Punicalagin and Ellagic Acid Demonstrate Antimutagenic ...

Punicalagin (PC) is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica ... BioMedResearchInternational+JournalMenuPDFJournaloverviewForauthorsForreviewersForeditorsTableofContentsSpecialIssuesSubmitBioMedResearchInternational/2014/ArticleArticleSectionsOnthispageAbstractIntroductionMaterialsandMethodsResultsDiscussionAcknowledgmentsSupplementaryMaterialsReferencesCopyrightRelatedarticlesResearchArticle|OpenAccessVolume2014|ArticleID467465|https://doi.org/10.1155/2014/467465MaryamZahin,IqbalAhmad,RameshC.Gupta,FarrukhAqil,"PunicalaginandEllagicAcidDemonstrateAntimutagenicActivityandInhibitionofBenzo[a]pyreneInducedDNAAdducts",BioMedResearchInternational,vol.2014,ArticleID467465,10pages,2014.https://doi.org/10.1155/2014/467465ShowcitationPunicalaginandEllagicAcidDemonstrateAntimutagenicActivityandInhibitionofBenzo[a]pyreneInducedDNAAdductsMaryamZahin,1,2IqbalAhmad,1RameshC.Gupta,2,3andFarrukhAqil2,41DepartmentofAgriculturalMicrobiology,AligarhMuslimUniversity,Aligarh202002,India2JamesGrahamBrownCancerCenter,UniversityofLouisville,Louisville,KY40202,USA3DepartmentofPharmacologyandToxicology,UniversityofLouisville,Louisville,KY40202,USA4DepartmentofMedicine,UniversityofLouisville,DeliaBaxterII,Room304B,580S.PrestonStreet,Louisville,KY40202,USAShowmoreAcademicEditor:DavorZeljezicReceived01Mar2014Accepted22Apr2014Published14May2014Punicalagin(PC)isanellagitanninfoundinthefruitpeelofPunicagranatum.WehavedemonstratedantioxidantandantigenotoxicpropertiesofPunicagranatumandshowedthatPCandellagicacid(EA)areitsmajorconstituents.Inthisstudy,wedemonstratetheantimutagenicpotential,inhibitionofBP-inducedDNAdamage,andantiproliferativeactivityofPCandEA.IncubationofBPwithratlivermicrosomes,appropriatecofactors,andDNAinthepresenceofvehicleorPCandEAshowedsignificantinhibitionoftheresultantDNAadducts,withessentiallycompleteinhibition(97%)at40 μMbyPCand77%inhibitionbyEA.AntimutagenicitywastestedbyAmestest.PCandEAdose-dependentlyandmarkedlyantagonizedtheeffectoftestedmutagens,sodiumazide,methylmethanesulfonate,benzo[a]pyrene,and2-aminoflourine,withmaximuminhibitionofmutagenicityupto90percent.Almostallthedosestested(50–500 μM)exhibitedsignificantantimutagenicity.AprofoundantiproliferativeeffectonhumanlungcancercellswasalsoshownwithPCandEA.Together,ourdatashowthatPCandEAarepomegranatebioactivesresponsibleforinhibitionofBP-inducedDNAadductsandstrongantimutagenic,antiproliferativeactivities.However,thesecompoundsaretobeevaluatedinsuitableanimalmodeltoassesstheirtherapeuticefficacyagainstcancer.1.IntroductionOverthepastfewdecades,tremendousoutcomeshavebeenresultedbyexploringantioxidantandantimutagenicpotentialofmedicinalplants.ItiswidelyacceptedthatoxidativemodificationofDNA,protein,lipids,andsmallcellularmoleculesbybothexogenousandendogenousreactiveoxygenspeciesincludingfreeradicalsandnonfreeradicalsplaysanimportantroleinawiderangeofcommondiseasesincludingcancerandagerelateddegenerativediseases[1,2].Thehumanbodypossessesinnatedefencemechanismstocounterfreeradicals.Plantsecondarymetabolitessuchasphenolics,flavonoids,andterpenoidsplayanimportantroleinthedefenceagainstfreeradicals[3].Moreover,thesenaturalantioxidantsmayreduceorinhibitthemutagenicpotentialofmutagens,promutagens,andcarcinogens[4,5].Therefore,thediscoveryandtheexplorationofcompoundspossessingantioxidant,antimutagenic,andanticancerpropertiesarenowfetchinggreatpracticalandtherapeuticsignificance.TheformationofDNAadducts(i.e.,carcinogenscovalentlyboundtoDNA)iswidelyconsideredaprerequisitefortheinitiationandprogressionofcancerdevelopment.ManycarcinogensareknowntoinducetheformationofDNAadducts[6]andthepresenceofDNAadductsinhumanshasbeenstronglycorrelatedwithanincreasedriskofcancerdevelopment[7].Forexample,humanstudieshaveshownahigheraccumulationoftissueDNAadductsincigarettesmokersthaninnonsmokersorindividualswhohaveneversmoked,indicatingthatDNAadductformationisaviabletargetforthetreatmentofcancer[8]. Benzo[a]pyrene(BP)isoneofthemostpotentandextensivelystudiedcarcinogens.Inacellularsystem,BPismetabolizedtotheelectrophilicmetabolite,benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE),thatattachescovalentlytoDNAbases,primarilydeoxyguanosine.InflammatoryresponsetochemicalcarcinogensandformationofDNAadductsaregenerallyconsideredaprerequisiteintheprocessofchemicalcarcinogenesis[9].AccumulationofDNAadductsresultingfromchronicexposuretolow-levelenvironmentalcarcinogenshasbeenusedasapossiblemeasureofexposuretocarcinogensandcancerriskassessment[10].NaturalantimutagensfromedibleandmedicinalplantsareofparticularimportancebecausetheymaybeusefulforinhibitionofDNAadductsleadingtohumancancerpreventionandhavenoundesirablexenobioticeffectsonlivingorganisms[11,12].Encouragingreportsonantimutagenicpropertiesofedibleplantshaveledtoincreaseinterestinsearchofnaturalphytoantimutagensfrommedicinalplants[13,14].AmongthemisPunicagranatum(pomegranate),whichhavebeenusedwidelyasantimicrobial,antioxidant,antimutagenic,andanticancer[15–17].Pomegranatehasbeenshowntopossesshighamountofellagitannins(ETs)suchaspunicalagin(PC),punicalin,gallagicacid,ellagicacid(EA),andEA-glycosides[18,19].Punicalaginandellagicacid(Figure1)emergedouttobethemostelaboratedgroupsofcompounds,knownfortheirpotentialroleinvariousbiologicalactivities.Likeotherpolyphenols,PC,EA,andtheirderivedmetabolitespossessawiderangeofbiologicalactivities,whichsuggestedthattheycouldhavebeneficialeffectsonhumanhealth.PCandEAhaveantioxidantfunctionsandpossessstronganti-inflammatory,antiproliferative,hepatoprotective,andantigenotoxicproperties[20–23].(a)(b)(a)(b)Figure1 Chemicalstructuresofpunicalaginandellagicacid.PCandEAalsoexhibitanticancerpropertiesinvitroandinvivo[17,24].However,studiesonantimutagenicpotentialonthesecompoundsarescanty.Therefore,consideringourresultsandpreviousfindingsbyotherworkers,weextendedourstudytoisolatethekeycompounds,PCfromP.granatumpeelextracts.InthisstudywedemonstrateantimutagenicpropertiesofPCandEAagainstthemutagenicityinducedbymutagens(sodiumazideandmethylmethanesulfonate)andpromutagens(BPand2AF)inAmesSalmonellaassay.Thisstudy,tothebestofourknowledge,isthefirsttoshowantimutagenicpropertiesagainstapanelofmutagens/carcinogensandprocarcinogens.WealsoexaminedprotectiveeffectofPCandEAagainstBP-inducedDNAadductsandantiproliferativeactivityagainstlungcancercellsinvitro.2.MaterialsandMethods2.1.BacterialStrainsandChemicalsTheSalmonellatyphimuriumstrainsTA97a,TA98,TA100,andTA102werekindlyprovidedbyProf.B.N.Ames,UniversityofCalifornia,Berkeley,USA.ThedetailsofthestrainsareprovidedintheSupplementaryTableS1(seeSupplementaryMaterialavailableonlineathttp://dx.doi.org/10.1155/2014/467465).Sodiumazide(NaN3)waspurchasedfromHiMediaLab.(Mumbai,India).D-glucose-6-phosphatedisodiumsalt,nicotinamideadeninedinucleotidephosphatesodiumsalt,sodiumphosphate,ammoniummolybdate,neocuproine,L-histidinemonohydrate,D-biotin,2-aminofluorene(2AF),benzo[a]pyrene,andellagicacidwerepurchasedfromSigma-Aldrich(St.Louis,MO,USA).Methylmethanesulfonate(MMS)andtrichloroaceticacidwerepurchasedfromSiscoResearchLaboratoriesPvt.Ltd.,Mumbai,India.Allotherreagentsusedtopreparebuffersandmediawereofanalyticalgrade.2.2.PreparationoftheExtractandIsolationofPunicalaginPunicagranatum(pomegranate)fruitspeelextracts(30%enrichedforpunicalagins)werepurchasedfromPharmanzaInc.(Gujarat,India).Theextractswerepreparedbydissolving10 gofpeelpowderin5 vol(50 mL)ofwater.Sampleswerethencentrifugedat6000 gfor10 minanddecantedandpooledextractsfromthreeextractionsweredriedunderreducedpressureusingRota-vaporat45°C.PCwasisolatedbyAmberliteXAD-16andC18columnchromatographyasdescribed[19].IsolatedPCwasatleast97%pureandessentiallyfreeofEAasdeterminedbyHPLC-UV.2.3.AntimutagenicityAssayTheSalmonellahistidinepointmutationassaydescribedbyMaronandAmes[25]wasusedtotesttheantimutagenicactivityofPCandEAasdescribedearlier[13,26].Inthepreincubationexperiment,testcompoundsandmutagen,eachhavingavolumeof0.1 mLofvaryingconcentrations,werepreincubatedat37°Cfor30 minandthen0.1 mLof1×107 CFU/mLdensityofthebacterialculturewasadded,followedbytheadditionof2.5 mLoftopagarat45°C(containing0.5%NaCland0.6%agar)supplementedwith0.5 mMhistidine-biotin.Theinfluenceofmetabolicactivationofpromutagens,BPand2AFwastestedusing500 μLofS9mixture(0.04 mgproteins/mLofmix).TheS9microsomefractionwaspreparedfromtheliversofratstreatedwithAroclor1254usingstandardprotocols[27].Thecombinedsolutionswerevortexedandpouredontominimalglucoseplates(40%glucosesolutionandVogelBonnermedium).Theplateswereincubatedat37°Cfor48 handthenumbersofhistidine-independentrevertantscolonieswerescored.Survivalofthebacteriawasroutinelymonitoredforeachexperiment.Parallelcontrolswererunwithcompoundsaloneatallconcentrationstotestthepossibletoxicity.Theconcentrationsofthetestsamplesforinvestigatingtheantimutagenicitywere50,100,200,and500 μM.PCandEAweretestedagainstmutagenssodiumazide(1.5 μg/0.1 mL/plate)andMMS(1 μg/0.1 mL/plate)aswellasagainstpromutagens,BPand2AFinTA97aandTA98(frameshiftmutation),TA100(basepairsubstitution),andTA102(transitionmutation)testerstrains(SupplementaryTableS1).AllthetestsamplesandmutagensweredissolvedinDMSO(finalconc.,0.01%).Ineachcase,therewasnotoxicityobservedandthenumbersofspontaneousrevertantswereidenticalwiththeDMSOvehiclecontrol.Non-toxicconcentrationswerecategorizedasthosewheretherewasawell-developedlawn,almostsimilarsizeofcolonies,andnostatisticaldifferenceinthenumberofspontaneousrevertantsintestandcontrolplates.Platesweresetupintriplicateforeachconcentrationandtheentireexperimentwasrepeatedthreetimes.Inhibitionofmutagenicitywasexpressedaspercentagedecreaseofreversemutationandcalculatedas where=numberofhistidinerevertantsinducedbymutagen,=numberofhistidinerevertantsinducedbymutageninthepresenceoftestcompound,and=numberofrevertantsinducedinnegativecontrol.2.4.MicrosomalBP-DNAAdductsst-DNA(300 μg/mL)waspreincubatedwith50 mMTris-HCl(pH7.5),1 mMMgCl2,2.5 mMglucose-6-phosphate,1 U/mLG6PDH,0.5 mMNADP+,andα-naphthoflavone-inducedratlivermicrosomalproteins(1 mg/mL)in1 mLfor10 min,inthepresenceofvehiclealoneandPCandEAat20and40 μM.BPdissolvedinDMSOwasaddedatafinalconcentrationof1 μM.Theincubationwascontinuedforanother30 minat37°CandthenreactionwasterminatedbytheadditionofEDTAandcentrifugation(9,000 g;10 min).DNAwasisolatedfromthesupernatantbyremovalofRNAandproteinsbydigestionswithRNasesAandT1andproteinaseKandaseriesofextractionswithphenol,phenol:Sevag(chloroform : isoamylalcohol,24 : 1),andSevag,followedbyprecipitationoftheDNAwithethanol[29].TheDNAconcentrationwasestimatedspectrophotometrically.2.5.AnalysisofDNAAdductsDNAadductswereanalyzedby32P-postlabelingasdescribedearlier[29].Briefly,10 μgofDNAwasdigestedwithmicrococcalnucleaseandspleenphosphodiesterase(MN/SPD).BeforefurthertreatmentwithnucleaseP1toenrichDNAadducts,analiquotwasremovedforevaluationofnormalnucleotidelevels.DNAadductsandnormalnucleotideswerelabelledwith[γ-32P]ATPandT4polynucleotidekinase.Labelledadductswereseparatedbymultidirectionalpolyethyleneimine(PEI)-celluloseTLCusingthefollowingsolvents:D1,1.0 Msodiumphosphate,pH6.0;D3,4 Mlithiumformate/7 Murea,pH3.5;D4,4 Mammoniumhydroxide/isopropanol(1 : 0.9);andD5,1.7 Msodiumphosphate,pH6.0.Normalnucleotideswereresolvedin180 mMsodiumphosphate,pH6.0,byone-directionalPEI-celluloseTLC.DNAadductsandnormalnucleotidesweredetectedandquantifiedbyPackardInstantImager.2.6.CellProliferationAssayandMeasurementsofCellViabilityInhibitionofcellproliferationbyPCandEAwasmeasuredwiththeMTTassay.HumanlungcancerA549andH1299cellswereobtainedfromATCC(Manassas,VA,USA)andmaintainedinDMEMsupplementedwith10%fetalcalfserum(FCS),1%penicillin/streptomycin.Cellswereplatedin96-wellcultureplates(5×103cells/well).After24 hincubation,cellsweretreatedwithvehiclealone(0.1%DMSO)andPCandEA(12.5–200 μg/mL)extractsfor48 h.Then,theculturemediumwasreplacedby100 μLoffreshmediumcontaining0.5 mg/mLMTT,andtheplateswereincubatedfor2 hat37°C.Themediumwasthenremovedandwasreplacedby200 μLofDMSOtosolubilizetheconvertedpurpledye.Theabsorbancewasmeasuredwithaspectrophotometermicroplatereaderatawavelengthof570 nm.2.7.StatisticalAnalysisTheresultsarepresentedastheaverageandstandarderrorofthreeexperimentswithtriplicateplates/dose/experiment.TheregressionanalysiswascarriedoutinMicrosoftExcel2007betweenpercentinhibitionofmutagenicityandlogvaluesofconcentrationsoftheplantextract.3.ResultsNaturalproductshaveattractedmuchattentionwithrespecttotheirbenefitstohumanhealthandprotectiveeffectsinvariousdiseasesincludingcancer[30].Wehavepreviouslydemonstratedtheantimicrobial[16],antioxidant,andantimutagenicpotentialandphytochemicalanalysisofPunicagranatum[15,19].InthisstudywedemonstratetheinhibitionofBP-inducedDNAadductsandantimutagenicandantiproliferativeactivitiesofPCandEA,thekeycomponentsofpomegranate.3.1.EvaluationofMutagenicityofTestedCompoundsThemutagenicityandantimutagenicityofacompoundcanbedetectedusingAmestestusingspecificindicatorstrainsofSalmonellatyphimurium[25].NotoxicityofPCandEAwasfoundattested50–500 μMconcentrationsasdepictedinTables1–8whentestedintheabsenceofS9fractioninAmesSalmonellatyphimuriumstrains.However,atfewconcentrationstherewasslightbutinsignificantincreaseintheHis+revertantscomparedtospontaneous.Nomutagenicactivityofeitherofthecompounds,PCorEA,wasdetectedwheninvestigatedonanyofSalmonellatesterstrains,TA97a,TA98,TA100,andTA102eitherwithorwithoutS9activationbyplateincorporationassay(Tables1–8).TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(2AF)1.5 μg aPunicalagin50 100 250 500 bPunicalagin+2AF50 (15.7) (20.5) (22.4) (38.7)100 (37.1) (37.5) (43.8) (54.8)250 (52.6) (59.2) (61.5) (72.8)500 (78.5) (81.4) (83.1) (81.6)  0.980.990.990.99 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;2AF:2-aminofluorene; :linearregressionanalysis.Table1 Effectofpunicalaginonthe2-aminofluoreneinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(BP)1.5 μg Punicalagin50 320.0±21.9100 250 500 Punicalagin+BP50 (15.1) (22.1) (30.6) (15.8)100 (34.3) (46.6) (48.3) (28.0)250 (53.5) (74.8) (64.0) (58.6)500 (76.7) (78.8) (84.5) (76.2)  0.990.940.990.99 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;BP:benzo[a]pyrene; :linearregressionanalysis.Table2 Effectofpunicalaginonthebenzo[a]pyreneinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(NaN3)1.5 μg Punicalagin50 100 250 500 Punicalagin+NaN350 (10.2) (13.3) (11.3) (18.0)100 (34.2) (30.0) (33.1) (17.6)250 (54.9) (52.2) (54.6) (42.2)500 (74.4) (65.3) (74.3) (59.8)  0.990.990.990.91 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;NaN3:sodiumazide; :linearregressionanalysis.Table3 EffectofpunicalaginsonthesodiumazideinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(MMS)1.0 μg Punicalagin50 100 250 500 Punicalagin+MMS50 (10.2) (29.4) (13.8) (17.5)100 (19.9) (46.3) (30.8) (30.8)250 (42.4) (59.2) (45.3) (50.8)500 (72.1) (70.9) (66.0) (74.1)  0.950.990.990.99 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;MMS:methylmethanesulfonate; :linearregressionanalysis.Table4 EffectofpunicalaginsonthemethylmethanesulfonateinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(2AF)1.5 μg aEllagicacid50 100 250 500 bEllagicacid+2AF50 (12.5) (19.4) (22.5) (17.5)100 (22.6) (39.6) (46.1) (45.9)250 (44.9) (56.3) (67.7) (74.1)500 (81.7) (80.0) (83.8) (89.0)  0.930.990.990.98 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;2AF:2-aminofluorene; :linearregressionanalysis.Table5 Effectofellagicacidonthe2-aminofluoreneinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(BP)1.5 μg aEllagicacid50 100 250 500 bEllagicacid+BP50 (14.1) (22.2) (21.8) (11.5)100 (36.0) (48.2) (42.8) (23.6)250 (71.8) (62.3) (70.0) (46.6)500 (83.5) (78.6) (88.9) (83.7)  0980.970.990.93 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;B[a]P:benzo[a]pyrene; :linearregressionanalysis.Table6 Effectofellagicacidonthebenzo[a]pyreneinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(NaN3)1.5 μg aEllagicacid50 100 250 500 bEllagicacid+NaN350 (21.1) (20.0) (11.4) (21.9)100 (39.4) (27.9) (25.6) (40.3)250 (53.6) (50.0) (46.4) (50.7)500 (72.1) (64.2) (65.9) (62.3)  0.990.980.990.97 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;NaN3:sodiumazide; :linearregressionanalysis.Table7 EffectofellagicacidonthesodiumazideinducedmutagenicityinSalmonellatyphimurium.TreatmentDose(μM)NumberofHis+revertantscolonies/plate(mean SE)TA97aTA98TA100TA102Spontaneous  Positivecontrol(MMS)1.5 μg aEllagicacid50 100 250 500 bEllagicacid+MMS50 (9.0) (22.2) (8.0) (18.5)100 (25.0) (34.0) (26.4) (30.3)250 (42.2) (50.0) (45.1) (44.3)500 (66.5) (69.0) (65.3) (72.4)  0.980.990.990.95 Negativecontrol;bpreincubationtest;valuesinparenthesisare%inhibitionofmutagenicity. ; and ;MMS:methylmethanesulfonate; :linearregressionanalysis.Table8 EffectofellagicacidonthemethylmethanesulfonateinducedmutagenicityinSalmonellatyphimurium.3.2.PCandEAAreHighlyAntimutagenicTheantimutagenicpotentialofPCandEAwasevaluatedusingAmesSalmonellatesterstrainsagainstdirectactingmutagens(NaN3andMMS),intheabsenceofS9,aswellasagainstpromutagens(BPand2AF)withAroclorinducedratliverS9.IntheabsenceoftestcompoundsthesemutagensinducedHis+revertants.WehavepreviouslydemonstratedthatthemethanolextractofPunicagranatumhasveryhighantimutagenicpotentialandcontainsPCandEAinadditiontoothertracecompounds[15].Toevaluatetheactiveprinciple,wefurthertested,inthisstudy,PCandEAforantimutagenicpotentialat50,100,250,and500 μMconcentrations.Thetestedconcentrationsbyplate-incorporationassayshowednosignoftoxicityandmutagenicitytoSalmonellatyphimuriumstrains,eitheraloneorinthepresenceofS9mix.BP-and2AF-inducedhighnumberofHis+revertantsintesterstrains.PCshowedasignificant()inhibitionofBP-and2AF-inducedmutagenicitytestedinthepresenceofS9mix.At500 μMconcentration,PCinhibited2AF-andBP-inducedmutagenicityintherangeof76.7%to85.0%(Tables1and2).TheeffectofPCwasdosedependentasdeterminedbyregressionanalysiswiththevaluesrangingbetween0.91and0.99.Similarly,PCatthehighesttestedconcentration(500 μM)showedsignificantantimutagenicityagainstNaN3andMMS.Itinhibitedsodiumazideinducedmutagenicityby74.4%inTA97afollowedbyTA100(74.3%),TA98(65.3%),andTA102(59.8%)strainsasdepictedinTable3.MMS(1 μg),whenincubatedwithTA97a,increasedtheHis+revertantsfrom142to449anditwasalmostcompletelyreducedto251(aninhibitionof72%)byPCat500 μM(50 μg/plate).TheeffectwassimilarinTA98,TA100,andTA102totheinhibitionofmutagenicityby71%,66%,and75%,respectively(Table4).TheantimutagenicityofEAagainstpromutagensBP-and2AF-inducedmutagenicitywasalsohighlysignificant()aspresentedinTables5and6.EAshoweddosedependentantimutagenicbehavioragainstbothBPand2AFandreducedHis+revertantsby78.6%to88.9%,respectively(Tables5and6).TheactivitywasdosedependentasdeterminedbyregressionanalysisbetweenEAdoseandantimutagenicresponsewithvaluesrangingbetween0.93and0.99.EAatadoseof500 μMshowedsignificant()antimutagenicactivityagainstTA97awithadecreaseinmutagenicityby72.1%followedbyTA100(65.9%),TA98(64.2%),andTA102(62.3%)againstNaN3inducedmutagenicity(Tables7and8).AsimilartrendofactivitywasobtainedagainstMMS-inducedmutagenicity.ThedecreaseinnumberofMMS-associatedHis+revertantswassignificant()inTA102(73.7%)followedbyTA98(69.0%),TA97a(66.5%),andTA100(65.3%),asdepictedinTables7and8.3.3.PCandEAInhibitMicrosomalBP-DNAAdductsBP(1 μM)resultedintheformationoftwomajoradductswhenincubatedwithratlivermicrosomesinthepresenceofst-DNA(Figure2).Theseadductsareproductsoftheinteractionof9-OH-benzo[a]pyrene-4,5-epoxideanddG(adduct1)andanti-BPDEanddG(adduct2)[31].NoadductsweredetectedinDNAincubatedwithvehiclealone(Figure2(a)).Incubationofst-DNAwithBP(1 μM),microsomes,andcofactorsinthepresenceof20and40 μMofPCandEAorvehicleproducedqualitativelythesameDNAadductprofilebuttheadductlevelsweredifferent.BothPCandEAinhibitedBP-DNAadductssignificantly.AscomparedwithBPalone(DNAadducts/108nucleotides;),PC(andDNAadducts/108nucleotides)andEA(andDNAadducts/108nucleotides)at20and40 μMconcentrations,respectively,resultedinsignificantinhibitionofBP-inducedDNAadducts(Figure2(b)).(a)(b)(a)(b)Figure2 (a)Representativeautoradiographsof32P-postlabellinganalysisofmicrosomal-benzo[a]pyrene(BP)DNAadductsinthepresenceofvehiclealone(2%DMSO),BP(1 μM)+vehicle,BP(1 μM)+punicalagin(PC)(40 μM),andBP(1 μM)+ellagicacid(EA)(40 μM).Adduct1,anti-benzo[a]pyrene-7,8-diol-9,10-epoxide-dG,andadduct2,9-OH-benzo[a]pyrene-4,5-epoxide-dG.PCandEAalonegroupwerenotincludedsincewedonotexpectanybackgroundBP-DNAadduct.OR,origin.(b)InhibitionofmicrosomalBP-inducedDNAadductsbyPCandEA.DNAadductswereanalyzedby32P-postlabelingassay.Datarepresentanaverage±standarderrorof4–6samples. ; .PartofthefigureisreprintedfromMutat.[28]Copyright(2014),withpermissionfromElsevier.3.4.AntiproliferativeActivityofPCandEATheantiproliferativeactivitiesofPCandEAweredeterminedbyMTTassayandpresentedinFigures3(a)and3(b).TestcompoundsshowedsignificantantiproliferativeactivityagainstbothlungcancerA549andH1299celllines.PCandEAshoweddosedependentactivityagainstbothofthecelllinesafter48 hr.However,theactivityofPCwassomewhatbetterthantheEA.PCdemonstratedsignificantlyhighactivityandinhibited57%and34%ofH1299andA549cellsat50 μg/mLconcentration,respectively.Similarly,atthesameconcentration(50 μg/mL),EAshowedthe34and39percentinhibitionagainstH1299andA549lungcancercelllines,respectively.(a)(b)(a)(b)Figure3 AntiproliferativeactivityofPCandEAagainstlungcancerH1299(a)andA549(b)cells.CellsweretreatedwitheithervehicleorPCandEAat12.5–200 μg/mLconcentrationsfor48 h.Dataareexpressedaspercentageofuntreatedcells,mean±SD( ).4.DiscussionPomegranate(PunicagranatumL.)fruitsarewidelyconsumedfreshandinprocessedformsasjuice,jam,andwineandhavebeenshowntohavevariousprotectiveeffects.WehaveshownthatthemethanolextractofPunicagranatumfruitpeelpossessesantimutagenic,antioxidant,DNAprotective,andantiproliferativeactivity[15,19].Thehuskofpomegranateisrichinellagitannins(ETs)suchasPC,punicalin,gallagicacid,EA,andEA-glycosides[18].Inthepresentstudy,PCandoneofitshydrolysedproductsEAweretestedfortheirprotectiveeffectsagainstBP-inducedDNAadductsandonthegenotoxicityinducedbyvariousmutagentsandpromutagensbyAmestest.Amesassayservesasaquickandconvenientassaytoestimatetheantimutagenicpotentialofacompoundwhichisalsoprescreeningofanticancercompoundsbecausestandardassaysonmiceandratsaretime-consumingandexpensive.Standardmutagens/carcinogensusedinthisstudywerewell-establishedmutagensinAmestest.Inourstudy,noneofthetestedcompoundsexhibitedanymutageniceffectintheAmestestintheabsenceofenzymaticmetabolism.ThissuggeststhatDNAdoesnotseemtobearelevanttargetforPCandEA,anditdidnotproduceDNAlesionsthatblockDNAsynthesis,leadingtotheinductionoftheSOSsystem[32].Avarietyofmechanismscanplayavitalroleinantimutagenicandanticarcinogenicactivityofphytocompounds.Thesemechanismsincludeinhibitionofcellproliferation,signaltransductionmodulation,scavengingoffreeradicals,inductionofdetoxificationenzymes,inductionofcell-cyclearrestandapoptosis,modulationofcytoskeletalproteinsthatplayakeyroleinmitosis,andtheinhibitionoftopoisomeraseIorIIactivity[33].IntheAmestest,PCandEAshowedantimutagenicity()againstmutagensandpromutagensinthepresenceofS9.SincethesebacterialstrainsareunabletometabolizeBPand2AF,toanappreciableextent,ametabolizingsystemofliverhomogenatefromAroclorinducedrats(S9)isincludedintheassaythathasbeenshowntoproduceanumberofreactiveintermediateslikeBP-4,5-oxideand9-hydroxy-BP-4,5-oxidefromBP[34].TheantimutageniceffectsofPCandEAcouldalsobeduetoinhibitoryeffectagainstthetestedmutagens.Similarly,theinhibitoryactivityinthepreincubationexperimentsagainst2AF-inducedmutagenicityimpliesthattheaddedmodulatorinterferedwiththemetabolicactivationofthepromutagenortendstointeractdirectlywiththeultimatemutagenicmetabolite.CytochromeP-450enzymesystemcatalysestheformationofN-hydroxyderivative,thatis,N-hydroxy-2-aminoflourenewhichprobablyinteractsdirectlywithDNA[35].Thus,thealterationinthestructureandfunctionofP-450enzymemayresultinalteredratesanddifferentialpathwaysofmetabolismofmutagensandcarcinogensandinsomecasesprovideprotectionagainstchemicallyinducedmutagenesis.Wehaverecentlydemonstratedthat,besideseffectonCYP1A1andinductionofglutathione,PCcaninhibitBPbydirectinhibition.Moreover,PCuponhydrolysisreleasesitsactivemetaboliteEA,whichhasbeenshowntoprotectDNAbycovalentlybinding[28].DNAadductformationrepresentsaneteffectofactivationanddetoxificationprocessesandcanbeusedtodetermineefficacyofchemopreventiveagents.Inthisstudy,efficacyofPCandEAwasdeterminedbyitsabilityinreducingBP-inducedDNAadductsinvitro.PCandEAwerefoundtoinhibitbothanti-BP-7,8-diol-9,10-epoxide-dGand9-OH-benzo[a]pyrene-4,5-epoxide-dG.However,inhibitionofanti-BPDEadductbyPCwasmorepronounced.TwomajorpathwayscanbeinvolvedintheinhibitionofBP-inducedDNAadducts(i)byinhibitingtheP450activityand/orenhancementofphaseIIenzymesand(ii)bydirectconjugationwithanti-BPDE.IthasbeenpreviouslydemonstratedthatPCdoesinhibitanti-BPDE-inducedDNAadductsandthusruledoutthescavengingofanti-BPDE.However,EAhasbeenshowntocovalentlyinteractwithanti-BPDEthroughitscatecholgroupsasdeterminedbyHPLC[36].Therefore,itislikelythatPCisindirectlyinvolvedinscavenginganti-BPDEthroughitscatecholcontainingmoieties,ellagicacid,andgallicacid.Apparently,thecatecholmoietiesoftheseconstituentswereprotectedintheconjugatedPCcomplex.ThisstudyshowedthehighantiproliferativeactivityofPCandEA.BothofthelungcancercelllinesA549andH1299showedalmostsimilarlevelofsensitivitytothetestedcompounds.TheantiproliferativeactivityofPCandEAagainstoral,colon,andprostatecancercelllineshasbeenpreviouslydemonstrated[17].Inanotherstudy,bothPCandEAwerefoundtoinduceapoptosisviamitochondrialpathwayincoloncancerCaco-2cellsbutnotinnormalcoloncells.EAarrestthecellcycleinSphasethroughdown-regulationofcyclinsAandB1andup-regulationofcyclinE.ItalsoinducesapoptosisviaintrinsicpathwaythroughBcl-XLdownregulation,mitochondrialreleaseofcytochromeC,andactivationofinitiatorcaspase9andeffectorcaspase3[37].Thus,ourdatacorroborateswiththeliterature[17]andclearlydemonstratestheantiproliferativepotentialofPCandEA.Insummary,thedataobtainedinthepresentstudyclearlydemonstratedthatPCandEAarethemajoractiveconstituentsofpomegranatewithpromisingantimutagenicandprotectiveagainstDNAdamage.Further,thesestudiesdemonstrate,forthefirsttime,thatbothPCandEApossessalmostsimilarlevelofantimutagenicpropertiesagainstavarietyofmutagensandcouldbeaviablecandidateforthefutureanticancerdrugs.ConflictofInterestsTheauthorsdeclarethatthereisnoconflictofinterestsregardingthepublicationofthispaper.AcknowledgmentsTheauthorsarethankfultotheUGC,NewDelhi,forfinancialassistanceintheformofresearchfellowshiptoMaryamZahinthroughAMU,Aligarh,India.ThisworkwaspartiallysupportedfromAgnesBrownDugganEndowmentfund.Dr.RameshGuptaholdstheAgnesBrownDugganChairinOncologicalResearch.TheauthorsalsothankDr.ManickaV.Vadhanamforusefuldiscussionduringpreparationofthepaper.SupplementaryMaterialsCharacteristicsofTesterStrains:AlltheSalmonellatyphimuriumbacterialstrainsusedintheAmestestcarryadefective(mutant)genethatpreventsthemfromsynthesizingtheessentialaminoacidhistidine.Themutantcolonies,whichcanmakehistidinearecalled"revertants".Revertantsareidentifiedascoloniesthatgrowinlowlevelsofhistidine.Frameshift,transitionandbase-pairsubstitutiondefectsarerepresentedtoidentifythetypes.ThepresenceoftheuvrA/Bmutationmakesthestrainsmoresensitivetothetestarticlesthatinducedamageinthismanner.TheuvrA/Bmutationispartofadeletionmutationextendingintoageneforbiotinsynthesis;therefore,thebiotinrequirementisaresultofthedeletionofthisregion.TheuvrA/BmutationisindicatedbysensitivitytoUVlight.Therfamutationchangesthepropertiesofthebacterialcellwallandresultsinthepartiallossofthelipopolysaccharide(LPS)barrierincreasingpermeabilityofcellstocertaintypesofchemicals.Therfamutationisindicatedbysensitivitytocrystalviolet.TheRfactorplasmid(pKM101)makesthestrainsmoreresponsivetoavarietyofmutagens.Thisplasmidcarriesanampicillinresistancegene.ThepAQ1plasmidcarriesatetracyclineresistancegene. SupplementaryTableReferencesJ.M.C.Gutteridge,“Invitedreview.Freeradicalsindiseaseprocesses:acompilationofcauseandconsequence,”FreeRadicalResearchCommunications,vol.19,no.3,pp.141–158,1993.Viewat:GoogleScholarM.Valko,D.Leibfritz,J.Moncol,M.T.D.Cronin,M.Mazur,andJ.Telser,“Freeradicalsandantioxidantsinnormalphysiologicalfunctionsandhumandisease,”InternationalJournalofBiochemistryandCellBiology,vol.39,no.1,pp.44–84,2007.Viewat:PublisherSite|GoogleScholarR.Govindarajan,M.Vijayakumar,andP.Pushpangadan,“Antioxidantapproachtodiseasemanagementandtheroleof“Rasayana”herbsofAyurveda,”JournalofEthnopharmacology,vol.99,no.2,pp.165–178,2005.Viewat:PublisherSite|GoogleScholarW.M.El-SayedandW.A.Hussin,“Antimutagenicandantioxidantactivityofnovel4-substitutedphenyl-2,2′-bichalcophenesandaza-analogs,”JournalofDrugDesign,DevelopmentandTherapy,vol.7,pp.73–81,2013.Viewat:GoogleScholarA.Cardador-Martínez,A.Albores,M.Bahetal.,“Relationshipamongantimutagenic,antioxidantandenzymaticactivitiesofmethanolicextractfromcommonbeans(PhaseolusvulgarisL),”PlantFoodsforHumanNutrition,vol.61,no.4,pp.161–168,2006.Viewat:PublisherSite|GoogleScholarK.Hemminki,“DNAadducts,mutationsandcancer,”Carcinogenesis,vol.14,no.10,pp.2007–2012,1993.Viewat:GoogleScholarK.Hemminki,M.Koskinen,H.Rajaniemi,andC.Zhao,“DNAadducts,mutations,andcancer2000,”RegulatoryToxicologyandPharmacology,vol.32,no.3,pp.264–275,2000.Viewat:PublisherSite|GoogleScholarD.H.Phillips,“DNAadductsinhumantissues:biomarkersofexposuretocarcinogensintobaccosmoke,”EnvironmentalHealthPerspectives,vol.104,supplement3,pp.453–458,1996.Viewat:GoogleScholarL.A.LoebandC.C.Harris,“Advancesinchemicalcarcinogenesis:ahistoricalreviewandprospective,”CancerResearch,vol.68,no.17,pp.6863–6872,2008.Viewat:PublisherSite|GoogleScholarR.W.L.Godschalk,F.-J.vanSchooten,andH.Bartsch,“AcriticalevaluationofDNAadductsasbiologicalmarkersforhumanexposuretopolycyclicaromaticcompounds,”JournalofBiochemistryandMolecularBiology,vol.36,no.1,pp.1–11,2003.Viewat:GoogleScholarL.R.Ferguson,“Antimutagensascancerchemopreventiveagentsinthediet,”MutationResearch:FundamentalandMolecularMechanismsofMutagenesis,vol.307,no.1,pp.395–410,1994.Viewat:PublisherSite|GoogleScholarM.Green,R.Thomas,L.Gued,andS.Sadrud-Din,“InhibitionofDES-inducedDNAadductsbydiallylsulfide:implicationsinlivercancerprevention,”OncologyReports,vol.10,no.3,pp.767–771,2003.Viewat:GoogleScholarF.Aqil,M.Zahin,andI.Ahmad,“Antimutagenicactivityofmethanolicextractsoffourayurvedicmedicinalplants,”IndianJournalofExperimentalBiology,vol.46,no.9,pp.668–672,2008.Viewat:GoogleScholarM.Khader,N.Bresgen,andP.M.Eckl,“AntimutageniceffectsofethanolicextractsfromselectedPalestinianmedicinalplants,”JournalofEthnopharmacology,vol.127,no.2,pp.319–324,2010.Viewat:PublisherSite|GoogleScholarM.Zahin,F.Aqil,andI.Ahmad,“BroadspectrumantimutagenicactivityofantioxidantactivefractionofPunicagranatumL.peelextracts,”MutationResearch:GeneticToxicologyandEnvironmentalMutagenesis,vol.703,no.2,pp.99–107,2010.Viewat:PublisherSite|GoogleScholarF.AqilandI.Ahmad,“AntibacterialpropertiesoftraditionallyusedIndianmedicinalplants,”MethodsandFindingsinExperimentalandClinicalPharmacology,vol.29,no.2,pp.79–92,2007.Viewat:PublisherSite|GoogleScholarN.P.Seeram,L.S.Adams,S.M.Henningetal.,“Invitroantiproliferative,apoptoticandantioxidantactivitiesofpunicalagin,ellagicacidandatotalpomegranatetanninextractareenhancedincombinationwithotherpolyphenolsasfoundinpomegranatejuice,”JournalofNutritionalBiochemistry,vol.16,no.6,pp.360–367,2005.Viewat:PublisherSite|GoogleScholarN.Seeram,R.Lee,M.Hardy,andD.Heber,“Rapidlargescalepurificationofellagitanninsfrompomegranatehusk,aby-productofthecommercialjuiceindustry,”SeparationandPurificationTechnology,vol.41,no.1,pp.49–55,2005.Viewat:PublisherSite|GoogleScholarF.Aqil,R.Munagala,M.V.Vadhanametal.,“Anti-proliferativeactivityandprotectionagainstoxidativeDNAdamagebypunicalaginisolatedfrompomegranatehusk,”FoodResearchInternational,vol.49,no.1,pp.345–353,2012.Viewat:PublisherSite|GoogleScholarJ.M.Landete,“Ellagitannins,ellagicacidandtheirderivedmetabolites:areviewaboutsource,metabolism,functionsandhealth,”FoodResearchInternational,vol.44,no.5,pp.1150–1160,2011.Viewat:PublisherSite|GoogleScholarC.-C.Lin,Y.-F.Hsu,andT.-C.Lin,“Effectsofpunicalaginandpunicalinoncarrageenan-inducedinflammationinrats,”AmericanJournalofChineseMedicine,vol.27,no.3-4,pp.371–376,1999.Viewat:GoogleScholarC.-C.Lin,Y.-F.Hsu,T.-C.Lin,andH.-Y.Hsu,“Antioxidantandhepatoprotectiveeffectsofpunicalaginandpunicalinonacetaminophen-inducedliverdamageinrats,”PhytotherapyResearch,vol.15,no.3,pp.206–212,2001.Viewat:PublisherSite|GoogleScholarP.-S.Chen,J.-H.Li,T.-Y.Liu,andT.-C.Lin,“FolkmedicineTerminaliacatappaanditsmajortannincomponent,punicalagin,areeffectiveagainstbleomycin-inducedgenotoxicityinChinesehamsterovarycells,”CancerLetters,vol.152,no.2,pp.115–122,2000.Viewat:PublisherSite|GoogleScholarD.Heber,“Multitargetedtherapyofcancerbyellagitannins,”CancerLetters,vol.269,no.2,pp.262–268,2008.Viewat:PublisherSite|GoogleScholarD.M.MaronandB.N.Ames,“RevisedmethodsfortheSalmonellamutagenicitytest,”MutationResearch,vol.113,no.3-4,pp.173–215,1983.Viewat:GoogleScholarM.Zahin,I.Ahmad,andF.Aqil,“AntioxidantandantimutagenicactivityofCarumcopticumfruitextracts,”ToxicologyinVitro,vol.24,no.4,pp.1243–1249,2010.Viewat:PublisherSite|GoogleScholarD.M.MaronandB.N.Ames,“RevisedmethodsfortheSalmonellamutagenicitytest,”MutationResearch,vol.113,no.3-4,pp.173–215,1983.Viewat:GoogleScholarF.Aqil,M.V.Vadhanam,andR.C.Gupta,“Enhancedactivityofpunicalagindeliveredviapolymericimplantsagainstbenzo[a]pyrene-inducedDNAadducts,”MutationResearch:GeneticToxicologyandEnvironmentalMutagenesis,vol.743,no.1-2,pp.59–66,2012.Viewat:PublisherSite|GoogleScholarR.C.Gupta,“32P-postlabelingfordetectionofDNAadducts,”inTechnologiesforDetectionofDNADamageandMutations,G.P.Pfeifer,Ed.,pp.45–61,PlenumPress,NewYork,NY,USA,1996.Viewat:GoogleScholarW.P.StewardandK.Brown,“Cancerchemoprevention:arapidlyevolvingfield,”BritishJournalofCancer,vol.109,pp.1–7,2013.Viewat:PublisherSite|GoogleScholarA.H.Fang,W.A.Smith,P.Vouros,andR.C.Gupta,“Identificationandcharacterizationofanovelbenzo[a]pyrene-derivedDNAadduct,”BiochemicalandBiophysicalResearchCommunications,vol.281,no.2,pp.383–389,2001.Viewat:PublisherSite|GoogleScholarJ.M.Arif,M.Kunhi,Y.M.Siddiquietal.,“Differentialmodulationofbenzo[a]pyrene-derivedDNAadductsinMCF-7cellsbymarinecompounds,”InternationalJournalofCancerPrevention,vol.1,pp.259–268,2004.Viewat:GoogleScholarE.Miadokova,I.Masterova,V.Vlckova,V.Duhova,andJ.Toth,“AntimutagenicpotentialofhomoisoflavonoidsfromMuscariracemosum,”JournalofEthnopharmacology,vol.81,no.3,pp.381–386,2002.Viewat:PublisherSite|GoogleScholarM.OsborneandN.Crosby,MetabolismBenzopyrenes,CambridgeUniversityPress,NewYork,NY,USA,1987.S.Kaur,I.S.Grover,M.Singh,andS.Kaur,“AntimutagenicityofhydrolyzabletanninsfromTerminaliachebulainSalmonellatyphimurium,”MutationResearch:GeneticToxicologyandEnvironmentalMutagenesis,vol.419,no.1–3,pp.169–179,1998.Viewat:PublisherSite|GoogleScholarJ.M.Sayer,H.Yagi,A.W.Wood,A.H.Conney,andD.M.Jerina,“Extremelyfacilereactionbetweentheultimatecarcinogenbenzo[a]pyrene-7,8-diol9,10-epoxideandellagicacid,”JournaloftheAmericanChemicalSociety,vol.104,no.20,pp.5562–5564,1982.Viewat:GoogleScholarM.Larrosa,F.A.Tomás-Barberán,andJ.C.Espín,“ThedietaryhydrolysabletanninpunicalaginreleasesellagicacidthatinducesapoptosisinhumancolonadenocarcinomaCaco-2cellsbyusingthemitochondrialpathway,”JournalofNutritionalBiochemistry,vol.17,no.9,pp.611–625,2006.Viewat:PublisherSite|GoogleScholarCopyrightCopyright©2014MaryamZahinetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.RelatedarticlesNorelatedcontentisavailableyetforthisarticle.MorerelatedarticlesNorelatedcontentisavailableyetforthisarticle.MorerelatedarticlesNorelatedcontentisavailableyetforthisarticle.PDFDownloadCitationCitationDownloadotherformatsMoreePubXMLOrderprintedcopiesOrderViews5411Downloads1523CitationsSharePostRelatedarticlesNorelatedcontentisavailableyetforthisarticle.ArticleoftheYearAward:Outstandingresearchcontributionsof2021,asselectedbyourChiefEditors. Readthewinningarticles.