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Hndw Pshn CopotonIntnton Jon o HyptnsonVom 2013, Atc ID 305202, 10 pshttp://dx.do.o/10.1155/2013/305202

Research ArticleDNA Damage and Augmented Oxidative Stress inBone Marrow Mononuclear Cells from Angiotensin-DependentHypertensive Mice

Bianca P. Campagnaro,1 Clarissa L. Tonini,1 Breno V. Nogueira,1,2 Dulce E. Casarini,3

Elisardo C. Vasquez,1,4 and Silvana S. Meyrelles1

1 Laboratory of ransgenes and Cardiovascular Control, Department of Physiological Sciences, Health Sciences Center,Federal University of Espirito Santo, Avenida Marechal Campos 1468, 29043-900 Vitoria, ES, Brazil

2 Department of Morphology, Health Sciences Center, Federal University of Espirito Santo, 29045-402 Vitoria, ES, Brazil3 Department of Nephrology, Federal University of Sao Paulo (UNIFESP), 04021-001 Sao Paulo, SP, Brazil4 College of Health Sciences, EMESCAM, 29043-900 Vitoria, ES, Brazil

Cospondnc shod ddssd to Svn S. Mys; [email protected].

Rcvd 26 Novm 2012; Accptd 16 Jny 2013

Acdmc Edto: Ncos Fdco Rnn

Copyht 2013 Bnc P. Cmpno t . Ts s n opn ccss tc dsttd nd th Ctv Commons AtttonLcns, whch pmts nstctd s, dstton, nd podcton n ny mdm, povdd th on wok s popyctd.

It hs n poposd tht th nonhmodynmc fcts o notnsn II mpotnt o th dm osvd n th two-kdny,on-cp (2K1C) novsc hyptnson mod. Mch vdnc conms tht notnsn II s dcty nvovd n NAD(P)Hoxds ctvton nd consqnt spoxd non podcton, whch cn dm DNA. T cnt stdy ws pomd toxmn th fcts o notnsn-II-dpndnt hyptnson n on mow mononc cs (BM-MNC); dhydothdmstnn ws sd to ssss ctv oxyn spcs (ROS) podcton, nd th comt ssy ws sd to ssss DNA mnttonn 2K1C hyptnsv mc 14 dys n ty cppn. In ths stdy w dmonsttd tht 2K1C hyptnsv mc hv nvtd ymphocyt cont, wh ndfnttd BM-MNC conts w dmnshd. 2K1C mc so showd n mntd ROSpodcton nd mkd BM-MNC DNA mntton. In concson, ndonos nn notnsn systm ctvton-ndcdt hyptnson s chctzd y xcssv ROS podcton n BM-MNC, whch mht cs mkd DNA dm.

1. Introduction

Hh ood pss s commony ond n ptnts wthchonc kdny dss nd novsc hyptnson s common om o scondy hyptnson nd qntysstnt to phmcooc ttmnt [1]. In th two-kdny,on cp (2K1C) Godtt mod, novsc hyptnsons ndcd y nt n ty stnoss, whch dcsn pson o th cppd kdny nd css ncsdnn s nd cctn notnsn II (An II) [2]. AnII, whch s th mn fcto pptd o th nn-notnsnsystm (RAS), hs mkd hmodynmc, cdc, nd nfcts, s pvosy osvd y o otoy n mc [25]. In ddton, t so xts tss-spcc sponss s t cn

ocy synthszd [68]. Athoh t s contovs, th

xstnc o ocon mow (BM) RAS hs n dmon-sttd n ts [9]. Bcs th BM s hhy onzd,compx on, tht s, th pncp hmtopotc tss ndts, ocy BM-omd An II my n tocn opcn pptd tht fcts physooc nd pthoochmtoposs [10].

Stds hv dmonsttd tht An II pys o noxdtv stss dvopmnt n th spontnosy hyp-tnsv t [11] nd n th novsc hyptnsv t[12]. Rctv oxyn spcs (ROS) py cc on RAS snn n BM cs [9, 13]. In ddton, stdsn xpmnt nms hv shown tht mntd ROS[1416], ptcy spoxd (O2

) [1720], cn ntct

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2 Intnton Jon o Hyptnson

wth DNA, whch sts n oxdtv dm nd DNAmntton-mdtd c njy [21].

kn toth, ths vdnc stony sppots thmpotnc o th 2K1C mn xpmnt mod tonvstt th nnc o hyptnson on DNA dm.To, n th psnt stdy, w tstd th hypothss

tht 2K1C-mdtd hyptnson ncss ROS podctonnd ndcs DNA dm n mn BM mononc cs(MNC).

2. Material and Methods

2.1. Animals. Expmnts w pomd n m C57BL/6(C57) mc, whch psnt sn nn n [22], whn23 on v, nd tht w d nd mntnd nth Lotoy o nsns nd Cdovsc Contonm cty (Vto, ES, Bz). T mc w d stndd chow dt nd povdd wt ad libitum. Anmsw hosd n ndvd pstc cs wth contodtmpt (22C) nd hmdty (60%) nd w xposd to 12 : 12 h ht-dk cyc. A o th xpmnt pocdsw pomd n ccodnc wth th Nton Insttts oHth (NIH) dns, nd stdy potocosw pvosyppovd y th Insttton Anm C nd Us Commt-t (CEUA-Emscm Potoco no. 010/2009).

2.2. Induction of 2K1C Renovascular Hypertension. W sd mos mod o 2K1C notnsn-dpndnt hyptnson,s pvosy dscd [3, 4, 23]. By, th nms wnsthtzd (ktmn/xyzn 91/9.1 m/k, i.p.) nd kpton htn pd tht mntnd th ody tmpt t37C to vod hypothm. T n ty ws xposdthoh topton nk ncson nd ws cysotd om th n vn, nvs, nd connctv tsss.A U-shpd stnss st cp wth 0.12 mm opnn wdthws pcd ond th n ty cos to th domnot, whch std n pt n pson occson[24]. T wond ws std, nd th nm cvd sn njcton o nzypncn nzthn (7 m/k, i.m.)oowd y covy nd c o 24 h. Conto mcndwnt th sm sc pocd xcpt o th nty cp pcmnt (Shm).

2.3. Hemodynamic Measurements. Fotn dys thn ty cppn (2K1C) o Shm optons, th nmsw nsthtzd wth comnton o ktmn/xyzn

(91/9.1 m/k, i.p.) nd th ht common cotd ty wsxposd nd sotd thoh cvc ncson. A ctht(0.040 mm OD 0.025 mm ID; Mco-Rnthn; BntScntc) ws d wth hpn soton (50 UI/mL sn)nd po to nston nto th ht cotd ty, whchws sctnosy tnnd nd oht ot t th npo th nck. Immdty sy, nms cvd sn nzypncn nzthn (7 m/k, i.m.) njcton.T ctht ws connctd to pss tnsdc (CoLotos, USA), whch ws pd nto pss-pocsso mp nd dt cqston systm (MP100,Bopc Systms, USA) o mn t pss (MAP)nd ht t (HR) codns. A 48 hos, MAP nd

HR dct codns w otnd wh th nms wconscos nd movn ond y n th c.

2.4. Plasma Ang II Level Measurement. A hmodynmcmsmnts, ood ws dwn thoh th t nnto ts contnn EDA nd pots nhto cock-

t (Podct no. P2714, Sm-Adch); th smps wcntd t 9.5 o 15 mn n td cnt(4C) to mov psm o t nyss. Psm An II wsqntd y vs phs hh-pomnc qd cho-mtophy (HPLC). By, pptds wnty sptdn vs phs Aqpo ODS 300 comn 7 m (4.6 250 mm) (Appd Boscncs, Fost Cty, CA, USA) sn n mo phs dnt om 5 to 35% (ctontn 0.1% phosphoc cd) o 40 mn sn 1.5 mL/mnow t. An III (320 n) ws ddd to ch smp sn ntn stndd, nd th pptds w dtctd t214 nm sonc. An II ws xtctd sn Sp-Pck-C18 comn chomtophy (Mpo, MA, USA) ndws ctvtd wth 5 mL mthno, 5 mL tthydon,5mL hxn, nd 10mL H2O (MQ). A ctvton,th smps w n thoh th comn nd td wththno : ctc cd : H2O(90:4:6,v/v).Tstphstcontnn An II ws vpotd n Spd Vc SC 110(Svnt Instmnts, Hoook, NY, USA) nd constttdwth 500 L 0.1% phosphoc cd n 5% ctont, td,nd njctd onto th HPLC nytc comn. Rtntontm ws sd to dnty pks o ntst, whch hd npvosy dtmnd y stndd pptd ton. T c-ctons w sd on pk , nd An II concnttonws xpssd s pmo/mL ood.

2.5. Bone Marrow Mononuclear Cell Isolation. Mc w

thnzd wth sodm thopnt ovdos (100 m/k,i.p.) nd mow smps w coctd om ms ndts tht hd n dssctd nd cnd o so tsss.A movn th pphyss nd nn ccss to thmow cvts, who BM ws shd ot sn 26- nd ttchd to 1 mL syn d wth DccosModd E Mdm (DMEM; Sm, St. Los, MO,USA). MNCs w sotd y dnsty-dnt cnt-ton; th BM sspnson n 4 mL DMEM ws odd on4 mL Hstopq 1083 (Sm-Adch) nd cntd o30 mn t 400 . T BM-MNC cton ws ssqntycoctd nd wshd n phospht-fd sn (PBS). Asm vom o th stn sspnson ws mxd wth

0.4% typn to pom c cont nd vty nyss.Lymphocyts nd ndfnttd cs w nyzd sn N chm.

2.6. DNA Damage Measurement with the Comet Assay. Bonmow MNC DNA dm ws nyzd y th kncomt ssy s dscd y Snh t . [25] wth mnomodctons. R mcoscop sds w pcotdwth 200 L 1.5% nom mtn pont os n dstdwt, t 60C (Sm-Adch), dd ovnht t oomtmpt, nd thn stod t 4C nt s. Ssqnty,

2 104 MNCs w mxd wth 100 L 1% ow mtn pontos n PBS t 37C (Invton, Spn) nd spd on

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Intnton Jon o Hyptnson 3

th os-cotd sds sn covsp. A n t4C o 20 mn, covsps w movd, nd th sds wmmsd n shy ppd cod yss soton (2.5 M NC,100 mM EDA, 10 mM s t pH 1010.5, wth shy ddd1% ton X-100 nd 10% DMSO) t 4C o 1h. A 5 mn wsh n cod dstd wt, th sds w pcd n

n ctophoss chm, whch ws thn d wth shkn f (300 mM NOH, 1 mM EDA, pH > 13) o20 mn t 4C. Ectophoss ws pomd t 300mA nd25 V o 30 mn. A o ths stps w condctd wthotdct ht to pvnt ddton DNA dm. T sdsw wshd th tms o 5 mn wth 0.4 M s f, pH7.5, o ntzton. Fny, 100 L 20 /mL o thdmomd (Sm-Adch) ws ddd to ch sd, covdwth covsp, nd nyzd t 20x mncton sn oscnc mcoscop (Oymps BX60, Untd Kndom)tht hd n qppd wth xctton (510550 nm) nd (590 nm) ts.

DNA dm ws vtd sn vs cssctono comts nto v vs ccodn to comt t sz om0 (ndmd wth no t) to 4 (mxmy dmd wthon t). T DNA dm xtnt ws xpssd n tynts (..). T hndd ndomy sctd cs (100 csom ch o th pct sds) w nyzd om chnm, nd th AU vs w ntd o ch nm,whch w vd to otn thn st p nm. Top dm ndx (DI) nd om 0, n whch o thcs w ndmd (300 cs 0), to 1200, n whch oth cs w mxmy dmd (300 cs 4) [26]. Tdm qncy (%) ws cctd sd on th nmo cs wth ts vss thos wthot ts [27] nd th %DNA dm ws th cton o ch dm v tvto o th comts tht w nyzd.

2.7. Intracellular Superoxide Anion Fluorescence Measurement.Nonoscnt dhydothdm (DHE) ws sd o nt-c O2

dtcton y ow cytomty. Hydothdn sy c pm nd s pdy oxdzd y spoxdto thdm, whch nds to DNA nd mps th doscnc sn. o stmt th O2

contnt n th csspnson, 105 BM-MNCs w stnd wth 160 M DHE,oowd y 30 mn ncton t 37C n th dk toctt dy odn. DHE-odd cs w ttd wth10mM H2O2 to oxdz th dy s postv conto. A5 mn o H2O2 ttmnt, th BM-MNCs w wshd wthPBS nd c ROS vs w nyzd mmdty wth

FACSCnto II ow cytomt (Bcton Dcknson, Sn Jn,CA, USA). n thosnd vnts w codd om chsmp, nd owd nd sd sctt ts w sd to sctsn cs om cmps nd ds. Spcc oscncntnsty ws xpssd s th mdn oscnc ntnstyom th v o t st th ptd xpmnts n ..Rdoscnc wsdtctdtwn 564 nd606 nm sn 585/42 ndpss t. T Dt w cqd ndnyzdsn BD FACSDv sow (BD).

2.8. Statistical Analysis. T dt psntd s p-snttv s o s th mns SEM. T ow cytom-ty dt xpssd s mdn oscnc ntnsty

able 1: Body, vntc, nd kdny whts o 2K1C nd Shmmc 14 dys n ty cppn.

PmtsShm

(5)2K1C

(5)

Body wht () 24 0.5 22 0.8

Vntc dy wht (m) 25 0.7 27 0.7

Cppd kdny dy wht (m) 37 1.4 25 4.2

Contt kdny dy wht (m) 40 2.0 44 1.7

Cppd kdny wht/ncppdkdny wht (m/m)

0.94 0.02 0.56 0.09

Vs th mns SEM. < 0.05 nd < 0.01 compd wth thShm nms (stdnts t-tst o ndpndnt smps).

(MFI) cocnt o vton (CV) o 3 ptd nd stts-tcy podc (Fdmn tst) msmnts o t stv ndpndnt nms. Nomty ws vtd sn thKomooov-Smnov tst. Sttstc nyss ws pomd

sn Stdnts -tst o compson o two ndpndntops, nd two-wy nyss o vnc (Anov) oowdy th Bonons post hoc tst ws sd o compsono mo thn 2 ops. T Mnn-Whtny tst ws sdto comp th nk sm o th MFIs om th oxdtvstss xpmnts. vs

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Figure 1: B phs dmonsttn stn mn t pss, ht t, nd psm notnsn II vs n conscos Shm ( = 5)nd novsc hyptnsv (2K1C, = 5) mc. Vs th mns SEM. < 0.05 nd < 0.01 vss th Shm op (Stdnts tst o ndpndnt smps).

ymphocyt conts (62%) wth smtnos dctonn ndfnttd c nm (18%) compd wth thconto nms.

3.3. DNA Damage Measurement with the Comet Assay. Tcomt ssy s vstnd snstv mthod oqntynndnyzn DNA mntton n ndvd cs tht cn sd to ssss oxdtv DNA dm. T sc pncpo th comt ssy s DNA ctophoss n n os

mtx. Bcs th mntd DNA mts, th cs ookk comt nd mcoscop wth hd contnn ntctDNA nd t contnn DNA mnts [24]. GnomcDNA mntton ncdnc ws vsy nyzd ccod-n to comt ppnc. o cdt An II-dpndnthyptnson fcts on BM-MNC, DNA dm ws scodnto v csss ccodn to t sz ndth tv t DNAcontnt ndcts th mont o DNA dm. A sncntpdomnnc o ow notoxcty vs 0 nd 1 n th Shmnms (v 0: 283.4 nd v 1: 402.4%) compd wthth 2K1C mc (v0: 74.4 ndv1: 71%) wsosvd.In contst, sv notoxcty vs 3 nd 4 pvd n th2K1C mc (v 3: 36 3.2 nd v 4: 27 6%) compd

wth th Shm mc (v 3: 7 0.4% nd v 4: 2 0.4%).Gnotoxcty vs dmonsttd s typc ms ndv vs n F 3. Moov, DNA mnttonws qntd sn th DNA dm ndx nd qncy(Fs 3() nd 3()). T 2K1C mc hd ncsd DNAdm s ndctd y hh dm ndx (Shm: 345 19 vss 2K1C: 80655 ..) nd qncy (Shm: 7 2 3%

vss 2K1C: 93 4%).

3.4. Ang II-Dependent Hypertension Induced ROS Production.T ov ndns d s to th nvstt th fctso An II-dpndnt hyptnson on ROS podcton nBM-MNC. DHE s mmn-pm oscntdy tht pdy ccmts n th cytopsm, wh t soxdzd y O2

, stn n d nc oscnc thtcn msd y ow cytomty. In F 4, psnt-tv hstoms (F 4()) nd v ( phs) O2

podcton vs shown. As dmonsttd y th htsh ndn th ph, DHEmdn oscncntnsty(MFI) vsw sncnty hh n th 2K1C thn n thShm op (16856 5809 vss 2051 336 .., < 0.01),ndctn ncsd ntc BM-MNC oxdtv stss.

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Figure 2: N chm nyss o on mow mononc cs om Anotnsn-II-dpndnt hyptnsv (2K1C, = 10) ndnomotnsv (Shm, = 10) mc. Vs o ymphocyts nd ndfnttd cs th mns SEM. < 0.05 vss Shm op(Stdnts tst o ndpndnt smps).

4. Discussion

T mn ndn o ths stdy ws mkd DNA mn-tton n BM-MNC om An II-dpndnt hyptnsvmc, most ky cs o mntd O2

podcton ndconsqnt oxdtv stss. Fth stds ncdn othxpmnt mods shod dsnd to dscmnt thtv nnc o hyptnson nd An II on ths pocss.

Rn ty cppn s ccompnd y ctvton o thRAS nd hmodynmc ttons [14, 2830]. Hh vso psm nn nd An II n 2K1C mc hv n osvdtwn 7 nd 14 dys cppn nd hv tnd to

nom vs y dy 28 [14, 28, 3032]. Bsd on ths os-vtons nd on pvos pcton om o otoy[2], w pomd ths stdy two wks n tycppn. As xpctd, 2K1C mc showd tophy o thcppd kdny nd hyptophy o th contt kdny.2K1C mc xhtd hh ood pss vs ccompndy tchycd, n mnt wth pvos stds [2, 5,24]. In ths stdy, w conmd th hh psm An II,coootn th concpt tht RAS ctvton pys pvoto n hyptnson dvopmnt n ths mn mod. Inddton to th psso nd postv chonotopc fcts, AnII so stmts cdomyocyt potn synthss [3336],whch n ddton to hyptnson my xpn th cdc

hyptophy tht w osvd n th 2K1C hyptnsvmc. On th oth hnd, t stds shod consd thmsmnt o potn vs o th vntcs s n ndxo hyptophy. kn toth, ths dt sst tht th2K1C mos xhts th mn ts o ndonos AnII-dpndnt hyptnson t ths tm pont.

In ddton to th systmc ctons o th RAS, mnytsss nd ons hv oc RAS, whch cn hvpcn, tocn, nd ntcn nctons [37]. BM s thmjo svo o dt on-spcc stm cs, ncdnndoth ponto cs (EPCs), hmtopotc stm cs(HSCs), nd msnchym stm cs (MSCs). In ths con-txt, th psnc o compt oc BM RAS tht fcts

physooc nd pthooc ood c podcton wshypothszd y Hzndo t . [38] nd hs cntyn conmd [9]. In o stdy, w ond mntd ym-phocyts nd dmnshd nms o ndfnttd BMcs n 2K1C hyptnsv mc. Consdn th psnc oRAS componnts n HSCs [39] nd stom/MSCs [9], t sson to popos tht RAS my so ocy ctvtdn BM o An II-dpndnt hyptnsv mc. Howv, thspossty st nds to conmd y ssqnt stds.

Accmtn vdnc ssts tht th oc RAS sctvy nvovd n BM cs poton, dfntton,nd dth. O not, An II fcts th nt BM-MNC poo,

sch s EPCs [40], HSCs [41, 42], nd MSCs [9]. As cntydmonsttd, An II consstnty dcss th nmo ctd EPCs thoh ctvton o A1 cptos ndndcton o poptoss [40]. In ddton nd consdntht 2K1C hyptnsv mc xht ndoth dysncton[23], An II cod ctvt nmmtoy cs o cytoknpodcton, whch my spons o c ctmntn nmmton [4347]. Moov, ths vsoctv pptddcty stmts ythoposs y mntn ythopo-tn homon podcton [48], whch ts ythocytdfntton [49], thoh A1 cptos [8, 41]. Accod-ny, d Cnh t . [50] nd Csss t . [48] potd thtnotnsn convtn nzym (ACE) nhtos nd A1

cpto ntonst ttmnts cs nm, dmonsttnhmtopotc sd fcts o RAS ocks nd ndctntht An II pys n mpotnt o n hmtoposs. kntoth, xpmnt vdnc ssts tht An II xhtsmpotnt hmtopotc fcts y stmtn ythod,myod, nd ymphod dfntton, stn n -mntd ymphocyt nm nd smtnosy dmnshdndfnttd c nm.

T s own vdnc tht ncsd oxdtv stss,whch sts n xcssv ROS nton, pys o ncdovsc dsss ncdn hyptnson, s cntyvwd y s nd oths [24, 5153]. Bcs th s nktwn ROS nd RAS snn [5355], ky mchnsm

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Figure 3: Bon mow mononc c DNA dm ssssd y th comt ssy. T phs dmonstt th v DNAdm ndx () nd qncy (). < 0.01 vss th Shm op (Stdnts tst o ndpndnt smps). Foscnt ms (c) typc comts dmonsttn ncsd DNA mntton n novsc hyptnsv (2K1C) mos compd wth nomotnsv(Shm) mos. T ns ph (c) dmonstts th v pcnts o DNA dm pcnts o ch notoxcty v, compnth 2K1C (d ccs, = 5) wth th Shm (mpty ccs, = 6) mc. < 0.01 vss th Shm op (two-wy Anov). Vs thmns SEM.

y whch An II nncs ht nd vss ncton cod v ts ty to ctvt ROS podcton [24, 56, 57].W osvd pononcd DHE MFI mntton n BM-MNC n th 2K1C hyptnsv mc compd wth Shmnomotnsv mc. T tonshp twn oxdtv stssnd ncsd ood pss hs n potd n mnyhyptnsv nm mods, ncdn th SHR[58], DOCA-st [59], th 2K1C [14], nd th 1K1C [60], whch hvxcssv O2

podcton d to mntd NAD(P)H ox-ds ctvty [6164]. Intstny, n th p47phox knockot

mos wth concnt 2K1C hyptnson mntd ROSpodcton occs v xpsson o ths NAD(P)H oxdssnt [14]. Ts nzym cn ctvtd y hmodynmcocs nd vsoctv onsts, o xmp, An II [6567],whch s pow vsoconstcto nvovd n hyptnsonpthonss tht ss ROS s n ntc snnmdto [66]. In ddton, t sms tht An II ndcsth ncs o ROS podcton n EPCs nd tht thsoxdtv stss cconts o th An II-mdtd dctono EPC nm, s ths fct cn ockd y cottmnt

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Figure 4: Efcts o novsc hyptnson on oxdtv stss n on-mow-mononc cs (BM-MNCs). () contns

psnttv hstoms o ntc oxdton o dhydothdm (DHE) to thdm y BM-MNC om hyptnsv (2K1C) nd nomotnsv (Shm) mos s vtd y ow cytomty. T ph smmzs th mdn oscnc ntnsty (MFI) vs oDHE-odd BM-MNC om 2K1C ( = 5) nd Shm ( = 5) mc. Vs th mdns cocnt o vton. < 0.01 vss thShm op (Mnn-Whtny tst).

wth n ntoxdnt [40] nd t ncss p91phox xps-son n EPCs, whch my contt to oxdtv stss[68].

As dscssd ov, th o o An II os yondconton cctoy homostss s dscssd ov n thmpct o thspptd n ROSpodcton, whch s stmtdy NAD(P)H oxds ctvton [69]. Rcnt xpmnt

stds hv shown tht, t hh concnttons, ROSs schs O2

cp o dct potn nd pd oxdton,whch css DNA mntton [70]. DNA dm, whchqnty occs n cs xposd to oxdtv stss [71],s om o c njy tht contts sncnty toth dvopmnt nd posson o cdovsc dsods[24, 64, 72].

T comt ssy hs n sd to dtmn DNA -mntton n ood cs n mn mods o spontnosthoscoss [73, 74] nd novsc hyptnson [24].Howv, ths s th st tm tht th comt ssy hs nsd to ssss DNA mntton n BM-MNC om 2K1Cmc. O sts cy dmonsttd mntd DNA

mntton n BM-MNC om th 2K1C mc compdwth th Shm mc. Amntd DNA dm hs son dmonsttd n oth nm mods o hyptnson,ncdn kdny cs om DOCA-st ts [75] nd mosnsd wth An II [21]. Fthmo, DNA dm csdy ROS occs mo commony n hyptnsv thn nnomotnsv ptnts nd cn dcd y ntoxdntds [76]. O not, n thpsd mos kdny, DNA dm- ws csd y An II, not y ndcd vsoconstcton,snc noth vsoconstcto dd not cs DNA dm[21]. Moov, An II ndcs nomc dm n ctdkdny cs most ky v oxdtv mchnsms, whch cn pvntd y A1 cpto ntonsts nd y ntoxdnts

[77]. T 2K1C mc hd ncsd O2 podcton, whch s

hhy ctv nd shot-vd dc tht s spons oROS nton nd cn ntct wth ny mocs, schs DNA [21, 78, 79]; ths, o dt sst tht ROS pys ky o y ndcn DNA oxdtv dm n ths mod oAn II-dpndnt hyptnson.

In concson, w dmonsttd tht t hyptn-son ndcd y ndonos RAS ctvton y cppn thn ty o two wks (th 2K1C mos mod) stsn mkd ncs n ROS podcton wth consqntBM-MNC DNA dm. W spct tht An II fctsmy d to cctn nd oc BM RAS; tho, othsystms my py cc pthoooc o n th DNAdm osvd n BM-MNC o 2K1C hyptnsv mc.kn nto ccont tht BM-dvd cs sponso mntnn, ntn, nd pcn dfnttd css consqnc o physooc c tnov o tssdm d to njy, th dt otnd y ths stdysstd tht comodts, spccy An II-dpndnt

hyptnson, hvto ptcy consdd tooostnspntton s ntndd, snc th dono tss (.., onmow) mht td n ts nctonty.

Acknowledgments

E. C. Vsqz nd S. S. Mys sppotd y thNton Conc o th Dvopmnt o Scnc ndchnooy (CNPq, Rs. 302582/2011-8 nd 302535/2009-8 Gnts, sp.) nd th Stt Ancy o th Dvopmnto Scnc nd chnooy (FAPES, R. FAPES/PRONEX012/2009).

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