Beneficial effects of hesperidin following cis diamminedichloroplatinum induced damage in heart of rats

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1 Originl Article Beneficil effects of hesperidin following cis dimminedichloropltinum induced dmge in hert of rts H Oguzturk, O Ciftci 1, A Cetin 2, K Ky 3, OM Disli 4, MG Turty, S Gürüz, N Bsk 5 Deprtments of Emergency Medicine, 1 Medicl Phrmcology, 2 Histology nd Emryology nd 4 Crdiovsculer Surgery, Fculty of Medicine, University of Inonu, 5 Deprtment of Phrmceuticl Toxicology, Fculty Phrmcy, University of Inonu, Mlty 44280, 3 Deprtment of Medicl Biochemistry, Grdute Institute of Helth Sciences, University of Inonu, Mlty, Turkey Astrct Bckground: Incresed oxidtive stress nd histopthologicl dmge hve een implicted in the crdiotoxicity tht limits the clinicl therpy of cispltin (CP) s n nti cncer drug. Ojectives: This study imed to investigte the protective effect of hesperidin (HP) ginst CP induced crdiotoxicity in rts. Mterils nd Methods: Rts were divided into four groups (n = 7/group), nd the first group served s the control group. Animls in Group CP nd Group CP + HP received single dose of CP (CP 7 mg/kg); nimls in Group HP nd Group CP + HP received 50 mg/kg/dy HP with gvge for 14 dys. At the end of dy 14, crdic tissue smples were histologiclly nd iochemiclly exmined. Results: In this experimentl study, thiorituric cid rective sustnces levels in the crdic tissue were significntly higher in the CP group, wheres glutthione (GSH), superoxide dismutse (SOD), nd CAT levels were significntly lower in this group. On the other hnd, GSH nd SOD levels in the CP + HP group were similr to the control group. There ws no significnt difference in crdic CAT levels etween Group CP nd Group CP + HP. Conclusion: Hesperetin tretment leds to decrese in oxidtive stress, nd ssocited histologicl dmge. The findings of the current study suggest tht HP hs protective effect ginst CP induced crdiotoxicity. Key words: Crdiotoxicity, cispltin, hesperidin Dte of Acceptnce: 03-Jul-2015 Introduction Cispltin (Cis dimminedichloropltinum [CP]) is pltinum-sed nti crcinogenic drug tht is commonly Address for correspondence: Dr. H Oguzturk, Deprtment of Emergency Medicine, Fculty of Medicine, University of Inonu, Mlty 44280, Turkey. E mil: oguzturk@hotmil.com Quick Response Code: Access this rticle online Wesite: used in the tretment of solid tumors. The therpeutic effect of CP increses prominently with incresing dose. [1] On the other hnd, the clinicl use of CP hs een limited due to incresed oxidtive stress, nd side effects (nephrotoxicity, crdiotoxicity) due to poptosis. [1,2] CP intercts with DNA, nd forms intr nd inter crosslinks. The resulting DNA dmge initites poptosis. [2] This is n open ccess rticle distriuted under the terms of the Cretive Commons Attriution-NonCommercil-ShreAlike 3.0 License, which llows others to remix, twek, nd uild upon the work non-commercilly, s long s the uthor is credited nd the new cretions re licensed under the identicl terms. For reprints contct: reprints@medknow.com DOI: / PMID: How to cite this rticle: Oguzturk H, Ciftci O, Cetin A, Ky K, Disli OM, Turty MG, et l. Beneficil effects of hesperidin following cisdimminedichloropltinum-induced dmge in hert of rts. Niger J Clin Prct 2016;19: Nigerin Journl of Clinicl Prctice Pulished y Wolters Kluwer - Medknow 99

2 Oguzturk, et l.: Hesperidin/cispltin induced crdiotoxicity Crdiotoxicity of the tretment in oncology usully limits its enefits. To dte, crdiotoxicity hs not received much ttention, s it hs not een considered s dose limiting fctor for CP; however, recent studies hve reported tht CP tretment leds to significnt crdiotoxic effects. [1 5] During the development of CP dependent crdiotoxicity, mild symptoms s well s severe symptoms (congestive hert filure, pericrditis, nd crdiomyopthy) cn e oserved. [3 5] Oxidtive stress plys n importnt role in the pthophysiology of CP crdiotoxicity. [4,5] The CP superoxide ion cn produce ctive oxygen species, such s hydroxyl rdicls, nd cn inhiit ctive ntioxidnt enzymes in norml tissues. [6] Therefore, vrious ntioxidnts including ginger extrct, propionyl L crnitine, DL lipoic cid, nd thimine pyrophosphte hve een tested to prevent CP crdiotoxicity. [2,4,5,7] On the other hnd, there re no studies on the efficcy of hesperidin (HP) ginst CP dependent crdiotoxicity. HP (3,5,7 trihydroxy 4 methoxyflvnone 7 rhmnoglycoside) is flvonoid glycoside tht is found in ornge nd lemon cultivrs, nd hs ntioxidnt properties. [8] This flvonoid hs nti inflmmtory, nti microil, nti crcinogenic, nti oxidnt effects, nd strengthens cpillries. [8,9] Thus, the im of the current study ws to nlyze the iochemicl nd histologicl effects of HP ginst CP induced crdiotoxicity nd resulting oxidtive stress. Mterils nd Methods Chemicls CP (10 mg/10 ml, code 1876A) ws otined from Fulding Phrmceuticls Plc (Wrwickshire, UK). All other chemicls were purchsed from Sigm Chemicl Co. (St. Louis, MO) nd were of nlyticl grde or of the highest grde ville. Animls nd tretments A totl of 28 helthy dult mle Sprgue Dwley rts (ged etween 2 nd 3 months nd weighing g) were otined from the Experimentl Animl Institute, Mlty, Turkey, for this experiment. Animls were housed in sterilized polypropylene rt cges, in 12 h light drk cycle, t n mient temperture of 21 C. Diet nd wter for them were given d liitum. Experiments were performed sed on niml ethics guidelines of the Institutionl Animls Ethics Committee. Rts were rndomly divided into four equl groups (n = 7/group). CP ws intrperitonelly (i.p.) dministered t the dose of 7 mg/kg with single injection. HP ws given in doses of 50 mg/kg/dy for 14 consecutive dys y gvges. Group 1 (control) served s the negtive control nd ws dministered isotonic sline (i.p.) nd corn oil (orlly) s vehicles. In Group 2 (CP group), CP ws dministered s single injection nd then the corn oil ws given for 14 dys. Rts in Group 3 (HP group) were treted with HP for 14 dys without CP. In Group 4, the rts were treted with CP nd HP (i.e. CP + HP group) together. Tissue smples were collected on dy 14 of CP tretment. The nimls were killed under ether nesthesi nd tissues (hert) were immeditely removed nd dissected over the ice cold glss. Tissue smples were stored t 86 C in deep freezer until nlysis. Biochemicl ssy The homogeniztion of tissues ws crried out in Teflon glss homogenizer with 150 mm KCl (ph 7.4) to otin 1:10 (w/v) dilution of the whole homogente. The homogentes were centrifuged t 18,000 g (4 C) for 30 min to determine thiorituric cid rective sustnces (TBARS), reduced glutthione (GSH) levels, nd ctlse (CAT) ctivities, nd t 25,000 g for 50 min, to determine Cu Zn superoxide dismutse (SOD) ctivities. The levels of homogenized tissue TBARS, s n index of lipid peroxidtion, were determined y thiorituric cid rection using the method of Ygi. [10] The product ws evluted spectrophotometriclly t 532 nm, nd results re expressed s nmol/g tissue. The GSH content of the testis homogente ws mesured t 412 nm using the method of Sedlk nd Lindsy. [11] The GSH level ws expressed s nmol/ml. SOD ctivity ws mesured y the inhiition of nitro lue tetrzolium (NBT) reduction due to O 2 generted y the xnthine/xnthine oxidse system. [12] One unit of SOD ctivity ws defined s the mount of protein cusing 50% inhiition in the NBT reduction rte. The product ws evluted spectrophotometriclly t 560 nm. Results were expressed s IU/mg protein. CAT ctivity of the tissues ws determined ccording to the method of Aei. [13] The enzymtic decomposition of H 2 O 2 ws followed directly y the decrese in sornce t 240 nm. The difference in sornce per unit time ws used s mesure of CAT ctivity. The enzyme ctivities re given in k/mg protein. GPx ctivity ws mesured y the method of Pgli nd Vlentin. [14] In the presence of GSH reductse nd NADPH, the oxidized GSH is immeditely converted to the reduced form with concomitnt oxidtion of NADPH to NADP. The decrese in sornce t 340 nm ws mesured. GPx ctivity is expressed s IU/mg protein. Tissue protein content ws determined ccording to the method developed y Lowry et l. [15] using ovine serum lumin s the stndrd. Histologicl evlution For the light microscopic evlution, hert smples were fixed in 10% formlin. The hert smples were processed y routine tissue techniques nd were emedded in prffin. Prffin emedded specimens were cut into 5 mm thick sections, mounted on slides, nd stined with hemtoxylin nd eosin. Sections exmined under Leic DFC280 light microscope y Leic Q Win nd Imge Anlysis System (Leic Micros Imging Solutions Ltd., Cmridge, U.K). 100 Nigerin Journl of Clinicl Prctice Jn-Fe 2016 Vol 19 Issue 1

3 Oguzturk, et l.: Hesperidin/cispltin induced crdiotoxicity An overll score of crdic dmge severity ws semi quntittively ssessed s follows: Eosinophilic stined nd pyknotic nuclei cells, congestion, nd necrosis. The microscopic score of ech tissue ws clculted s the sum of the scores given to ech criterion. Scores were given s: 0 none; 1 mild; 2 moderte; nd 3 severe for ech criterion. Sttisticl nlysis ws performed using SPSS 13 (SPSS Inc. Chicgo IL, USA) nd MedClc softwre MedClc Softwre, (Acciln Ostend, Belçik). All groups were compred y the nonprmetric Kruskl Wllis test. Exct P vlues were given where ville, nd P = ws ccepted s sttisticlly significnt. All results were expressed s men ± stndrd devition. Results Histologicl results In the control [Figure 1] nd HP groups [Figure 1], hert tissues showed norml histologicl ppernce. In the CP group, eosinophilic stined nd pyknotic nuclei cells, hemorrhge, nd degenertion were detected [Figure 2 nd ]. On the other hnd, these histologicl chnges were prtilly decresed with HP tretment, nd the therpeutic effects of HP were seen in the histologicl exmintion. Moreover, it ws detected tht the necrotic field [Figure 2c] in crdic tissue hd significnt ppernce compred to control nd other experimentl groups. However, these chnges were not seen in other tretment groups (HP nd CP + HP groups) nd HP tretments prevented these effects of CP in CP + HP groups. On the other hnd, crdic dmge decresed in the CP + HP group. Crdic dmge ws not completely meliorted in the CP + HP group [Figure 3 nd ]. Histopthologicl scores re shown in Tle 1. TBARS level in hert tissue. The current study showed tht in CP + HP group significntly incresed the SOD, GSH levels in hert tissue, compred to the CP group. As seen in Tle 2, CP tretment led to mrked increse in TBARS level in the crdic tissue compred to other groups. However, CP tretment led to significnt decrese in SOD, GSH, nd CAT levels. There ws no significnt difference in TBARS nd CAT levels etween the HP group nd the control group, wheres GSH nd SOD levels were mrkedly higher in the HP group. In ddition, when the CP + HP group ws compred to the control group, chnges tht were cused y CP tretment were closer to the control group, nd except for CAT levels, the effects of CP were ttenuted. Tle 1: Histopthologicl scores of groups Groups Histopthologicl scores (men±se) Control CP CP+HP HP 1.00± ± ±0.36c 1.29±0.29 The men differences etween the vlues ering different superscript letters within the sme column re sttisticlly significnt. (, nd c: P=0.0002). SE=Stndrd error; CP=Cispltin; HP=Hesperidin Tle 2: The levels of SOD, CAT, GSH, nd TBARS in rt hert tissue (n=7, men±sd) Control CP HP CP+HP TBARS nmol/g tissue GSH nmol/ml SOD IU/mg protein CAT k/mg protein 7.40± ± ±1.28c 10.2±1.20c 84.0± ± ±6.18c 80.5± ± ± ±0.91c 7.03± ± ± ± ±0.006 Mens ering different superscripts within the sme line were significntly (P 0.01) different. SD=Stndrd devition; CP=Cispltin; HP=Hesperidin; SOD=Superoxide dismutse; GSH=Glutthione; CAT=Ctlse; TBARS=Thiorituric cid rective sustnces Biochemicl results As shown in Tle 2, the results of the current study reveled tht use of CP resulted in significnt increse in hert tissue TBARS level. In ddition, the dministrtion of HP with CP diminished the CP induced increse in the Figure 1: Control () nd hesperidin () groups: Norml histologicl ppernce of crdic cells, H nd E, 20 c Figure 2: Cispltin group: () Eosinophilic stined nd pyknotic nuclei cells. () Hemorrhge (rrows) H nd E, 10. (c) Necrosis (rrows) H nd E, 20 Nigerin Journl of Clinicl Prctice Jn-Fe 2016 Vol 19 Issue 1 101

4 Oguzturk, et l.: Hesperidin/cispltin induced crdiotoxicity The present study reveled tht HP inhiited CP induced oxidtive dmge y incresing SOD nd GSH levels, nd decresing TBARS levels. These findings indicte the crdioprotective role of HP due to its ntioxidnt effect nd re consistent with other studies indicting the protective effect of HP on the crdiovsculr system. [8,17] Figure 3: Cispltin + hesperidin groups: () Eosinophilic stined nd pyknotic nuclei cells (rrows). (): Hemorrhge (rrows), H nd E, 20 Discussion Crdiotoxicity of the therpy in oncology often limits its enefits. Therefore, monitoring nd erly prevention of nti crcinogenic tretment crdic complictions is of clinicl interest. CP is used in the tretment of mny solid tumors. Dose limittion due to side effects is one of the mjor prolems in CP tretment. Therefore, vrious studies re conducted to reduce the side effects of CP, which is frequently used in chemotherpy. The current study nlyzed the oxidtive nd histologicl dmge in crdic tissue tht is cused y CP. Oxidtive stress cn e defined s the imlnce etween rective oxygen species nd the cpcity of ntioxidnt defense systems. While the pthogenesis of CP induced crdiotoxicity is not fully explined, the contriution of DNA dmge due to oxidtive stress is known. Previous studies support the current study, nd it hs een shown tht CP leds to crdiotoxicity y inducing crdic enzymes nd histopthologicl lesions. [2] Secondry to the CP triggered lipid peroxidtion in crdic memrnes, this dmging effect cuses chnges in TBARS, GSH, nd SOD levels, ctivities of certin protective enzymes, nd levels of ntioxidnt molecules. [1,3 5] The present study reveled tht single dose CP tretment led to mrked increse in TBARS level in the crdic tissue, nd led to significnt decrese in SOD, CAT, nd GSH levels. Due to its effects on oxidnt nd ntioxidnt systems, CP cuses significnt level of oxidtive stress in the crdic tissue. HP hs ttrcted ttention in recent studies, especilly regrding its cpcity to prevent the effects of free rdicls, nd its high ntioxidnt properties. Antioxidnt molecules such s HP hve protective effects ginst the oxidtive dmge cused y drugs nd other dmging gents. [16,17] The strong ntioxidnt ctivity of HP contriutes to the prevention of vrious types of oxidtive dmge, toxicity, nd other diseses. [17] The reserchers of the present study did not oserve significnt difference in GSH levels etween the CP + HP group nd the control group. However, compred to the CP group, GSH levels were significntly higher in Group 3 nd Group 4. GSH is nonenzymtic ntioxidnt, nd is key component of the ntioxidnt defense system. At the sme time, GSH ensures protection of the memrne integrity. [7] In the present study, high GSH levels in Group 3 nd Group 4 re consistent with the previous studies indicting its crdioprotective fetures. [3,7] In the present study, elevted GSH levels supported the memrne integrity in myocrdil cells, nd protected them from oxidtive dmge, thus, preventing vrious pthologies within the myocrdium. Incresed TBARS levels nd decresed GSH levels in the crdic tissue due to CP tretment support the notion tht CP triggers oxidtive dmge tht led to crdic dmge. In ddition, recent studies hve focused on the protective effect of HP on the crdiovsculr system. [8] Deng et l. reported tht HP inhiits pressure dependent dysfunction nd firosis, nd crdic hypertrophy, nd decreses incresed oxidtive stress nd poptosis. According to the findings of the present study, HP protects hert muscle cells from dmge, nd ttenutes crdic dmge nd toxicity. Previous studies hve shown tht CP inhiits cellulr protein synthesis, nd cuses poptosis fter mitochondril dmge. [18,19] CP lso cuses significnt histopthologicl chnges in hert tissue. [1 5] According to the findings regrding the histopthologicl chnges, CP, together with firous tissue rections, cuses degenertion nd necrosis in crdiomyocytes. Mssive degenertive chnges hve een reported in study on the histopthologicl chnges in the crdic system tht re cused y CP. [1] The numer of studies on this topic is limited, nd the findings re consistent with the current study with regrds to tissue dmge criteri (e.g., necrosis, degenertion, nd hemorrhge). [1 3] This condition cn e interpreted s the disruption of the lnce etween the oxidnt system, which is induced y CP, nd the ntioxidnt system. In ddition, the present findings suggest tht the comintion of CP nd HP decreses histopthologicl dmge. These findings re consistent with the findings y Al Mjed et l. [1] Conclusion HP tretment led to decrese in oxidtive stress nd ssocited histologicl dmge. The present findings indicte 102 Nigerin Journl of Clinicl Prctice Jn-Fe 2016 Vol 19 Issue 1

5 Oguzturk, et l.: Hesperidin/cispltin induced crdiotoxicity tht HP tretment supports reduction in hert dmge due to CP tretment. HP tretment led to iochemicl nd histologicl improvements in the hert tissue. HP tretment led to decrese in TBARS levels in the crdic tissue, nd n increse in SOD nd GSH levels. The current study represents the first study on the protective effect of HP ginst CP induced crdiotoxicity. While the results indicte the protective effect of HP in CP dependent crdic dmge, more comprehensive prospective studies re required. Future clinicl studies will contriute to the potentil clinicl use of HP. Finncil support nd sponsorship Nil. Conflicts of interest There re no conflicts of interest. References 1. Al Mjed AA, Syed Ahmed MM, Al Yhy AA, Aleis AM, Al Rejie SS, Al Shnh OA. Propionyl L crnitine prevents the progression of cispltin induced crdiomyopthy in crnitine depleted rt model. Phrmcol Res 2006;53: Attyh AM, Ismil SH. Protective effect of ginger extrct ginst cispltin induced heptotoxicity nd crdiotoxicity in rts. Irqi J Phrm Sci 2012;21: Hussein A, Ahmed AA, Shoumn SA, Shrwy S. Ameliorting effect of DL lipoic cid ginst cispltin induced nephrotoxicity nd crdiotoxicity in experimentl nimls. Drug Discov Ther 2012;6: El Awdy el SE, Moustf YM, Ao Elmtty DM, Rdwn A. Cispltin induced crdiotoxicity: Mechnisms nd crdioprotective strtegies. Eur J Phrmcol 2011;650: Demkow U, Stelmszczyk Emmel A. Crdiotoxicity of cispltin sed chemotherpy in dvnced non smll cell lung cncer ptients. Respir Physiol Neuroiol 2013;187: Sdzuk Y, Shoji T, Tkino Y. Effect of cispltin on the ctivities of enzymes which protect ginst lipid peroxidtion. Biochem Phrmcol 1992;43: Coskun R, Turn MI, Turn IS, Gulpoglu M. The protective effect of thimine pyrophosphte, ut not thimine, ginst crdiotoxicity induced with cispltin in rts. Drug Chem Toxicol 2014;37: Deng W, Jing D, Fng Y, Zhou H, Cheng Z, Lin Y, et l. Hesperetin protects ginst crdic remodelling induced y pressure overlod in mice. J Mol Histol 2013;44: Grg A, Grg S, Zneveld LJ, Singl AK. Chemistry nd phrmcology of the Citrus ioflvonoid hesperidin. Phytother Res 2001;15: Ygi K. Simple ssy for the level of totl lipid peroxides in serum or plsm. Methods Mol Biol 1998;108: Sedlk J, Lindsy RH. Estimtion of totl, protein ound, nd nonprotein sulfhydryl groups in tissue with Ellmn s regent. Anl Biochem 1968;25: Sun Y, Oerley LW, Li Y. A simple method for clinicl ssy of superoxide dismutse. Clin Chem 1988;34: Aei H. Ctlse. In: Bergmeyer HU, editor. Methods of Enzymtic Anlysis. New York: Acdemic Press; p Pgli DE, Vlentine WN. Studies on the quntittive nd qulittive chrcteriztion of erythrocyte glutthione peroxidse. J L Clin Med 1967;70: Lowry OH, Roserough NJ, Frr AL, Rndll RJ. Protein mesurement with the Folin phenol regent. J Biol Chem 1951;193: Menze ET, Tdros MG, Adel Tw AM, Khlif AE. Potentil neuroprotective effects of hesperidin on 3 nitropropionic cid induced neurotoxicity in rts. Neurotoxicology 2012;33: Trivedi PP, Kushwh S, Tripthi DN, Jen GB. Crdioprotective effects of hesperetin ginst doxoruicin induced oxidtive stress nd DNA dmge in rt. Crdiovsc Toxicol 2011;11: Jung M, Hotter G, Viñs JL, Sol A. Cispltin upregultes mitochondril nitric oxide synthse nd peroxynitrite formtion to promote renl injury. Toxicol Appl Phrmcol 2009;234: Chirino YI, Pedrz Chverri J. Role of oxidtive nd nitrostive stress in cispltin induced nephrotoxicity. Exp Toxicol Pthol 2009;61: Nigerin Journl of Clinicl Prctice Jn-Fe 2016 Vol 19 Issue 1 103