Apricot Melanoidins Prevent Oxidative Endothelial Cell Death by Counteracting Mitochondrial

12 Pages·2012·4.87 MB·English

Apricot Melanoidins Prevent Oxidative Endothelial Cell
Death by Counteracting Mitochondrial Oxidation and
Membrane Depolarization
Annalisa Cossu1., Anna Maria Posadino1., Roberta Giordo1, Costanza Emanueli5, Anna
Maria Sanguinetti3, Amalia Piscopo4, Marco Poiana4, Giampiero Capobianco6, Antonio Piga3*,
Gianfranco Pintus1,2*
1DepartmentofBiomedicalSciences,UniversityofSassari,Sassari,Italy,2CentreofExcellenceforBiotechnologyDevelopmentandBiodiversityResearch,Universityof
Sassari, Sassari, Italy, 3Department of Agriculture, University of Sassari, Sassari, Italy, 4Department of Biotechnologies for Agricultural Food and Environmental
Monitoring,MediterraneanUniversityofReggioCalabria,FeodiVito,ReggioCalabria,Italy,5LaboratoryofVascularPathologyandRegeneration,RegenerativeMedicine
Section,SchoolofClinicalSciences,BristolHeartInstitute,UniversityofBristol,Bristol,UnitedKingdom,6GynecologicandObstetricClinic,UniversityofSassari,Sassari,
Italy
Abstract
The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals
containedinfreshplantmaterial.However,whetherprocessedplantfoodsprovidethesamebenefitsasunprocessedones
isanopenquestion.Melanoidinsfromheat-processedapricotswereisolatedandtheirpresenceconfirmedbycolorimetric
analysis and browning index. Oxidative injury of endothelial cells (ECs) is the key step for the onset and progression of
cardiovascular diseases (CVD), therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-
induced oxidative mitochondrial damageand celldeath was explored in human ECs. The redox state of cytoplasmic and
mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP), while the
mitochondrial membrane potential (MMP) was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen
peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-
induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly
counteractedandultimatelyabolishedhydrogenperoxide-inducedintracellularoxidation,mitochondrialdepolarizationand
celldeath.Inthisregard,ourcurrentresultsclearlyindicatethatmelanoidinsderivedfromheat-processedapricots,protect
humanECs against oxidativestress.
Citation: Cossu A, Posadino AM, Giordo R, Emanueli C, Sanguinetti AM, et al. (2012) Apricot Melanoidins Prevent Oxidative Endothelial Cell Death by
CounteractingMitochondrialOxidationandMembraneDepolarization.PLoSONE7(11):e48817.doi:10.1371/journal.pone.0048817
Editor:FerencGallyas,UniversityofPecsMedicalSchool,Hungary
ReceivedJune4,2012;AcceptedOctober1,2012;PublishedNovember8,2012
Copyright: (cid:1) 2012 Cossu etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:TheworkwassupportedbygrantsfromtheItalianMinistryofEducation,UniversitiesandResearch‘‘PRIN05/08’’,RegioneSardegnaLRn.7-2007and
BancodiSardegnaFoundation.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:GianfrancoPintusisaPLOSONEAcademicEditor.Thisdoesnotaltertheauthors’adherencetoallthePLOSONEpoliciesonsharingdata
andmaterials.
*E-mail:[email protected](GP);[email protected](AP)
.Theseauthorscontributedequallytothiswork.
Introduction of the NEB reaction high molecular-weight heterogeneous
polymers called melanoidins are formed [2]. Melanoidins are
Aninversecorrelationbetweenadietrichinplantfoodsandthe widelydistributedinprocessedfoodsandmayhavevariousinvitro
occurrenceofcardiovasculardiseases(CVD)hasbeenreportedin functionalproperties,includingantioxidant[3,4],antihypertensive
several epidemiological studies [1]. The vasculoprotective effect [5] and metal-binding activities [6]. The antioxidant activity of
associatedtofruitandvegetableconsumptionisthoughttobedue melanoidins is of particular interest since it can influence the
to fresh plant-contained phytochemicals, including antioxidant oxidativeandshelflifeofseveralfoodsduringstorage[7,8].Inline
substancessuchasphenoliccompounds,carotenoidsandvitamins
with the observed antioxidant activity, some biological effects,
[1]. However, a remarkable amount of the food intake in the
including cell protection against oxidative damage, have been
human diet comes from processed foodstuffs, and whether
reported for coffee, biscuit and prune melanoidins [9–11].
processed plant-foods provide less benefit than unprocessed ones
However, because of the huge complexity of both reactions and
remains an area ofinquiry.
productsduringtheirchemicalpathwayofformation,onlypartial
One of the main food unit operations is based on thermal structuresofmelanoidinshavebeenelucidatedsofar[2].Thusitis
treatments. Heat-based food transformations often result in non- very difficult to address a specific health effect to a distinctive
enzymaticbrowning(NEB),whichoccursthroughsugarsthermal melanoidin chemical structure; therefore a deep and accurate
degradation,or,underacidicconditions,bytheMaillardReaction explorationisneededformelanoidinsderivedfromdifferentfoods.
(MR)betweensugarsandorganicacids[2].Duringthelaststage
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ApricotMelanoidinProtectagainstOxidativeDeath
Apricotfruitsareconsideredasarichsourceofphytochemicals, dried fruits, thus confirming melanoidins presence in the extract
which are mainly polyphenols and carotenoids [12,13]. Phenolic obtained from the processed samples (Fig. 1C). Among all the
compounds,inparticular,byactingasantioxidants,arethoughtto melanoidins fractions, Fraction I showed the highest amount of
provide various invivo health benefits including hepato- and NEBPs as measured at 420nm (data not shown), and was
cardio-protective effects [14,15]. The antioxidant properties of therefore chosen to be tested for its antioxidant activity on cells
polyphenolsinapricotshavebeenstudiedinrelationtoripening, exposedtooxidative stress.
cultivarandpureepreparation[13,16,17],andcontrastingresults
about the antioxidant activity of fresh apricot fruits have been Hydrogen Peroxide Induces Mitochondrial Damage and
often reported [18,19]. However, 40–45% of the total world Cell Death
productionofapricotsisprocessed,mainlybydryingandthermal
Inordertomimicoxidativedamageweinvestigatedtheeffectof
treatment[20].Similartoourpreviousfindingonprunes[3],we
different doses of H O on ECs death measured as cell viability
foundthatdryingapricotsathighprocessingtemperaturesresulted 2 2
in a significant increase of antioxidant activity, even though the
phenol content was significantly reduced [21]. We hypothesized
that the increased in antioxidant activity observed in the dried
apricots might have been due to the formation of NEB products
(NEBPs), after drying (e.g. melanoidins). Thus, as reported for
prunes[3],melanoidinsappeartobetheprevailingcontributorsto
themaintainedantioxidantactivityofdriedapricotinvitro.Inthis
regard, although the antioxidant properties of melanoidins have
been studied invitro for several years their potential antioxidant
effects on human biological systemsremains largely unknown.
The finding that oxidative stress is a common feature in many
aspectsof CVDpathogenesis [22],suggeststhat itscounteraction
with antioxidants may prevent disease occurrence or ameliorate
a patient’s pathological condition. For this reason a great deal of
attention is now focusing on naturally occurring antioxidants as
potential candidates for CVD prevention and/or treatment.
Endothelial cells (ECs) play a crucial role in the integration and
modulationofsignalswithinthevascularwall[23]andperturba-
tionofsuchhomeostasisbyoxidativedamageisthetriggerforthe
development of CVD [24]. We have previous reported that
melanoidins obtained from prunes protect human ECs from
hydrogen peroxide-induced oxidative stress and cell death [11],
but whether such kind of cellular protection is also provided by
melanoidinsisolatedfromapricotsiscompletelyunknown.Indeed,
chemical characteristics, both quantitative and analytical, of
compounds that participate in melanoidins formation in prunes
and apricots are known to differ and thus different melanoidins
mayoriginate from theirprocessing[21,25,26].
Hence, the present work was undertaken with the intent to
investigate whether the food melanoidins isolated from dried
apricots might protect human ECs against H O -induced
2 2
oxidative stress andcelldamage.
Results
Thermal Treatment Increases NEB in Processed Apricots
A significant increase in antioxidant capacity was elicited by
fruitdrying,whichstronglycorrelatedwiththeparalleledincrease
ofcolorfoundinthesamepolyphenolicextract(Fig.1A).Colour
was indeed dramatically changed by the drying process with
a significant reduction of tonality (Fig. 1B). In particular, the
blanching of sample resulted in a significantly lower reduction of
thedrying-inducedvariationofcolor,whichisexpressedasahue
angleintheHunterscale[27,28].Infact,amorepronouncedshift
toaredderanddeeperzoneintheHunterscaleisevidentinthe
dried, control fruits, with respect to the blanched ones, which Figure 1. Changes in chemical parameters elicited by fruit
might depend on the enzymatic browing contribution [29]. processing.(A)Changesinantioxidantactivityandcolorexpressedas
However, in blanched fruits where no enzymatic browning is hue variation (tan - 1 b*/a*). (B) Changes in color expressed as hue
variation (tan - 1 b*/a*). (C) Changes in color expressed as browning
present[30],theobservedcolorvariationcanbeexplainedonlyby
index (Abs at 420nm g21dm of fruit). Fresh, fresh fruits; M,
theformationofNEBduringthedryingprocess(Fig.1B).Browing
melanoidins; B, blanching. Data are the mean 6 SE from four or five
index analysis of the melanoidin fractionsisolated from fresh and measurements.(A–C)a;b,significantlydifferentfromthefreshsample.
processed apricots indicated that NEBPs were present only in doi:10.1371/journal.pone.0048817.g001
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ApricotMelanoidinProtectagainstOxidativeDeath
and mitochondrial metabolic activity (MMA). As expected, 2 hr-
treatment of ECs with H O resulted in a dose-dependent
2 2
reduction of cell survival as indicated by the significant decrease
in the number of viable cells in comparison with the untreated
control group (Fig. 2A). Consistently a significant decline of the
MMA was concomitantly observed with the increasing doses of
H O (Fig.2B).Theoxidant-inducedcellimpairmentwasindeed
2 2
associatedwithasuperimposablelossofmitochondrialmembrane
potential (MMP), clearly indicating mitochondria implication in
the hydrogen peroxide-induced cellular damage (Fig. 2B). Based
on these experiments, around 50% of H O -induced mitochon-
2 2
drialimpairmentandcelldeathwasobservedat100mMofH O ,
2 2
andthereforethisconcentrationwasusedtomimicoxidativestress
in thenextexperiments.
Apricot Melanoidins Protect Endothelial Cells from
Hydrogen Peroxide-induced Mitochondrial Damage and
Cell Death
We first testedapricot melanoidins forpotential toxicity inour
human endothelial cells model. Based on previous observations
concerningmelanoidinsfromothersourcesoffoodsuchascoffee
[9] and biscuit [10], we tested apricot-melanoidin at the
concentrations of 2, 6 and 12mg/ml at 48hrs of stimulation,
thenmelanoidin-treatedcellswerecomparedtountreatedonesfor
both cell viability and MMA. Results shown in figure 3, which
depict respectively lactate dehydrogenase (LDH) release and 3-
(4,5-dimethythiazol2-yl)-2,5-diphenyltetrazoliumbromide(MTT)
reduction, clearly indicate that apricot melanoidins did not
producetoxiceffectsforthecellsundertheemployedexperimen-
talconditions.Wehaveprevious demonstratedthatpretreatment
of human ECs with prune melanoidins [11] exerts remarkable
protection against oxidative-induced cell death. However, mela-
noidins from different foods may have different structures and
activities [2].Indeed,theexactsequence of reactionsfromwhich
melanoidins originated, as well as their chemical structures, in
different food remain largely unknown [2]. We therefore asked
whether, aswiththose isolated fromprunes,melanoidins isolated
from apricots could exert a protective effect on the observed
H O -induced cell impairment. To this end, cells were treated
2 2
with apricot melanoidins for 6 hrs and then H O was added
2 2
during the last 2 hrs of incubation to induce oxidative stress. As
shown in figure 4A melanoidins pretreatment was able to dose-
dependently counteract the decrease in cell viability induced by
100mM H O . Failure of oxidant in eliciting MMP and MMA
2 2
impairment in melanoidin-pretreated cells strongly indicates
,
a protective effect of these compounds against oxidative stress
and mitochondrial-mediated ECs death(Fig.4B–C).
Hydrogen Peroxide Induces Oxidation of Both Cytosolic
and Mitochondrial Compartments
Tofurtherinvestigatethemolecularmechanismsunderpinning
apricot melanoidins protection, we use two human EC lines
constitutionally expressing the redox-sensing green fluorescent Figure 2. Hydrogen peroxide induces mitochondrial damage
protein(roGFP)inboththecytosolic(cyto-roGFP)andmitochon- andcelldeath.Dose-dependenteffectofhydrogenperoxide(H O )
2 2
drial (mito-roGFP) compartment (Fig. 5A–E). In particular, the on (A) cell viability, (B) mitochondrial metabolic activity and (C)
mitochondrial membrane potential. Data are the mean 6 SE of four
fluorescence photo shown in figure 5B corresponds to ECV304
experiments.(A–C)*,significantlydifferentfromthecontrol.
cells expressing the cyto-roGFP, while the fluorescence image
doi:10.1371/journal.pone.0048817.g002
shown in figure 5D represents ECV304 cells expressing mito-
roGFP. Expression of the mito-roGFP in the mitochondrial
WhilethetwoimagesdisplayedinpanelsAandCoffigure5,
compartmentisconfirmedbytheimageshowninfigure5F,which
depict the merged photos of ECV304 cells expressing the
depicts the merged photo of ECV304 cells expressing the mito-
cytoplasmic (A) and the mitochondrial (C) form of the roGFP
roGFP (D) and ECV304 cells stained with the mitochondrial
(green),alongwiththebright-field(40Xmagnification).FromA–
marker MitoTracker Red(E).
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ApricotMelanoidinProtectagainstOxidativeDeath
Figure 3. Apricots melanoidins are not toxic for endothelial
cells. Effect of different concentrations of melanoidins on (A) cell
viabilityand(B)mitochondrialmetabolicactivity.Dataarethemean6
SEoffourexperiments.
doi:10.1371/journal.pone.0048817.g003
F, the nuclei are stained with hoechst (blue). These two cell lines
allowed us tospecifically followpotential changesof thecytosolic
and mitochondrial redox state during our experimentation. As
shown in Figure 6A–B, the treatment of roGFP expressing cells
with H O concentration-dependently shifted the intracellular
2 2,
redox status toward a more oxidative condition in both the mitochon-
drial and cytosolic compartment, indicating that under our
experimental conditions roGFP has a significant dynamic range
andrespondslinearlytoincreasingdosesofawell-knownoxidant.
Apricot Melanoidins Protect Cytosolic and Mitochondrial
Compartments from Hydrogen Peroxide-induced
Figure4.Melanoidinsprotectendothelialcellsfromhydrogen
Oxidative Redox Changes
peroxide-inducedmitochondrialdamageandcelldeath.Dose-
We next wanted to determine whether the cellular protection dependenteffectofmelanoidinsonhydrogenperoxide(H O )-induced
2 2
elicited by melanoidins was due to the counteraction of H2O2- (A) cell death, (B) mitochondrial metabolic activity and (C) mitochon-
induced intracellular oxidation. To this end, roGFP expressing drialmembranepotential.Dataarethemean6SEoffourexperiments.
cellsweretreatedwithmelanoidinsfor6 hrsand100mMofH O (A–C)*,significantlydifferentfromthecontrol.
2 2 doi:10.1371/journal.pone.0048817.g004
was added during the last 2 hrs of incubation. At the end of the
experiment both mito- and cyto-roGFP fluorescence were
induced by 100mMH O maintaining an intracellular redox
2 2
recorded. Data shown in figure 7 A–B indicate that melanoidins
statesimilartothatofcontrolcells.Fluorescentmicroscopyresults
were able to dose-dependently inhibit intracellular oxidation
confirming the protective effect of apricot-melanoidins on H O -
2 2
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ApricotMelanoidinProtectagainstOxidativeDeath
Figure5.ECV304cellslinesconstitutivelyexpressingthecytoplasmic(cyto-roGFP)andmitochondrial(mito-roGFP)formofroGFP.
Cellsweregrowninglasschamberslidesatconcentrationstoallow50–70%confluencein24hrs.Onthedayofexperiments,cellswerewashedwith
PBSthreetimes,counterstainedwiththemitochondrialmarkerMitoTrackerRedandthenuclearmarkerHoechst,fixedwith4%paraformaldehyde
andmountedforfluorescencemicroscopyvisualization.Images(A)and(C)depictrespectivelymergedphotosofECV304cellsexpressingthecyto-
andmito-roGFP(green)protein,Hoechststaining(blue)andbright-field(40X,NA=1.00).Images(B)and(D)depictrespectivelymergedphotosof
ECV304cellsexpressingthecyto-andmito-roGFP(green)protein,counterstainedwithHoechst(blue)(100X,NA=1.35)).Thefigure5F,depictsthe
mergedphotoofECV304cellsexpressingthemito-roGFPprotein(D)andECV304cellsstainedwiththemitochondrialmarkerMitoTrackerRed(E).
(100X,NA=1,35)).
doi:10.1371/journal.pone.0048817.g005
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ApricotMelanoidinProtectagainstOxidativeDeath
Figure 6. Hydrogen peroxide induces oxidation of both
cytosolic and mitochondrial compartments. Dose-dependent
effect of hydrogen peroxide (H2O2) on mitochondrial (mito-roGFP) Figure 7. Melanoidins protect human endothelial cells from
andcytoplasmic(cyto-roGFP)ro-GFPoxidation.Dataarethemean6SE hydrogen peroxide-induced intracellular oxidative stress.
offourexperiments.(A–B)*,significantlydifferentfromthecontrol. Dose-dependent effect of melanoidins on H O -induced cytoplasmic
2 2
doi:10.1371/journal.pone.0048817.g006 (cyto-roGFP)andmitochondrial(mito-roGFP)roGFPoxidation.Dataare
themean6SEoffourexperiments.(A–B)*,significantlydifferentfrom
thecontrol.
induced intracellular oxidation are reported in figure 8. A yellow
doi:10.1371/journal.pone.0048817.g007
fluorescencepattern,whichistheoverlapping ofthemito-roGFP
(green)andMitoTrackerRed,isclearlyvisibleinbothcontroland
Discussion
H O -treated cells pretreated with apricot-melanoidins (Fig 8A
2 2
andD).Onthecontrary,H O -inducedmitochondrialdamageis
2 2 Oxidative cellular damage during lifetime is emerging as an
clearly evident in oxidatively stressed cells, which lack theabove-
important factor in the onset and development of many
mentioned yellow pattern. H2O2-treated cells, have indeed clear
pathological conditions including CVD [22]. Although endoge-
mitochondrial damage, and therefore unable to take the Red
nousantioxidantsplayanimportantroleinprotectingcellsagainst
MitoTracker dye inside (Fig. 8B). As indicated in figure 8C
oxidativeinsults,additionalantioxidants(e.g.dietaryantioxidants)
melanoidin alone did not produce intracellular oxidative stress in
appear to be required to prevent or to protect living cells from
cultured ECs. Quite similar results are shown concerning ECs
oxidation [1]. In this context, health benefits exerted by plant-
expressingthecyto-roGFP(Figure8E–H),althoughaclearyellow
derived compounds and extracts have been mainly ascribed to
pattern is not visible due to the expression of roGFP in the
their antioxidant potential and the resulting capability to
cytoplasm. However, a bigger and clearer, punctate red patter is
counteractoxidative-induceddamage[13].However,duringfood
evident in both control and H O -treated cells pretreated with
2 2 processing and storage, chemical reactions among food compo-
apricot-melanoidins(Fig.8EandH),ascomparedwiththeH2O2-
nentsleadtobothdestructionandformationofphytonutrients[8],
treated ones (Fig. 8F). This is due to the compromised
thereforewhetherprocessedplantfoodsprovidethesamebenefits
mitochondrial function in H O -treated. No oxidative damage
2 2 as those ascribed to unprocessed ones is uncertain. For instance,
has beenproduced bymelanoidins alone (Fig.8G).
melanoidins are heterogeneous polymeric structures formed
during food processing in the last stage of the MR whose effects
on human health are largely unknown.
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ApricotMelanoidinProtectagainstOxidativeDeath
Figure8.Melanoidins protecthumanendothelialcellsfromhydrogenperoxide-inducedintracellular oxidativestress.Cellswere
growninglasschamberslidesatconcentrationstoallow50–70%confluencein24hrs.Onthedayofexperiments,cellsweretreatedwithapricot
melanoidinsfor6hrsandthenH O wasaddedduringthelast2hrsofincubationtoinduceoxidativestress.ThencellswerewashedwithPBSthree
2 2
times, counterstained with the mitochondrial marker MitoTracker Red and the nuclear marker Hoechst, fixed with 4% paraformaldehyde and
mountedforfluorescencemicroscopyvisualization.Images(A–D)depictmergedphotosofECV304cellsexpressingthemito-roGFP(green)protein,
counterstained with both MitoTraker Red and Hoechst. Images (E–H) depict merged photos of ECV304 cells expressing the cyto-roGFP (green)
protein,counterstainedwithbothMitoTrakerRedandHoechst.FromA–H,magnificationwas20XandNA0,70).
doi:10.1371/journal.pone.0048817.g008
Using a previously published procedure [11,21,25], fresh forms of melanoidins may originate from the processing of these
apricots were processed by standardized drying and heating twofruits [21,25,26].
conditions,thenbothfreshandprocessedfruitswerecharacterized FluorescenceimagingofROSinlivecellshasbeenwidelyused
on the basis of commonly recognized parameters namely the to assess oxidative stress in different cellular compartments and
variationofcolorandthebrowningindex,whicharebothsignsof under various experimental conditions [32]. However, many of
NEBPsformation[27,29].Aspreviouslyreportedforprunes[11], the methods so far employed to determine the levels of
the processing-induced elevation of antioxidant capacity was intracellular ROS suffer from various pitfalls [32]. A new
accompaniedbyasignificantincreaseinthecolor,asindicatedby approachwasthereforeusedinthisworktofollowH2O2-induced
the decrease of hue angle; suggesting NEBPs may be responsible intracellular oxidation and its possible counteraction by apricot
for the rise in chain-breaking activity observed post- fruit melanoidins. By employing two ECs lines [11], which constitu-
transformation. Indeed, the decrease of hue angle value, which tionallyexpresstheredox-sensitiveproteinsmito-andcyto-roGFP
represents the color variation, is an important parameter for the (Fig. 5), we were able to selectively follow changes of the redox
presenceofNEBPsandisrelatedtothebrownpigmentformation stateinthecytosolicandmitochondrialcompartment.Duetothe
caused by NEB reaction during fruit processing [29]. Confirma- ability to measure the ratio between the oxidized (GSSG) and
tory data of NEBPs formation during apricot processing comes reduce form of glutathione (GSH), these two cells lines provided
from the results obtained after sample blanching. Blanching is ausefultoolforassessingthevariationofintracellularredoxstate,
a common treatment used to prevent enzymatic browning [30], showing significant dynamic range and linear response to in-
creasing doses of the well-known oxidant, H O . Of note, the
and therefore variation of color under these experimental 2 2
increased oxidative conditions induced by the applied doses of
conditions can be explained only by the formation of processing-
oxidantwereparalleledbyacorrespondingdose-dependentrisein
elicited NEBPs. Results depicting the absorbance at 420nm,
mitochondrialdamageandcellulardeath,suggestingarelationship
which represents the browning index, are further confirmatory
between these H2O2-induced phenomena. Under these experi-
data related to NEB due to processing-elicited NEBPs formation
mental conditions, apricot melanoidins were able to counteract
[29]. Importantly, these data indicate that NEBPs were presents
H O -induced oxidation, maintaining the intracellular redox
only in the melanoidins fraction extracted from dried samples. 2 2
conditions similar to that of control cells. Consistent with this
Indeed,aswithwholefreshfruitsamples,NEBPswereabsenton
antioxidant effect is the observed dose-associated protection
themelanoidinsfractionisolatedfromfreshfruit,confirmingboth
exerted by apricot melanoidins against the H O -induced
the processing-induced NEBPs formation and the successfull 2 2
mitochondrial impairment and cell death, indicating a tight link
isolation of melanoidin components by the extraction technique
between theirantioxidant activityandcellular protection.
employed.
Toourknowledge,thisisthefirstreportontheprotectiveeffect
Giventhepivotalroleplayedbytheendotheliumincardiovas-
of apricot melanoidins against oxidative-induced cell death.
cular homeostasis and the involvement of oxidative-induced EC
Moreover, using a novel genetically engineered fluorescence
dysfunctioninCVDpathogenesis[24],itwasreasonableforusto
protein to ratiometrically assess the intracellular redox state in
use a human ECs line to investigate the effect of apricot
living cells, our data confirm and reinforce previously published
melanoidins on H O -induced oxidative damage. A pathology-
2 2 observations using coffee [9], biscuit [10] and prune melanoidins
associated rise of ROS can trigger mitochondrial membrane
[11]. Melanoidins indeed, appear to work as antioxidants by
permeability promoting the dissipation of MMP and ultimately
positively modulating the GSSG/GSH ratio in favor of the
cell death [31]. Also in our experimental model of oxidative cell
reduced form, and thus favorably preparing the cell to face
death we found a superimposable loss of both MMP and MMA,
oxidative insult. In addition, we also detailed the mechanism of
whichclearlyindicatedtheimplicationofmitochondriainthecell
cellularprotectionaffordedbymelanoidins,whichclearlyinvolves
death induced byH O .
2 2 protection against intra-mitochondrial oxidation and oxidative-
In this context, we have previously reported that melanoidins induced mitochondrial impairment assessed as MMA and MMP
isolated from prunes exert remarkable protection against oxida- depolarization.Webelievethisworkaddsnewinsightconcerning
tive-induced ECs death [11], but whether melanoidins from theeffectofprocessedplantfoodsoncellularphysiology.Indeed,
apricots possess the same functional proprieties was so far melanoidins from different sources could have different effects,
unknown. Only the partial structure of melanoidins have been andbecauseofthelackofknowledgeinthisfield,itisimperative
elucidated so far and the chemical precursor species responsible thatvariousmelanoidinsbeevaluatedunderdifferentexperimen-
remain largely undefined [2], thus melanoidins from different tal conditions to determine their effects. Although further studies
foodsmayhavedifferentstructuresandactivities.Inthiscontext, are required to better characterize the molecular mechanism of
we believe our results may be of importance, since they indicate melanoidin protection, our findings support the general observa-
forthefirsttimethatmelanoidinsisolatedfromdriedapricots,as tion that natural antioxidants from fruits and vegetable can have
previously reported from those of prunes [11], exert a significant a cardiovascular protective effect against oxidative stress. To our
cellular protection against H O -induced oxidative stress and knowledge, plasma, organs and tissue levels of melanoidins in
2 2
mitochondrial-mediatedECdeath.Inparticular,consideringthat peoplearesofarunknown,thereforewhethertheprotectiveeffect
compounds that participate in melanoidin formation in prunes exertedbymelanoidinsinculturedcellsmaybetranslatedinvivo
and apricots differ both analytically and quantitatively, alternate remains to be elucidated. However, there is circumstantial
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ApricotMelanoidinProtectagainstOxidativeDeath
evidence of melanoidin absorption in vivo [33], and consistent piecesoffruit,wasfractionIV.Theyieldofeachfraction(asgper
with this observation, their antioxidant activity in human 100g ofdried fruit) was recorded.
volunteers has indeed been reported [34–36]. In addition, data
obtained with gravimetric techniques allow estimation of a daily Determination of Non-enzymatic Browning (NEB)
intakeofabout10gofmelanoidinsinaWesterndiet[33].These NEB was assessed by both browing index and color variation.
observations indicate that melanoidins may reach invivo con- TheformationofbrownpigmentduetotheNEBreactioncanbe
centrations comparable to the ones we used in vitro, suggesting estimated as a brown index from spectrophotometric readings at
that our results could be representative of a physiologically 420nm[29].Forthisreasontheselectedsamplesweresubjected
relevant invivomechanism. toaspectrophotometricreadinginabsorbancemodeat420nmin
a 1 cm glass cuvette (Beckman DU 640 spectrophotometer). The
Materials and Methods samples were appropriately diluted in water to give absorbance
valuesof,1.Thesevalueswereusedtogiveanabsorbancevalue
Chemicals
pergdmofeachdilutedfraction.Fivemeasurementsweremade
UnlessstatedinthetextallthereagentsusedwerefromSigma foreachsample.Thecolorimetricanalysishasbeencarriedoutas
(Sigma, StLouis, MO). proposed by Mastrocola and Lerici [27]. The peel colour
measurement were assessed with a tristimulus colorimeter
Sampling, Dehydration and Blanching (Chromameter-2 Reflectance, Minolta, Osaka, Japan), fitted with
TheexperimentswereconductedontheCafonaapricotvariety, a CR-300 measuring head. The colour tonality was expressed as
which has been chosen for its very high content in polyphenols. L,a*,b*Hunterscaleparameters,and‘‘a’’and‘‘b’’wereusedto
Thefruitswerepurchasedlocallyatanoptimumstageofripening computehueangle(tan-1b*/a*)[28,39].Themeasurementshave
andthoseshowingdefectswerediscarded.Fruitsweresize-graded, beenalwaysdoneonthesamesetof10fruitsselectedatthestart
so that size difference would not affect drying times. Fruits were of theexperiment, inorder tominimisefruit colourvariability.
cutinhalfalongthesuturelinewithaknifeandthestonecarefully
removed by hand. At the end of this procedure, the fruits were Cells Culture Treatments
immediately checked to eliminate those that had been damaged ECV304isanendothelialcelllineestablishedfromtheveinof
and then, pre-treated and dried at 75uC as previously described an apparently normal human umbilical cord. This cell line has
[21]. Before analysis, the dried fruit was packed in co-extruded beenproposedasasuitablemodelforprovidingnovelinsightsinto
plastic bags and kept in a freezer 220uC. The blanching was the mechanisms governing ECs biology under both physiological
executedaspreviouslyreportedbyboilingtheselectedsamplesin and pathological conditions [11,40,41]. ECV304 were provided
water at 90uC for3 minutes[37]. by the European Collection of Animal Cell Cultures (ECACC
Salisbury,UK).CellsweregrowninmediumM199supplemented
Determination of Total Antioxidant Activity with10%fetalbovineserum(Invitrogen,Carlsbad,CA),100mg/
The polyphenol fraction, which was used to assess the m1 penicillin, and 100mg/m1 streptomycin (Invitrogen,). Cells
antioxidant activity, color variation and browing index, was were maintained ina standard culture incubator withhumidified
extracted as previously reported [26]. Antioxidant activity was aircontaining5%CO at37uC.Thedaybeforeeachexperiment,
2
evaluatedusingtheradicalDPPHmethodaspreviouslydescribed cells were plated in 24-well plates (Corning, Lowell, MA) at
in detail[26] andexpressed as-Abs23 min21g21of dm. a concentration of 100,000 cells per well and pretreated with
melanoidins for 6 hrs before oxidative stress was induced in the
Melanoidins Extraction last 2 hrs, by treatment with the indicated concentration of
The extraction of melanoidins was carried out in triplicate, hydrogen peroxide (H2O2). In accord with our previously study
followingapreviouslypublishedmethod[3,11,38].Indetail,100g usingprunemelanoidinsonhumanECs[11],thedosesof2,6and
of pitted and ground apricots were defatted with CHCl while 12mg/mlwere tested inour vascularmodel.
3
stirring. After solvent evaporation, the operation was repeated
twice. Solvent traces were eliminated by rotary evaporation. Cell Viability and Metabolic Assay
200mlofbi-distilledwaterwereaddedtothisresidualsolid,and Cellviability,fortreatedanduntreatedcells,wasassessedafter
the resulting slurry was sonicated for 30 minutes at 40uC. The 24hrs by checking the leakage of the cytoplasmatic lactate
water fraction was collected and the operation repeated on the dehydrogenase(LDH)fromcellswithadamagedmembrane.The
solid phase. The two water fractions were combined and amount of LDH released in the medium by death cells was
centrifugedat8400gfor15minutesat15uC,andthesupernatant assessedusingthekitCytoTox-ONETM(Promega,Madison,WI).
wasthenevaporatedundervacuumatthemaximumtemperature A standard curve with definite amounts of cells (200m/well) was
of 50uC (fraction I). The residual solid was added to that of made,andthereleaseofLDHinthemediumwasmeasuredafter
fractionIanddissolvedin200mlofethanol/water (60:40V/V), the application of lysis solution (4ml/well). Plates containing
andtheresultingslurrywasthensonicatedfor30minutesatroom samples were removed from the incubator and equilibrate to
temperature.Thisoperationwasrepeated.Thetwoethanol:water 22uC,andthenthereleaseofLDHfromdeathcellswasmeasured
fractionswerecombinedandcentrifugedat8400gfor15minutes by supplying lactate, NAD+, and resazurin as substrates in the
at15uC,andthesupernatantwasthenevaporatedundervacuum presenceoftheenzymediaphorase.Generationofthefluorescent
at the maximum T of 50uC (fraction II). The residual solid was resorufin product, which is proportional to the amount of LDH,
added to that of fraction II and dissolved in 200mL of 2- was measured using a GENios plus micro-plate reader (Tecan)
propanol/water (50:50V/V), and the resulting slurry was then withexcitationandemissionof560nmand590nm,respectively.
sonicatedfor60minutesatroomtemperature.Thisoperationwas Byusingthestandardcurve,theamountofLDHreleaseintreated
repeated. The two propanol/water fractions were combined and anduntreatedcellwasconversedinnumberofcellsperwell.The
centrifugedat8400gCfor15minutesat15u,andthesupernatant Mitochondrial Metabolic Activity was assessed as previously
was then evaporated under vacuum at the maximum T of 50uC reported in 96-well plates (BD Falcon) by using the colorimetric
(fraction III). The remaining solid fraction, which consisted of assayMTT(Promega,Madison,WI)[42].Thiscolorimetricassay
PLOSONE | www.plosone.org 9 November2012 | Volume 7 | Issue 11 | e48817
ApricotMelanoidinProtectagainstOxidativeDeath
measures the reduction of the yellow 3-(4,5-dimethythiazol2-yl)- Measurement of Mitochondrial Membrane Potential
2,5-diphenyl tetrazolium bromide by mitochondrial succinate Measurementofmitochondrialmembranepotential(MMP)was
dehydrogenase. The yellow tetrazole compound enters the cells performed with the JC-1 stain (Invitrogen), a lipophilic cation
and passes into the mitochondria where it is reduced to an fluorescent dye that accumulates in mitochondria in a MMP-
insoluble, purple colored, formazan product. The reduction of dependent manner, showing red fluorescent JC-1 aggregates
MTT in isolated cells and tissues is regarded as an indicator of (590 nmemissions)athigherMMP.WhenMMPdecreases,JC-1
‘‘cellredoxactivity’’[43].AlthoughevidenceexiststhattheMTT aggregates depart from mitochondria and change to green
reduction in mammalian cells is also catalyzed by a number of fluorescent JC-1 monomers (535 nm emissions). Therefore, the
non-mitochondrial enzymes, this reaction is attributed mainly to ratio of the red signal to the green can been used to detect the
mitochondrial enzymes and electron carriers [43–45]. After
occurrenceofMMPdepolarizationintheearlystagesofcelldeath
treatments cells were added with 20ml MTT solution (5 mg/ml)
duetomitochondrialdamage[49,50].Aftertreatmentscellswere
inmediumM199andincubatedat37uCinacellincubatorfor60 incubatedatroomtemperatureinthedarkwith5 mg/mlJC-1in
minutes. At the end of the incubation period, the medium was
HBSS for 30 minutes. The cells were then washed twice with
removed and the cell monolayer was washed twice with HBSS.
HBSS and fluorescence levels were immediately acquired with
Theconverteddyewassolubilizedwithacidicisopropanol(0.04N
excitation and emission wavelengths set at 535 and 590nm,
HClinabsoluteisopropanol),andplateswereanalyzedat570nm
respectively, for red fluorescence, and 485 and 535nm, re-
usingaGENiosplusmicro-platereader(Tecan)withbackground
spectively, forgreen fluorescence. Measurements were performed
subtraction at 650nm. Results were expressed as percent of
in clear 24-well plates (Corning, Lowell, MA) on a fluorescence
untreated control cells.
plate reader GENios plus (Tecan, Ma¨nnedorf, CH) from the
upper side using multiple reads per well (the read pattern was
Determination of the Intracellular Redox State
square,andthenumberofreadswas262).Foreachsample,the
Intracellular redox state was investigated by using the redox- results were calculated as the ratio (red/green) of fluorescence of
sensinggreenfluorescentprotein(roGFP),whichreportstheredox
sample,averagedafterthefluorescencevalueshadbeencorrected
stateoftheGSH/GSSGpoolinvivoinbothplantandmammalian
forthebackground andprotein content.
cells[46,47].PlasmidscodingforroGFPexpressionwereobtained
startingfrompCVU55762-roGFP2(kindlyprovidedbyDr.Andreas
Staining and Fluorescence Visualization
J. Meyer, University of Heidelberg, Germany). Cyto-roGFP was
MitoTracker Red CMXRos (Invitrogen, catalog # M7512) is
obtained by restriction cloning using BamHI and NotI restriction
the oxidized form of a dye that can be taken up into the
enzymes into pcCDNA3 vector (Invitrogen); mito-roGFP2 was
mitochondria oflive cells utilizing theiruniquely highmembrane
obtained by cloning a PCR amplification product into pCMV/
potential. This dye is retained in the mitochondria after fixation
myc/mito(Invitrogen)usingPstIandXhoIsites.Plasmidscontain-
andthereforecanbeusedtolabel/stainmitochondriafollowedby
ing cytoplasmic roGFP2 (cyto-RoGFP) and a mitochondrial
additionalimmunocytochemistry.Forthestaining,cellshavebeen
targeted roGFP (mit-RoGFP2) were transfected in HCV304 by
grown on glass coverslips inserted inside the multi-wells. At the
using the lipofectamine 2000 reagent following the provider
endofexperiments,theculturemediahasbeenremovedandthe
protocol (Invitrogen). Transfected cells were selected using
cells monolayer washed with pre-warmed PBS. Then the pre-
0.8 mg/mL of G418 in the media for 3 to 4 weeks. Positive
warmed solution containing the MitoTrackerH probe has been
stably transfectants were selected by serial dilution of G418-
addedtoeachwell(finalprobeconcentrationof300nM)andthe
resistant clones which constitutively expressed both cyto- and
mito-RoGFP2 under a fluorescence microscope (Olympus XI70). cells have been incubated at 37uC for 30 minutes. After the
staining was complete, cells were fixed with a solution of 4%
RoGFP has two fluorescence excitation maxima at 400 (oxidized
form)and485nm(reducedform)anddisplayrapidandreversible formaldehyde in complete growth medium at 37uC for 15
ratiometric changes in fluorescence in response to changes in minutes,andthepermeabilizedfor10minutesinPBScontaining
ambient redox potential. The ratios of fluorescence from 0.2%TritonHX-100.Hoechst33342(SIGMA,catalog#B2261),
excitation at 400 and 485nm indicate the extent of oxidation ispartofafamilyofbluefluorescentdyescommonlyusedtostain
and thus the redox potential while canceling out the amount of DNA. After fixation/permeabilization, the Hoechstdye has been
indicator and the absolute optical sensitivity [47]. In place of addedtothecellsatafinalconcentrationof0.12 mg/ml.Thedye
confocal imaging analysis we used a recently developed fluorom- hasbeenlefttoincubatewiththecellsfor15minutesandthenthe
eter-based method for monitoring roGFP oxidation [11,48]. cells monolayer has been washed for five times with PBS before
Fluorescence measurements were performed in clear 24-well visualization. Fluorescence visualization of fixed cells has been
plates (Corning, Lowell, MA) on a fluorescence plate reader performedonaOlympusBX51microscope,usinga20,40,and
GENios plus (Tecan,Ma¨nnedorf,CH) fromtheupper sideusing 1006objectiveswithNumericalAperture(NA)of0,70,1,00and
multiple reads per well (the read pattern was square, and the 1,35 respectively.
number of reads was 262). Cells were excited by using 400 and
485nm filters and fluorescence values were measured using Statistical Analysis
535nm emission filter. For background correction emission Data were expressed as means 6 S.E.M. of three or four
intensities were determined for non-transformed cells (4 discs different experiments. One-way analysis of variance (ANOVA)
each experiment) exposed to same excitation wavelengths under followedbyapost-hocNewman-KeulsMultipleComparisonTest
the same conditions. These values were averaged and subtracted were used to detect differences of means among treatments with
fromthefluorescencevaluesofroGFP2.Thedegreeofoxidation significancedefinedasP,0.05.Statisticalanalysiswasperformed
of the roGFP2 was estimated from the ratios of light intensities using GraphPad Prism version 5.00 for Windows, GraphPad
obtained during 1-min intervals under 400- and 485-nm Software, San DiegoCalifornia USA.
excitation. Treatment-induced variations of roGFP2 oxidation
were estimated by comparison with roGFP oxidation in control
untreated cells.
PLOSONE | www.plosone.org 10 November2012 | Volume 7 | Issue 11 | e48817