Protective Effect of Quercetin on Melphalan-Induced Oxidative Stress and Impaired Renal and Hepatic...

Research ArticleProtective Effect of Quercetin on Melphalan-Induced OxidativeStress and Impaired Renal and Hepatic Functions in Rat

Ebenezer Tunde Olayinka Ayokanmi OreOlaniyi Solomon Ola and Oluwatobi Adewumi Adeyemo

Biochemistry Unit Department of Chemical Sciences Ajayi Crowther University PMB 1066 Oyo 211213 Oyo State Nigeria

Correspondence should be addressed to Ebenezer Tunde Olayinka etolayinkaacuedung

Received 30 August 2014 Accepted 23 November 2014 Published 11 December 2014

Academic Editor Paolo Pronzato

Copyright copy 2014 Ebenezer Tunde Olayinka et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

One major challenge with the use of anticancer agents is the phenomenon of drug-induced toxicity Melphalan (MPLN) is analkylating anticancer agent while quercetin (QCT) is an antioxidantWe investigated the protective role of quercetin againstMPLN-induced toxicity Twenty-five male Wistar rats (160ndash170 g) were randomized into five treatment groups (I) control (II) MPLN(02mgkg bw) (III) pre-treated withQCT (20mgkg bw) for 7 days followed byMPLN (02mgkg bw) for 7 days (IV) cotreatedwith QCT (20mgkg bw) and MPLN (02mgkg bw) for 7 days and (V) QCT (20mgkg bw) alone MPLN caused a significantincrease in plasma bilirubin urea and creatinine by 1222 1023 and 188 respectively (119875 lt 005) Similarly plasma ALP ALTAST and 120574-GT activities increased significantly by 579 1443 713 and 3072 respectively relative to control However pre orcotreatment with QCT ameliorated the levels of renal and hepatic function indices Hepatic ascorbic acid and GSH and activities ofglutathione-S-transferase SOD and catalase decreased significantly by 362 188 465 344 and 552 respectively followedby increase inMDAcontent by 465 relative to control Pre- and cotreatmentwithQCT reestablished the hepatic antioxidant statusand lipid peroxidation Overall quercetin protected against MPLN-induced renal and hepatic toxicity in rats

1 Introduction

Melphalan (4-[bis(2-chloroethyl)amino]-L-phenylalanine)Figure 1(a) is a phenylalanine derivative of nitrogen mus-tard It is a bifunctional alkylating agent and one of the mostaggressive antineoplastic drugs indicated for multiple mye-loma and ovarian cancer [1] Melphalan (MPLN) is classifiedas a cell cycle phase-nonspecific alkylating agent [2] Itsmechanism of action involves inhibition of DNA and RNAsynthesis through formation of interstrand cross-links withDNA Following oral administration it is distributed mainlyto the liver where microsomal glutathione-S-transferase(GST) plays a significant role in its metabolism [3] Amongthe reported toxicities elicited by melphalan are hemato-logical suppression [4] hepatotoxicity [5ndash7] renal toxicity[8 9] and bone marrow suppression [10]

One of the cytotoxic mechanisms of alkylating agents istheir ability to generate free radicals and trigger oxidative

stress in vivo [11] This may also be associated with the toxici-ties elicited by these drugs as a result of suppression of cellularantioxidant defense [12ndash14] A free radical is a reactive atomor group of atoms that has one or more unpaired electronsThey are produced in the body by natural biological processesor introduced from an exogenous source such as drugs andenvironmental toxicants [15] Excessive production of freeradicals which are not neutralised may result in lipid pro-teins and DNA oxidation and ultimately in cell damage [15]

The liver is themain site of drugmetabolism andmetabo-lites generated in the liver and in some cases free drugmolecules are also distributed to the kidneys thus exposingthese organs to drug-induced toxicities These tissues havehowever evolved an array of antioxidant defense systems toprotect against the harmful effect of drug metabolites andfree radicals [15 16] Antioxidants are substances that inhibitoxidation or reactions promoted by oxygen peroxides or freeradicals or their actions [16] These include the nonenzymic

Hindawi Publishing CorporationChemotherapy Research and PracticeVolume 2014 Article ID 936526 8 pageshttpdxdoiorg1011552014936526

2 Chemotherapy Research and Practice

HO

O

N

Cl

ClNH2

(a)

OHO

OH

OH

OH

OH

O

(b)

Figure 1 Structure of (a) melphalan and (b) quercetin

antioxidants like reduced glutathione (GSH) ascorbic acid(AA) and vitamin E among others Enzymic antioxidantsinvolved in this protection include glutathione-S-transferase(GST) glutathione peroxidase (GPx) glutathione reductase(GR) superoxide dismutase (SOD) and catalase (CAT) [1718]

Quercetin (3573101584041015840-pentahydroxyflavone) Figure 1(b)is one of themost widely distributed flavonoids (of the flavon-ols subclass) in plants [19] It is abundant in plants plantproducts and foods such as red wine onions green teaapples broccoli berries ginkgo biloba and buckwheat tea[20] QCT has been exhibited in several studies as a potentantioxidant with a very strong free radical scavenging capac-ity [21 22] It also possesses a number of pharmacologi-cal activities including antidiabetic anti-inflammatory andimmunostimulatory and protection of low density lipopro-tein against oxidation [22ndash24] Previous reports suggest thatQCT also possesses the capacity to effectively inhibit theproliferation of cancer cells [25ndash27] Moreover it is knownto improve chemotherapeutic efficacy of certain alkylatingagents [28] In addition recent studies demonstrated thatquercetin could effectively attenuate drug-induced toxicityand oxidative stress in vivo [29ndash31]

One of the major challenges often encountered with theuse of anticancer agents is the incident of drug-inducedtoxicity Nevertheless it is alleged that the administration ofantioxidants alongwith anticancer agentsmayhelp relieve thetoxic side effects elicited by these agents Consequently thepresent study was designed to investigate the protective effectof quercetin pretreatment and cotreatment on melphalan-induced renal and hepatic toxicity in rat models

2 Materials and Methods

21 Chemicals and Reagents Melphalan tablets were a prod-uct of Excella GmbH Nurnberger Germany Quercetin glu-tathione 1-chloro-24-dinitrobenzene (CDNB) 55-dithio-bis-2-nitrobenzoic acid (DTNB) epinephrine and hydrogenperoxide (H

2O2) were all purchased from Sigma Chemical

Company (London UK) Kits for alanine transferase (ALT)aspartate aminotransferase (AST) alkaline phosphatase(ALP) gamma glutamyl transpeptidase (GGT) urea creati-nine and total bilirubinwere products of Randox laboratoriesLtd (Antrim UK) All other reagents used were of analyticalgrade and of the highest purity

Table 1 Experimental design

Treatmentdarrgroups Treatments

Duration rarr Days 1ndash7 Days 8ndash14I (CTRL) mdash Control distilled waterII (MPLN) mdash 02mgKg bw MPLN

III (MPLN + QCT-P) 20mgKg bwQCT 02mgKg bw MPLN

IV (MPLN + QCT-C) mdash 02mgKg bw MPLN +20mgKg bw QCT

V (QCT-A) mdash 20mgKg bw QCTCTRL control MPLN melphalan QCT quercetin QCT-P quercetin-pretreated QCT-C quercetin-cotreated QCT-A quercetin-alone bw bodyweight

22 Animal Selection and Care Twenty-five maleWistar ratsweighing between 160 and 170 g were obtained from TheAnimal Holding Unit Department of Chemical SciencesAjayi Crowther University Oyo Nigeria The rats were accli-matized under laboratory conditions prior to experimentThe animals were housed in wire-meshed cages and providedwith food and water ad libitumThe animals weremaintainedunder standard conditions of temperature and humidity with12-hour lightdark cycles They were fed with commercial ratdiet (Ladokun Feeds Nigeria Ltd) Handling of the experi-mental animalswas in conformitywith theNational Institutesof Healthrsquos Guide for the Care andUse of Laboratory Animals(NIHpublicationnumber 85-23 revised 1985USDepartmentof Health Education and Welfare Bethesda MA)

23 Animal Grouping and Drug Treatments The animalswere randomly assigned into five experimental groups (IndashV)of five animals each The animals of each group were treatedas presented in Table 1 The doses for MPLN and QCT weredecided based on the available literature [32 33] and weredelivered in one mL solution of distilled water once daily byoral intubation

24 Animal Sacrifice and Collection of Blood and Liver Sam-ples Blood sampleswere collected from each animal throughretro orbitals plexus into heparinized tubes (Li heparin)Animals were sacrificed and the liver was carefully excisedfrom each animal for preparation of cytosolic fraction

Chemotherapy Research and Practice 3

25 Preparation of Plasma and Cytosolic Fractions Plasmawas obtained by centrifugation of whole blood sample at4000 rpm for 5 minutes using a Cencom bench centrifugeThe plasma obtained was stored at minus4∘C for subsequentplasma assays The liver excised from each rat was blottedof blood stains rinsed in ice-cold 115 KCl and homog-enized in 4 volumes of ice-cold 001M potassium phos-phate buffer (pH 74) The homogenates were centrifuged at12500 g for 15min at minus4∘C (Eppendorf UK) and the super-natants termed the postmitochondrial fractions (PMF) werealiquoted and used for subsequent biochemical assays

26 Determination of Plasma and Liver Protein Content Theprotein concentration in the plasma and liver homogenatewas determined by the Biuret method of Gornall et al [34]using bovine serum albumin as standard

27 Assay of Plasma Biomarkers of Renal Toxicity Plasmaurea and creatinine were determined with Randox diagnostickits Method for creatinine assays was based on colorimetricalkaline picratemethods [35]with creatinine-picrate complexmeasured at 492 nm Plasma urea determination was basedon the Fenton reaction [36] with the diazine chromogenformed absorbing strongly at 540 nm

28 Assay of Plasma Biomarkers of Hepatotoxicity Plasmatotal bilirubin (TBILI) determinationwas done using Randoxdiagnostic kits based on the dimethyl sulphoxide method byTietz [36] The dimethyl sulphoxide forms a coloured com-pound withmaximum absorption at 550 nm Plasma alkalinephosphatase (ALP) alanine aminotransferase (ALT) aspar-tate aminotransferase (AST) and gamma glutamyl trans-ferase (120574-GT) activities were determined using Randoxdiagnostic kits ALP activity was determined in accordancewith the principles of Tietz et al [37] The p-nitrophenolformed by the hydrolysis of p-nitrophenyl phosphate confersyellowish colour on the reactionmixture and its intensity canbe monitored at 405 nm to give a measure of enzyme activityDetermination of plasma ALT and AST activities was basedon the principle described by Reltman and Frankel [38] ALTactivity was measured by monitoring the concentration ofpyruvate hydrazone formedwith 24-dinitrophenylhydrazineat 546 nmAST activity wasmeasured bymonitoring the con-centration of oxaloacetate hydrazone formed with 24-dini-trophenylhydrazine at 546 nm 120574-GT activity was determinedfollowing the principle described by Szasz [39]The substrateL-120574-glutamyl-3-carboxy-4-nitroanilide in the presence ofglycylglycine is converted to 5-amino-2-nitrobenzoate by120574-GT measured at 405 nm The increase in absorbance isproportional to 120574-GT activity

29 Assay for Nonenzymatic Antioxidants in the Liver Hep-atic reduced glutathione level was determined according tothe method of Jollow et al [40] The chromophoric productresulting from the reaction of Ellmanrsquos reagent with thereduced glutathione 2-nitro-5-thiobenzoic acid possesses amolar absorption at 412 nm which was read in a spectropho-tometer Reduced GSH is proportional to the absorbance

Table 2 Protective effects of quercetin on melphalan-inducedchanges in the levels of plasma creatinine urea and bilirubin levelsin rats

Treatment Creatinine(mgdL) Urea (mgdL) Bilirubin

(mgdL)CTRL 098 plusmn 013 5136 plusmn 023 036 plusmn 001

MPLN 214 plusmn 011(188)lowast

1039 plusmn 019(1023)lowast


MPLN + QCT-P 176 plusmn 009lowastdagger 769 plusmn 064lowastdagger 055 plusmn 002lowastdagger

MPLN +QCT-C 152 plusmn 008lowastdagger 958 plusmn 084lowastdagger 051 plusmn 001lowastdagger

QCT-A 138 plusmn 008lowastdagger 742 plusmn 130lowastdagger 046 plusmn 001lowastdagger

Data represent the means plusmn SD for five rats in each group lowastsignificantlydifferent from the CTRL daggersignificantly different from MPLN Values in pa-renthesis represent percentage () increase

at 412 nm The ascorbic acid concentration was determinedaccording to the method of Jagota and Dani [41] AA in bio-logical samples reacts with Folinrsquos reagent an oxidizing agentto give a blue color which has its maximum absorption at760 nm

210 Assay of Hepatic Antioxidant Enzymes Hepatic glu-tathione S-transferase (GST) activity was determined bythe method described by Habig et al [42] using 1-chloro-24-dinitrobenzene (CDNB) as substrate The procedure ofMisra and Fridovich [43] was used for the determination ofhepatic superoxide dismutase (SOD) activity by measuringthe inhibition of autooxidation of epinephrine at pH 102 and30∘CHepatic catalase activity was determined by themethoddescribed by Sinha [44] based on the reduction of dichromatein acetic acid to chromic acetate when heated in the presenceof hydrogen peroxide (H

2O2) The chromic acetate produced

is measured spectrophotometrically at 570 nm

211 Assay of Hepatic Level of Lipid Peroxidation The extentof lipid peroxidation (LPO) in the liver was estimated by themethod of Varshney and Kale [45] The method involved thereaction between malondialdehyde (MDA product of lipidperoxidation) and thiobarbituric acid to yield a stable pinkchromophore with maximum absorption at 532 nm

212 Statistical Analysis The results were expressed as meansof 5 replicates plusmn SD Data obtained were subjected to one-wayanalysis of variance (ANOVA) followed by Duncan multiplerange test for comparison between control and treated rats inall groups using SigmaPlot statistical application package 119875values less than 005 were considered statistically significant

3 Results

31 Plasma Biomarkers of Renal Toxicity Table 2 shows theprotective effects of QCT on MPLN-induced changes inplasma creatinine and urea in rats Administration of MPLNcaused a significant increase in the plasma level of creatinineand urea by 188 and 1023 respectively when compared


Table 3 Protective effects of quercetin on melphalan-inducedchanges in the plasma activities of ALT AST ALP and 120574-GT in rats

Treatment ALT (UL) AST (UL) ALP (UL) 120574-GT (UL)CTRL 21 plusmn 024 174 plusmn 230 257 plusmn 241 14 plusmn 016





MPLN +QCT-P 34 plusmn 140lowastdagger 204 plusmn 230lowastdagger 319 plusmn 228lowastdagger 45 plusmn 023lowastdagger

MPLN +QCT-C 28 plusmn 056lowastdagger 195 plusmn 387lowastdagger 331 plusmn 228lowastdagger 42 plusmn 025lowastdagger

QCT-A 27 plusmn 074lowastdagger 184 plusmn 159lowastdagger 279 plusmn 228lowastdagger 32 plusmn 019lowastdagger


Table 4 Protective effect of quercetin on melphalan-inducedchanges in hepatic SOD and CAT activities in rats

Treatment SOD (Units)CAT (120583moleH2O2consumedminmg

protein)CTRL 838 plusmn 018 023 plusmn 002MPLN 55 plusmn 022 (344)lowast 011 plusmn 001 (522)lowast

MPLN + QCT-P 704 plusmn 017lowastdagger 019 plusmn 001lowastdagger

MPLN + QCT-C 748 plusmn 018lowastdagger 018 plusmn 001lowastdagger

QCT-A 784 plusmn 011lowastdagger 022 plusmn 002lowast

Data represent the means plusmn SD for five rats in each group lowastsignificantlydifferent from the CTRL daggersignificantly different from MPLN Values in pa-renthesis represent percentage () decrease

with the control Pre- and cotreatmentwithQCT significantlyprotected against the increase in plasma level of creatinineand urea in rats

32 Plasma Biomarkers of Hepatotoxicity Plasma bilirubinlevel reduced by 1222 following MPLN administration(Table 2) Similar trends were also observed for the activitiesof the liver enzymes ALT AST ALP and 120574-GT in the plasmaof experimental animals The activities of ALT AST ALPand 120574-GT in the plasma of MPLN-treated rats increasedsignificantly by 1443 713 579 and 3072 respec-tively compared to the values of the control animals (Table 3)However quercetin pre- and cotreatment significantly ame-liorated the MPLN-induced increase in plasma bilirubin aswell as plasma ALT AST ALP and 120574-GT in rats

33 Activity of Antioxidant Enzymes (SOD CAT and GST)Table 4 represents the protective effect of quercetin onMPLN-induced reduction in the activities of SOD and CATin the liver of rats Hepatic SOD and CAT activities were sig-nificantly reduced in the MPLN-treated group by 344 and522 respectively when comparedwith values of the controlgroup Hepatic GST activity was also significantly reducedby 465 when compared to control (Figure 4) Howeverpretreatment and cotreatment with quercetin significantlyameliorated the activities of hepatic SOD CAT and GST

0

2

4

6

8

10

12

14

CTRL MPLN MPLN +QCT-P

MPLN +QCT-C

QCT-A

AA

(120583g

mL)

lowast

dagger

lowast

lowastdagger

Figure 2 Protective effects of quercetin on melphalan-inducedchanges in hepatic AA level in rats Data represent the means plusmn SDfor five rats in each group lowastsignificantly different from the CTRLdaggersignificantly different fromMPLN

0123456789


MPLN +QCT-C

QCT-A

lowast

lowastdagger lowastdagger

lowastdagger

GSH

(120583g

g liv

er)

Figure 3 Protective effect of quercetin on melphalan-inducedchanges in the levels of GSH in rats Data represent the means plusmn SDfor five rats in each group lowastsignificantly different from the CTRLdaggersignificantly different fromMPLN

34 Nonenzymic Antioxidants (AA and GSH) Hepatic AAlevel also reduced significantly by 362 following oraladministration ofMPLN to rats (Figure 2) A similar decreasein hepatic GSH (by 188) was also observed (Figure 3) in theMPLN-treated animals relative to control The level of thesenonenzymatic antioxidants was enhanced following pre- andcotreatment with quercetin

35 Lipid Peroxidation The MPLN-induced reduction inhepatic antioxidant status was accompanied by a significantincrease in the level of lipid peroxidation (as indicated by theMDA content) as shown in Figure 5 The level of lipid per-oxidation was significantly increased inMPLN-treated groupby 465 when compared with the control Pretreatmentand cotreatment with quercetin significantly attenuated thisincrease in hepatic MDA when compared with melphalangroup


02468

1012141618


MPLN +QCT-C

QCT-A

GST

(nm

olm

inm

g pro

tein

)

lowast

lowastdaggerlowastdagger

lowastdagger

Figure 4 Protective effect of quercetin on melphalan-inducedchanges in hepatic GST activity in rats Data represent the means plusmnSD for five rats in each group lowastsignificantly different from theCTRLdaggersignificantly different fromMPLN

0

100

200

300

400

500

600


MPLN +QCT-C

QCT-A

MD

A (n

mol

mg p

rote

in) lowast


lowastdagger

Figure 5 Protective effect of quercetin on melphalan-inducedchanges in the level of lipid peroxidation (MDA) in rats Datarepresent the means plusmn SD for five rats in each group lowastsignificantlydifferent from the CTRL daggersignificantly different fromMPLN

4 Discussion

The present study evaluates renal and hepatotoxic effect ofmelphalan (MPLN) inWistar rats and possible protection bythe flavonoid antioxidant quercetin (QCT) The plasma bio-markers of renal function creatinine and urea were consid-ered in this study Creatinine and urea aremetabolic productswhich are removed from circulation by the kidney to preventtheir accumulation Increase in plasma level of these sub-stances is regarded as an indication of loss of renal function[46 47] Data from this study suggest that alkylating agentscaused a loss of renal function and this is consistent withprevious reports [48 49]We observed that QCT amelioratedthe levels of plasma creatinine and ureawhich is an indicationof renal protection This also confirms the protective roleof QCT against drug-induced renal toxicity as previouslyreported [50 51] The liver is an organ involved in thebiotransformation of drugs and other hepatotoxicants Theplasma level of bilirubin and activities of the liver enzymesALT AST ALP and 120574-GT are considered reliable indicesof hepatotoxicity [52 53] In this study MPLN caused a

significant increase in the plasma bilirubin level and activitiesof ALT AST ALP and 120574-GT ALT and AST are primarilylocated in the cytoplasm and mitochondria of hepatocytes[54] Increase in plasmaALTandASTmayhave resulted fromleakage from damaged hepatocytes (hepatocellular injury)[55] Bilirubin is found in the liver bile intestines and thereticuloendothelial cells of the spleen while ALP and 120574-GTare associated with the cell membrane [56] Plasma bilirubinand activities of ALP and 120574-GT are found to increase inconditions associated with hepatobiliary injury (decrease inhepatic clearance of bilirubin) and overproduction or leakageof ALP and 120574-GT [56] In this study pre- and cotreatmentwith quercetin protected against increase in plasma levels ofbilirubin ALT AST ALP and 120574-GT which is an indication ofhepatoprotection by QCT Our observation also corroboratesprevious findings showing the hepatoprotective activity ofQCT [30 57]

Several studies have established a connection betweenhepatotoxicity and oxidative stress [58ndash60] thus promptingthe consideration of the effect of MPLN on major enzymicas well as nonenzymic antioxidant systems of rats Activitiesof enzymic antioxidants SOD CAT and GST are vital to themaintenance of the cellular redox balance [61] In this studyMPLN significantly decreases the activity of SOD CAT andGST in the liver of rats SOD catalyzes the reaction involving arapid dismutation of superoxide radical to hydrogen peroxideand dioxygen while CAT converts the hydrogen peroxideformed in this process and other cellular processes into waterand molecular oxygen [62] Reduction in the activities ofSOD and CAT by MPLN may expose the liver to oxidativestress [37] Reduction in hepatic SOD activity is an indicationof oxidative stress [51] Similarly decrease in the activityof CAT in the liver of MPLN rats may have resulted fromaccumulation of superoxide anion radical due to reductionin hepatic SOD activity [52] GST is an enzyme found inmost tissues and it is involved in the detoxification of ingestedxenobiotics in the liver [63 64] and also forms a vital com-ponent of the antioxidant defense mechanism [64 65]

The non-enzymic antioxidant molecules AA and GSHplay a crucial role in cellular redox balance Both AA andGSH are involved in scavenging ROS and are the first line ofdefense against oxidation [15] AA functions in the aqueousenvironments of the body and is involved in the regenerationof tocopherol from tocopherol radicals in membranes andlipoproteins [66 67] One of the major roles of glutathioneagainst oxidative stress includes acting as a cofactor for sev-eral enzymic antioxidants like glutathione peroxidase (GPx)glutathione-S-transferase and others also scavenging hy-droxyl radical and singlet oxygen and regeneration of otherantioxidants such as vitamins C and E back to their activeforms [68] Disturbance in the cellular redox status of AA andGSH has been reported to enhance oxidative stress and tissueinjury [46] Pre- and cotreatment with QCT significantlyimproved the levels of AA and GSH in rats which supportsprevious reports by Mishra et al [30] and Dong et al [31]

Increase in tissueMDA content (from oxidation of unsat-urated fatty acids) is a commonly used marker of oxidativestress [15] Lipid peroxidation is initiated by the attack of a freeradical on fatty acid [18] and leads to cell and tissue damage


The observed significant increase in the concentration ofMDA in the liver of MPLN-treated animals may be relatedto decreased antioxidant protection from free radicals [31]

5 Conclusion

In conclusion we report that QCT has the capacity to protectagainst MPLN-induced hepatotoxicity and oxidative stressprobably through scavenging the free radicals

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

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[2] D A Volpe and M K Warren ldquoMyeloid clonogenic assays forcomparison of the in vitro toxicity of alkylating agentsrdquo Toxicol-ogy in Vitro vol 17 no 3 pp 271ndash277 2003

[3] J Zhang Z Ye and Y Lou ldquoMetabolism of melphalan by ratlivermicrosomal glutathione S-transferaserdquoChemico-BiologicalInteractions vol 152 no 2-3 pp 101ndash106 2005

[4] SM Devine R Sanborn E Jessop et al ldquoFludarabine andmel-phalan-based conditioning for patients with advanced hema-tological malignancies relapsing after a previous hematopoieticstem cell transplantrdquo Bone Marrow Transplantation vol 28 no6 pp 557ndash562 2001

[5] M Kresse Mechanism of liver cell injury by the cytostatic drugmelphalan [Dissertation] 2005

[6] P D King and M C Perry ldquoHepatotoxicity of chemotherapyrdquoThe Oncologist vol 6 no 2 pp 162ndash176 2001

[7] M Kresse M Latta G Kunstle et al ldquoKupffer cell-expressedmembrane-bound TNF mediates melphalan hepatotoxicity viaactivation of both TNF receptorsrdquo Journal of Immunology vol175 no 6 pp 4076ndash4083 2005

[8] V Sahni D Choudhury and Z Ahmed ldquoChemotherapy-asso-ciated renal dysfunctionrdquoNature Reviews Nephrology vol 5 no8 pp 450ndash462 2009

[9] N Leung J M Slezak E J Bergstralh et al ldquoAcute renalinsufficiency after high-dose melphalan in patients with pri-mary systemic amyloidosis during stem cell transplantationrdquoAmerican Journal of Kidney Diseases vol 45 no 1 pp 102ndash1112005

[10] O O Shevchuk K A Posokhova A S Sidorenko et alldquoThe influence of enterosorption on some haematological andbiochemical indices of the normal rats after single injection ofmelphalanrdquo Experimental Oncology vol 36 no 2 pp 94ndash1002014

[11] C Gorrini I S Harris and TWMak ldquoModulation of oxidativestress as an anticancer strategyrdquoNature Reviews DrugDiscoveryvol 12 no 12 pp 931ndash947 2013

[12] S Khan J J Ramwani and P J OrsquoBrien ldquoHepatocyte toxicity ofmechlorethamine and other alkylating anticancer drugsnrdquo Bio-chemical Pharmacology vol 43 no 9 pp 1963ndash1967 1992

[13] J Chandra A Samali and S Orrenius ldquoTriggering and modu-lation of apoptosis by oxidative stressrdquo Free Radical Biology andMedicine vol 29 no 3-4 pp 323ndash333 2000

[14] K A Conklin ldquoDietary polyunsaturated fatty acids impacton cancer chemotherapy and radiationrdquo Alternative MedicineReview vol 7 no 1 pp 4ndash21 2002

[15] J M C Gutteridge ldquoLipid peroxidation and antioxidants asbiomarkers of tissue damagerdquo Clinical Chemistry vol 41 no 12pp 1819ndash1828 1995

[16] R K Murray D K Granner P A Mayer and V W RodwellHarpers Biochemistry 25th edition 2000

[17] T P A Devasagayam J C Tilak K K Boloor K S Sane SS Ghaskadbi and R D Lele ldquoFree radicals and antioxidants inhuman health current status and future prospectsrdquo Journal ofAssociation of Physicians of India vol 52 pp 794ndash804 2004

[18] M Valko C J Rhodes J Moncol M Izakovic and M MazurldquoFree radicals metals and antioxidants in oxidative stress-induced cancerrdquo Chemico-Biological Interactions vol 160 no 1pp 1ndash40 2006

[19] C A Rice-Evans N J Miller and G Paganga ldquoStructure-anti-oxidant activity relationships of flavonoids and phenolic acidsrdquoFree Radical Biology and Medicine vol 20 no 7 pp 933ndash9561996

[20] K Murota and J Terao ldquoAntioxidative flavonoid quercetinimplication of its intestinal absorption and metabolismrdquo Ar-chives of Biochemistry and Biophysics vol 417 no 1 pp 12ndash172003

[21] L Stephen-Cole ldquoQuercetin a review of clinical applicationsrdquoClinical Sciences vol 40 pp 234ndash238 1998

[22] H Liu L Zhang and S Lu ldquoEvaluation of antioxidant andimmunity activities of quercetin in isoproterenol-treated ratsrdquoMolecules vol 17 no 4 pp 4281ndash4291 2012

[23] J Oslash Moskaug H Carlsen M Myhrstad and R BlomhoffldquoMolecular imaging of the biological effects of quercetin andquercetin-rich foodsrdquoMechanisms of Ageing and Developmentvol 125 no 4 pp 315ndash324 2004

[24] M A Abdelmoaty M A Ibrahim N S Ahmed and MA Abdelaziz ldquoConfirmatory studies on the antioxidant andantidiabetic effect of quercetin in ratsrdquo Indian Journal of ClinicalBiochemistry vol 25 no 2 pp 188ndash192 2010

[25] J A Choi J Y Kim J Y Lee et al ldquoInduction of cell cyclearrest and apoptosis in human breast cancer cells by quercetinrdquoInternational Journal of Oncology vol 19 no 4 pp 837ndash8442001

[26] C S Ong E Tran T T T Nguyen et al ldquoQuercetin-inducedgrowth inhibition and cell death in nasopharyngeal carcinomacells are associated with increase in Bad and hypophosphory-lated retinoblastoma expressionsrdquo Oncology Reports vol 11 no3 pp 727ndash733 2004

[27] A M Seufi S S Ibrahim T K Elmaghraby and E E HafezldquoPreventive effect of the flavonoidquercetin on hepatic cancerin rats via oxidantantioxidant activity molecular and histo-logical evidencesrdquo Journal of Experimental amp Clinical CancerResearch vol 28 no 1 article 80 2009

[28] K Sak ldquoChemotherapy and dietary phytochemical agentsrdquoChemotherapy Research and Practice vol 2012 Article ID282570 11 pages 2012

[29] E Atef and A El-Baky ldquoQuercetin protective action on oxida-tive stress sorbitol insulin risistance and cells function inexpermintal diabetic ratsrdquo International Journal of Pharmaceu-tical Studies and Research vol 2 no 2 pp 11ndash18 2011

[30] S K Mishra P Singh and S K Rath ldquoProtective effect ofquercetin on chloroquine-induced oxidative stress and hepa-totoxicity in micerdquo Malaria Research and Treatment vol 2013Article ID 141734 10 pages 2013


[31] Y S Dong J L Wang D Y Feng et al ldquoProtective effect ofquercetin against oxidative stress and brain edema in an exper-imental rat model of subarachnoid hemorrhagerdquo InternationalJournal of Medical Sciences vol 11 no 3 pp 228ndash290 2014

[32] R C Park J Blom P J Disaia L D Lagasse and J ABlessing ldquoTreatment of women with disseminated or recurrentadvanced ovarian cancer with melphalan alone in combinationwith 5-fluorouracil and dactinomycin or with the combinationof cytoxan 5-fluorouracil and dactinomycinrdquo Cancer vol 45no 10 pp 2529ndash2542 1980

[33] P Krishnappa K Venkatarangaiah V S Kumar S RajannaR Kashi and P Gupta ldquoAntioxidant and prophylactic effectsof Delonix elata L stem bark extracts and flavonoid isolatedquercetin against carbon tetrachloride-induced hepatotoxicityin ratsrdquo BioMed Research International vol 2014 Article ID507851 14 pages 2014

[34] A G Gornall C J Bardwawill and M M David ldquoDetermi-nation of serum proteins by means of the biuret reactionrdquo TheJournal of Biological Chemistry vol 177 no 2 pp 751ndash766 1949

[35] E R Jaffe ldquoOxidative hemolysis or ldquowhat made the red cellbreakrdquordquo The New England Journal of Medicine vol 286 no 3pp 156ndash157 1972

[36] N W Tietz Clinical Guide to Laboratory Tests WB SaundersPhiladelphia Pa USA 3rd edition

[37] N W Tietz E L Pruden and O Siggaard-Andersen ldquoLiverfunctionrdquo in Tietz Textbook of Clinical Chemistry A C Burtisand E R Ashwood Eds pp 1354ndash1374WB Saunders LondonUK 1994

[38] S Reltman and S A Frankel ldquoColorimetric method for thedetermination of serum ALT and ASTrdquo American Journal ofClinical Pathology vol 28 pp 56ndash63 1957

[39] G Szasz ldquoA kinetic photometric method for serum 120574-glutamyltranspeptidaserdquo Clinical Chemistry vol 15 no 2 pp 124ndash1361969

[40] D J Jollow J R Mitchell N Zampaglione and J R GilletteldquoBromobenzene induced liver necrosis Protective role of glu-tathione and evidence for 34 bromobenzene oxide as thehepatotoxic metaboliterdquo Pharmacology vol 11 no 3 pp 151ndash169 1974

[41] S K Jagota and H M Dani ldquoA new colorimetric techniquefor the estimation of vitamin C using folin phenol reagentrdquoAnalytical Biochemistry vol 127 no 1 pp 178ndash182 1982

[42] W H Habig M J Pabst and W B Jakoby ldquoGlutathione Stransferases The first enzymatic step in mercapturic acid for-mationrdquo Journal of Biological Chemistry vol 249 no 22 pp7130ndash7139 1974

[43] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972

[44] A K Sinha ldquoColorimetric assay of catalaserdquo Analytical Bio-chemistry vol 47 no 2 pp 389ndash394 1972

[45] R Varshney and R K Kale ldquoEffects of calmodulin antagonistson radiation-induced lipid peroxidation in microsomesrdquo Inter-national Journal of Radiation Biology vol 58 no 5 pp 733ndash7431990

[46] W K Han and J V Bonventre ldquoBiologic markers for the earlydetection of acute kidney injuryrdquo Current Opinion in CriticalCare vol 10 no 6 pp 476ndash482 2004

[47] G S George M E Wakasi and E Egoro ldquoCreatinine and urealevels as critical markers in end-stage renal failurerdquo Research

and Review Journal of Medical and Health Sciences vol 3 no 1pp 41ndash44 2014

[48] M U Rehman M Tahir F Ali et al ldquoCyclophosphamide-induced nephrotoxicity genotoxicity and damage in kidneygenomic DNA of Swiss albino mice the protective effect ofEllagic acidrdquoMolecular and Cellular Biochemistry vol 365 no1-2 pp 119ndash127 2012

[49] MU RehmanMTahir F Ali et al ldquoA case report of acute renalfailure induced by melphalan in a patient with ovarian cancerrdquoGan To Kagaku Ryoho vol 34 pp 201ndash205 1986

[50] T Aktoz M Kanter Y H Uz C Aktas M Erboga and IH Atakan ldquoProtective effect of quercetin against renal toxicityinduced by cadmium in ratsrdquo Balkan Medical Journal vol 29no 1 pp 56ndash61 2012

[51] M I Yousef S A M Omar M I El-Guendi and L AAbdelmegid ldquoPotential protective effects of quercetin and cur-cumin on paracetamol-induced histological changes oxidativestress impaired liver and kidney functions and haematotoxicityin ratrdquo Food and Chemical Toxicology vol 48 no 11 pp 3246ndash3261 2010

[52] L Boone D Meyer P Cusick et al ldquoSelection and inter-pretation of clinical pathology indicators of hepatic injury inpreclinical studiesrdquo Veterinary Clinical Pathology vol 34 no 3pp 182ndash188 2005

[53] A Singh T K Bhat and O M Sharma ldquoClinical biochemistryof hepatotoxicityrdquo Journal of Clinical Toxicology supplement 4pp 1ndash19 2011

[54] D E Amacher ldquoA toxicologistrsquos guide to biomarkers of hepaticresponserdquo Human and Experimental Toxicology vol 21 no 5pp 253ndash262 2002

[55] J Ozer M Ratner M Shaw W Bailey and S Schomaker ldquoThecurrent state of serumbiomarkers of hepatotoxicityrdquoToxicologyvol 245 no 3 pp 194ndash205 2008

[56] S K Ramaiah ldquoA toxicologist guide to the diagnostic interpre-tation of hepatic biochemical parametersrdquo Food and ChemicalToxicology vol 45 no 9 pp 1551ndash1557 2007

[57] C De David G Rodrigues S Bona et al ldquoRole of quercetin inpreventing thioacetamide-induced liver injury in ratsrdquo Toxico-logic Pathology vol 39 no 6 pp 949ndash957 2011

[58] X Y Guo G F Sun and Y C Sun ldquoOxidative stress fromfluoride-induced hepatotoxicity in ratsrdquo Fluoride vol 36 no 1pp 25ndash29 2003

[59] T Yamamoto R Kikkawa H Yamada and I Horii ldquoIdentifica-tion of oxidative stress-related proteins for predictive screeningof hepatotoxicity using a proteomic approachrdquo The Journal ofToxicological Sciences vol 30 no 3 pp 213ndash227 2005

[60] JMOh Y S Jung B S Jeon et al ldquoEvaluation of hepatotoxicityand oxidative stress in rats treated with tert-butyl hydroperox-iderdquo Food and Chemical Toxicology vol 50 no 5 pp 1215ndash12212012

[61] G Notas I E Koutroubakis and E A Kouroumalis ldquoOxi-dants and antioxidants in liver diseaserdquo in Antioxidants NewResearch H V Panglossi Ed pp 2ndash48 Nova Science NewYork NY USA 2012

[62] W Droge ldquoFree radicals in the physiological control of cellfunctionrdquo Physiological Reviews vol 82 no 1 pp 47ndash95 2002

[63] D Trachootham W Lu M A Ogasawara N R Valle and PHuang ldquoRedox regulation of cell survivalrdquo Antioxidants andRedox Signaling vol 10 no 8 pp 1343ndash1374 2008

[64] R Morgenstern J Zhang and K Johansson ldquoMicrosomalglutathione transferase 1 mechanism and functional rolesrdquoDrug Metabolism Reviews vol 43 no 2 pp 300ndash306 2011


[65] P J Sherratt and J D Hayes ldquoGlutathione S-transferasesrdquo inEnzyme Systems That Metabolise Drugs and Other XenobioticsC Ioannides Ed pp 219ndash252 John Wiley amp Sons ChichesterUK 2002

[66] J Lee N Koo and D B Min ldquoReactive oxygen species agingand antioxidative nutraceuticalsrdquo Comprehensive Reviews inFood Science and Food Safety vol 3 no 1 pp 2ndash21 2004

[67] S Kojo ldquoVitamin C basic metabolism and its function as anindex of oxidative stressrdquo Current Medicinal Chemistry vol 11no 8 pp 1041ndash1064 2004

[68] R Masella R di Benedetto R Varı C Filesi and C Giovan-nini ldquoNovel mechanisms of natural antioxidant compounds inbiological systems Involvement of glutathione and glutathione-related enzymesrdquo Journal of Nutritional Biochemistry vol 16 no10 pp 577ndash586 2005

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


HO

O

N

Cl

ClNH2

(a)

OHO

OH

OH

OH

OH

O

(b)

Figure 1 Structure of (a) melphalan and (b) quercetin

antioxidants like reduced glutathione (GSH) ascorbic acid(AA) and vitamin E among others Enzymic antioxidantsinvolved in this protection include glutathione-S-transferase(GST) glutathione peroxidase (GPx) glutathione reductase(GR) superoxide dismutase (SOD) and catalase (CAT) [1718]

Quercetin (3573101584041015840-pentahydroxyflavone) Figure 1(b)is one of themost widely distributed flavonoids (of the flavon-ols subclass) in plants [19] It is abundant in plants plantproducts and foods such as red wine onions green teaapples broccoli berries ginkgo biloba and buckwheat tea[20] QCT has been exhibited in several studies as a potentantioxidant with a very strong free radical scavenging capac-ity [21 22] It also possesses a number of pharmacologi-cal activities including antidiabetic anti-inflammatory andimmunostimulatory and protection of low density lipopro-tein against oxidation [22ndash24] Previous reports suggest thatQCT also possesses the capacity to effectively inhibit theproliferation of cancer cells [25ndash27] Moreover it is knownto improve chemotherapeutic efficacy of certain alkylatingagents [28] In addition recent studies demonstrated thatquercetin could effectively attenuate drug-induced toxicityand oxidative stress in vivo [29ndash31]

One of the major challenges often encountered with theuse of anticancer agents is the incident of drug-inducedtoxicity Nevertheless it is alleged that the administration ofantioxidants alongwith anticancer agentsmayhelp relieve thetoxic side effects elicited by these agents Consequently thepresent study was designed to investigate the protective effectof quercetin pretreatment and cotreatment on melphalan-induced renal and hepatic toxicity in rat models

2 Materials and Methods

21 Chemicals and Reagents Melphalan tablets were a prod-uct of Excella GmbH Nurnberger Germany Quercetin glu-tathione 1-chloro-24-dinitrobenzene (CDNB) 55-dithio-bis-2-nitrobenzoic acid (DTNB) epinephrine and hydrogenperoxide (H

2O2) were all purchased from Sigma Chemical

Company (London UK) Kits for alanine transferase (ALT)aspartate aminotransferase (AST) alkaline phosphatase(ALP) gamma glutamyl transpeptidase (GGT) urea creati-nine and total bilirubinwere products of Randox laboratoriesLtd (Antrim UK) All other reagents used were of analyticalgrade and of the highest purity

Table 1 Experimental design

Treatmentdarrgroups Treatments

Duration rarr Days 1ndash7 Days 8ndash14I (CTRL) mdash Control distilled waterII (MPLN) mdash 02mgKg bw MPLN

III (MPLN + QCT-P) 20mgKg bwQCT 02mgKg bw MPLN

IV (MPLN + QCT-C) mdash 02mgKg bw MPLN +20mgKg bw QCT

V (QCT-A) mdash 20mgKg bw QCTCTRL control MPLN melphalan QCT quercetin QCT-P quercetin-pretreated QCT-C quercetin-cotreated QCT-A quercetin-alone bw bodyweight

22 Animal Selection and Care Twenty-five maleWistar ratsweighing between 160 and 170 g were obtained from TheAnimal Holding Unit Department of Chemical SciencesAjayi Crowther University Oyo Nigeria The rats were accli-matized under laboratory conditions prior to experimentThe animals were housed in wire-meshed cages and providedwith food and water ad libitumThe animals weremaintainedunder standard conditions of temperature and humidity with12-hour lightdark cycles They were fed with commercial ratdiet (Ladokun Feeds Nigeria Ltd) Handling of the experi-mental animalswas in conformitywith theNational Institutesof Healthrsquos Guide for the Care andUse of Laboratory Animals(NIHpublicationnumber 85-23 revised 1985USDepartmentof Health Education and Welfare Bethesda MA)

23 Animal Grouping and Drug Treatments The animalswere randomly assigned into five experimental groups (IndashV)of five animals each The animals of each group were treatedas presented in Table 1 The doses for MPLN and QCT weredecided based on the available literature [32 33] and weredelivered in one mL solution of distilled water once daily byoral intubation

24 Animal Sacrifice and Collection of Blood and Liver Sam-ples Blood sampleswere collected from each animal throughretro orbitals plexus into heparinized tubes (Li heparin)Animals were sacrificed and the liver was carefully excisedfrom each animal for preparation of cytosolic fraction























3 Results























0

2

4

6

8

10

12

14


MPLN +QCT-C

QCT-A

AA

(120583g

mL)

lowast

dagger

lowast

lowastdagger


0123456789


MPLN +QCT-C

QCT-A

lowast


lowastdagger

GSH

(120583g

g liv

er)





02468

1012141618


MPLN +QCT-C

QCT-A

GST

(nm

olm

inm

g pro

tein

)

lowast


lowastdagger


0

100

200

300

400

500

600


MPLN +QCT-C

QCT-A

MD

A (n

mol

mg p

rote

in) lowast


lowastdagger


4 Discussion








5 Conclusion




References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































3 Results























0

2

4

6

8

10

12

14


MPLN +QCT-C

QCT-A

AA

(120583g

mL)

lowast

dagger

lowast

lowastdagger


0123456789


MPLN +QCT-C

QCT-A

lowast


lowastdagger

GSH

(120583g

g liv

er)





02468

1012141618


MPLN +QCT-C

QCT-A

GST

(nm

olm

inm

g pro

tein

)

lowast


lowastdagger


0

100

200

300

400

500

600


MPLN +QCT-C

QCT-A

MD

A (n

mol

mg p

rote

in) lowast


lowastdagger


4 Discussion








5 Conclusion




References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































0

2

4

6

8

10

12

14


MPLN +QCT-C

QCT-A

AA

(120583g

mL)

lowast

dagger

lowast

lowastdagger


0123456789


MPLN +QCT-C

QCT-A

lowast


lowastdagger

GSH

(120583g

g liv

er)





02468

1012141618


MPLN +QCT-C

QCT-A

GST

(nm

olm

inm

g pro

tein

)

lowast


lowastdagger


0

100

200

300

400

500

600


MPLN +QCT-C

QCT-A

MD

A (n

mol

mg p

rote

in) lowast


lowastdagger


4 Discussion








5 Conclusion




References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















02468

1012141618


MPLN +QCT-C

QCT-A

GST

(nm

olm

inm

g pro

tein

)

lowast


lowastdagger


0

100

200

300

400

500

600


MPLN +QCT-C

QCT-A

MD

A (n

mol

mg p

rote

in) lowast


lowastdagger


4 Discussion








5 Conclusion




References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion




References













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Protective Effect of Quercetin on Melphalan-Induced Oxidative Stress and Impaired Renal and Hepatic...

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Transcript of Protective Effect of Quercetin on Melphalan-Induced Oxidative Stress and Impaired Renal and Hepatic...