quercetin affects erythropoiesis and heart mitochondrial function in

Research ArticleQuercetin Affects Erythropoiesis and Heart MitochondrialFunction in Mice

Lina M Ruiz1 Celia Salazar1 Erik Jensen2 Paula A Ruiz3 William Tiznado4

Rodrigo A Quintanilla1 Marlen Barreto1 and Alvaro A Elorza25

1Center of Biomedical Research Faculty of Health Sciences Universidad Autonoma de Chile Ricardo Morales 33698910132 Santiago Chile2Center for Biomedical Research Faculty of Biological Sciences and Faculty of Medicine Universidad Andres BelloRepublica 217 8370146 Santiago Chile3iEng Solutions Ltd London N12 0DR UK4Department of Chemical Sciences Faculty of Exact Sciences Universidad Andres Bello Republica 275 8370146 Santiago Chile5Millennium Institute of Immunology and Immunotherapy Santiago Chile

Correspondence should be addressed to Lina M Ruiz linaruizuautonomacl and Alvaro A Elorza alvaroelorzaunabcl

Received 30 January 2015 Revised 8 May 2015 Accepted 11 May 2015

Academic Editor Victor M Gonzalez

Copyright copy 2015 Lina M Ruiz et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Quercetin a dietary flavonoid used as a food supplement showed powerful antioxidant effects in different cellularmodels Howeverrecent in vitro and in vivo studies in mammals have suggested a prooxidant effect of quercetin and described an interaction withmitochondria causing an increase in O

2

∙minus production a decrease in ATP levels and impairment of respiratory chain in livertissue Therefore because of its dual actions we studied the effect of quercetin in vivo to analyze heart mitochondrial functionand erythropoiesis Mice were injected with 50mgkg of quercetin for 15 days Treatment with quercetin decreased body weightserum insulin and ceruloplasmin levels as compared with untreated mice Along with an impaired antioxidant capacity in plasmaquercetin-treated mice showed a significant delay on erythropoiesis progression Heart mitochondrial function was also impaireddisplaying more protein oxidation and less activity for IV respectively than no-treated mice In addition a significant reductionin the protein expression levels of Mitofusin 2 and Voltage-Dependent Anion Carrier was observed All these results suggestthat quercetin affects erythropoiesis and mitochondrial function and then its potential use as a dietary supplement should bereexamined

1 Introduction

The generation of reactive oxygen species (ROS) due tonormal cell metabolism and the accumulative damage theycause to DNA proteins and lipid membranes have beenassociated with the development of many acquired diseasesand agingThus antioxidant therapies especially through theintake of nutraceutical pills as food supplement have becomepopular in our communities However in vivo studies of theantioxidant properties of dietary flavonoids have shown someparadoxical effects on human health [1] making importantto investigate further and deeper the mechanism of action ofthese supplements

Quercetin is one of themost abundant dietary flavonoidswith the highest antioxidant capability [2 3] modulating theexpression of different antioxidant enzymes such a catalaseand superoxide dismutase and increasing the intracellu-lar levels of glutathione [4ndash6] Furthermore multiple anddiverse functions have been ascribed to quercetin such asan antihypertensive [7] anticoagulant [8] antiatherogenic[7] antibacterial [9] and antiproliferative [10] In last yearsconflicting biological effects of quercetin have been reportedwhich might be related to its metabolites its dose and thecellular redox state [11ndash14] During ROS scavenging processquercetin gets oxidized and it further reacts with glutathioneand the protein thiol groups leading to consumption of

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 836301 12 pageshttpdxdoiorg1011552015836301

2 Oxidative Medicine and Cellular Longevity

glutathione an increase in cytosolic calcium concentrationand lactate dehydrogenase leakage [11]

Quercetin may interact directly with mitochondrialmembranes [15 16] affecting its fluidity This has also beenassociated with mitochondrial dysfunction through the inhi-bition of respiratory chain or uncoupling [15 16] In additionquercetin affects mitochondrial calcium regulation increasesO2∙minus production and induces the opening of mitochondrial

transition pore (mPTP) in HCT116 cells [17] The abovestudies suggest quercetin could promote oxidation affectingmitochondrial function raising concerns about the validityof quercetin as an antioxidant [17]

At molecular level quercetin is potent iron chelatorwhich is shown to alter the expression of proteins involved iniron absorption affecting iron homeostasis [18 19] Iron is anessential element in each of the four mitochondrial electrontransfer complexes either as FeS clusterrsquos or hemersquos compo-nent [20] Iron deficiency leads to altered cell metabolism[20] and anemia [21 22] Red blood cell development isthe most iron requiring process for oxygen transport aswell as the most active cell generator system involving bothproliferation and differentiation from hematopoietic stemcells which are also dependent onmitochondrialmetabolism[21 23] Additionally iron deficiency-induced anemia canhave deleterious effects on heart [24] and cardiomyopathydevelopment is related with mitochondrial dysfunction dueto ROS excess [25 26] Both erythropoiesis and the cardiactissue are then suitable and attractive targets for quercetinmechanistic studies

Our working hypothesis is that quercetin interferes withmitochondrial function exacerbating mitochondrial ROSgeneration and altering the physiology of tissues highlydependent on iron metabolism and mitochondrial functionsuch as the erythroid and cardiac tissue We are interestedin addressing the in vivo prooxidant effect of quercetin onmitochondrial function in these tissues

Adaptive responses of mitochondria to maintain cellrsquosbioenergetics capacity under stressful conditions involve theremodeling of mitochondrial respiratory complexes to buildup or down supramolecular structures called supercom-plexes and themitochondrial fusion and fission events calledmitochondrial dynamics The former will allow a bettersubstrate channeling to preclude extreme production of ROSfrom the normal respiratory chain function [27ndash29] Thelatter will control energy expenditure and metabolic repro-gramming [30ndash33] Upregulation of Mitofusin 2 (MFN2)protein involved inmitochondrial fusion and thenwith elon-gated mitochondria has been associated with a protectiverole against apoptosis hypoxia and ROS [32 34 35] aswell as with higher oxidative capacity by regulating in partthe respiratory complex proteins expression [35ndash38] MFN2expression is regulated in turn by the peroxisome proliferatoractivated receptor-gamma coactivator-1120572 (PGC-1120572) under avariety of conditions characterized by energy expenditure[39] PGC-1120572 is cofactor that participates in the regulationof mitochondrial biogenesis and activation of peroxisomeproliferator activated receptor-120574 (PPAR 120574) pathway [40 41]

Our results showed that quercetin clearly affected mito-chondrial function inmice Interestingly quercetin decreased

erythropoiesis and reduced the expression levels of mito-chondrial proteins that control mitochondrial dynamicsThese results proposed that the antioxidant properties ofquercetin need to be reevaluated given their widespread use[42]

2 Materials and Methods

21 Animals and Experimental Design Male C57BL6 mice(12 months of age) were daily administered intraperitoneally(ip) with 50mgkg quercetin (Cat Q4951 Sigma) or withvehicle (5 DMSO) and PBS for control animals during 15days Animals were daily checked for weight and health con-ditions Right after the treatments animals were submitted todifferent tests indicated below and then sacrificed for heartand bone marrow dissections Ip injections of quercetin inrodents have been reported previously in [43ndash47] and thedose of 50mgkg of quercetin has been reported in [47ndash50]

22 Strength Test To evaluate strength resistance and exer-cise abilities mice performed the Kondzielarsquos test and aweightlifting test Briefly Kondzielarsquos inverted screen is a testfor muscle strength using all four limbs in which eachmousewas placed in the center of a screen and then rotated toan inverted position with the mousersquos head declining firstthe mice falling time was recorded The performance of eachmouse in the inverted screen was scored as follows Fallingbetween 1ndash10 sec = 1 11ndash25 sec = 2 26ndash60 sec = 3 over 60 sec= 4 [51] On the other hand the weightlifting is a test forforelimbs muscle strength The weightlifting assessed theability to raise seven weights ranging from 20 g to 98 g (2033 46 59 72 85 and 98 g) First the mouse was allowed toraise the lightestweight (20 g) for 3 sec andup to 3 timesAftera 10 sec rest in between each lift the second and third raiseswere performed to move onto the next heaviest weight Thetrial finishes when the mouse fails to lift or hold the weightafter three attempts recording the maximum timeweightachieved The score calculations was made according to [51]and normalized by the body weight

23 Bone Marrow Analyses Bone marrow was isolated fromboth legs and immunostained with the antibodies phy-coerythrin- (PE-) conjugated anti-TER119 and fluoresceinisothiocyanate- (FITC-) conjugated anti-CD71 according to[30 52] Progression of erythropoiesis was then assessed byflow cytometry [30]

24 Total Antioxidant Capacity Antioxidant capacity wasevaluated using OxiSelect Total Antioxidant Capacity (TAC)Assay Kit (Cat STA-360 Cell Biolabs Inc) according tomanufacturerrsquos instructions Results are expressed as ldquo120583MCopper Reducing Equivalentsrdquo and compared with controland quercetin-treated samples

25 Insulin Determination For the quantitative determina-tion of insulin in mouse plasma was used the Mouse InsulinELISA kit (Cat 80-INSMS-E01 APLCO) according with themanufacturerrsquos instructions [53]

Oxidative Medicine and Cellular Longevity 3

26 Western Blot Mitochondrial proteins were preparedfrom fresh isolated mitochondria solubilized in 20mMHEPES 2mMEDTA 05Triton-X100 150mMNaCl 1mMPMSF and a HALT protease cocktail inhibitor [30] Thesewere then buffered and fractionated in 8 Bis-Tris polyacry-lamide gel with MOPS and a 5mM sodium bisulfite runningbuffer before being transferred onto a 02 120583m PVDF mem-branewithNUPAGE transfer buffer in the semidry apparatusThe antibodies PGC1-120572+120573 (Cat ab72230 Abcam) MFN2(Cat ab50843 Abcam) VDAC1Porin (Cat ab15895Abcam) ceruloplasmin (Cat ab8813 Abcam) and 120573-actin(Cat ab8227 Abcam) were used according to a previousstudy [30 32] Detection of carbonyl groups introduced intoproteins by oxidative stress was performed with the OxyBlotProtein Oxidation Detection Kit (Cat S7150 Millipore)according to [30 32] The quantity of ceruloplasmin wasevaluated in plasma samples mixing 5120583L of plasma 5 120583Lof sample buffer and 15 120583L of H

2O The sample was boiled

at 95∘C for 10min and centrifuged and run in a proteinelectrophoresis and blotted in PVDF membrane for cerulo-plasmin immunodetection [30]

27 Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Mitochondria were isolated by differential centrifu-gation Mitochondrial proteins were solubilized with theNativePAGE Sample Prep Kit and 80 120583g per well were loadedonto a 3ndash12 polyacrylamide gradient NativePAGE NovexBis-Tris Gel (Invitrogen Carlsbad CA) [30]

28 In-Gel Activity Assay (IGA) Complex I in-gel activity(CI-IGA) was detected by incubating BN-PAGE gels rightafter electrophoresis in 100mM Tris-HCl pH 74 with1mgmL nitro blue tetrazolium and 014mMNADH at roomtemperature for 60min in the dark with gentle rocking[54] Complex IV in-gel activity (CIV-IGA) was detected byincubating the gel with 01 (wv) 331015840-diaminobenzidine01 (wv) cytochrome c and 24 unitsmL catalase in 1mMTris-HCl pH 74 at 37∘C for 6 h in the dark with gentlerocking [28 55] Complex II in-gel activity (CII-IGA) wasdetected by incubating the gel in 5mMTris-HCl pH 74 with20mM sodium succinate 02mM phenazine methosulfateand 25mgmL nitro blue tetrazolium [30 56]

29 Statistics Statistical analyses were performed with Ori-gin Pro8 with a significance level set at 119875 le 005 UnpairedStudentrsquos 119905-test was used when comparing 2 average values

3 Results

31 Effects of Quercetin on Metabolism and Strength Supportin Mice Evidence suggests that quercetin is able to modulatemetabolism in mice [57 58] to evaluate the metabolicstatus of quercetin-treated mice we measured body weightand plasma insulin levels Mice body weight was measureddaily for 15 days decreasing significantly up to 25 uponthe treatment with quercetin at 50mgkg as comparedwith control mice (Figure 1(a)) Along with the weight lossquercetin-treatedmice had significantly lower plasma Insulin

levels (05 ngmL plusmn 01) than control ones (21 ngmL plusmn 06)(Figure 1(b)) confirming an altered metabolism Normalaverage plasma insulin level in mice is 06 plusmn 01 ngmL [59]and the range of native insulin level could be found between01 and 29 ngmL

Previous reports of epidemiological and clinical studiesin humans showed that treatment with quercetin improvedcardiovascular health [58] To test cardiovascular benefitsinduced by quercetin we evaluate if quercetin treatmentaffects exercise abilities enhancing muscle strength and resis-tance by the weightlifting capability test for forelimbs musclestrength (Figure 1(c)) and the Kondzielarsquos inverted test forresistance and strength (Figure 1(d))Our studies showed thatquercetin-treated mice performed similarly on both strengthtests than control mice

32 Quercetin Treatment Affects Iron Metabolism and Ery-thropoiesis The ferroxidase ceruloplasmin can be used asmarkers of iron deficiency [60 61] Ceruloplasmin is essentialfor iron homeostasis by favoring cellular iron release [62] andhas been described to decrease upon iron deficiency [61ndash63]In our study the levels of ceruloplasmin were significantlyreduced in 50 (119875 lt 005) as compared with controlmice (Figures 2(a) and 2(b)) Furthermore the plasmarsquos TotalAntioxidant Capacity (TAC) was evaluated showing a no sig-nificant decrease upon the quercetin treatment (Figure 2(c))

The erythropoiesis process is highly dependent on irontransport and metabolism Flow cytometry on bone mar-row isolated cells from quercetin-treated mice displayeda significant delay on erythropoiesis progression as com-pared with control mice Immature erythroid populations R1(CD71med-TER119low) and R2 (CD71high-TER119low) showeda significant increase as compared with vehicle-treatedmice (Figures 3(a) and 3(b)) On the other hand theiron dependent cell populations of erythropoiesis for hemeand hemoglobin biosynthesis R3 (CD71high-TER119high) R4(CD71med-TER119high) and R5 (CD71low-TER119high) wereall of them significantly decreased in quercetin-treated mice(Figures 3(a) and 3(b)) These observations suggest thatquercetin treatment may induce anemia given the significantdelay on erythropoiesis progression (Figures 2 and 3)

33 Effect of Quercetin on Heart Mitochondrial FunctionPrevious work showed the prooxidant effect of quercetinin vitro in the HCT116 human colon tumor cells and livertissue [15 17] In addition heart function is highly dependenton aerobic metabolism and then mitochondrial functionthat is the oxidative phosphorylation system (OXPHOS) forenergy generation [25 26] We analyzed the effect quercetinon heart mitochondrial function analyzing respiratory com-plexes activity in response to quercetin treatment In-gelactivity (IGA) was tested for mitochondrial Complexes III and IV as well as for supercomplex rearrangementsusing heart mitochondrial proteins from quercetin- andvehicle-treated mice For Complex I no significant changeswere observed at the level of monomeric Complex I norsupercomplex rearrangement (Figures 4(a) and 4(b)) Similar


100

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wei

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)

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ghtli

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t

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ght

(d)

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lowast

95th percentile

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25th percentile

5th percentile

998686 Mean (998686)

(e)

Figure 1 Effects of quercetin treatment on weight and plasma insulin levels in mice Quercetin modulates mice metabolism (a) Mice weretreated with quercetin (grey circles) for 15 days and body weight was evaluated Quercetin-treated mice lost around 25 of body weight ascompared with untreated mice (black squares) lowast119875 lt 005 control mice (119899 = 11) quercetin-treated mice (119899 = 9) (b) Insulin plasma levelswere measured using mouse insulin ELISA Quercetin-treated mice showed a significant decrease in the plasma levels of insulin as comparedwith control mice lowast119875 lt 005 (c) and (d) Quercetin treatment affects metabolic performance and exercise abilities (c) Weightlifting test offorelimbs muscle strength test and (d) Kondzielarsquos inverted test muscle strength and resistance using all four limbs (d) Quercetin-treatedmice performed similarly on both strength tests than control mice (c d) The score obtained in the strength test was normalized by the bodyweight Average values were analyzed by two-sample 119905-test (119875 lt 005) control mice (119899 = 11) and quercetin-treated mice (119899 = 9) Significantdifferences (lowast) were detected between control and quercetin-treated mice (e) Box chart shows the 25th and 75th percentiles The whiskersshow the 5th and 95th percentiles Additional values are show in box chart including the minimum (lowast) median mean () maximum (lowast)the 1st and 5th percentiles and 95th percentiles


Control Quercetin

Cp

IgG

(a)

Control Quercetin

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998686 Mean (998686)

(b)

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Quercetin

Cop

per r

educ

ing

equi

vale

nts (120583

M)

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Median

Minimum value

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lowast

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75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(c)

Figure 2 Effect of quercetin treatment on mice iron metabolism (a) Plasma ferritin levels from quercetin-treated mice were measured byELISA as it was described in Section 2 Treatment with quercetin did not affect ferritin levels as compared with untreated mice (b) TotalAntioxidant Capacity was measured in plasma samples from quercetin-treated mice using TAC Oxyselect kit and results are expressed asldquo120583MCopper Reducing Equivalentsrdquo The value of the Copper Reducing Equivalents is directly proportional to the total antioxidant capacity(c) Mice were treated for 15 days with quercetin and then ceruloplasmin plasma levels were measured by western blot (d) Densitometryanalysis of ceruloplasmin levels performed with the ImageJ software Ceruloplasmin plasma levels of mice treated with quercetin significantdecreased 57 compared to untreated mice lowast119875 lt 005 compared to control Control mice 119899 = 11 quercetin-treated mice 119899 = 9 Each bar(box charts) represents the mean plusmn SD analyzed by two-sample 119905-test (119875 lt 005) Significant differences (lowast) were found between control andquercetin-treated mice lowast119875 lt 005

results were obtained for Complex II (Figures 4(e) and 4(f))However at the level of Complex IV (Figures 4(c) and4(d)) quercetin treatment significantly reduced the activityof monomeric Complex IV (not being part of the super-complexes CICIII

2CIV1minus4

) Altogether these observations

indicate that quercetin significantly altered mitochondrialfunction through deregulation of Complex IV activity

The effect of quercetin on heart mitochondrial functionwas also studied at the level of mitochondrial biogen-esis and mitochondrial dynamics Protein expression for


1054

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1013 102 103 1042

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Ter119-PE

Ter119-PE

Control

Quercetin

(a)

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0

Cel

l (

)

R1 R2 R3 R4 R5Erythroid populations

lowast

lowast

lowast

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lowast

ControlQuercetin

(b)

Figure 3 Erythropoietic progression in bonemarrow cells is altered in quercetin-treatedmice (a) FACS plots (a) Five erythroid populationsfrom immature to mature (R1 R2 R3 R4 and R5) are distinguished by CD71 and TER119 expression levels in freshly isolated bone marrowthrough flow cytometry (CD71med-TER119low CD71high-TER119low CD71high-TER119high CD71med-TER119high and CD71low-TER119high) (b)Quantitative analysis of cell progression CTR mice 119899 = 11 (red bars) quercetin-treated mice 119899 = 9 (green bars) Each bar represents themean plusmn SD analyzed by two-sample 119905-test (119875 lt 005) Significant differences (lowast) were found between control and Quercetin-treated micelowast119875 lt 005 compared to control

PGC-1120572 MFN2 and VDAC was analyzed by immunoblots(Figure 5) PGC-1120572 the master regulator of mitochondrialbiogenesis is upregulated upon energy expenditure anddemand [39] Mitochondrial dynamics (MtDy) given bythe balance between fusion and fission events control notonly mitochondrial morphology but rather mitochondrialfunction mitochondrial turnover and bioenergetics MFN2a mitochondrial fusion protein located on the outer mito-chondrial membrane has been shown to be upregulatedupon stressful conditions [36 37] The Voltage-DependentAnion Carrier (VDAC) also performs several key functionsincluding regulating the shape and structure ofmitochondriainteraction with hexokinase and apoptosis signaling [64]The immunoblot results showed quercetin treatment did

not affect the expression of PGC-1120572 On the other handVDAC andMFN2 protein expression levels were significantlydecreased in quercetin-treated mice (Figures 5(a) and 5(c))Mitochondrial dysfunction is normally correlated with anincrease in the reactive oxygen species (ROS) To evaluateredox status in heart mitochondria protein oxidation wasassessed by the OxyBlot methodology [65] Treatment withquercetin showed a significant increase in mitochondrialprotein oxidation as compared with control (Figures 5(b) and5(d))

Altogether our results suggest that in vivo quercetin treat-ment is associated with a severe mitochondrial dysfunctiondrastically affecting erythropoiesis and heart mitochondria


Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

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06

05

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03

02

01

00

In g

el ac

tivity

com

plex

I (a

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CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

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04

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01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

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02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)

Figure 4 Quercetin treatment affects complexes OXPHOS properties in mice isolated mitochondria Heart mitochondria isolated fromquercetin-treated mice were digitonin solubilized and fractionated by BN-PAGE and then followed by Complex I (a) Complex II (b) andComplex IV (c) in-gel activity assays (a) IGAComplex I densitometry analysis for supercomplex andmonomer fraction (b) IGAComplex IVdensitometry analysis for supercomplex and monomer fraction Images show three independent experiments Each bar represents the meanplusmn SD analyzed by two-sample 119905-test (119875 lt 005) Abbreviations used are as follows blue native polyacrylamide gel electrophoresis (BN-PAGE)Complex I (CI) Complex IV (CIV) in-gel activity assay (IGA) and supercomplexes (SC) SC1CICIII

2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

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rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

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00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)

Figure 5 Effect of quercetin treatment on the expression of PGC-1120572 Mitofusin 2 VDAC and protein oxidation (a) Detection of carbonylgroups was performed with the OxyBlot Protein Oxidation Detection Kit (c) Densitometry quantification of carbonyl groups was madewith the ImageJ software Carbonylation of proteins was normalized by Ponceau staining and Complex V (CV) expression (b) Expressionof mitochondrial proteins Protein expression of MFN2 PGC-1120572 and VDAC1 was analyzed in heart isolated mitochondria from controland quercetin-treated mice 120573-actin was used as a loading control (d) Densitometry analysis MFN2 PGC-1120572 and VDAC1 expressions werenormalized by 120573-actin expression Each bar represents the mean plusmn SD analyzed by two-sample 119905-test (119875 lt 005) Control mice 119899 = 11quercetin-treated mice 119899 = 9 Each bar represents the mean plusmn SD analyzed by two-sample 119905-test (119875 lt 005) Significant differences (lowast) werefound between control and quercetin-treated mice 119875 lt 005

4 Discussion

In vitro and in vivo studies showed that quercetin may exertdual antioxidant and prooxidant properties that depend ontissue and cellular redox state [11ndash14] This study shows thatquercetin clearly affects heart mitochondrial function anderythropoiesis in mice We observed the prooxidant capacityof this polyphenolic flavonoid which induces higher levels ofcarbonylated (oxidized) heart mitochondrial proteins wheninjected in mice (Figures 5(a) and 5(c)) Associated with thisprooxidant property quercetin treatment induced a signif-icant decrease in the activity of the monomeric ComplexIV (Figures 4(e) and 4(f)) In addition quercetin treatmentclearly decreased MFN2 and VDAC levels (Figures 5(b) and5(d)) All these results are in agreement with the effects ofquercetin on mitochondria previously observed in vitro [1516] Previous evidence suggests that quercetin interacts withthe mitochondrial inner membrane inducing an inhibition

of respiratory chain and decreasing ATP levels [15] Thisis relevant because quercetin enters into cytosol and mayalso reach the mitochondria [4 66] Flavonoids can alsoinduce apoptosis in association with prooxidant activitiesinducing the mitochondrial transition pore (mPTP) [67 68]Quercetin induces the opening of mPTP mediated by Fe andCu [17] to release Ca2+ accumulated in mitochondria [15]

Larocca et al reported in bone marrow from humansadult patients suffering acute leukemia that quercetin couldinhibit leukemic cell growth without suppressing normalhematopoiesis [69] In that study bone marrow isolatedfrom the patients (in vitro experiments) was treated withquercetin every two days during two weeks and the abilityof human CD34+ cells to form both BFU-E and CFU-GMwas not affected by quercetin but the quercetin concentrationwas just 2 times 10minus5 M (approximately 6mgkg) [69] Bakheetreported that quercetin was not cytotoxic (DNA strandbreaks) to bone marrow at the tested doses of 50mgkg


and 100mgkg In addition bone marrow ROS productionlipid peroxidation and GSHGSSG ratio (reduced and oxi-dized glutathione) did not show significant variation aftertreatment of mice [70] However these experiments weremade for a short time two days The effect that we observedon the bone marrow from quercetin-treated mice (in vivoexperiments) was an evident and significant delay on theerythropoiesis progression (Figure 3) which suggests thedevelopment of anemia In the present study a decreasedactivity of Complex IV inmice treated with quercetin is clearThis is well correlated with decreased levels of ceruloplasmin(Figure 2) Our previous research [30] exposed that mildcopper deprivation in mice is correlated with a decreasedprotein expression and activity of Complex IV at the levelof OXPHOS supercomplexes along with a decrease in theceruloplasmin levels Our present results are suggesting apotential interaction between quercetin and copper ionswhich is in accordance with in vitro observations made byPękal et al [71] Laccases are multicopper oxidases struc-turally and functionally similar to ceruloplasmin Laccasesand ceruloplasmin exhibit reactivity in the hemoglobin-flavonoid system Then quercetin as a flavonoid in thepresence of ceruloplasmin may oxidize hemoglobin affectingthe viability of red blood cells [72] Galati et al reported thatthe generated phenoxyl radicals from flavonoid oxidation areresponsible for oxidation of oxy-hemoglobin directly in redcells and lysis of red cells It is known that hemoglobin isprone to oxidative damage and unable to transport molecularoxygen [73]

Quercetin has been proposed to increase mitochondrialbiogenesis through the regulation of PGC-1120572 pathway Daviset al treated mice (and HepG2 cells) with quercetin at125mgkg and 25mgkg for one week and showed thatquercetin increases brain andmusclemitochondrial biogene-sis through the activation of PGC-1120572 and sirtuin 1 increasingthe levels of mtDNA and cytochrome c in HepG2 cells andmice [74] On the other hand Casuso et al supplementedrats by oral gavage with 25mgkg of quercetin combinedwith exercise during 6-week [13] observing that quercetinsupplementation during exercise compromises both theexercise and the quercetin effects on brain mitochondrialcontent by disrupting the SIRT1-PGC-1120572 pathway Quercetinimpedes exercise-induced adaptations in the brainQuercetininduced oxidative damage which in the sedentary conditionis counteracted by modulating antioxidant activity [13] Inthis work we observed that quercetin treatment for twoweeks at 50mgkg did not affect the expression of PGC-1120572in heart In contrast VDAC andMFN2 protein levels showeda significant decrease in quercetin-treated mice (Figures 5(b)and 5(d)) These results suggest that quercetin affects mito-chondrial dynamics compromising metabolism substrateoxidation and the oxidative phosphorylation system [38]Alterations in the expression of MFN-2 have been reportedto cause a parallel change in protein expression levels ofComplexes I III and IV [34] These effects could be relatedwith our observations reported here where quercetin affectsComplex IV activity

Quercetin-treated mice displayed a weight loss andlower insulin levels which confirmed an altered metabolism

(Figure 1) Vessal et al show an antidiabetic effect of quercetinin diabetic rats [75] Li et al show a decrease in insulin levelsand weight loss in fructose-induced hyperinsulinemia [57]Xia et al proposed that under a nutritional balanced situationquercetin exerts prooxidant effects affecting cognition [12]Thus depending on the diet quercetin might have protectiveor detrimental effects on cell physiology

5 Conclusion

Quercetin is widely used as a dietary supplement in healthypeople as an antioxidant However our results suggest thatquercetin intake affects mitochondrial function in cardiactissue and also erythropoiesis which is a warning about thenutraceutical use of this compound

Conflict of Interests

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

Acknowledgments

The authors would like to thank the financial support byFONDECYT Grants nos 3110171 (Lina M Ruiz) 11130192(Lina M Ruiz) 1140358 (William Tiznado) and 1140968(Rodrigo A Quintanilla) and Nucleo-UNAB DI-209-12N(Alvaro A Elorza) Cochilco-Fondecyt 1100995 (Alvaro AElorza) and IMII P09-016-F (Alvaro A Elorza)

References

[1] J A Ross and CM Kasum ldquoDietary flavonoids bioavailabilitymetabolic effects and safetyrdquo Annual Review of Nutrition vol22 pp 19ndash34 2002

[2] K B Pandey and S I Rizvi ldquoPlant polyphenols as dietaryantioxidants in human health and diseaserdquo Oxidative Medicineand Cellular Longevity vol 2 no 5 pp 270ndash278 2009

[3] E Osorio E G Perez C Areche et al ldquoWhy is quercetin a bet-ter antioxidant than taxifolinTheoretical study of mechanismsinvolving activated formsrdquo Journal of Molecular Modeling vol19 no 5 pp 2165ndash2172 2013

[4] F Arredondo C Echeverry J A Abin-Carriquiry et al ldquoAftercellular internalization quercetin causes Nrf2 nuclear translo-cation increases glutathione levels and prevents neuronaldeath against an oxidative insultrdquo Free Radical Biology andMedicine vol 49 no 5 pp 738ndash747 2010

[5] R J Nijveldt E van Nood D E C van Hoorn P G Boelens Kvan Norren and P A M van Leeuwen ldquoFlavonoids a review ofprobable mechanisms of action and potential applicationsrdquoTheAmerican Journal of Clinical Nutrition vol 74 no 4 pp 418ndash425 2001

[6] AW Boots G RMM Haenen and A Bast ldquoHealth effects ofquercetin from antioxidant to nutraceuticalrdquo European Journalof Pharmacology vol 585 no 2-3 pp 325ndash337 2008

[7] F Perez-Vizcaino D Bishop-Bailley F Lodi et al ldquoTheflavonoid quercetin induces apoptosis and inhibits JNK activa-tion in intimal vascular smooth muscle cellsrdquo Biochemical andBiophysical Research Communications vol 346 no 3 pp 919ndash925 2006


[8] R Bucki T J J Pastore F Giraud J-C Sulpicejand and P AJanmey ldquoFlavonoid inhibition of platelet procoagulant activityand phosphoinositide synthesisrdquo Journal of Thrombosis andHaemostasis vol 1 no 8 pp 1820ndash1828 2003

[9] T P T Cushnie and A J Lamb ldquoAntimicrobial activity offlavonoidsrdquo International Journal of Antimicrobial Agents vol26 no 5 pp 343ndash356 2005

[10] N Gulati B Laudet V M Zohrabian R Murali and MJhanwar-Uniyal ldquoThe antiproliferative effect of Quercetin incancer cells is mediated via inhibition of the PI3K-AktPKBpathwayrdquoAnticancer Research vol 26 no 2 pp 1177ndash1181 2006

[11] AW Boots H Li R P F Schins et al ldquoThe quercetin paradoxrdquoToxicology and Applied Pharmacology vol 222 no 1 pp 89ndash962007

[12] S-F Xia Z-X Xie Y Qiao et al ldquoDifferential effects ofquercetin on hippocampus-dependent learning and memoryin mice fed with different diets related with oxidative stressrdquoPhysiology amp Behavior vol 138 pp 325ndash331 2015

[13] R Casuso E Martınez-Lopez F Hita-Contreras et al ldquoThecombination of oral quercetin supplementation and exerciseprevents brain mitochondrial biogenesisrdquo Genes amp Nutritionvol 9 pp 1ndash8 2014

[14] E J Choi K-M Chee and B H Lee ldquoAnti- and prooxidanteffects of chronic quercetin administration in ratsrdquo EuropeanJournal of Pharmacology vol 482 no 1ndash3 pp 281ndash285 2003

[15] D J Dorta A A Pigoso F E Mingatto et al ldquoThe interactionof flavonoidswithmitochondria effects on energetic processesrdquoChemico-Biological Interactions vol 152 no 2-3 pp 67ndash782005

[16] D J Dorta A A Pigoso F E Mingatto et al ldquoAntioxidantactivity of flavonoids in isolated mitochondriardquo PhytotherapyResearch vol 22 no 9 pp 1213ndash1218 2008

[17] U De Marchi L Biasutto S Garbisa A Toninello and MZoratti ldquoQuercetin can act either as an inhibitor or an inducerof the mitochondrial permeability transition pore a demon-stration of the ambivalent redox character of polyphenolsrdquoBiochimica et Biophysica Acta vol 1787 no 12 pp 1425ndash14322009

[18] M Lesjak R Hoque S Balesaria et al ldquoQuercetin inhibitsintestinal iron absorption and ferroportin transporter expres-sion in vivo and in vitrordquo PLoS ONE vol 9 no 7 Article IDe102900 2014

[19] R Hoque The effects of quercetin on iron metabolism [Doctoralthesis] Diabetes amp Nutritional Sciences Division School ofMedicine Kingrsquos College London University of London 2014

[20] R Lill B Hoffmann S Molik et al ldquoThe role of mitochondriain cellular iron-sulfur protein biogenesis and iron metabolismrdquoBiochimica et BiophysicaActaMolecular Cell Research vol 1823no 9 pp 1491ndash1508 2012

[21] J L Spivak ldquoThe anaemia of cancer death by a thousand cutsrdquoNature Reviews Cancer vol 5 no 7 pp 543ndash555 2005

[22] M Fontenay S Cathelin M Amiot E Gyan and E SolaryldquoMitochondria in hematopoiesis and hematological diseasesrdquoOncogene vol 25 no 34 pp 4757ndash4767 2006

[23] A D Sheftel A-S Zhang C Brown O S Shirihai and PPonka ldquoDirect interorganellar transfer of iron from endosometo mitochondrionrdquo Blood vol 110 no 1 pp 125ndash132 2007

[24] N Hegde M W Rich and C Gayomali ldquoThe cardiomyopathyof iron deficiencyrdquo Texas Heart Institute Journal vol 33 no 3pp 340ndash344 2006

[25] J Marın-Garcıa and M J Goldenthal ldquoThe mitochondrialorganelle and the heartrdquo Revista Espanola de Cardiologıa vol55 no 12 pp 1293ndash1310 2002

[26] G Karamanlidis C F Lee L Garcia-Menendez et al ldquoMito-chondrial complex i deficiency increases protein acetylationand accelerates heart failurerdquo Cell Metabolism vol 18 no 2 pp239ndash250 2013

[27] E Maranzana G Barbero A I Falasca G Lenaz and M LGenova ldquoMitochondrial respiratory supercomplex associationlimits production of reactive oxygen species from complex irdquoAntioxidants and Redox Signaling vol 19 no 13 pp 1469ndash14802013

[28] R Acın-Perez P Fernandez-Silva M L Peleato A Perez-Martos and J A Enriquez ldquoRespiratory active mitochondrialsupercomplexesrdquo Molecular Cell vol 32 no 4 pp 529ndash5392008

[29] R Acin-Perez and J A Enriquez ldquoThe function of the res-piratory supercomplexes the plasticity modelrdquo Biochimica etBiophysica Acta Bioenergetics vol 1837 no 4 pp 444ndash4502014

[30] L M Ruiz E L Jensen R I Bustos et al ldquoAdaptive responsesof mitochondria to mild copper deprivation involve changes inmorphology OXPHOS remodeling and bioenergeticsrdquo Journalof Cellular Physiology vol 229 no 5 pp 607ndash619 2014

[31] J D Wikstrom K Mahdaviani M Liesa et al ldquoHormone-induced mitochondrial fission is utilized by brown adipocytesas an amplification pathway for energy expenditurerdquoTheEMBOJournal vol 33 no 5 pp 418ndash436 2014

[32] R I Bustos E L Jensen L M Ruiz et al ldquoCopper deficiencyalters cell bioenergetics and induces mitochondrial fusionthrough up-regulation of MFN2 and OPA1 in erythropoieticcellsrdquo Biochemical and Biophysical Research Communicationsvol 437 no 3 pp 426ndash432 2013

[33] A Vazquez-Martin B Corominas-Faja S Cufi et al ldquoThemitochondrial H+-ATP synthase and the lipogenic switch Newcore components of metabolic reprogramming in inducedpluripotent stem (iPS) cellsrdquo Cell Cycle vol 12 no 2 pp 207ndash218 2013

[34] S Pich D Bach P Briones et al ldquoThe Charcot-Marie-Tooth type 2A gene product Mfn2 up-regulates fuel oxidationthrough expression of OXPHOS systemrdquo Human MolecularGenetics vol 14 no 11 pp 1405ndash1415 2005

[35] M Picard O S Shirihai B J Gentil and Y Burelle ldquoMito-chondrial morphology transitions and functions implicationsfor retrograde signalingrdquo American Journal of PhysiologymdashRegulatory Integrative andComparative Physiology vol 304 no6 pp R393ndashR406 2013

[36] M LiesaM Palacın andA Zorzano ldquoMitochondrial dynamicsin mammalian health and diseaserdquo Physiological Reviews vol89 no 3 pp 799ndash845 2009

[37] M Liesa and O S Shirihai ldquoMitochondrial dynamics in theregulation of nutrient utilization and energy expenditurerdquo CellMetabolism vol 17 no 4 pp 491ndash506 2013

[38] D Bach S Pich F X Soriano et al ldquoMitofusin-2 deter-mines mitochondrial network architecture and mitochondrialmetabolism a novel regulatory mechanism altered in obesityrdquoJournal of Biological Chemistry vol 278 no 19 pp 17190ndash171972003

[39] A Zorzano ldquoRegulation of mitofusin-2 expression in skeletalmuscleThis paper is one of a selection of papers publishedin this Special Issue entitled 14th International Biochemistry


of Exercise ConferencemdashMuscles as Molecular and MetabolicMachines and has undergone the Journalrsquos usual peer reviewprocessrdquoApplied Physiology Nutrition andMetabolism vol 34no 3 pp 433ndash439 2009

[40] B N Finck and D P Kelly ldquoPeroxisome proliferator-activatedreceptor 120574 coactivator-1 (PGC-1) regulatory cascade in cardiacphysiology and diseaserdquo Circulation vol 115 no 19 pp 2540ndash2548 2007

[41] Z Wu P Puigserver U Andersson et al ldquoMechanisms con-trolling mitochondrial biogenesis and respiration through thethermogenic coactivator PGC-1rdquo Cell vol 98 no 1 pp 115ndash1241999

[42] K Vanhees L de Bock R W L Godschalk F J van Schootenand S B vanWaalwijk van Doorn-Khosrovani ldquoPrenatal expo-sure to flavonoids implication for cancer riskrdquo ToxicologicalSciences vol 120 no 1 pp 59ndash67 2011

[43] A M Sabogal-Guaqueta J I Munoz-Manco J R Ramırez-Pineda M Lamprea-Rodriguez E Osorio and G P Cardona-Gomez ldquoThe flavonoid quercetin ameliorates Alzheimerrsquos dis-ease pathology and protects cognitive and emotional functionin aged triple transgenic Alzheimerrsquos disease model micerdquoNeuropharmacology vol 93 pp 134ndash145 2015

[44] P Galindo S Gonzalez-Manzano M J Zarzuelo et al ldquoDiffer-ent cardiovascular protective effects of quercetin administeredorally or intraperitoneally in spontaneously hypertensive ratsrdquoFood amp Function vol 3 no 6 pp 643ndash650 2012

[45] E Schultke R W Griebel and B H J Juurlink ldquoQuercetinadministration after spinal cord trauma changes S-100120573 levelsrdquoCanadian Journal of Neurological Sciences vol 37 no 2 pp 223ndash228 2010

[46] S-T Chan Y-C Lin C-H Chuang R-J Shiau J-W Liaoand S-L Yeh ldquoOral and intraperitoneal administration ofquercetin decreased lymphocyte DNA damage and plasmalipid peroxidation induced by TSA in vivordquo BioMed ResearchInternational vol 2014 Article ID 580626 9 pages 2014

[47] H K Park S J Kim D Y Kwon J H Park and Y C KimldquoProtective effect of quercetin against paraquat-induced lunginjury in ratsrdquo Life Sciences vol 87 no 5-6 pp 181ndash186 2010

[48] G H Heeba and M E Mahmoud ldquoDual effects of quercetin indoxorubicin-induced nephrotoxicity in rats and its modulationof the cytotoxic activity of doxorubicin on human carcinomacellsrdquo Environmental Toxicology 2014

[49] J Renugadevi and S M Prabu ldquoQuercetin protects againstoxidative stress-related renal dysfunction by cadmium in ratsrdquoExperimental and Toxicologic Pathology vol 62 no 5 pp 471ndash481 2010

[50] S K Richetti M Blank K M Capiotti et al ldquoQuercetin andrutin prevent scopolamine-induced memory impairment inzebrafishrdquo Behavioural Brain Research vol 217 no 1 pp 10ndash152011

[51] R M J Deacon ldquoMeasuring the strength of micerdquo Journal ofVisualized Experiments no 76 p e2610 2013

[52] A Elorza B Hyde H K Mikkola S Collins and O S ShirihaildquoUCP2 modulates cell proliferation through the MAPKERKpathway during erythropoiesis and has no effect on hemebiosynthesisrdquo The Journal of Biological Chemistry vol 283 no45 pp 30461ndash30470 2008

[53] W Duan Z Guo H JiangMWare X-J Li andM PMattsonldquoDietary restriction normalizes glucose metabolism and BDNFlevels slows disease progression and increases survival inhuntingtin mutant micerdquo Proceedings of the National Academy

of Sciences of the United States of America vol 100 no 5 pp2911ndash2916 2003

[54] C Jung C M J Higgins and Z Xu ldquoMeasuring the quantityand activity of mitochondrial electron transport chain com-plexes in tissues of central nervous system using blue nativepolyacrylamide gel electrophoresisrdquo Analytical Biochemistryvol 286 no 2 pp 214ndash223 2000

[55] L I Grad and B D Lemire ldquoRiboflavin enhances the assemblyof mitochondrial cytochrome c oxidase in C elegans NADH-ubiquinone oxidoreductase mutantsrdquo Biochimica et BiophysicaActa Bioenergetics vol 1757 no 2 pp 115ndash122 2006

[56] I Wittig R Carrozzo F M Santorelli and H Schagger ldquoFunc-tional assays in high-resolution clear native gels to quantifymitochondrial complexes in human biopsies and cell linesrdquoElectrophoresis vol 28 no 21 pp 3811ndash3820 2007

[57] J-M Li W Wang C-Y Fan et al ldquoQuercetin preserves 120573-cell mass and function in fructose-induced hyperinsuline-mia through modulating pancreatic AktFoxO1 activationrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 303902 12 pages 2013

[58] S Khurana K Venkataraman A Hollingsworth M Piche andT C Tai ldquoPolyphenols benefits to the cardiovascular systemin health and in agingrdquo Nutrients vol 5 no 10 pp 3779ndash38272013

[59] M J Ryan G R McLemore Jr and S T Hendrix ldquoInsulinresistance and obesity in a mouse model of systemic lupuserythematosusrdquoHypertension vol 48 no 5 pp 988ndash993 2006

[60] C Thomas and L Thomas ldquoBiochemical markers and hema-tologic indices in the diagnosis of functional iron deficiencyrdquoClinical Chemistry vol 48 no 7 pp 1066ndash1076 2002

[61] L Viatte J-C Lesbordes-Brion D-Q Lou et al ldquoDeregulationof proteins involved in iron metabolism in hepcidin-deficientmicerdquo Blood vol 105 no 12 pp 4861ndash4864 2005

[62] N E Hellman and J D Gitlin ldquoCeruloplasmin metabolismand functionrdquoAnnual Review of Nutrition vol 22 pp 439ndash4582002

[63] B N Patel R J Dunn S Y Jeong Q Zhu J-P Julien andS David ldquoCeruloplasmin regulates iron levels in the CNS andprevents free radical injuryrdquoThe Journal of Neuroscience vol 22no 15 pp 6578ndash6586 2002

[64] A Raghavan T Sheiko B H Graham and W J CraigenldquoVoltage-dependant anion channels novel insights into isoformfunction through genetic modelsrdquo Biochimica et BiophysicaActa Biomembranes vol 1818 no 6 pp 1477ndash1485 2012

[65] I Dalle-Donne M Carini M Orioli et al ldquoProtein car-bonylation 24-dinitrophenylhydrazine reacts with both alde-hydesketones and sulfenic acidsrdquo Free Radical Biology andMedicine vol 46 no 10 pp 1411ndash1419 2009

[66] M M Baccan O Chiarelli-Neto R M S Pereira and B PEsposito ldquoQuercetin as a shuttle for labile ironrdquo Journal ofInorganic Biochemistry vol 107 no 1 pp 34ndash39 2012

[67] M Salvi A M Brunati G Clari and A Toninello ldquoInteractionof genistein with the mitochondrial electron transport chainresults in opening of themembrane transition porerdquo Biochimicaet Biophysica ActamdashBioenergetics vol 1556 no 2-3 pp 187ndash1962002

[68] H S Yoon S C Moon N D Kim B S Park M H Jeongand Y H Yoo ldquoGenistein induces apoptosis of RPE-J cellsby opening mitochondrial PTPrdquo Biochemical and BiophysicalResearch Communications vol 276 no 1 pp 151ndash156 2000


[69] L M Larocca L Teofili G Leone et al ldquoAntiproliferativeactivity of quercetin on normal bone marrow and leukaemicprogenitorsrdquo British Journal of Haematology vol 79 no 4 pp562ndash566 1991

[70] S A Bakheet ldquoAssessment of anti-cytogenotoxic effects ofquercetin in animals treated with topotecanrdquo Oxidative Medi-cine and Cellular Longevity vol 2011 Article ID 824597 8 pages2011

[71] A Pekal M Biesaga and K Pyrzynska ldquoInteraction ofquercetin with copper ions complexation oxidation and reac-tivity towards radicalsrdquo BioMetals vol 24 no 1 pp 41ndash49 2011

[72] A C Mot C Coman C Miron G Damian C Sarbu and RSilaghi-Dumitrescu ldquoAn assay for pro-oxidant reactivity basedon phenoxyl radicals generated by laccaserdquo Food Chemistry vol143 pp 214ndash222 2014

[73] G Galati O Sabzevari J X Wilson and P J OrsquoBrien ldquoProox-idant activity and cellular effects of the phenoxyl radicals ofdietary flavonoids and other polyphenolicsrdquoToxicology vol 177no 1 pp 91ndash104 2002

[74] J M Davis E A Murphy M D Carmichael and B DavisldquoQuercetin increases brain and muscle mitochondrial biogen-esis and exercise tolerancerdquo American Journal of PhysiologymdashRegulatory Integrative and Comparative Physiology vol 296 no4 pp R1071ndashR1077 2009

[75] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology C Toxicology amp Pharmacology vol135 no 3 pp 357ndash364 2003

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


glutathione an increase in cytosolic calcium concentrationand lactate dehydrogenase leakage [11]

Quercetin may interact directly with mitochondrialmembranes [15 16] affecting its fluidity This has also beenassociated with mitochondrial dysfunction through the inhi-bition of respiratory chain or uncoupling [15 16] In additionquercetin affects mitochondrial calcium regulation increasesO2∙minus production and induces the opening of mitochondrial

transition pore (mPTP) in HCT116 cells [17] The abovestudies suggest quercetin could promote oxidation affectingmitochondrial function raising concerns about the validityof quercetin as an antioxidant [17]

At molecular level quercetin is potent iron chelatorwhich is shown to alter the expression of proteins involved iniron absorption affecting iron homeostasis [18 19] Iron is anessential element in each of the four mitochondrial electrontransfer complexes either as FeS clusterrsquos or hemersquos compo-nent [20] Iron deficiency leads to altered cell metabolism[20] and anemia [21 22] Red blood cell development isthe most iron requiring process for oxygen transport aswell as the most active cell generator system involving bothproliferation and differentiation from hematopoietic stemcells which are also dependent onmitochondrialmetabolism[21 23] Additionally iron deficiency-induced anemia canhave deleterious effects on heart [24] and cardiomyopathydevelopment is related with mitochondrial dysfunction dueto ROS excess [25 26] Both erythropoiesis and the cardiactissue are then suitable and attractive targets for quercetinmechanistic studies

Our working hypothesis is that quercetin interferes withmitochondrial function exacerbating mitochondrial ROSgeneration and altering the physiology of tissues highlydependent on iron metabolism and mitochondrial functionsuch as the erythroid and cardiac tissue We are interestedin addressing the in vivo prooxidant effect of quercetin onmitochondrial function in these tissues

Adaptive responses of mitochondria to maintain cellrsquosbioenergetics capacity under stressful conditions involve theremodeling of mitochondrial respiratory complexes to buildup or down supramolecular structures called supercom-plexes and themitochondrial fusion and fission events calledmitochondrial dynamics The former will allow a bettersubstrate channeling to preclude extreme production of ROSfrom the normal respiratory chain function [27ndash29] Thelatter will control energy expenditure and metabolic repro-gramming [30ndash33] Upregulation of Mitofusin 2 (MFN2)protein involved inmitochondrial fusion and thenwith elon-gated mitochondria has been associated with a protectiverole against apoptosis hypoxia and ROS [32 34 35] aswell as with higher oxidative capacity by regulating in partthe respiratory complex proteins expression [35ndash38] MFN2expression is regulated in turn by the peroxisome proliferatoractivated receptor-gamma coactivator-1120572 (PGC-1120572) under avariety of conditions characterized by energy expenditure[39] PGC-1120572 is cofactor that participates in the regulationof mitochondrial biogenesis and activation of peroxisomeproliferator activated receptor-120574 (PPAR 120574) pathway [40 41]

Our results showed that quercetin clearly affected mito-chondrial function inmice Interestingly quercetin decreased

erythropoiesis and reduced the expression levels of mito-chondrial proteins that control mitochondrial dynamicsThese results proposed that the antioxidant properties ofquercetin need to be reevaluated given their widespread use[42]

2 Materials and Methods

21 Animals and Experimental Design Male C57BL6 mice(12 months of age) were daily administered intraperitoneally(ip) with 50mgkg quercetin (Cat Q4951 Sigma) or withvehicle (5 DMSO) and PBS for control animals during 15days Animals were daily checked for weight and health con-ditions Right after the treatments animals were submitted todifferent tests indicated below and then sacrificed for heartand bone marrow dissections Ip injections of quercetin inrodents have been reported previously in [43ndash47] and thedose of 50mgkg of quercetin has been reported in [47ndash50]

22 Strength Test To evaluate strength resistance and exer-cise abilities mice performed the Kondzielarsquos test and aweightlifting test Briefly Kondzielarsquos inverted screen is a testfor muscle strength using all four limbs in which eachmousewas placed in the center of a screen and then rotated toan inverted position with the mousersquos head declining firstthe mice falling time was recorded The performance of eachmouse in the inverted screen was scored as follows Fallingbetween 1ndash10 sec = 1 11ndash25 sec = 2 26ndash60 sec = 3 over 60 sec= 4 [51] On the other hand the weightlifting is a test forforelimbs muscle strength The weightlifting assessed theability to raise seven weights ranging from 20 g to 98 g (2033 46 59 72 85 and 98 g) First the mouse was allowed toraise the lightestweight (20 g) for 3 sec andup to 3 timesAftera 10 sec rest in between each lift the second and third raiseswere performed to move onto the next heaviest weight Thetrial finishes when the mouse fails to lift or hold the weightafter three attempts recording the maximum timeweightachieved The score calculations was made according to [51]and normalized by the body weight

23 Bone Marrow Analyses Bone marrow was isolated fromboth legs and immunostained with the antibodies phy-coerythrin- (PE-) conjugated anti-TER119 and fluoresceinisothiocyanate- (FITC-) conjugated anti-CD71 according to[30 52] Progression of erythropoiesis was then assessed byflow cytometry [30]

24 Total Antioxidant Capacity Antioxidant capacity wasevaluated using OxiSelect Total Antioxidant Capacity (TAC)Assay Kit (Cat STA-360 Cell Biolabs Inc) according tomanufacturerrsquos instructions Results are expressed as ldquo120583MCopper Reducing Equivalentsrdquo and compared with controland quercetin-treated samples

25 Insulin Determination For the quantitative determina-tion of insulin in mouse plasma was used the Mouse InsulinELISA kit (Cat 80-INSMS-E01 APLCO) according with themanufacturerrsquos instructions [53]








3 Results








100

90

80

70

60

500 2 4 6 8 10 12 14 16

Body

wei

ght (

)

Days

ControlQuercetin

lowast


lowastlowastlowast

(a)

Control Quercetin

10

9

8

7

6

5

4

3

2

1

0

minus1

minus2

Insu

lin (n

gm

L)

lowast

(b)

Control Quercetin

10

8

6

4

2

0Wei

ghtli

fting

scor

ebo

dy w

eigh

t

(c)

Control Quercetin

24

22

20

18

16

14

12

10

08

06

04

02

Kond

ziel

a sco

reb

ody

wei

ght

(d)

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(e)



Control Quercetin

Cp

IgG

(a)

Control Quercetin

30000

25000

20000

15000

10000

5000

0

Relat

ive c

erul

opla

smin

leve

l (au

)

lowast

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(b)

Control

Plasma

Quercetin

Cop

per r

educ

ing

equi

vale

nts (120583

M)

1000

900

800

700

600

500

400

300

200

100

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(c)



2CIV1minus4





1054

104

103

102

101

1013 102 103 1042

1013 102 103 1042

CD71

-Fitc

1054

104

103

102

101

CD71

-Fitc

Ter119-PE

Ter119-PE

Control

Quercetin

(a)

25

20

15

10

5

0

Cel

l (

)


lowast

lowast

lowast

lowast

lowast

ControlQuercetin

(b)






Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results








100

90

80

70

60

500 2 4 6 8 10 12 14 16

Body

wei

ght (

)

Days

ControlQuercetin

lowast


lowastlowastlowast

(a)

Control Quercetin

10

9

8

7

6

5

4

3

2

1

0

minus1

minus2

Insu

lin (n

gm

L)

lowast

(b)

Control Quercetin

10

8

6

4

2

0Wei

ghtli

fting

scor

ebo

dy w

eigh

t

(c)

Control Quercetin

24

22

20

18

16

14

12

10

08

06

04

02

Kond

ziel

a sco

reb

ody

wei

ght

(d)

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(e)



Control Quercetin

Cp

IgG

(a)

Control Quercetin

30000

25000

20000

15000

10000

5000

0

Relat

ive c

erul

opla

smin

leve

l (au

)

lowast

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(b)

Control

Plasma

Quercetin

Cop

per r

educ

ing

equi

vale

nts (120583

M)

1000

900

800

700

600

500

400

300

200

100

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(c)



2CIV1minus4





1054

104

103

102

101

1013 102 103 1042

1013 102 103 1042

CD71

-Fitc

1054

104

103

102

101

CD71

-Fitc

Ter119-PE

Ter119-PE

Control

Quercetin

(a)

25

20

15

10

5

0

Cel

l (

)


lowast

lowast

lowast

lowast

lowast

ControlQuercetin

(b)






Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

90

80

70

60

500 2 4 6 8 10 12 14 16

Body

wei

ght (

)

Days

ControlQuercetin

lowast


lowastlowastlowast

(a)

Control Quercetin

10

9

8

7

6

5

4

3

2

1

0

minus1

minus2

Insu

lin (n

gm

L)

lowast

(b)

Control Quercetin

10

8

6

4

2

0Wei

ghtli

fting

scor

ebo

dy w

eigh

t

(c)

Control Quercetin

24

22

20

18

16

14

12

10

08

06

04

02

Kond

ziel

a sco

reb

ody

wei

ght

(d)

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(e)



Control Quercetin

Cp

IgG

(a)

Control Quercetin

30000

25000

20000

15000

10000

5000

0

Relat

ive c

erul

opla

smin

leve

l (au

)

lowast

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(b)

Control

Plasma

Quercetin

Cop

per r

educ

ing

equi

vale

nts (120583

M)

1000

900

800

700

600

500

400

300

200

100

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(c)



2CIV1minus4





1054

104

103

102

101

1013 102 103 1042

1013 102 103 1042

CD71

-Fitc

1054

104

103

102

101

CD71

-Fitc

Ter119-PE

Ter119-PE

Control

Quercetin

(a)

25

20

15

10

5

0

Cel

l (

)


lowast

lowast

lowast

lowast

lowast

ControlQuercetin

(b)






Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control Quercetin

Cp

IgG

(a)

Control Quercetin

30000

25000

20000

15000

10000

5000

0

Relat

ive c

erul

opla

smin

leve

l (au

)

lowast

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(b)

Control

Plasma

Quercetin

Cop

per r

educ

ing

equi

vale

nts (120583

M)

1000

900

800

700

600

500

400

300

200

100

Median

Minimum value

Maximum valuelowast

lowast

95th percentile

75th percentile

25th percentile

5th percentile

998686 Mean (998686)

(c)



2CIV1minus4





1054

104

103

102

101

1013 102 103 1042

1013 102 103 1042

CD71

-Fitc

1054

104

103

102

101

CD71

-Fitc

Ter119-PE

Ter119-PE

Control

Quercetin

(a)

25

20

15

10

5

0

Cel

l (

)


lowast

lowast

lowast

lowast

lowast

ControlQuercetin

(b)






Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1054

104

103

102

101

1013 102 103 1042

1013 102 103 1042

CD71

-Fitc

1054

104

103

102

101

CD71

-Fitc

Ter119-PE

Ter119-PE

Control

Quercetin

(a)

25

20

15

10

5

0

Cel

l (

)


lowast

lowast

lowast

lowast

lowast

ControlQuercetin

(b)






Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control Quercetin

SC1

CI

(a)

Control Quercetin

09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

I (a

u)

CISC

(b)

CIV

SC1

SC2

Control Quercetin

(c)

Control Quercetin

lowast09

08

07

06

05

04

03

02

01

00

In g

el ac

tivity

com

plex

IV (a

u)

SC1SC2CIV

(d)

CII

Control Quercetin

(e)

Control Quercetin

10

08

06

04

02

00

In g

el ac

tivity

com

plex

II (a

u)

CII

(f)


2CIV1minus4

SC2CIII2CIV1minus4


MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MFN2

VDAC1

Control Quercetin

120573-actin

PGC-1120572

(a)

Control Quercetin

Ponceau

CV

120572-DNP

(b)

ControlQuercetin

18

16

14

12

10

08

06

04

02

00Relat

ive p

rote

in le

vel120573

-act

in n

orm

aliz

ed

MFN2 VDAC1

lowastlowast

PGC-1120572

(c)

Control Quercetin

10

08

06

04

02

00

Mito

chon

dria

l pro

tein

oxi

datio

n (a

u)

Oxyblot

lowast

(d)


4 Discussion









5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion




Acknowledgments


References






















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















quercetin affects erythropoiesis and heart mitochondrial function in

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