Role of Zucchini and Its Distinctive Components in the Modulation of Degenerative Processes

22 Pages·2017·2.41 MB·English

nutrients
Article
Role of Zucchini and Its Distinctive Components in
the Modulation of Degenerative Processes:
Genotoxicity, Anti-Genotoxicity, Cytotoxicity and
Apoptotic Effects
DamiánMartínez-Valdivieso1,RafaelFont2,ZahiraFernández-Bedmar3,*,TaniaMerinas-Amo3,
PedroGómez1,ÁngelesAlonso-Moraga3andMercedesdelRío-Celestino1 ID
1 DepartmentofGenomicsandBiotechnology,IFAPA(AndalusianInstituteofAgriculturalResearchand
Training,Fisheries,FoodandEcologicalProduction)CenterLaMojonera,CaminoSanNicolás,
1LaMojonera,04745Almería,Spain;[email protected](D.M.-V.);
[email protected](P.G.);[email protected](M.d.R.-C.)
2 DepartmentofFoodandHealth,IFAPACenterLaMojoneraCaminoSanNicolás,1LaMojonera,
04745Almería,Spain;[email protected]
3 DepartmentofGenetics,UniversityofCórdoba,CampusRabanales,GregorMendelBuilding,
14071Córdoba,Spain;[email protected](T.M.-A.);[email protected](Á.A.-M.)
* Correspondence:[email protected]@gmail.com;Tel.:[phone]
Received:4May2017;Accepted:11July2017;Published:14July2017
Abstract: Zucchini (Cucurbita pepo subsp. pepo) is a seasonal vegetable with high nutritional and
medicalvalues. Manyusefulpropertiesofthisfruitareattributedtobioactivecompounds. Zucchini
fruits(“Yellow”and“LightGreen”varieties)andfourdistinctivecomponents(lutein,β-carotene,
zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, β-carotene, zeaxanthin
and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate
the safety and suitability of their use, different assays were carried out: (i) genotoxicity and
anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide;
(ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to
evaluatethepro-apoptoticeffect. Resultsshowedthat: (i)allthesubstanceswerenon-genotoxic;
(ii)allthesubstanceswereanti-genotoxicexceptthehighestconcentrationoflutein;(iii)“Yellow”
zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC > 0.1 mg/mL and
50
0.2 mg/mL, respectively); and (iv) “Light Green” zucchini skin induced internucleosomal DNA
fragmentation, β-carotene being the possible molecule responsible for its pro-apoptotic activity.
Tosumup,zucchinifruitcouldplayapositiveroleinhumanhealthandnutritionduetothisfruit
anditscomponentsweresafe,abletoinhibitsignificantlytheH O -induceddamageandexhibit
2 2
anti-proliferativeandpro-apoptoticactivitiestowardHL60(humanpromyelocyticleukemiacells)
tumor cells. The information generated from this research should be considered when selecting
potentialaccessionsforbreedingprogrampurposes.
Keywords:cancer;carotenoid;chemoprevention;Cucurbita;cytotoxicity;DNA-protection;tumorcells
1. Introduction
It is currently accepted that diet affects the overall process of carcinogenesis by different
mechanisms:itsconstituentsmaycontaincancer-causingsubstancesaswellasmanycancerpreventive
agents[1]. Inappropriatedietetichabitsareestimatedtobethecauseofmorethanonethirdofcancer
deaths[2].
Nutrients2017,9,755;doi:10.3390/nu9070755 www.mdpi.com/journal/nutrients
Nutrients2017,9,755 2of22
Morethan20,000speciesofplantsareusedintraditionalmedicines,allegedtobeallpotential
reservoirs for new drugs [3] and are considered a potential source of chemical constituents with
antitumorandcytotoxicactivities[4]. Protectiveelementsinacancerpreventiondietincludeselenium,
folicacid,vitaminB-12,vitaminD,chlorophyllandantioxidantssuchasthecarotenoids(α-carotene,
β-carotene,lycopene,lutein,andcryptoxanthin)[5,6].
Summer squash (Cucurbita pepo subsp. pepo) is a seasonal vegetable that contains a number of
beneficialmicronutrientssuchasminerals,carotenoids,vitaminC,phenoliccompounds,etc.[7–10].Ithas
beenusedintraditionalfolkmedicinetotreatcoldsandalleviateaches,duetoitsantioxidant/anti-radical,
anti-carcinogenic,anti-inflammatory,antiviral,antimicrobialandanalgesicactivities[11–15].
Studiesofgenotoxicity/anti-genotoxicityandcytotoxicityarerapidmethodstoassessthesafety
andpossiblebeneficialeffectsofsinglecompoundsorcomplexmixturesandfoods[16–18].TheSomatic
MutationandRecombinationTest(SMART)ofDrosophilamelanogasterisasuitableeukaryotictool
forgenotoxicityandanti-genotoxicitystudies[19]andisaone-generationtestbasedonthelossof
heterozygosityoftwosuitablerecessivemarkers(mwhandflr),duetodifferentgenotoxicevents(i.e.,
mitoticrecombination,mutationandchromosomalaberration). HL60(humanpromyelocyticleukemia
cells) have been used in cytotoxicity assays in order to determine the tumoricide activity of some
vegetablematrices[20].
ThereareafewstudieswherethecytotoxicactivityofC.pepohasbeenchecked.Shokrzadehetal.[14]
reported different cytotoxic activities of the C. pepo leaf extracts on normal and cancer cell lines.
Wangetal.[15] observed a significant dose-dependent inhibitory effect against HeLa and HepG
cell growth of C. pepo fruits extracts. Menéndez et al. [11] also reported a significant decrease of
thegrowthofprostatichyperplasiabyextractofC.peposeedsatthetestedconcentrations(400and
200mg/kg). However,nostudieshavebeencarriedouttodemonstratetheinvivoactivityofzucchini
fruitandaboutthemechanismsofactionoftheirantioxidantcompounds.
In the case of the genotoxic or anti-genotoxic activities of antioxidant compounds such as the
ascorbicacid(thereducedformofdehydroascorbicacid),mostofthereporteddata[21–23]indicate
thatitisanti-mutagenic.Althoughantioxidantprotectionisassumedforascorbicacidagainstoxidative
damage,thedetailsofthisprotectionarenotyetcompletelyunderstood.Infact,undercertainconditions,
ascorbicacidseemstohaveco-genotoxicactivityinsteadofthenormalanti-genotoxicaction[24].
Studiesaboutcarotenoidshavealsobeenperformedinrelationtodecreasecancerrisk. Thus,
the chemopreventive action of β-carotene is effective mainly at the beginning of the carcinogenic
processorintheinitialstagesofitspromotion,inhibitingtheformationofpre-neoplasticlesionsin
someinvitroandinvivoexperimentalmodels[25–27]. Luteinandzeaxanthin,naturallyoccurring
carotenoids,haveshowntoreducetheriskofcataractsandage-relatedmaculardegeneration[28].
Zeaxanthinisrelatedwiththereductionofmelanomacellsviability[29]. Luteincausedsignificant
DNAdamageinhumanretinalpigmentepithelialcells[30].
Theaimofthisworkwastogiveanaddedvaluetothesimplenutritionalvalueofthezucchini
fruit, alongwithsomeofactivecomponents, carotenoids(lutein, β-carotene, andzeaxanthin)and
vitaminC,withrespecttoDNAintegrity,andtoestablishthepossibleinvivogenotoxicandinvitro
cytotoxiceffects. Inordertomeasuretheseproperties,differentassayswerecarriedout: genotoxicity
andanti-genotoxicity(invivoanalysisusingtheDrosophilamelanogastermodel),andcytotoxicityand
pro-apoptoticactivity(invitroassaysusingthepromyelocyticleukemiaHL60cellline). Inthisway,
thebeneficialhealtheffectsoftheconsumptionofzucchinicouldbeexplainedandusedforbreeding
programpurposes.
2. MaterialsandMethods
2.1. PlantMaterial
TwodifferentvarietiesofC.pepobelongingtozucchinimorphotypewereevaluatedinthiswork:
“LightGreen”(elongatedinshapewithlightgreenskin)and“Yellow”(elongatedinshapewithyellow
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skin). They were representativesof zucchinicommercialcultivars currently offered inthe market.
SeedsofthesevarietiesweregerminatedonwetfilterpaperinPetridishesatroomtemperaturefor
2–4daysinthedark,afterwhichtheyweretransplantedintorock-woolcubes(GrodanBV,Roermond,
TheNetherlands)inagreenhouse. Plantsweretransferredto1mlargerock-woolslabsatadensity
oftwoplants/slabwhendevelopedthreetofourleaves. Plantsweregrowninagreenhouseinthe
AndalusianInstituteofAgriculturalResearchandTraining,Fisheries,FoodandEcologicalProduction
(IFAPA)CenterinLaMojonera,Almería,Spain(36◦47(cid:48)19(cid:48)(cid:48) N,02◦42(cid:48)11(cid:48)(cid:48) W,142ma.s.l.) fromMarchto
June2011followingstandardlocalculturalpracticesforbothplantnutritionandinsectpestanddisease
control. Six fruits of each variety were harvested at an immature stage, and processed preserving
epicarp and mesocarp of each fruit separately, packaged in polypropylene plastic containers and
storedat−80◦C.Samplewaslyophilizedusingfreezedrierequipment(TelstarLyoQuest,Barcelona,
Catalonia,Spain)at−55◦Cundervacuum(133×10−3 mbar)for96hpersample. Then,samples
weregroundandfrozenat−80◦Cforfurtherextractionsandbiologicalanalyses.
2.2. AntioxidantCompounds
The compounds used in this study were purchased from Fluka (lutein: Cat. Number 07168
andβ-carotene: Cat. Number22040,Milan,Italy),Extrasynthese(zeaxanthin: Cat. Number0307S,
Genay,France)andSigma-Aldrich(ascorbicacid: Cat. Number255564anddehydroascorbicacid: Cat.
Number261556,St. Louis,MO,USA).Thecarotenoidsweredissolvedinethanolpriortoadditionto
thecorrespondingculturemedia,i.e.,inwaterforflytreatment,orinRPMI(RoswellParkMemorial
Institute)1640mediumforHL60cellcultureatthetimeoftheexperiment. Thefinalconcentrationof
ethanolwas1%intheculturemedia.
2.3. DeterminationoftheCarotenoidContent
Allmanipulationswereperformediniceandundersubduedartificiallightconditionswithheadspaces
of containers flushed with oxygen free nitrogen to help prevent carotenoid degradation. Individual
carotenoid concentration was determined by reverse phase high performance liquid chromatography
(HPLC) after saponification following the methods described by Martínez-Valdiviesoetal.[10].
The carotenoids were extracted from the rehydrated sample with 5 mL ethanol containing 1 mg/mL
butylatedhydroxytoluene(BHT)usingaPolytronhomogenizer(PolytronKinematica,Newark,NJ,USA).
Samples were saponified in order to hydrolyze esterified carotenoids that mightcomplicate the
chromatographicdeterminations[31]. Onemilliliterofa40%KOHmethanolicsolution(w/v)was
addedtoeachtube,andthesamplesweresaponifiedfor10minat85◦C.Thesampleswerecooledin
anicebath,and2mLofice-coldwaterwasadded. Thesuspensionswereextractedtwicewith2mLof
hexanebyvigorousvortexingfollowedbya2000rpmcentrifugationfor10minatroomtemperature.
TheupperhexanelayerswerepooledandevaporatedtodrynessinaSavantSpeedVacapparatus
andresuspended(Waltham,MA,USA).Immediatelybeforeinjectionthecarotenoidsweredissolved
in800µLofanacetonitrile/methanol/dichloromethane(45:20:35v/v/v)solution,filteredthrough
a 0.22 µm polytetrafluoroethylene (PTFE) syringe filter (Millipore, Billerica, MA, USA) directly to
samplevials,and10µLwereinjectedintothechromatograph. Theinitialmobilephaseconsistedof
acetonitrile/methanol(97:3,v/v/v)containing0.05%(v/v)triethylamine. Weusedalineargradientof
dichloromethanefrom0to10%in20minattheexpenseofacetonitrile,andthenthedichloromethane
was kept constant at 10% until the completion of the runs. The flow rate was 1.0 mL/min while
thecolumntemperaturewas30◦C.TheanalyseswerecarriedoutonaHPLCapparatusequipped
withbinarypump,in-linevacuumdegasser,autosamplerinjector,aWatersSymmetryC18column
(4.6mm×150mm,5µmparticlesize),(Waters,Milford,MA,USA)anda996diodearraydetector
(Waters,Milford,MA,USA)supportedbytheEmpowerchromatographymanagercomputingsystem
(Waters)wasusedtodetectcoloredcarotenoidsat450nm. Compoundswereidentifiedbycomparison
ofretentiontimes,co-injectionwithknownstandards,andcomparisonoftheirultraviolet(UV)-visible
spectrawithauthenticstandards. Quantificationwascarriedoutbyexternalstandardization. Full
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standardcurveswereconstructedwithfivedifferentconcentrationsforeachcarotenoidintriplicate.
Thecurvespassedthroughorwereveryneartheorigin,werelinearandbracketedtheconcentrations
expectedinthesamples. Resultswereexpressedonadryweight(DW)basis.
Oncethecontentoftheselectedantioxidantcompoundswasevaluatedintheepicarpandthe
mesocarpofC.pepofruit,thegenotoxicity,cytotoxicityandapoptosisassayswereperformed.
2.4. ExtractionandAnalysisofVitaminC
ThevitaminCanalysiswascarriedoutwithfreezedriedlyophilizedsamplesstoredat−80◦C.
Fivegramsofsampleswerehomogenizedin10mLofMeOH/H O(5:95)pluscitricacid(21g/L)
2
withEDTA(0.5g/L)and4mMNaF.HomogenateswerethenfilteredthroughcheeseclothandC18
Sep-Pakcartridges(Waters,Milford,MA,USA).Ascorbicacid(AA)anddehydroascorbicacid(DHA)
contentsweredeterminedfollowingthemethodsdescribedbyZapataandDufour[32,33]. HPLC
analyses were performed after derivatization of DHA into the fluorophore 3-(1,2-dihydroxyethyl)
furol[3,4-b]quinoxaline-1-one(DFQ),with1,2-phenylenediaminedihydrochloride(OPDA).Samples
of 20 µL were analyzed by using a Merck-Hitachi (Tokyo, Japan). The analyses were carried out
onaHPLCapparatusequippedwithbinarypump,in-linevacuumdegasser,autosamplerinjector,
a Waters and a 996 diode array detector (Waters, Milford, MA, USA) supported by the Empower
chromatographymanagercomputingsystem(Waters). SeparationsofDFQandAAwereachieved
onaKromasil100C18column(250mm×4mm;5µmparticlesize;Tecnokroma,Barcelona,Spain).
ThemobilephasewasMeOH/H O(5:95, v/v)containing5mMcetrimideand50mMpotassium
2
dihydrogen phosphate at pH 4.5. The flow rate was 0.9 mL/min. The detector wavelength was
initiallysetat348nmandafterelutionofDFQ,thewavelengthwasmanuallyshiftedto261nmfor
AA detection. Standard solutions, column conditioning and derivatization procedures have been
previouslydescribedbyGiletal.[34].
2.5. GenotoxicityandAnti-GenotoxicityTests
TheprinciplesandbasicproceduresfortheDrosophilawingspottesthavebeendescribedby
Graf et al. [19], and in previous works of our group [16,17]. Two strains of flies carrying wing
geneticmarkersontheleftarmofchromosome3: multiplewinghair(mwh,3-0.3)andflare(flr3,3-38)
areused. Thetransheterozygouslarvaewereobtainedbycrossingmwh/mwhmalesandflr3/TM3
(ThirdMultiple3),BdS (Beadedserrate)virginfemales. Hybrideggsderivedfromcrossingoptimally
fertile flies were collected over an 8 h period. Larvae emerged 72 ± 4 h later were cleaned from
remaining feeding medium with distilled water, and subsequently transferred to treatment vials.
Thesevialscontained0.85gofDrosophilaInstantMedium(Formula4–24,CarolinaBiologicalSupply,
Burlington,NC,USA)wettedwith4mLoftheepicarpandmesocarpofC.pepoandtheirantioxidant
compoundssolutionsatphysiologicalconcentrationsforDrosophilamelanogaster: 0.25and8mg/mL
ofepicarpandmesocarpofeachvarietyandthecorrespondentconcentrationsofpurecompounds
basedonthepreviouslydetaileddetermination(0.039and0.615µMforlutein,0.0003and0.0689µM
for β-carotene, 0.0001 and 0.105 µM for zeaxanthin, and 0.003 and 0.107 mM for dehydroascorbic
acid). Concurrentnegativecontrolswiththesolventalone(water)andpositivecontrolswithhydrogen
peroxide(120mM)werealsorun.
Anti-genotoxicitytestswerecarriedoutbymixingthemutagen(hydrogenperoxide,120mM)
withthecompoundssolution. Afteremergence,adultflieswerecollectedfromthetreatmentvials
andstoredin70%ethanol. Thewingsoftheflieswereremovedunderastereomicroscopeusinga
pairofentomologicaltweezers,similarnumberofmalesandfemales-wingsweremountedinFaure’s
solutiononmicroscopeslidesandinspected,under400×magnification,forthepresenceofclones
ofcells. Themutantcloneswereclassifiedintothreetypes: (1)smallsinglespots,containingoneor
twocells;(2)largesinglespots,containingthreeormorecells;and(3)twinspots,containingadjacent
mwhandflr3 cells[19]. Theappearanceoftwinspotsindicatedtherecombinogenicactivityofthe
chemotherapeuticagent(Figure1).
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Nutrients 2017, 9, 755 5 of 21

Figure 1. Types of spots found in the Drosophila melanogaster spot wing test: (A) wild phenotype
Figure 1. Types of spots found in the Drosophila melanogaster spot wing test: (A) wild phenotype
(normal trichomes); (B) simple mwh spot; (C) large mwh spot; and (D) twin spot.
(normaltrichomes);(B)simplemwhspot;(C)largemwhspot;and(D)twinspot.
2.6. Data Evaluation and Statistical Analysis
2.6. DataEvaluationandStatisticalAnalysis
Differences between quality compounds present in epicarp and mesocarp from “Yellow” and
Differencesbetweenqualitycompoundspresentinepicarpandmesocarpfrom“Yellow”and
“Light Green” zucchini were assessed by Student´s t-test. Data normality was tested prior to
“LightGreen”zucchiniwereassessedbyStudent´st-test. Datanormalitywastestedpriortoanalysis.
analysis. SPSS Version 10.0 software was used to perform all statistical analyses (SPSS, Chicago, IL,
SPSSVersion10.0softwarewasusedtoperformallstatisticalanalyses(SPSS,Chicago,IL,USA).
USA).
The frequencies of spots per fly of treated series were compared with its corresponding
The frequencies of spots per fly of treated series were compared with its corresponding
negativecontrolseries(water)toanalyzeSomaticMutationandRecombinationTest(SMART)data.
negative control series (water) to analyze Somatic Mutation and Recombination Test (SMART) data.
Formaximumpower,statisticalanalysesweredoneexclusivelyforthetotalnumberofspotsrecovered.
For maximum power, statistical analyses were done exclusively for the total number of spots
Thenon-parametricU testofMann, WhitneyandWilcoxonwasusedtoavoidweakpositiveand
recovered. The non-parametric U test of Mann, Whitney and Wilcoxon was used to avoid weak
inconclusiveresultsonSMARTandtoachieveawell-definedstatisticaldiagnosiswhetheragiven
positive and inconclusive results on SMART and to achieve a well-defined statistical diagnosis
treatmentshouldberegardedasgenotoxicornot[35]. Theinhibitionpercentages(IP)werecalculated
whether a given treatment should be regarded as genotoxic or not [35]. The inhibition percentages
usingthetotalspotsperwingwiththefollowingformula[16,36]:
(IP) were calculated using the total spots per wing with the following formula [16,36]:
IP=IP( (=G (e(Gnoentooxtionxeinaelo anleon−e G− eGneontootxoinxeinpel pulsuasc aticvteivceo cmopmopuonudn)/dG)/Geneontootxoixnienea laolnoen)e×) ×1 10000
2.7. CellCultureandCytotoxicityAssay
2.7. Cell Culture and Cytotoxicity Assay
HL60cellswereculturedinsuspensionat37◦CinRPMI1640medium(BE12-167F,BioWhittaker,
HL60 cells were cultured in suspension at 37 °C in RPMI 1640 medium (BE12-167F,
Verviers,Belgium)supplementedwith10%fetalbovineserum(DE14-801F,BioWhittaker),Glutamine
BioWhittaker, Verviers, Belgium) supplemented with 10% fetal bovine serum (DE14-801F,
(G7513,Sigma)andanantibiotic-antimycoticsolution(A5955,Sigma)inahumidifiedatmosphere
BcoionWtahiniitntagke5r%), GClOuta.mCinuel t(uGr7e5d13H, LSi6g0mcae)l lan(2d ×an 1a0n5ticbeiolltsi/c-manLt)imwyacsottirce asoteludtifoonr (7A259h5w5, iSthigmepai)c ianr pa,
2
hmuemsoidcaifripeda antdmdosifpfehreernet cocnomtapinoiunngd 5s%a CtOth2.e Ccuolntucerendtr HatLio6n0s ceinll (w2 h×i c1h05 tcheelyls/amreL)f owuansd trienattehde fofrru 7i2t:
hep wiciathrp ep(0ic.0a1r5p–, 0m.2e5somcga/rpm aLn)da nddiffmereensot ccaormpp(0o.u0n15d–s0 a.5t tmhge /cmonLc)enfrtoramtio“nYse lilno ww”hiCch. pthepeoy, aarned foeupnicda irnp
tahned frmueits:o ecpaircpar(p0. 0(03.10–105.–50m.25g /mmgL/m)fLr)o mand“L migehstocGarrepe n(0”.0C1.5p–e0p.5o, mlugt/eminL()0 f.r0o0m39 –“0Y.e2l5loµwM”) ,Cβ. -pceaproo, taennde
e(0p.i0c2a3r–p0 .a4n66d µmMe)s,ozceaarxpa n(0th.0i3n1–(00..052 2m–0g./8m7L9)µ fMro)m,d e“hLyigdhrto aGscroeerbni”c Cac. idpe(p0o.,0 3lu41te–i0n. 6(801.0m03M9–).0A.25m µixMo)f,
ββ--ccaarrootteennee, (z0e.0a2x3a–n0th.4i6n6a µnMd)d, ezheyadxaronatshcionr (b0i.c0a2c2i–d0a.8t7t9h eµMsa)m, deechoyndcernotarsactoiorbnsica abcoidve (0in.0d3i4c1a–te0d.6w81a msaMls)o.
A mix of β-carotene, zeaxanthin and dehydroascorbic acid at the same concentrations above
preparedtocheckifthecytotoxiceffectswereinadditiveorsynergicway.
indicated was also prepared to check if the cytotoxic effects were in additive or synergic way.
Thereactivityoftrypanblue(93595,Sigma),avitaldye,isbasedonthefactthatthechromophore
The reactivity of trypan blue (93595, Sigma), a vital dye, is based on the fact that the
isnegativelychargedanddoesnotinteractwiththecellunlessthemembraneisdamaged. Therefore,
chromophore is negatively charged and does not interact with the cell unless the membrane is
allthecellsthatexcludethedyeareviable. Non-viablecellsstainedpurple-violet,whereasviableones
dreammaaingeedd. uTnhstearienfeodr.eT, raylpl anthbel uceewllsa stahdadt edextcolucdelel ctuhlteu rdeysea taar1e: 1vriaatbiol,ea. nNdo2n0-µvLiabolfec ecllelslus spsetanisnioedn
purple-violet, whereas viable ones remained unstained. Trypan blue was added to cell cultures at a
1:1 ratio, and 20 µL of cell suspension was loaded into a Neubauer chamber. The cells were counted
Nutrients2017,9,755 6of22
was loaded into a Neubauer chamber. The cells were counted under an inverted microscope at
100× magnification. Aftertheincubationperiod, agrowthcurvewasestablishedandIC values
50
(concentrationoftestcompoundcausing50%inhibitionofcellgrowth)wereestimated. Curvesare
expressed as survival percentage with respect to controls growing at 72 h. All data represent the
averageofatleastthreeindependentexperiments.
2.8. AssessmentofPro-ApoptosisbyDNAFragmentationandAnnexinV-PI
The HL60 tumoral cell line was used with the aim of analyse the apoptotic induction. HL60
cells(1.5×106 cells)weretreatedwiththesameconcentrationsofthedifferentcompoundsfor5h.
Anon-treatedcellscontrolwasincludedforeachexperiment. ForDNAfragmentation,treatedcells
werecollected,werecentrifugedat4000rpmfor5minandwashedwithPBS.TheDNAwasextracted
usingacommercialkit(MBL243,Dominionmbl,Córdoba,Spain)andtheresultingtotalDNAwas
treatedwithRNAseinordertoeliminatefalsepositivefor30minat37◦C.Samplesof1.5µgofDNA
weremixedwithloadingbufferandloadedontoapre-solidified2%agarosegelcontainingethidium
bromide. Theagarosegelswererunat50Vfor90mininTBE(Tris/Borate/EDTA)bufferandthen
observedandphotographedunderUVlight. ForAnnexinV-PItest,AnnexinVApoptosisDetection
Kit (Canvax Biotech S.L., Cordoba, Spain) was used. Briefly, cells were resuspended in 100 µL of
Bindingbuffersolution1×andsubsequently,5µLAnnexinV-FITC(Fluoresceinisothiocyanate)and
10µLpropidiumiodidewereadded,andsampleswereincubatedfor10minatroomtemperaturein
darkness,andflowcytometry(BeckmanCoulterInc.,Brea,CA,USA)wasthenusedtoanalyzethe
rateofapoptosis.
3. Results
3.1. QuantitationofAntioxidantCompounds
Themeancontentvaluesofthedifferentactivecompoundsinepicarpandmesocarpofthetwo
summer squash varieties are listed in Table 1. The two varieties differed in the content of all the
compoundsanalyzed. The“Yellow”zucchinicarotenoidcontentwassignificantlyhigherthan“Light
Green”zucchiniinbothtissues,exceptzeaxanthininmesocarp. Itshouldbenotedthatthelutein
epicarpcontentof“Yellow”zucchiniisseventimeshigherthan“LightGreen”zucchini. Further,inthe
lattervariety,β-carotenewasnotdetectedinepicarp. Dehydroascorbicacidcontentof“LightGreen”
zucchini was similar in both tissues, but mesocarp of “Yellow” zucchini was tenfold higher than
epicarp. Inaddition,dehydroascorbicacidmesocarpcontentof“Yellow”zucchiniwassignificantly
higherthan“LightGreen”zucchini.
Table1.Epicarpandmesocarpmeancontentoflutein,β-carotene,zeaxanthinanddehydroascorbic
acidfromtwozucchinivarieties(“Yellow”and“Lightgreen”)expressedinmg/kgdryweight.
Yellow Light
Epicarp
Lutein 1036.9a 135b
β-carotene 99.5a n.d.b
Zeaxanthin 18.6a 1.7b
DHA 369.3b 592a
Mesocarp
Lutein 362.7a 63.2b
β-carotene 31.6a 4.0b
Zeaxanthin 1.9a 1.7a
DHA 3200a 683.3b
MeanswithinthesamerowfollowedbythesameletterarenotsignificantlydifferentusingStudent’stestatp<0.05.
n.d.:notdetected.
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3.2. GenotoxicityandAnti-GenotoxicityAnalysisofC.pepoandTheirComponents
The results of chronic treatment of D. melanogaster transheterozygous larvae with zucchini
epicarp and mesocarp and their different antioxidant compounds, along or combined with one
fixedconcentration(120mM)ofhydrogenperoxideintheDrosophilawingspotassay(SMART)are
presentedinTables2and3. Thepositivecontrolhydrogenperoxide(H O )behavedasagenotoxin
2 2
inducingoxidativestressandDNAdamage,givingtheexpectedresult. Itexhibitedatotalmutation
rate(0.52mutantclones/wing),whichquadrupledthenegativecontrol(distilledwater)rate(0.13).
Thisratioensurestheaccuracyofthegenotoxicityandanti-genotoxicityconcurrentassays[37].
Table2. Genotoxicity(singletreatment)ofepicarpandmesocarpoftwovarietiesofC.pepoandits
activecompounds(lutein,β-carotene,zeaxanthinanddehydroascorbicacid)intheDrosophilaWing
SpotTest.
ClonesPerWing(NumberofSpots)1
SmallSpots LargeSpots
Compound N (1–2Cells) (>2Cells) TwinSpotsm=5 TotalSpotsm=2 Mann-WhitneyTest3
m=2 m=5
NegativeControl(H2O) 40 0.08(3)2 0.00(0) 0.05(2) 0.13(5)
Epicarp
Yellow(mg/mL)
0.25 40 0.15(6) 0.00(0) 0.03(1) 0.18(7) n.s.
8 40 0.00(0) 0.00(0) 0.03(1) 0.03(1) n.s.
LightGreen(mg/mL)
0.25 40 0.10(4) 0.03(1) 0.00(0) 0.13(5) n.s.
8 40 0.05(2) 0.00(0) 0.00(0) 0.05(2) n.s.
Mesocarp
Yellow(mg/mL)
0.25 40 0.08(3) 0.03(1) 0.00(0) 0.10(4) n.s.
8 40 0.05(2) 0.00(0) 0.00(0) 0.05(2) n.s.
LightGreen(mg/mL)
0.25 40 0.03(1) 0.03(1) 0.00(0) 0.05(2) n.s.
8 40 0.03(1) 0.03(1) 0.00(0) 0.05(2) n.s.
SingleCompounds
Lutein(µM)
0.039 40 0.15(6) 0.08(3) 0.00(0) 0.23(9) n.s.
0.615 40 0.33(13) 0.08(3) 0.05(2) 0.45(18) n.s.
β-carotene(µM)
0.0003 36 0.08(3) 0.00(0) 0.00(0) 0.08(3) n.s.
0.0689 40 0.03(1) 0.03(1) 0.00(0) 0.05(2) n.s.
Zeaxanthin(µM)
0.0001 40 0.05(2) 0.00(0) 0.00(0) 0.05(2) n.s.
0.105 40 0.10(4) 0.03(1) 0.03(1) 0.15(6) n.s.
DHA(mM)
0.003 40 0.10(4) 0.03(1) 0.00(0) 0.13(5) n.s.
0.107 40 0.15(6) 0.03(1) 0.00(0) 0.18(7) n.s.
1 StatisticaldiagnosisbyFreiandWürgler[38]. N:numberofwings; 2 Numberofclonesorspotsperwing;
3 Theinconclusiveresultswereresolvedusingthenon-parametricU-TestMann-WhitneyandWilcoxon. n.s.:
non-significant.DHA:Dehydroascorbicacid.m:multiplicationfactor.
NocompoundresultedgenotoxicintheSMARTatthetestedconcentrations(Table2). Therewas
nosignificantincreaseinthenumbersofsmallsinglespotsandtotalspotsinthecaseofzucchiniepicarp
andmesocarp,andsinglecompoundsatthestudieddoses. Mutationrateswereevenlowerthanthe
negativewatercontrol. Theonlycasewithhighbutnon-significantmutationratewasthehighest
concentration of lutein with 0.45 total spots/wing for 0.615 µM. Zeaxanthin and dehydroascorbic
acidinducedanon-significantincreaseofmutationfrequenciesatthehighesttestedconcentrations
(0.15totalspots/wingat0.105µM,and0.18at0.107mM,respectively)afterlarvalfeeding. Thus,inthe
wingspottestinvivomodel,carotenoidsanddehydroascorbicacidarenotgenotoxic.
Nutrients2017,9,755 8of22
Table3.Anti-genotoxicity(combinedtreatment)ofepicarpandmesocarpoftwovarietiesofC.pepo
anditsactivecompounds(lutein,β-carotene,zeaxanthinanddehydroascorbicacid)intheDrosophila
WingSpotTest.
ClonesPerWing(NumberofSpots)1
SmallSpots LargeSpots
Compounds N (1–2Cells) (>2Cells) TwinSpotsm=5 TotalSpotsm=2 Mann-WhitneyTest3
m=2 m=5
NegativeControl(H2O) 40 0.08(3)2 0.00(0) 0.05(2) 0.13(5)
PositiveControl
40 0.38(15) 0.13(5) 0.03(1) 0.52(21) *
(H2O2120mM)
Epicarp
Yellow(mg/mL)
0.25 16 0.13(5) 0.00(0) 0.00(0) 0.13(5) n.s.
8 40 0.15(6) 0.03(1) 0.00(0) 0.18(7) n.s.
LightGreen(mg/mL)
0.25 40 0.13(5) 0.05(2) 0.03(1) 0.20(8) n.s.
8 40 0.05(2) 0.03(1) 0.03(1) 0.10(4) n.s.
Mesocarp
Yellow(mg/mL)
0.25 18 0.00(0) 0.00(0) 0.00(0) 0.00(0) n.s.
8 40 0.38(15) 0.00(0) 0.03(1) 0.40(16) n.s.
LightGreen(mg/mL)
0.25 40 0.05(2) 0.03(1) 0.00(0) 0.08(3) n.s.
8 40 0.05(2) 0.00(0) 0.00(0) 0.05(2) n.s.
SingleCompounds
Lutein(µM)
0.039 32 0.06(2) 0.06(2) 0.00(0) 0.13(4) n.s.
0.615 32 0.25(8) 0.16(5) 0.06(2) 0.47(15) *
β-carotene(µM)
0.0003 12 0.00(0) 0.00(0) 0.00(0) 0.00(0) n.s.
0.0689 38 0.08(3) 0.03(1) 0.00(0) 0.11(4) n.s.
Zeaxanthin(µM)
0.0001 40 0.03(1) 0.00(0) 0.03(1) 0.05(2) n.s.
0.105 30 0.07(2) 0.00(0) 0.00(0) 0.07(2) n.s.
DHA(mM)
0.003 40 0.08(3) 0.00(0) 0.00(0) 0.08(3) n.s.
0.107 40 0.10(4) 0.03(1) 0.00(0) 0.13(5) n.s.
1 StatisticaldiagnosisbyFreiandWürgler[36]. N:numberofwings; 2 Numberofclonesorspotsperwing;
3Theinconclusiveandpositiveresultswereresolvedusingthenon-parametricU-TestMann-WhitneyandWilcoxon.
n.s.:non-significant;*:significant(p≤0.05).m:multiplicationfactor.
Table3showstheresultsoftheanti-genotoxicityassays. Theresultsobtainedinthecombined
treatmentoflarvaewithzucchiniepicarpandmesocarp(0.25and8mg/mL)showednon-significant
differences when compared to the negative control. Then, the SMART test showed that zucchini
epicarp and mesocarp, and bioactive compounds were able to detoxify the genotoxic activity of
hydrogenperoxidealthoughnodoseeffectwasobservedingeneral(Table3). Themutationfrequency
inβ-carotene, zeaxanthinanddehydroascorbicacidwasevensmallerthanthatobtainedwiththe
negative control. Only the highest concentration of lutein showed significant differences with the
negativecontrol(0.47for0.615µM).
The inhibition percentage (IP) of genotoxicity by epicarp and mesocarp of “Yellow” zucchini
was75%and100%at0.25mg/mLand65%and23%at8mg/mL,respectively. Inthecaseofthe
epicarpandmesocarpof“LightGreen”zucchiniadose-dependentIPwasobservedbeingthehighest
detoxifyingactivityby90%at8mg/mLofepicarp. Thefruitantioxidantcompoundswouldactas
anti-mutagensagainsthydrogenperoxideinSMART.Thelowestconcentrationof“Yellow”zucchini
mesocarp(0.25mg/mL)showedthehighestIP(100%). Thebioactivezucchiniselectedcompounds
exhibitedanIPthatrangedfrom13%to100%(onaverage75%)(Figure2).
ItshouldbenotedthattheSMARTtestissuitabletodetecttheanti/pro-mutageniceffectsofsome
chemicals[2,16,17,24,39].Theresultsreportedhereareofinterestwheninvestigatingthedifferentways
inwhichsummersquashandtheirantioxidantcompoundscaninterfereinvivowiththemechanisms
ofgenotoxicagents.
Nutrients 2017, 9, 755 9 of 21
hydrogen peroxide although no dose effect was observed in general (Table 3). The mutation
frequency in β-carotene, zeaxanthin and dehydroascorbic acid was even smaller than that obtained
with the negative control. Only the highest concentration of lutein showed significant differences
with the negative control (0.47 for 0.615 µM).
The inhibition percentage (IP) of genotoxicity by epicarp and mesocarp of “Yellow” zucchini
was 75% and 100% at 0.25 mg/mL and 65% and 23% at 8 mg/mL, respectively. In the case of the
epicarp and mesocarp of “Light Green” zucchini a dose-dependent IP was observed being the
highest detoxifying activity by 90 % at 8 mg/mL of epicarp. The fruit antioxidant compounds would
act as anti-mutagens against hydrogen peroxide in SMART. The lowest concentration of “Yellow”
zucchini mesocarp (0.25 mg/mL) showed the highest IP (100 %). The bioactive zucchini selected
compounds exhibited an IP that ranged from 13% to 100% (on average 75%) (Figure 2).
It should be noted that the SMART test is suitable to detect the anti/pro-mutagenic effects of
some chemicals [2,16,17,24,39]. The results reported here are of interest when investigating the
different ways in which summer squash and their antioxidant compounds can interfere in vivo
Nutrients2017,9,755 9of22
with the mechanisms of genotoxic agents.
"Yellow" Epicarp 0.25 mg/mL
8 mg/mL
0.25 mg/mL
"Light green" Epicarp
8 mg/mL
"Yellow" Mesocarp 0.25 mg/mL
8 mg/mL
"Light green" Mesocarp 0.25 mg/mL
8 mg/mL
Lutein 0.039 µM
0.615 µM
β-carotene 0.0003 µM
0.0689 µM
Zeaxanthin 0.0001 µM
0.105 µM
DHA 0.003mM
0.107 mM
0 20 40 60 80 100 120
Inhibition Percentage (%)

Figure 2. Inhibition percentages (IP) of zucchini epicarp and mesocarp and its bioactive compounds
Figure2.Inhibitionpercentages(IP)ofzucchiniepicarpandmesocarpanditsbioactivecompounds
againagsta Hin2sOtH2-inOdu-cineddu dcaemdadgaem inag theein DtrhoesoDprhoislao pwhiinlag wspinogt tsepsott. test.
2 2
3.3. Effects on Tumoral Growth of HL60 Cells and Apoptosis
3.3. EffectsonTumoralGrowthofHL60CellsandApoptosis
Cultured mammalian cells provide an important tool for evaluating the cytotoxicity of
Culturedmammaliancellsprovideanimportanttoolforevaluatingthecytotoxicityofcompounds
compounds with potential therapeutic activity [40]. Trypan blue assessment was used to check the
withpotentialtherapeuticactivity[40]. Trypanblueassessmentwasusedtocheckthecytotoxicity
cytotoxicity of epicarp and mesocarp of two summer squash varieties and the main antioxidant
ofepicarpandmesocarpoftwosummersquashvarietiesandthemainantioxidantcompoundsof
compounds of the fruit.
thefruit.
A wide range of concentrations was used for every compound. Figures 3 and 4 show the
Awiderangeofconcentrationswasusedforeverycompound. Figures3and4showtherelative
relative tumor growth inhibition activity of the assayed substances. The results are expressed as
tumor growth inhibition activity of the assayed substances. The results are expressed as survival
survival percentage with respect to the controls (% tumoral viability). The shapes of the curves
percentagewithrespecttothecontrols(%tumoralviability).Theshapesofthecurvesweredifferentfor
were different for each case. Epicarp and mesocarp of “Yellow” zucchini showed the most
eachcase. Epicarpandmesocarpof“Yellow”zucchinishowedthemostnegativeslopeandthehighest
negattuimveo rsilcoipdea laenffde ctthine thhieghtreyspta tnubmluoeriecxidclauls ieoffnecats siany t(hICe try=p0a.0n8 bmluge/ mexLcalunsdio0n.2 amssga/ym (LI,Cre50s =p e0c.0ti8v ely).
50
mg/mL and 0.2 mg/mL, respectively). Epicarp and mesocarp of “Light Green” zucchini also
Epicarp and mesocarp of “Light Green” zucchini also showed a negative slope, but not reaching
showICed .aT nheegaretisvteo sflcoopme,p bouutn ndost arsesaacyheidngd IiCd5n0.o Tthree arcehstI oCf coamttphoeutnesdtse dascsoanyceedn dtriadt inoonts r,eaaltchho IuCg5h0 astl ight
50 50
the tested concentrations, although slight cytotoxic activities were observed in some of them
cytotoxicactivitieswereobservedinsomeofthem(zeaxanthin,dehydroascorbicacid,β-caroteneand
themixofthecompounds)(Figure4). Wedidnotobserveanycytotoxiceffectofluteinatthechosen
concentrations. Takingtheseobservationstogether,itappearsthatthecytotoxicactivityofzucchini
epicarpandmesocarpcannotberelatedtotheantioxidanteffectofasingleantioxidantcompound,
butrathertothetotalinteractionsbetweendifferentcompoundsofsuchacomplexmixture.
Nutrients 2017, 9, 755 10 of 21
(zeaxanthin, dehydroascorbic acid, β-carotene and the mix of the compounds) (Figure 4). We did
not observe any cytotoxic effect of lutein at the chosen concentrations. Taking these observations
together, it appears that the cytotoxic activity of zucchini epicarp and mesocarp cannot be related to
the antioxidant effect of a single antioxidant compound, but rather to the total interactions between
different compounds of such a complex mixture.
Nutrients2017,9,755 10of22
Figure 3. Effects of “yellow” and “Light green” zucchini epicarp ((A) and (C) respectively) and
mesocarp((B)and(D)respectively)onviabilityofHL60(humanpromyelocyticleukemiacells)tumoral
Figure 3. Effects of “yellow” and “Light green” zucchini epicarp ((A) and (C) respectively) and
cells.Cellviabilitywasassessedafter72hbytrypanblueexclusiontestassay.Thedataareexpressed
mesocarp ((B) and (D) respectively) on viability of HL60 (human promyelocytic leukemia cells)
aspercentagewithrespecttocontrol(mean±SDfromthreeindependentexperiments). TheIC
tumoral cells. Cell viability was assessed after 72 h by trypan blue exclusion test assay. The data are50
values(Inhibitoryconcentration50)areshowedinred.SD:standarddeviation.
expressed as percentage with respect to control (mean ± SD from three independent experiments).
The IC50 values (Inhibitory concentration 50) are showed in red. SD: standard deviation.
Oncecytotoxicityassaysofzucchiniepicarpandmesocarpandsomeofitsantioxidantcompounds
wereOpnecref ocrymteodto,xaivciitsyib laesasassyasy ooff DzuNcAchfirnaig mepeinctaartipo nawnda smcaersroiecdaropu tainndo rsdoemrteo oinfv ietsst igaantteiowxhideathnetr
ctohmepmoeucnhdasn iwsmeruen pdeerrfgoorimngedth, ea cvyitsoitbolxei caitsysawya osfm DeNdiAat efdravgimaaenpotapttioosnis w. Tahse cdarergireadd aotuiot nino fogredneorm toic
inDvNeAstiignattoe owlighoetnhuecrl etohseo mmaelchfraangimsmen utsnidsearghoailnlmg atrhke ocfyatpotoopxtiocsitisy. wThaiss mDNedAiaftreadg mviean ataptoiopntoesnisd. pTohinet
dwegarsaadnaatliyozne dofb ygecnoonmveicn tiDoNnaAl aignatroo soeliggeolneuleccletroospohmoarle sfirsa.gTmheenHtsL 6is0 cae lhlalilnlmeawraks otrfe aapteodpftoorsi5s.h Twhiitsh
DdNeAsa mfraegcmonecnetnattriaotnio ennsdapsoinintth wetarsy panaanlbylzueedc byyto ctooxnivceitnytaiosnsaayl. aAgasrsohsoew gneli neFleicgturroepsh5oarnedsis6., tThheeh HigLh6e0st
cceolln lcinene twraatsio tnrseaotfed“ Lfoigrh 5t hG wreietnh” dzeu scacmhien cioenpciecnartprasthioonws eads ians tlhigeh ttryinptaenrn buluclee ocysotomtoaxlifcriatyg masesnatya.t Aiosn
slhaodwdenr ipna Fttigerunreosf 5D aNnAd 6f,o trhHe Lh6ig0hterseta tceodncceenlltsr.atTiohniss dofi d“Lniogthot cGcurereinn” tzhueccchoinntir oelpaicnadrps osmhoewloedw ear
scliognhcte innttreartniouncsle.oAsodmoasle f-rdaegpmenednteanttioenff leacdtdisers upgagtteesrtne do.f “DYNelAlo wfo”r zHuLc6c0h itnrie,a“tLedig chetllgsr. eTehni”s zduidcc nhoint i
omcceusro cinar tph,eβ c-ocnartorotel naen,dlu stoemine, zloeawxearn cthoinnceanntdraDtiHonAs.d Aid dnoostes-dhoepwenandyenint teefrfencutc ilse ossuogmgeasltferdag. “mYeenlltoawtio”n
zluacdcdheirnpi, a“tLteirgnh.t Tghreeerne”s uzlutsccohfiAnni nmeexsioncVar/pP, rβo-pciadrioutmenieo, dluidteein(P, Iz)eaasxsaanyt(hFiing uanred7 D)sHhAow deidd sniogtn sifihcoawnt
adniyff eirnetnercnesucbleetowsoemenatlh ferangemgaetnivtaeticoonn tlraodldaenrd ptahtetehring.h Tehstec roenscuelntstr oatfi oAnnsnoefxYine llVo/wPrzoupcicdhiuinmi Eipodiciadrep ,
(PLIi)g hatssGarye e(nFizguucrceh 7in) isehpoiwcaerdp asingdniβfi-ccaanrot tdenifefesraemncpelse sb,eintwdieceant inthgea nneignadtuivcee dcoanptorpotlo asnisd. the highest
concentrations of Yellow zucchini Epicarp, Light Green zucchini epicarp and β-carotene samples,
indicating an induced apoptosis.