control) 0 2 mmol/l +6 mg/l Cu +6 mg/l Cu 4 mmol/l 6 mmol/l 8 mmol/l 0 mmol/l 12 mmol/l Fig. 2. The nti-superoxie nion (ASA, U/g protein) (A) n nti-hyroxyl ril (AHR, U/g protein), (B) tivities in enteroytes ulture with meium ontining gre levels of Glu for 72 h, followe y exposure to 6 mg/l Cu for 24 h. Vlues re mens of six replites, with their stnr errors.,,, Men vlues with unlike letters were signifintly ifferent (P < 0 05) Tle 5. Effet of ifferent onentrtions (U/mg protein) of glutmte on totl superoxie ismutse (T-SOD), tlse (CAT), glutthione peroxise (GPx), glutthione S-trnsferse (GST), glutthione reutse (GR) tivities n GSH ontent (nmol/mg protein) in opper-expose grss rp enteroytes* (Men vlues with their stnr errors, n 6) T-SOD CAT GPx GST GR GSH Groups Men SEM Men SEM Men SEM Men SEM Men SEM Men SEM Ctrl/Ctrl e e 0 5 Ctrl/Cu e mmol/l Glu/Cu ,e , , mmol/l Glu/Cu 4 10, , mmoll Glu/Cu mmol/l Glu/Cu mmol/l Glu/Cu e mmol/l Glu/Cu e ,,,,e Men vlues within olumn with unlike supersript letters were signifintly ifferent (P < 0 05). * The ells were pre-trete with ifferent onentrtions (0, 2, 4, 6, 8, 10, 12 mmol/l) of Glu for 72 h, followe y exposure to 6 mg/l of Cu for 24 h. ompre with the Ctrl/Ctrl group (P < 0 05). Dietry Glu tretment (Glu/Cu) signifintly prevente the up-regultion of CAT mrna expression (P < 0 05). The Ctrl/Cu tretment use signifint erese in GR, GPx n GST mrna expression in the intestine when ompre with the Ctrl/Ctrl (P < 0 05). Pretretment with Glu prevente own-regultion of the GR, GPx n GST mrna expression (P < 0 05). The effets of Glu on Nrf2 n Kep1 mrna expression in the intestine of fish following Cu exposure re presente in Fig. 3. Fish expose to Cu showe erese in Nrf2 mrna expression of intestine s ompre with the Ctrl/Ctrl group (P < 0 05), n Glu mrkely inhiite own-regultion of Cu-inue Nrf2 mrna expression (P < 0 05). Exposure to Cu signifintly inrese Kep1 trnsript unnes in intestine of grss rp ompre with the untrete ontrol (P < 0 05). Pre-tretment with Glu erese Kep1 mrna expression (P < 0 05). Effets of glutmte on ntioxint enzyme genes n NF-E2-relte nuler ftor 2 signlling moleule in vitro Reltive gene expressions of CAT, GPx, GST, GR, Nfr2 n Kep1 in enteroytes were presente in Fig. 4. Cu exposure lone signifintly inrese CAT n GPx mrna expression (P < 0 05). Pre-tretment with Glu resulte in signifint inrese of levels of GPx mrna s ompre with the Cu stress tretment. However, GST n GR mrna expression were signifintly erese y Cu stress lone, n Glu mrkely inhiite Cu-inue own-regultion of GST n GR mrna expression (P < 0 05). The reltive expression level of Nrf2 in enteroytes ws signifintly own-regulte y Cu stress lone (P < 0 05). Pre-tretment with Glu efore Cu exposure signifintly epresse Cu-inue own-regultion of Nrf2 mrna (P < 0 05). Cu exposure signifintly inrese the Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

7 76 J. Jing et l. reltive mrna expression levels of Kep1 in enteroytes (P < 0 05). Pre-tretment of ells with Glu prtilly prevente the inrese in Cu-inue reltive mrna expression of Kep1 (P < 0 05). Disussion Cu is n essentil nutrient, whih hs numerous funtions in ellulr iohemistry (suh s oftor for mny ifferent enzymes) (45). However, Cu t higher onentrtions my e toxi. The toxiity of Cu is suggeste to e minly use y oxitive stress in fish (46). It ws emonstrte tht Cu exposure oul inue oxitive stress in zerfish heptoytes (14), rinow trout gill ell (46) n rp enteroytes (16). Oxitive mge in ulture ells hs een ssesse y LDH relese (47). The extent of ell mge ws ssesse y mesuring the relese of the ytosoli enzyme LDH from mge ells to the thing meium (48). The present stuy emonstrte tht Cu Reltive expression units CAT GR GPx GST Nrf2 Kep1 Fig. 3. Reltive gene expression (shows to e reltive to Ctrl/Ctrl ( ), whih ws set t 1 0, in ritrry units) of tlse (CAT ), glutthione reutse (GR), glutthione peroxise (GPx ), glutthione S-trnsferse (GST ), NF-E2-relte nuler ftor 2 (Nrf2 ) n Kelh-like ECH-ssoite protein 1 (Kep1) inthe intestine of young grss rp (Ctenophryngoon iell) fe iets ontining ifferent Glu levels for 56, followe y exposure to 0 7 mg/l Cu for 96 h. Vlues re mens of six replites, with their stnr errors.,, Men vlues with unlike letters were signifintly ifferent (P < 0 05)., Ctrl/Cu;, Glu/Cu. exposure signifintly inrese LDH levels in the meium, initing severe enteroyte mge. The olorimetri ssy using MTS n rpily quntify the ell viility of fish enteroyte (44,49). AKP serves s n enteroyte ifferentition mrker n is onsiere to e involve in the sorption of nutrients (50). In the present stuy, Cu exposure signifintly erese ell viility n AKP tivity. Interestingly, tretment of Cu-expose enteroytes with some onentrtions of Glu epresse LDH relese, n elevte ell viility n AKP tivity. MDA n PC ontents were wiely use s iohemil initors of oxitive mge (51). Exposure of enteroytes to 6 mg/l Cu ws shown to use signifint inrese in MDA n PC ontents, suggesting tht Cu exposure use lipi n protein oxitive mge in enteroytes, whih my e ttriute to the inrese of ROS proution. ROS re proue in response to metls (52). Inresing the levels of ROS n le to severe ell injury or eth (53). Superoxie nions n use wie rnge of oxitive mge within the ell (51). The present stuy showe tht MDA n PC ontents were erese in ells trete with Glu in ose-epenent mnner. The result suggeste tht Glu my exert potent protetion of the intestine ginst Cu-inue oxitive mge. To our knowlege, no stuy hs investigte the protetive effets of Glu ginst metl toxiity. This is the first report emonstrting tht Glu oul ttenute Cu-inue ellulr mge. To investigte the potentil protetive effets of Glu ginst Cu-inue oxitive mge, the ntioxint enzymes suh s SOD, CAT, GPx, GST, GR n GSH ontent were etermine. The SOD is the first enzyme to respon ginst O 2 rils n importnt enogenous ntioxints for protetion ginst oxitive stress (54). CAT hs een implite s n essentil efene ginst the potentil toxiity of hyroxyl rils (55). The GSH-epenent enzymes (GST, GPx n GR) re le to ountert peroxitive mge (56). GSH is the mjor enogenous ntioxint svenger tht protets ells from oxitive stress, n GSH/GSSG represents the mjor ellulr reox uffer n therefore is representtive initor for the reox environment of the ell (57). In the present stuy, signifint erese in SOD n GR tivities n GSH ontent ws foun 4 Reltive expression units 3 2 1,,,,,,,,,,,,, 0 CAT GR GPX GST Nrf2 Kep1 Fig. 4. Reltive expression (shows to e reltive to Ctrl/Ctrl, whih ws set t 1 0, in ritrry units) of tlse (CAT ), glutthione reutse (GR ), glutthione peroxise (GPx), glutthione S-trnsferse (GST ), NF-E2-relte nuler ftor 2 (Nrf2 ) n Kelh-like ECH-ssoite protein 1 (Kep1) mrna in enteroytes ulture with meium ontining gre levels of Glu for 72 h, followe y exposure to 6 mg/l Cu for 24 h. Vlues re mens of six replites, with their stnr errors.,,, Men vlues with unlike letters were signifintly ifferent (P < 0 05).,Control;, 0 mmol/l;, 2 mmol/l;, 4 mmol/l;, 6 mmol/l;, 8 mmol/l;, 10 mmol/l;, 12 mmol/l. Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

8 Glu meliortes Cu-inue oxitive injury 77 in ells expose to Cu. The result emonstrte tht Cu-inue enteroyte oxitive mge my e prtly relte to isturne of the ntioxint system. Glu pre-tretment prevente Cu-inue erese of these ntioxint enzyme tivities. This my suggest tht the ntioxitive effet of Glu my e ttriute to its ility to mintin the tivity of ril svenging enzymes. A vriety of itrte yle intermeites suh s fumrte, oxloette, mlte n suinte were shown to moulte lipi peroxie proution y interting with enogenous iron ions, thus justing the iron reox yle n, susequently, free-ril genertion (58). Glu n form α-ketoglutrte y emintion n enter into the itrte yle to promote the proution of itrte yle intermeites (3). Hene, the reution in lipi peroxies with n inrese in GSH on Glu pre-tretment oul e euse of the ntioxint effet of itrte yle intermeites forme from Glu. Further, GSH ws proue from exogenous Glu (2). However, Cu exposure signifintly elevte CAT n GPx tivities. Glu pre-tretment of enteroytes further inrese GPx tivities. The reson might e ttriute to n ptive mehnism ginst stress. The enteroytes were iretly expose to Cu; inrese CAT n GPx tivities were require to inhiit the Cu-inue oxitive stress. The ASA n AHR tivity re two inexes use to evlute the totl pity of svenging superoxie n hyroxyl ril, respetively (22). To investigte how Glu inhiite the Cu-inue oxitive mge in fish, we etermine the ASA n AHR tivities. Our results showe tht Cu exposure signifintly erese the ASA n AHR tivities in enteroytes. Glu pre-tretment prevente the Cu-inue erese of ASA n AHR tivities in ose-epenent mnner. These results inite tht the protetive effets of Glu on Cu-inue oxitive mge my, t lest in prt, e euse of the inrese ASA n AHR tivities. The tivities of ntioxint enzymes n e ffete y the mrna levels in fish (27). To further eluite whether Glu regulte ntioxint enzyme tivities t the gene level in fish, the mrna levels of ntioxint enzyme of enteroytes fter eing hllenge ginst Cu were investigte. The results of the urrent stuy emonstrte tht Cu exposure signifintly inrese mrna levels of CAT n GPx in enteroytes. Glu pre-tretment loke the inrese in CAT mrna expression inue y Cu. However, Glu pre-tretment further elevte mrna expression of GPx gene inue y Cu. The ntioxint enzyme gene expression exhiite sme pttern with their respetive enzyme hnges. The regultion of ntioxint gene mrna levels my result from tivting the ntioxint-relte signlling moleules. Nrf2 is mster regultor of the ntioxint response through regulting the trnsription of ntioxint gene in fish, inluing SOD, CAT, GPx, GR n GST (26). The present stuy showe tht Cu exposure ownregulte the Nrf2 gene expression in enteroytes. The result is very well in greement with our previous report, whih showe tht Cu exposure mrkely erese the ining of nuler Nrf2 to ARE (59). In ition, the negtive effets of Cu-inue ntioxint gene mrna expression my e prtly srie to erese in Nrf2 nuler trnslotion. Kep1 is ientifie s n Nrf2-ining protein tht prevents Nrf2 trnslotion to the nuleus n promotes the uiquitintion-protesoml egrtion of Nrf2 (26). Our stuy lso showe tht Cu inues the up-regultion of Kep1 mrna level in enteroytes (6). Glu pre-tretment elevte Nrf2 mrna expression n loke the inrese in Kep1 mrna expression (6). These results suggeste tht the ntioxint effet of Glu is meite t lest in prt y Nrf2 signlling pthwys in fish. To te, no informtion is ville out the effet of Glu on Nrf2 gene mrna expression in fish enteroytes. The unerlying mehnism nees further investigtion. On the sis of the enefiil effets of Glu ginst Cu-inue oxitive mge in the enteroytes, it ws resonle to hypothesise tht Glu n protet fish ginst Cu-inue intestinl oxitive mge in vivo. The present stuy showe tht Cu exposure oul inue intestinl oxitive stress in grss rp. Similr results were oserve tht Cu exposure oul inue oxitive stress in juvenile Epinephelus oioies intestine (60) n pu Pirtus mesopotmius liver (61). The ntioxint enzyme tivities of intestine were elevte in response to Glu supplementtion. The GR, GPx n GST mrna unne in intestine ws enhne y Glu pre-tretment. The positive effets of Glu on ntioxint enzyme mrna expression my e prtly srie to promote Nrf2 nuler trnslotion y own-regulting Kep1 mrna expression. In the present stuy, signifint inrese in mrna levels of Nrf2 ws oserve in the grss rp intestine with Glu supplementtion. In ontrst, Glu pre-tretment signifintly erese Kep1 mrna expression. These results were in greement with the present stuy sttements in vitro. Stuies from rts lso inite tht Glu supplementtion llevites oxitive mge inue y isoproterenol (7). However, it is of interest tht the in vivo GPx tivity in intestine ws erese y Cu exposure, whih ws the reverse of the pttern oserve in enteroytes. The reson for these results is unler. The ifferenes my e euse, in this stuy, the enteroytes were iretly expose to Cu, wheres the intestine is iniretly expose to Cu. In other wors, euse the enteroytes were iretly expose to Cu, inrese GPx tivity ws require to inhiit the Cu-inue oxitive stress. The previous stuy in grss rp showe tht ietry Cu ontent (5 25 mg/kg), eyon requirement (4 78mg/kg), inue intestinl oxitive stress, ut inrese GPx tivity (9). In ition, higher oses of Cu ( mg/kg) inue intestinl oxitive stress n erese the GPx tivity t the sme time (9). Therefore, the enteroytes iretly expose to ertin mount of Cu y ulture in vitro n orl ministrtion in vivo n oth inue oxitive stress, s well s inrese GPx tivity in fish intestine. In onlusion, Cu exposure oul inue oxitive mge, resulting in lipi peroxition, protein oxition n ntioxint enzymes tivity ltertions in intestine n the enteroytes of grss rp. Cu exposure own-regulte the mrna unne of GR, GPx n GST in the intestine of grss rp. Dietry n meium pre-supplementtion with Glu oul llevite Cu-inue oxitive mge in fish intestine n the enteroytes, respetively. The protetive effets of Glu on Cu-inue oxitive mge re ssoite with up-regulting the expression of ntioxint enzymes gene y regulting mrna unne of signlling moleule Nrf2. It provies Downloe from IP ress: , on 01 My 2018 t 03:41:21, sujet to the Cmrige Core terms of use, ville t

9 78 J. Jing et l. theoretil sis for reuing oxitive stress of Cu in ulture fish y mens of nutrition. Aknowlegements The uthors woul like to express their sinere thnks to the personnel of these tems for their kin ssistne. This stuy ws finnilly supporte y the Youth Fountion Progrm of the Eution Deprtment of Sihun Provine, Chin (grnt no. 14ZB0021) n the Applie Bsi Reserh Progrms of Siene n Tehnology Commission Fountion of Sihun Provine, Chin (grnt no. 2015JY0067). J. J. n X.-Y. W. onute the tril, performe the RT-PCR experiments n wrote the mnusript. Y. Z. n X.-Q. Z. ontriute to the esign of the stuy. L. F. n W.-D. J. ssiste in the mnusript preprtion. Y. L. ssiste with ll t nlysis. P. W. ssiste with the tril. The uthors elre tht there re no onflits of interest. Referenes 1. Rezei R, Kne DA, Tekwe CD, et l. (2013) Dietry supplementtion with monosoium glutmte is sfe n improves growth performne in postwening pigs. Amino Ais 44, Rees PJ, Burrin DG, Stoll B, et l. 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