The exposure to formaldehyde causes renal dysfunction, inflammation and redox imbalance in rats.

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The

exposure

to

formaldehyde

causes

renal

dysfunction,

in

ﬂammation

and

redox

imbalance

in

rats

Camila

de

Oliveira

Ramos

a

,

Clarissa

Rodrigues

Nardeli

a

,

Keila

Karine

Duarte

Campos

a

,

Karina

Braga

Pena

a

,

Dafne

Fernandes

Machado

a

,

Ana

Carla

Balthar

Bandeira

b

,

Guilherme

de

Paula

Costa

c

,

André

Talvani

c

,

Frank

Silva

Bezerra

a,

*

a

LaboratoryofExperimentalPathophysiology(LAFEx),DepartmentofBiologicalSciences(DECBI),CenterofResearchinBiologicalSciences(NUPEB),Federal UniversityofOuroPreto(UFOP),OuroPreto,MG,Brazil

b_Laboratory_of_Metabolic_Biochemistry_(LBM),_Department_of_Biological_Sciences_(DECBI),_Center_of_Research_in_Biological_Sciences_(NUPEB),_Federal

UniversityofOuroPreto(UFOP),OuroPreto,MG,Brazil

c

LaboratoryofImmunobiologyofInﬂammation(LABIIN),DepartmentofBiologicalSciences(DECBI),CenterofResearchinBiologicalSciences(NUPEB), FederalUniversityofOuroPreto(UFOP),OuroPreto,MG,Brazil

ARTICLE INFO

Articlehistory:

Received8November2016

Receivedinrevisedform10February2017 Accepted23February2017 Keywords: Formaldehyde Inﬂammation Redoximbalance Renaltissue ABSTRACT

Twenty-eight Fischermaleratsweredividedintofourgroups: controlgroup(CG),exposed tothe ambientair,andgroupsexposedtoformaldehyde(FA)atconcentrationsof1%(FA1%),5%(FA5%)and10% (FA10%).Kidneyfunctionwasassessedbydosageofuricacid,creatinineandurea.Morphometrywas performedonthethicknessofthelumenofBowman'scapsuleanddiameterofthelumenoftherenal tubules.Weevaluatedtheredoximbalancethroughthecatalaseandsuperoxidedismutaseactivityas wellasoxidativedamagebylipidperoxidation.InﬂammatorychemokinesCCL2,CCL3andCCL5were analyzed byenzymeimmunoassays. Therewas anincreaseinthe concentrationofureain FA10% comparedwithCGandFA1%.Thelevelsofcreatinine,renallumenandlipidperoxidationincreasedinall FA-treatedgroupscomparedwithCG.TheconcentrationofuricacidinFA10%waslowercomparedwith allothergroups.TherewasanincreaseinthespaceofBowman'scapsuleinFA5%andFA10%compared withCGandFA1%.However,thesuperoxidedismutaseactivitywashigherinFA5%comparedwithother groupswhileCCL5washigherinFA1%comparedwithCG.Theexposuretoformaldehydeinashort periodof timeleadstochanges in thekidneyfunction,inﬂammation andmorphology, aswellas promotedtheincreaseofsuperoxidedismutaseactivityandoxidativedamage.

1.Introduction

Formaldehyde(FA)isacolorlessgas,highlysolubleinwaterand irritates in its pure form. FA is a contaminating compound commonly found in the environment due to its wide use in industriessuchastheproductionofbuildingmaterials, textiles, sterilizationofproducts,plasticsandcosmetics(Bakaretal.,2015; Checkowayetal.,2015;Ciftcietal.,2015).FAcanalsobefoundin cigarette smoke, in car emissions, fuel oil and natural gas, contributing to increased air pollution (Zararsiz et al., 2007b). Theexposure toFAis increasingly common,eitherby environ-mental or laboratory conditions, where professionals and/or studentsin themedical ﬁeldare constantlyexposed(Schroeter

et al., 2014; Zararsiz et al., 2007b). FA is also endogenously

producedbytheL-methioninemetabolism,histamine,methanol andmethylalanine,beingakeyintermediateforthebiosynthesis ofpurinesandotheraminoacids(Checkowayetal.,2015;Gulec etal.,2006).

In2006,theInternationalAgencyforResearchonCancer(IARC) described FA as a carcinogen (IARC, 2006). In addition,several studieshaveshownthatchronicexposuretoFAmayalsoresultin sensoryirritation,salivation,dyspnea,headache,insomnia, seiz-uresand neurodegenerativedisorders (Bakar etal.,2015;Gulec etal.,2006).ThetoxicitycausedbytheexposuretoFAbyaerobic metabolismandbyinﬂammationcanleadtotheproductionand thereleaseofreactiveoxygenspecies(ROS)(Birbenetal.,2012; Gulecetal.,2006;Saitoetal.,2005).Atlowconcentrations,ROS have physiological functions in cellular processes, but in high amounts,theycancauseadversechangesinthecellcomponents, includingproteins,lipidsanddeoxyribonucleicacid(DNA)(Birben

* Correspondingauthor.

E-mailaddress:franksbezerra@hotmail.com(F.S.Bezerra).

http://dx.doi.org/10.1016/j.etp.2017.02.008

ContentslistsavailableatScienceDirect

Experimental

and

Toxicologic

Pathology

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etal.,2012;Saitoetal.,2005).Achangeinthebalancebetween oxidant/antioxidantinfavorofoxidantsisnamedoxidativestress (Birbenetal.,2012).Toprotectagainstthedeleteriouseffectsof ROS,thecellspresentacomplexenzymaticantioxidantdefense systemincludingsuperoxidedismutase(SOD),catalase(CAT),and glutathioneperoxidase(GPx)(Gulecetal.,2006;Matsuokaetal., 2010).

StudieshaveshownthatprolongedexposuretoFAcanresult in degeneration and necrosis of proximal tubule kidney and consequentlyimpairedurinarysystem(IARC, 2006;Kumetal.,

2007;Zararsiz et al., 2007b).Furthermore, theexposure toFA

induces a number of pathophysiological conditions, including inﬂammatorydiseases byinterfering in theconcentrationof T CD3+_cells,_natural_killer_(NK)_cells,_TNF,_IL-6_and_IL1_–

_b

₍ Lino-dos-Santos-Francoetal.,2011;Moroetal.,2016;Seowetal.,2015). The kidney is one of the most sensitive organs to the inflammation and is an important source of chemokines and cytokinesin the tubularepitheliumdueits close contactwith highbloodflow(Grunz-Borgmannetal.,2015).Thus,theaimof the study was to analyze the oxidative effects on renal inflammatory response in Fischer rats exposed to different concentrationsofFA.

2.Materialsandmethods 2.1.Animals

Twenty-eightmaleFischerrats,between10and12weeksof ageandbody mass 180–200g fromtheExperimentalNutrition LaboratoryoftheFederalUniversityofOuroPreto,wereusedin this study. The animals were kept in boxes with environment temperature,controlledlightandhumidity(212C,12-hcycles oflight/dark,5010%,respectively)receivingcommercialdietfor rat and water, both ad libitum. This study was performed in accordancewithstandardsofanimal protectionand theethical principlesoftheBrazilianSocietyofScienceinLaboratoryAnimals and approved by the Ethics Committee on Animal Use of this university(Protocol2011/01).

2.2.Exposuretoformaldehyde(FA)

AnimalswereexposedtoFAbyanultrasonicnebulizer(Unique Group, Indaiatuba, São Paulo, Brazil) coupled to an inhalation chamber of 30L (25cm30cm40cm). Three groups of 7 animalswereexposed todifferentconcentrations of FA(1%5% and10%)andacontrolgroupexposedtoambientair.Theexposure proceededfor20min,3timesaday(morning,afternoon,evening) for5consecutivedays(Maiellaroetal.,2014;Murtaetal.,2016). After 24h of the experimental protocol, the animals were euthanatizedwithan overdoseofKetamine (50–75mg/kg) and Xylazine(5_–10mg/kg)intraperitoneally.

2.3.Homogenizedtissue

Aftereuthanasia,therightkidneywasremovedand homoge-nized in 1mL of pH 7.5 potassium phosphate buffer and centrifugedat10.000gfor10minat4C.Thesupernatantwas collectedandstoredinafreezerat 80Cforbiochemicalanalyses. 2.4.Renalfunction

Blood was obtained by cardiac puncture and collectedinto tubes. After, the samples were centrifuged at 10.000rpm for 10min.Serumwascollectedforanalysesofuricacid,creatinine andurea.Analyseswerespectrophotometricallyperformedusing commercialkits(Bioclin1,BeloHorizonte,Brazil).

2.5.Histologyandmorphometricanalyses

Theleftkidneywasremovedandimmersedinﬁxativesolution containing 4% formaldehyde for 48h. Serial sections were performed to four micrometers thick, which were stain with hematoxylin-eosin(HE) forhistopathologicalanalyses.The vari-ablesanalyzedwere:Bowman’sspaceandthelumenoftherenal tubules. Bowman's space was calculated by the area of the Bowman'scapsuleandtheglomerulartuftandsubtractedfromthe secondoftheﬁrstarea.Glomeruliwereevaluatedintheafferent arterymeasuringhencetheequatorialportionoftheglomerulus. The lumen of the renal tubules was calculated by tracing the boundaries thereofby ImageJ1 software (National Institutesof Health,Bethesda,Maryland,USA)

2.6.Theactivityofcatalase(CAT)

CATactivitywasmeasuredasthedecreasedrateofhydrogen peroxidetoanabsorbanceof240nmrepresentedbyU/mgofthe protein(Aebi,1984).Proteincontentwasperformedonsamplesof tissuehomogenatebythemethodofBradford(Bradford,1976). 2.7.Theactivityofsuperoxidedismutase(SOD)

SOD activity was measured in the tissue according to the Marklundmethod(MarklundandMarklund,1974)whichisbased ontheenzyme'sabilitytoinhibittheautoxidationofpyrogallol. Theabsorbancewasread intheELISAreaderat570nm.Protein contentwasperformedonsamplesofthehomogenatetissueby themethodofBradford(Bradford,1976).

2.8.Analysisofoxidativedamage

Lipid peroxidation was determined by testing reactive sub-stancesthiobarbituricacid(TBARS)describedbyBuege(Buegeand Aust,1978).Thehomogenizedtissuewascentrifugedfor10minat 13.000rpmandthesupernatantwasreadinaspectrophotometer at 535nm. The concentration was represented in nmols per milligramoftheprotein(nmol/mgprotein).

2.9.Immunoassays

The renal parenchyma was used for the evaluation of the inﬂammatory chemokines CCL2, CCL3 and CCL5. The immuno-assayswereperformedin96-wellplates bytheadditionof the 100

m

Lofamonoclonalantibodytoprotein(orpeptide)ofinterest, diluted in PBS containing 0.1% bovine serum albumin-BSA (SIGMA).After 12hat roomtemperatureincubation,theplates wereblockedwith300

m

L/wellofaPBS/1%BSAsolutionfor1hat 37C.Sampleswereappliedinavolumeof100

m

Ltoeachwell.The avidin-HRP(1:2000)andthesubstrateABTSliquidwereusedat the end of the reaction beforethe reading an ELISA reader at 490nm.AllchemokineELISAkitswerepurchasedfromPeprotech (RibeirãoPreto,Brazil)andperformedaccordingtothe manufac-turerrecommendations.

2.10.Statisticalanalysis

Thenormaldistributionofeachvariablewasassessedusingthe Kolmogorov-Sminorvtestandpresentedasmeanstandarderror ofmean(SEM).Forcomparisonamonggroups,aone-wayANOVA followed by Tukey's post-test was used. We used the Kruskal-Wallis test followed by Dunn's post-test for discrete data and expressed themas median,minimum and maximumvalues.In bothcases,thedifferencewasconsideredsigniﬁcantwhenpvalue was <0.05. The statistical analyses were performed using

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GraphPadsoftware(GraphPadInStatversion5.00forWindows7, GraphPadSoftware,SanDiego,CAUSA).

3.Results 3.1.Renalfunction

Renal function was assessed through the analysis of urea concentration(mg/dL),creatinine(mg/dL)anduricacid(mg/dL)in serum(Fig.1).There was anincrease ofureaconcentrationsin FA10% compared with CG and FA1% (p=0.001) (Fig. 1A). The creatininelevelsincreasedinalltreatedgroupscomparedwithCG (p=0.003)(Fig.1B).However, theconcentrationof uric acidin FA10% was lowercompared withtheothergroups (p<0.0001) (Fig.1C).

3.2.Morphometry

Inthisstudy,therewasanincreaseinBowman'sspaceinFA5% andFA10%comparedwithCGandFA1%(p<0.0001)(Fig.2A).The lumenoftherenaltubuleswaselevatedinallgroupsexposedto formaldehyde compared with CG (p<0.0001) (Fig. 2B). Fig. 3

shows the increase in Bowman's space FA10% and thickness increaseinthelumenoftherenaltubulestherenaltubuleFA1%. 3.3.Redoximbalance

Theredoximbalancewasassessedthroughthemeasurementof theactivityofantioxidantenzymesSODandCAT.TheSODactivity

increasedinFA5%comparedwiththeothergroups(p=0.0083). TheCATactivitydidnotchangesigniﬁcantlyamongthegroupsof animalsexposed toformaldehyde(p=0.714).Lipid peroxidation was measuredthrough theformation of thiobarbituric reactive substances (TBARS) in renal homogenized tissue to check the possible oxidative damage caused by different formaldehyde concentrationsinthekidneys.TherewasanincreaseofTBARSinall treatedgroupscomparedwithCG(p<0.0001).Furthermore,the lipid peroxidation was signiﬁcantly higher in FA10% compared withFA1(p<0.0001)(Table1).

3.4.Kidneychemokinemeasurement

ThechemokinesCCL2,CCL3andCCL5weremeasuredthrough enzymeimmunoassaysinrenaltissuehomogenate.Highlevelsof CCL2 were observed in FA1% and FA5% compared with CG (p=0.0044).HighlevelsofCCL3werealsoobservedinFA1%and FA5%comparedwithCG.Ontheotherhand,thelevelsofCCL3was lowerinFA10%comparedwithGC,FA1%andFA5%(p<0.0001). Finally,thekidneylevelsofCCL5chemokinewashigherinFA1% comparedwithCG,FA5%,andFA10%(p<0.0001)(Table2). 4.Discussion

Inthisstudy,weanalyzedrenalfunction,oxidativeeffectsand inﬂammatoryresponseinratsexposedtodifferentconcentrations offormaldehydefor5consecutivedays.Clinicalandexperimental studieshaveshownthetoxiceffectsofFAintheurinarysystem (Zararsizetal.,2007b,2006).Inourpresentstudyanincreasein

Fig.1.EffectsoftheexposuretoFAinlevelsofbiomarkersthekidneyfunction.Theletter(a)representsasigniﬁcantdifferencecomparedtoCG.Letters(b,c)representsa signiﬁcantdifferencescomparedtoFA1%andFA5%.DatawereexpressedasmeanSEMandwereanalyzedbyone-wayANOVAfollowedTukey’sposttest(p<0,05).

Fig.2. Morphometricanalysesinrenaltissueofratsexposedtodifferentconcentrationsofformaldehyde.(A)AnalysisofBowman’sspaceofratsexposedtodifferent concentrationsofformaldehyde.(B)Analysisoflumenoftherenaltubulesofrenaltissueofratsexposedtodifferentconcentrationsofformaldehyde.Theletter(a)represents asigniﬁcantdifferencecomparedtoCG.Theletter(b)representsasigniﬁcantdifferencecomparedtoFA1%.Datawereexpressedasmedian,minimumandmaximumvalues andwereanalyzedbyKruskal-WallistestfollowedbyDunn'sposttest.(p<0,05).

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theBowman’sspaceandinthelumenoftherenaltubulesofthe exposedgroupwasobservedwhichcorroborateswithZararsizand colleagues’study.TheseauthorsexaminedthetoxicityofFAinthe kidneysandtheprotectiveeffects of

V

3essential fattyacids against these toxic effects in Wistar rats during 14days and observed a glomerular and tubular degeneration as well as a remarkableexpansioninthedistaltubulesindicatingrenaltissue injuryinthoseFAexposedanimals(Zararsizetal.,2006).Inthis way,another studydemonstrating the protectiveeffects of the melatoninonrenaldamagereinforcedthepresenceofglomerular degeneration,vacuoleformation,vasculardilationandcongestion inthoseratsexposuretoFAfor14days,everyotherday(Zararsiz etal., 2007a).Bakar and colleaguesalso studiedtheprotective effects of theproanthocyanidinand the vitamin E in therenal damage induced by the exposure to FA in rats and reported

epithelial damageof theglomerulus and themembrane of the renaltubules,hypertrophiccellsinthetubulesandpyknoticnuclei incellsoftheloopofHenle(Bakaretal.,2015).Together,these ﬁndingsshowthattheexposuretoFAleadstothedifferentlevels of lesion in the renal tissue which, therefore, would result in releasingofthevasopressoragents,decreasingthevasodilatation andincreasingROSandoxidativestresslevels(Bakaretal.,2015; Kunaketal.,2015;Zararsizetal.,2006).

In our study we observed an increase in serum urea and creatinine in animals exposed to higher concentration of FA comparedwiththenon-exposedgroup.Therelationshipbetween ureaandcreatininecanbe,inparticular,usefulwhenthere are sharpfallsintheglomerularﬁltrationrate.Theureaisreabsorbed bytherenaltubuleafterthe_ﬁltrationprocesswhichisnotapplied for the creatinine. A pathological condition (eg. heart failure,

Fig.3.Photomicrographsofkidneysectionsstainedwithhematoxylinandeosin.Barr=50mm.AnalysisofBowman’sspaceandthelumenoftherenaltubulesofrenaltissue ofratsexposedtodifferentconcentrationsofformaldehyde.RTshowsincreasedlumenoftherenaltubuleinFA1%andBSshowsincreasedBowman’sspaceinFA10% comparedtoCG.

Table1

ActiviesofSOD,CATandTBARScontentinkidneyssamplesfromCG,FA1%,FA5%and FA10%.

CG FA1% FA5% FA10% SOD (U/mgptn) 11.761.37 13.301.07 20.733.25a, b 13.260.84c CAT (U/mgptn) 3.390.23 3.470.24 3.090.50 3.630.30 TBARS(nmol/mg ptn) 0.270.01 1.190.20a 1.830.26a 1.870.06a, b

Thelettersrepresentsignificantdifferencesamonggroups.Theletter(a)represents asignificantdifferencecomparedtoCG.Theletter(b)representsasignificant differencecomparedtoFA1%andtheletter(c)representsasignificantdifference comparedtoFA5%.DataareexpressedasmeanSEMandwereanalyzedby one-wayANOVAfollowedbyTukey'spost-test(p<0.05).

Table2

LevelofCCL2,CCL3andCCL5ofthekidneyparenchymasamplesfromCG,FA1%, FA5%andFA10%.

CG FA1% FA5% FA10%

CCL2(pg/ mL) 196648.12 227445.41a 227162.45a 208475.61 CCL3(pg/ mL) 388.2012.51 48514.17a 44025.16a 31412.88a,b,c CCL5(pg/ mL) 401.7016.33 788.649.55a 46622.79b 537.5055.31b

Thelettersrepresentsignificantdifferencesamonggroups.Theletter(a)represents asignificantdifferencecomparedtoCG.Theletter(b)representsasignificant differencecomparedtoFA1%andtheletter(c)representsasignificantdifference comparedtoFA5%.DataareexpressedasmeanSEMandwereanalyzedby one-wayANOVAfollowedbyTukey'spost-test(p<0.05).

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dehydration, feverish and chemical toxicity conditions) which stimulatestubularreabsorptionofsodiumdeterminesanincrease intheproportionofurea/creatinine.Inaddition,oxidationofFAfor formic acid is catalyzed by various enzymes such as NAD-dependentdehydrogenaseformaldehyde,xanthineoxidase, cata-laseandperoxidase.Then,theincreaseofureamaybeassociated with the high production of these enzymes used for the detoxiﬁcation of the FA (Kum et al., 2007; Teng et al., 2001). Somestudieshavesuggestedthatcreatinine,theendmetaboliteof creatine phosphate, besides its antioxidant properties, is an important marker of nephrotoxicity. Increased serum levels of creatininestronglysuggestfailureinkidneyfunctionduetothe exposuretoFA(Kunaketal.,2015;Milovanovicetal.,2015).

Theuricacidistheendproductofthemetabolicpathwayof purines and is considered a natural antioxidant with chelating propertiesinthepresenceofmetalintoxication.Inaddition,uric acidhasascavengercapacityofReactiveNitrogenSpecies(RNS) andthesuperoxideanion,therebyhelpingtoblocktheformation ofthehighlyreactiveperoxynitriteoxidant(Whitemanetal.,2002; Yangetal.,2015).

Areductioninserumlevelsoftheuricacidinthegroupexposed to a higher concentration of FA was observed in our study, suggestingthattheroleofuricacidasscavengersinrenaltissue duetooxidativedamagegeneratedbyFA.

TheROS generation occursduring theconventional cellular respiratory process, being potentiated in the presence of exogenous chemical agencies, such as FA (Bakar et al., 2015;

Birben et al., 2012; Saito et al., 2005). Indeed, there is a

physiologicalbalancebetweenthereactivespeciesand antioxi-dantdefensesysteminthebody,butanychangeordisturbancein this equilibrium can start an oxidative stress process (Birben etal.,2012;Camposetal.,2013;Matsuokaetal.,2010;Zararsiz etal.,2006).Theoxidativestresscanbecontrolledbyendogenous and enzymatic mechanisms performed by CAT, SOD and glutathione(GSH)(Matsuokaetal.,2010;Zararsizetal.,2006). KumandcolleaguesdidnotobservedifferencestoSODandCAT levelsintheanimalsexposedtoxyleneandFA(Kumetal.,2007). However,inthestudyofZararsizetal.therewasareductionin SODactivityingroupsexposedtoFA(Zararsizetal.,2007b)while CATactivitywasobservedinanotherstudywherethediaphragm muscleandtracheawereevaluatedinFischerratsexposedtoFA (Limaet al.,2015).Ourstudyshowednosigniﬁcantchangesin CATamongallthegroups,however,asigniﬁcantdifferenceinSOD amongFAgroupswasobserved.ThesedatasuggestthattheFAis abletobreaktheantioxidantdefensemechanismsinthekidneys and lead to the formation of oxidative stress (Zararsiz et al., 2007b).

FAdevelopscytotoxiceffectbyformingacrosslinkingbetween proteinsandDNAresultingindamagethatcouldleadtocanceror celldeath,andtheformationofreactivespeciescanacceleratethis process(Saitoetal.,2005;Zararsizetal.,2007b).Theincreaseof TBARSisanindicationofoxidativedamagetothelipid peroxida-tion process and thus a marker widely used to demonstrate oxidativestress(Bakaretal.,2015;Limaetal.,2015;Zararsizetal., 2006).ThisincreaseofTBARScanresultinchangesinthestructure and permeability of the cell membrane and consequently stimulatethereleaseofcytotoxiccompoundsasmalondialdehyde (MDA) (Campos et al., 2013; Lima et al., 2015). Our results corroboratepreviousstudiesinwhichanincreaseinMDAlevelsin groups of animals exposed to FA was observed, suggesting an associationofoxidativedamage in kidneytissue causedby the toxicityofFA(Bakaretal.,2015;Ciftcietal.,2015;Limaetal.,2015; Zararsizetal.,2006).

Finally, the renal damage is usually accompanied by an important and localinﬂammatoryprocess which promotes the inﬁltrationofcellsmainlyconductedbychemokines.Chemokines

aresmallchemotacticcytokinesactivatedbyinflammatorycells andwiththecapacitytorecognizeavarietyofleukocytes(Jung etal.,2015;vanderVeenetal.,2009).Theincreaseinthelevelsof chemokinessuchasCCL2,CCL3andCCL5hasbeenassociatedwith theinfluxofcellssuchasmonocytes,lymphocytesandeosinophils intotissuesandfluids(Capellietal.,2005;ContiandDiGioacchino, 2001).Supportedbytheseconcepts,inthisstudyweobservedhigh levelsofCCL2,CCL3andCCL5intherenalparenchymaofanimals exposedtolowerFAconcentrations.Otherstudieshavepreviously demonstrated that increased CCL2, CCL3 and CCL5 in the parenchymawerestronglyassociatedwiththeworseninginthe renalin_flammatoryinjury(Andersetal.,2003;Keepersetal.,2007; Nishiharaetal.,2013).Inconclusion,ourresultsdemonstratedthat theexposuretoformaldehydeinashortperiodoftimeleadsto changesinthekidneyfunction,inflammationandmorphology,as wellaspromotedtheincreaseofsuperoxidedismutaseactivityand oxidativedamage.

Conﬂictsofinterest

Therearenoconﬂictsofinterest. Acknowledgments

ThisworkwassupportedbytheNationalCouncilforScientiﬁc and Technological Development (N 461495/2014-7 3), Minas Gerais ResearchSupport Foundation(FAPEMIG) (NCDS e APQ-01253-13). and the Federal University of Ouro Preto (UFOP) (23.109.006271/2014-70 2).WethanktheLaboratory of Experi-mentalNutrition(LABNEXeUFOP)andtheLaboratoryofMetabolic Biochemistry(LBMeUFOP).ATisincreditwiththeCNPqforthe fellowshipofresearchproductivity.

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