research article intracellular oxidant activity...

Research ArticleIntracellular Oxidant Activity Antioxidant Enzyme DefenseSystem and Cell Senescence in Fibroblasts with Trisomy 21

Viacutector Rodriacuteguez-Sureda12 Aacutengel Vilches2 Olga Saacutenchez23

Laura Audiacute14 and Carmen Domiacutenguez12

1Center for Biomedical Network Research on Rare Diseases (CIBERER) 08028 Barcelona Spain2Biochemistry and Molecular Biology Research Center for Nanomedicine Vall drsquoHebron Research Institute 08035 Barcelona Spain3Maternal and Child Health and Development Network II (SAMID II) Institute of Health Carlos III 28029 Madrid Spain4Paediatric Endocrine Service Vall drsquoHebron University Hospital 08035 Barcelona Spain

Correspondence should be addressed to Carmen Domınguez carmendominguezvhirorg

Received 18 December 2014 Accepted 19 February 2015

Academic Editor Ersin Fadillioglu

Copyright copy 2015 Vıctor Rodrıguez-Sureda et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Downrsquos syndrome (DS) is characterized by a complex phenotype associated with chronic oxidative stress and mitochondrialdysfunction Overexpression of genes on chromosome-21 is thought to underlie the pathogenesis of the major phenotypic featuresof DS such as premature aging Using cultured fibroblasts with trisomy 21 (T21F) this study aimed to ascertain whether animbalance exists in activities mRNA and protein expression of the antioxidant enzymes SOD1 SOD2 glutathione-peroxidaseand catalase during the cell replication process in vitro T21F had high SOD1 expression and activity which led to an interenzymaticimbalance in the antioxidant defense system accentuated with replicative senescence Intracellular ROS production and oxidizedprotein levels were significantly higher in T21F compared with control cells furthermore a significant decline in intracellular ATPcontent was detected in T21F Cell senescence was found to appear prematurely in DS cells as shown by SA-120573-Gal assay and p21assessment though not apoptosis as neither p53 nor the proapoptotic proteins cytochrome c and caspase 9 were altered in T21FThese novel findings would point to a deleterious role of oxidatively modified molecules in early cell senescence of T21F therebylinking replicative and stress-induced senescence in cultured cells to premature aging in DS

1 Introduction

Downrsquos syndrome (DS) is a developmental abnormality char-acterized by the presence of a third (partial or total) copy ofchromosome 21 Trisomy 21 is the most common genomicaneuploidy with an incidence of approximately 1 in 700 livebirths [1] DS is characterized by well-defined and distinctivephenotypic features including craniofacial dysmorphismmental retardation almost constant muscular hypotoniaand joint laxity Further potential abnormalities and theclinical picture include heart defects digestive malforma-tions congenital cataract short stature sensory deficienciesHirschsprungrsquos diseaseWestrsquos syndrome seizures propensityto leukemias and other blood diseases autoimmune andendocrine disorders premature aging and Alzheimer-typeneuronal pathology by the third to the fourth decade of life

[2ndash5] Although the DS phenotype has become better knownin the last fifty years the underlying pathogenic mechanismsof this complex syndrome and how the additional chromo-some 21 causes this array of diseases remain to be elucidatedOverexpression of a number of genes located in chromosome21 has been considered to be the central point of the DSphenotype since increased synthesis of these gene productscould cause an imbalance in various biochemical pathwaysresulting in dysregulation of physiologic networks

Reactive oxygen species (ROS) are generated in aerobicorganisms during physiologic or physiopathologic oxidativemetabolism These species are harmful if not neutralized bythe antioxidant enzymedefense system Firstly CuZn-super-oxide dismutase-1 (SOD1 EC 15111) a major cytoplasmicantioxidant enzyme catalyzes the dismutation of superoxide

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 509241 17 pageshttpdxdoiorg1011552015509241

2 Oxidative Medicine and Cellular Longevity

radicals to molecular oxygen and hydrogen peroxide whichin turn is converted by either glutathione peroxidase (GPxEC 11119) or catalase (CAT EC 11116) to water and oxygenthus providing a combined enzymatic action against oxygentoxicity An imbalance in the ratio of SOD to GPx and CATresults in the accumulation of H

2O2which may participate

in the Fenton reaction resulting in the formation of noxioushydroxyl radicals As the gene coding for the enzyme SOD1is localized to 21q221 almost all tissues of DS patientsvarious trisomy 21 cultured cells [6] and the brains of DSpatients [7] overexpress this gene Overexpression of SOD1as a consequence of gene dosage in trisomy 21 cells canlead to increased oxidative stress (OS) caused by an alteredSOD(GPx+CAT) activity ratio that may upset the oxidant-antioxidant balance thereby resulting in oxidative damageto molecules and vital cellular targets In fact evidence for amultiple prooxidant state in young DS patients would appearto support the hypothesis that ROS overgeneration couldpotentially contribute to some pathologic manifestations ofDS especially accelerated aging [8 9] However this hypoth-esis has never been unequivocally tested since cells overex-pressing SOD1 have been shown to produce less H

2O2[10]

which would lend support to the hypothesis proposed byLiochev and Fridovich [11] that more efficient superoxideanion dismutation in DS would prevent high H

2O2levels

since excess SOD1 can catalyze surrogate reactions therebygenerating hydroxyl radicals fromH

2O2 Furthermore trans-

genic mice designed to overexpress SOD1 turned out to beeven more resistant to oxygen toxicity than controls [12]Another study showed that SOD1 overexpression increasedthe survival of transplanted neurons [13]

The net intracellular oxidant activity depends on theoverall balance between ROS generation and the capacity ofcells to buffer these highly reactive species however whenthis capacity is exceeded oxidative stress is produced andconsequently ROS cause damage to macromolecules such asprotein lipids and DNA Oxidative stress is implicated in thepathogenesis of diverse chronic or acute diseases as well asin aging [14] and has been associated with DS and its majorphenotypic features such as premature aging [15]

Normal human fibroblasts cultured in vitro have limitedproliferation potential and eventually become senescent asa result of serial passages commonly known as replicativesenescence giving rise to the idea that cellular senescencemight be a determinant of human aging In vitro cellularsenescence is thus regarded as a useful model for elucidat-ing molecular mechanisms that underlie organismal agingfurthermore replicative cell senescence is continually beingshown to represent a validmodel of in vivo aging [16] In addi-tion to replicative senescence cellular senescence can alsobe induced by various forms of stress including oxidativestress thus cultured fibroblasts with trisomy 21 can serve asan excellent in vitro tool for aging research since they canbecome oxidative stress-induced senescent cells in whichfurthermore replicative senescence can be induced

The hypothesis of this study was that fibroblasts withtrisomy 21 (T21F) have high SOD expression and activitywhich lead to an imbalance in the antioxidant enzymatic sys-tems thereby provoking overgeneration of ROS and cellular

oxidative damage that could pathophysiologically underliepremature cell senescence in DS In addition to antioxidantenzyme activity alterations mitochondrial dysfunction [17]previously reported in DS would affect cell energy pro-duction and increase intracellular oxidant activity whichcombined could accelerate cellular senescence in T21F

This work aimed to assess intracellular oxidant activity ofcultured T21F and ascertain whether an imbalance exists inthe activities mRNA and protein expression of antioxidantenzymes SOD1 SOD2 GPx and CAT at low (3ndash7) and highpassages (8ndash12) during the cell replication process in vitro Inan attempt to determine whether oxidative stress is relatedto premature senescence in T21F senescence-associated 120573-galactosidase activity (SA-120573-Gal) and the expression of pro-teins p53 and p21 were analyzed at low and high passages dur-ing in vitro cell replication To our knowledge this is the firststudy relating ROS generation reduced ATP levels oxidativestress-induced molecular damage and cellular replicativesenescence in fibroblasts from DS the results of this studyshed new light on the complex relationship between oxidativestress and premature senescence in DS

2 Materials and Methods

21 Reagents and Materials Chemicals were of analyticalgrade and purchased fromSigma (St LouisMOUSA) unlessotherwise stated 221015840-azobis-2-methyl-propanimidamide di-hydrochloride (AAPH) 1-butanol 35-di-tert-4-butylhydrox-ytoluene 4-methylumbelliferone (4-MU) 4-MU-120573-D-galac-topyranoside 4-MU-120573-D-glucuronide 1133-tetraethox-ypropane 2-thiobarbituric acid adenosine 51015840-triphosphateassay mix acetonitrile diethylenetriaminepentaacetic acid(DTPA) dimethyl sulphoxide (DMSO) Dulbeccorsquos ModifiedEagle Medium (DMEM) Eaglersquos Minimal Essential Medium(MEM) fetal calf serum (FCS) Hankrsquos buffered salt solution(HBSS) sodium chloride sodium dodecyl sulfate (SDS)somatic cell ATP releasing reagent tert-butyl hydroperoxide(TBHP) TrissdotHCl Triton X-100 and Tween-20

Penicillin streptomycin 5-(and-6-)-chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetate acetyl ester probe (CM-H2DCFDA) MitoSOX Red Mitochondrial Superoxide Indi-

cator MitoTracker Green FM and Hoechst 33342 werepurchased from Invitrogen (Carlsbad CA USA) Cell Prolif-eration Kit II (XTT) was purchased from Roche Applied Sci-ence (Basel Switzerland) 96-well and 12-well clear-bottomculture plates were from Corning (Corning NY USA) SODactivity determination kit (RANSOD) was from Randox(Crumlin UK) and GPx activity determination kit (cGPx-340) was from Bioxytech (Deltaclon Madrid Spain) Thefollowing antibodies were used for immunoblotting SOD1(sc-30080) and SOD2 (sc-11407) were purchased from SantaCruz Biotechnology (Santa Cruz CA USA) Dinitrophenyl-KLH (A-6430) was from Invitrogen p21 Waf1Cip 1 (2947)cytochrome c (4272) and cleaved caspase 9 (9505) werefrom Cell Signaling (Danvers MA USA) p53 (P8999) 120573-actin (A3854) rabbit IgG-HRP (A0545) and mouse IgG-HRP (A2304) were from Sigma-AldrichMammalian protein

Oxidative Medicine and Cellular Longevity 3

extraction reagent (M-PER) Halt Protease Inhibitor Cock-tail and SuperSignal West Pico were from Pierce Biotechnol-ogy (Rockford IL USA) Immobilon-P was purchased fromMillipore Corporation (Billerica MA USA) RNeasy MiniKit and RNase-Free DNase Set were from Qiagen (HildenGermany) RevertAid H minus first strand cDNA synthesiskit was from Fermentas (St Leon-Rot Germany) TaqManGene Expression Assays (predesigned probe and primersets) were from Applied Biosystems (Foster City CA USA)and senescence detection kit was from BioVision (MountainView CA USA) Human p21 Waf1Cip1 and Human p 53ELISAkits were fromCell Signaling (DanversMAUSA) andBiovendor (Brno Czech Republic) respectively

22 HumanDermal Fibroblast Cultures fromDownrsquos Syndromeand Control Fetuses Abdominal skin biopsies from 10human fetuses at 9ndash22 weeks of gestation products of legalterminations were collected with informed parental con-sent within 12 h postmortem from the Fetal Tissue Bankat the Hospital Universitari Vall drsquoHebron (fetaltissuepathvhebronnet) Five fetuses were confirmed as having Downrsquossyndrome three 47XY + 21 and two 47XX + 21 The fiveremaining fetuses two diagnosed of complex cardiac malfor-mation (46XX) one anhydramnioswith previousmembranerupture (46XY) and two occipital encephalocele (46XX and46XY) were used as controls (CF)

Primary fibroblasts were obtained from skin explants ina 25 cm2 culture flask with Eaglersquos Minimal Essential Medi-um (MEM) supplemented with nonessential amino acids10 fetal calf serum (FCS) and antibiotics (100 IUmLpenicillin and 100mgmL streptomycin) The flasks weremaintained at 37∘C in a 95 humid air 5 CO

2atmosphere

Fibroblasts were released by enzymatic digestion with trypsinand subcultured in a 75 cm2 plastic culture flask with 12mLof medium Cultures were fed by changing the mediumevery 3 days After reaching confluence cells were subjectedto different passages and fold increase in the cell countduring each passage was calculated or washed and the pelletresuspended in 1mL MEM with 10 FCS and DMSO andfrozen (24 h at minus80∘C and then in liquid nitrogen) Cellswere thawed and reincorporated into the cultures for ensuingexperiments at different passages for which fibroblasts weregrown inmedium supplemented with 10 FCSWe observedthat cultured FT21 aged more prematurely than controlfibroblasts since beyond passage 11 they showed progres-sive morphologic characteristics typical of senescent cells(enlarged vesicle-rich cytoplasm diminished cell density andincreased times to reach confluence) which rendered theirculture inviable beyond passage 13 Therefore the followingpopulation doubling (PD) cut-offs were used to differentiatecell aging in two stages nonsenescent cells (8ndash16 PD lowpassages (LP)) and aged fibroblasts (24ndash32 PD high passages(HP)) Cells were seeded at all times in 96-well culturemicroplates and incubated for 48 hours to allow them toattach to the plates

23 Prooxidant Challenge Cells were grown in 96-well cul-ture plates at 104 cellswell for ROS assessment and 5 sdot 103

cellswell for ATP and cell viability On the day of the exper-iment the culture medium was removed and the cells werewashed twice with 100 120583L of Hankrsquos buffered salt solution(HBSS Sigma) Cells were then incubated for 1 hour at 37∘Cwith either 100 120583L of HBSS (untreated cells) 100 120583L of 10mMAAPH in HBSS (AAPH-treated cells) or 100 120583L of 100 120583MTBHP in HBSS (TBHP-treated cells) washed as above andprocessed for the determination of cell viability ATP andROS (see below)

24 Cell Viability Cell viability was assessed with the CellProliferation Kit II (XTT) In brief cells grown on 96-wellculture plates were washed twice with 100 120583L of HBSS toremove culture medium Then 100 120583L of HBSS and 50 120583L ofthe reaction mixture were added to each well Plates wereincubated for 4 hours at 37∘C and absorbance was read at460 nm with a reference filter of 680 nm

25 Cell ATP Content Intracellular ATP determination wasperformed by a bioluminescence assay based on the ATP-dependent luciferin-luciferase reaction [18] A new internalcalibration standard of ATP was prepared each day in a rangefrom 1 to 100 120583g ATPmL prior to readings To determinethe cellular ATP content cells grown on 96-well whiteclear-bottom plates were first incubated for 3 minutes with25 120583L somatic cell ATP releasing reagent and then for 3minutes with 25120583L of sterile water The plates were placedin a Luminoskan RS luminometer (Thermo Fisher ScientificWaltham MA USA) and 50 120583L of the luciferase-containingbuffer (adenosine 51015840-triphosphate assay mix) was added toeach well just before measurement of the light emitted whichis proportional to the ATP concentration As the differentbatches of luciferase yielded varying linear responses readingvalues in each microplate were converted into percentageswith the control values of CF under the same conditions(untreated AAPH and TBHP) considered to be 100

26 Measurement of Reactive Oxygen Species IntracellularROS levels were assessed using the CM-H

2DCFDA probe

Cells grown in 96-well flat clear-bottom black polystyrenemicroplates were washed twice with 150120583L of HBSS toremove culture mediumThen 50 120583L of the probe (721 120583M inHBSS) was added to each well and the plates were incubatedfor 30 minutes at 37∘C Excess probe was discarded and100 120583L of the prooxidant solutions prepared in HBSS wasadded (see above) After the incubation cells were lysedby adding 50 120583L of 05 Triton X-100 in PBS The plateswere left protected from light for 3 minutes in a shakerand fluorescence was read at 538 nm with an excitationfilter of 485 nm Cellular levels of ROS were also visualizedunder microscope using CM-H

2DCFDA fluorescence live

cell imaging cell fluorescence images were captured using anepifluorescence microscope equipped with a digital cameraand image processing software (NIS-Elements Nikon Instru-ments Europe Amstelveen Netherlands)

27 Mitochondrial Superoxide Generation Mitochondrialsuperoxide levels were assessed using the MitoSOX probe


Cells grown in 24-well flat clear-bottom polystyrene micro-plates were washed twice with 1mL of prewarmed HBSSto remove culture medium Then 1mL of the probe (5 120583Min HBSS) was added to each well and the plates wereincubated for 10 minutes at 37∘C in a 5 CO

2atmosphere

protected from light After this period cells were washed andimages were taken using a Nikon Eclipse Ti epifluorescencemicroscope using a TRITC filter set

28 Enzyme Activities Total SOD activity was assayed usingthe RANSOD kit with minor modifications In brief fibrob-lasts were lysed in SOD buffer (500mM TrissdotHCl 10mMDTPA pH 82) by sonication at 4∘C and centrifuged for10min at 10000 g SOD activity was assayed in 15 120583L of thesupernatant and the results are expressed as Umg protein(one unit of SOD is that which causes 50 of the INTreduction rate under the assay conditions)

GPx activity was assayed using the cGPx-340 kit withminor modifications In brief fibroblasts were lysed in GPxbuffer (50mM TrissdotHCl 5mM EDTA 1mM DTT pH 75)by sonication at 4∘C and centrifuged for 10min at 10000 gGPx activity was assayed in 70 120583L of the supernatant using anundiluted sample and two samples diluted at 12 and 14 andthe results are expressed as mUmg protein (one unit of GPxis that which produces 1 120583mol of NADP+ per minute at pH80 and 25∘C in a coupled reaction with reduced glutathioneglutathione reductase and tert-butyl hydroperoxide)

Catalase was assayed by the Aebi method [19] In brieffibroblasts were lysed in CAT buffer (200mM KH

2PO4

K2HPO4 pH 7) by sonication at 4∘C and centrifuged for

10min at 10000 g CAT activity was assayed in 20 120583L ofthe supernatant by adding 1mL of 20mM H

2O2in CAT

buffer Absorbance of the samples was then recorded for 45seconds and the rate ofNADPHconsumption perminutewascalculated Results were expressed as Umg protein (one unitof CAT is that which decomposes 1 120583mol of H

2O2per minute

at pH 70 and 25∘C)120573-glucuronidase (120573-Gluc EC 32131) and 120573-galactos-

idase (120573-Gal EC 32123) were determined using the flu-orogenic substrates 4-methylumbelliferyl-120573-D-glucuronide(346mM in 40mM acetic-acetate buffer pH 35) and 4-methylumbelliferyl-120573-D-galactopyranoside (2mM in 01Mcitrate-phosphate buffer adjusted to pH 4 or 6 02M NaCl)respectively Fibroblasts were lysed by sonication at 4∘C indistilled water 25120583L of each sample (diluted 110 in water)was incubated with 100 120583L of the substrate at 37∘C for 30minutes The reaction was then stopped by adding 125mL of100mM glycine-NaOH buffer (pH 104) and the fluorescencewas read in a F-2500 FL spectrophotometer (excitation365 nm emission 450 nm) After blank subtraction activ-ity was calculated with respect to a standard curve of 4-methylumbelliferone and expressed in nmol per minute andmg of protein in the lysate Protein content was determinedaccording to themethod of Lowry [20] adapted to the samplecharacteristics or with the BCA assay [21]

29 Western Blot Western blotting was performed usingantibodies against SOD1 (15000) SOD2 (12000) p21

(1 1000) p53 (1 2500) cytochrome c (1 1000) cleavedcaspase 9 (1 1000) and 120573-actin (1400000) Cells werelysed by sonication inM-PER containing protease inhibitorsand 20120583g of protein was applied to each well Sampleswere separated on 15 SDS-polyacrylamide gels and thentransferred onto PVDF membranes Blots were blocked for1 hour at 37∘C in TBS (10mM TrissdotHCl 150mM NaCl pH74) with 5 powdered milk and then incubated overnightat 4∘C with the primary antibodies (1 powdered milk and001 (vv) Tween 20 in TBS) Blots were washed three timesinTBS-Tween (005 (vv) Tween-20 inTBS) and three timesin TBS and then incubated with a horseradish peroxidase-conjugated secondary antibody in incubation buffer for2 hours at room temperature After washing immuno-complexes were developed using an enhanced horseradishperoxidase-luminol chemiluminescence reagent (SuperSig-nal West Pico) according to the manufacturerrsquos instructions

210Quantitative Real-TimePCR formRNAExpressionAnaly-ses Gene expression was analyzed with quantitative real-time PCR (1 ng cDNA for each reaction) using a Prism 7000SequenceDetection System (Applied Biosystems) Total RNAwas isolated from cultured fibroblasts (1 sdot 106 cellscolumn)using the RNeasy Mini Kit and genomic DNA was removedusing the RNase-Free DNase Set RNA (200 ng for eachreaction) was used for cDNA synthesis with random hex-amer primers using the RevertAid H minus first strandcDNA synthesis kit TaqMan Gene Expression Assays (pre-designed probe and primer sets) were obtained fromAppliedBiosystems SOD1 (assay ID Hs00166575 m1) SOD2 (assayID Hs00167309 m1) and GPx (assay ID Hs00829989 gH)mRNA expression levels were normalized to the levelsof human DNA-directed RNA-polymerase II (assay IDHs00172187 m1) and human120573-actin (HumanACTBEndoge-nous Control) Relative expression was calculated by thedouble delta Ct method [22] using the Sequence DetectionSoftware (v 123) from Applied Biosystems

211 Lipoperoxidation (LPx) and Protein Carbonyl Groups(PGC) in Cell Lysates Cell lysates of fibroblasts were pre-pared by resuspending the extracted cells in distilled waterfollowed by sonification (10 sec three times in ice) Cellu-lar MDA concentration was determined as its diethylthio-barbituric acid adduct (TBA-MDA) after reverse-phase iso-cratic HPLC separation of the MDA-TBA complex as previ-ously described in detail [23 24]

Protein carbonyl content was analyzed by Western blotanalysis Cell lysates in M-PER were denatured with SDSand incubated with dinitrophenyl-hydrazine for 5 minutesat room temperature Samples were then neutralized andreduced by addition of 2-mercaptoethanol and loaded onto9 polyacrylamide gels After electroblotting oxidized pro-teins were detected by an anti-dinitrophenyl-KLH antibody

212 Detection of Senescence-Associated 120573-Galactosidase SA-120573-Gal expression in fibroblasts was examined cytochemicallywith a commercial senescence detection kit which is basedon the method described by Dimri and coworkers [25]


Low passages

0

2

4

6

8

10

Untreated AAPH TBHP

DCF

(RFU

)

T21FCF

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

2

4

6

8

10

DCF

(RFU

)

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Figure 1 ROS production determined by dichlorofluorescein oxidative stress assay in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls(CF) (119899 = 5) Fibroblasts were grown in 96-well microplates and incubated for one hour with Hankrsquos buffered salt solution (untreated) orsupplemented with 10mM AAPH or 100120583M TBHP ROS production was evaluated at low (a) and high (b) passages with H2DCFA-DAValues are expressed as mean plusmn SEM of six separate experiments performed in quadruplicate Statistical differences between CF and T21Fwere analyzed by Studentrsquos 119905-test lowastlowastlowast119875 lt 0001

Briefly cells grown on 12-well culture plates (7sdot103 cellswell)were fixed with 3 formaldehyde washed and incubated for16 hours at 37∘Cwith a staining solution containing 5-bromo-4-chloro-3-indolyl-120573-D-galactopyranoside (X-Gal) Nucleiwere then counterstained for 10 minutes at 37∘C with thenuclear dye Hoechst 33342 (Invitrogen) and cells observedin a Nikon Eclipse Ti epifluorescence microscope (NikonInstruments Europe Amstelveen Netherlands) Aminimumof five random fields per well were photographed at 100xunder both bright field and UV light for senescent cellidentification and cell counting respectively

213 Statistical Analysis Results are expressed asmean plusmn stand-ard error of the mean (SEM) Statistical analyses were per-formed using GraphPad Prism version 500 for Windows(GraphPadSoftwareSanDiego CAUSA httpwwwgraph-padcom) Studentrsquos 119905-test was performed throughout unlessotherwise stated A 119875 value lt005 was considered statisticallysignificant Western blotting densitometric analysis was per-formed using Quantity One 469 software (Bio-RadMadridSpain)

3 Results

31 Intracellular ROS Production by Fibroblasts with Trisomy21 and Control Fibroblasts To ascertain the degree of oxida-tive stress in cultured fibroblasts with trisomy 21 and theirsensitivity against a prooxidant challenge we determinedintracellular ROSproduction and cell viability at low andhighpassages To this end FT21 and control cells were exposed tothe prooxidant chemical substances AAPH and TBHP at theconcentration that produced maximum ROS induction withminimum cytotoxicity The results showed that fibroblastviability at low passages was not significantly affected by

Table 1 Effect of prooxidants on cell viability

Treatment Low passages High passagesCF T21F CF T21F

Untreated 1000 plusmn 3 1000 plusmn 3 1000 plusmn 3 1000 plusmn 2AAPH 978 plusmn 2 991 plusmn 3 980 plusmn 3 951 plusmn 2TBHP 930 plusmn 2 937 plusmn 2 961 plusmn 2 890 plusmn 2a

Control (119899 = 5) and trisomy 21 (119899 = 5) fibroblasts were grown in 96-wellmicroplates and incubated for one hour with Hankrsquos buffered salt solution(HBSS) (untreated) or supplemented with 10mM AAPH or 100 120583M TBHPCell viability was determined by XTT assay and expressed as a percentageof XTT reduction compared with untreated fibroblasts Values are expressedas mean plusmn SEM of six separate experiments performed in quadruplicateStatistical differences between low- and high-passage groups were analyzedby Studentrsquos 119905-test a119875 lt 001

the prooxidant treatments however at high passages T21Fproved to be more sensitive to prooxidant challenge partic-ularly with TBHP which significantly reduced cell viability(Table 1)

Cells were incubated with the CM-H2DCFDA probe and

its subsequent oxidation was quantified by measuring thefluorescence of the probersquos adduct to detect ROS generation(Figure 1) Basal ROS production at low passages was foundto be 30 higher in T21F (119875 lt 0001) than in controlfibroblasts ROS induction by AAPH was 23 (119875 lt 0001)greater in T21F than in CF Both cell types in response toTBHP showed more intense ROS production which was fargreater in T21F than in CF (119875 lt 0001) (Figure 1(a)) ROSgeneration in replicatively aged T21F was 48 higher thanin CF (119875 lt 0001) rising significantly during replicativesenescence (Figure 1(b)) from 30 in T21F cells at LP to 48at HP (119875 lt 001) and from 23 to 34 in AAPH-treated


Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)

Untreated TBHPCF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(b)

Figure 2 ROS and mitochondrial superoxide anion detection by fluorescence microscopy in fibroblasts with trisomy 21 (T21F) (119899 = 5)and controls (CF) (119899 = 5) Fibroblasts were grown in 96-well microplates and incubated for one hour with Hankrsquos buffered salt solution(untreated) or supplemented with 100 120583MTBHPThe observed green fluorescent signal corresponds to the oxidation of the CM-H2DCFDAprobe in response to intracellular ROS production in (a) The red fluorescent signal corresponds to the oxidation of the MitoSOX probe inresponse to superoxide radical production in themitochondria (b) Images were taken with a fluorescencemicroscope at a total magnificationof 200x

cells (119875 lt 005) whereas no differences were found in TBHP-treated fibroblasts during in vitro aging

ROS generation was also visualized by conventional epi-fluorescencemicroscopy in cultured fibroblasts (Figure 2(a))Representative fluorescent microscope images showedstronger CM-H

2DCFDA staining in T21F than in CF and

the fluorescence was even more intense at high passages thefluorescent micrographs of CM-H

2DCFDA-loaded cells also

revealed higher fluorescence intensities in cells treated withthe prooxidant TBHP than in nontreated fibroblasts

With the aim of detecting mitochondrial superoxideanion generation the MitoSOX RedMitochondrial Superox-ide Indicator fluorescent probe was used The probe rapidlypenetrates cell mitochondria and is oxidized by superoxideanions and not by other ROS or reactive nitrogen speciesTheoxidized probe can be visualized by fluorescence microscopysince it emits bright red fluorescence upon binding to thenuclear DNA Both cell types (T21F and CF) at LP and

HP were treated with TBHP (microscopic images shown inFigure 2(b)) Microscopic observation of greater red fluo-rescence in T21F than in CF revealed higher mitochondrialsuperoxide anion production

32 Intracellular ATP Levels in T21F Levels of intracellularATP at LP were lower in T21F fibroblasts in basal conditions(minus13 119875 lt 005) and ATP depletion was accentuated whencells were treated with 10mM AAPH (minus40 119875 lt 0001) orwith 100 120583M TBHP (minus17 119875 lt 0001) (Figure 3(a)) In basalconditions intracellular ATP content atHPwas lower in T21Fcompared with CF (minus13 119875 lt 005) and ATP depletion wasmore marked when cells were treated with either of the twoprooxidants AAPH (minus18 119875 lt 0001) or TBHP (minus15 119875 lt001) (Figure 3(b))

33 Oxidative Stress Markers in T21F Western blot analysiswas performed to visualize oxidatively modified cellular


Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)

lowastlowastlowastlowast lowastlowast

CFT21F

Untreated AAPH TBHP

(b)

Figure 3 Intracellular ATP levels in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5) Fibroblasts were grown in 96-wellmicroplates and incubated for one hour with Hankrsquos buffered salt solution (untreated) or supplemented with 10mMAAPH or 100120583MTBHPIntracellular ATP content at low (a) and high (b) passages was expressed as a percentage of mean values obtained in the control group foreach treatment Results are expressed as mean plusmn SEM of six separate experiments performed in quadruplicate Statistical differences betweenCF and T21F were analyzed by Studentrsquos 119905-test lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

proteins in T21F versus CF and ascertain whether theseoxidized proteins varied throughout the cell aging processin vitro (Figure 4) Despite the variable degree of proteinoxidation observed in fibroblasts from DS and control cellsT21F showed significantly more oxidized proteins than CFat both low (14 119875 lt 005) and high passages (9 119875 lt005) Furthermore our results showed that throughout invitro cell aging both control (20 increase 119875 lt 001) andT21F (14 increase 119875 lt 005) accumulated oxidized proteins(Figures 4(a) and 4(b)) While concentrations of cellularmalondialdehyde (MDA) the most representative indicatorof lipid peroxidative damage appeared to rise with in vitrocell aging this increase was significant only in T21F (22119875 lt 001) (Figure 4(c))

34 Antioxidant Enzyme Activities in T21F SOD GPx andCAT activities were determined to ascertain whether animbalance existed in the antioxidant enzymatic systemsbetween FT21 and CF and if so whether it persistedin successive passages Total SOD activity from T21F cellhomogenates at LP was 35 higher than in CF (119875 lt 005)and 48 higher in fibroblasts at HP (119875 lt 001) (Figures 5(a)and 5(b)) Intracellular GPx activity of T21F at LP was 13lower than in the control group and 31 lower than in CF atHP (Figures 5(a) and 5(b)) Total CAT activity of T21F cellhomogenates at LP was 17 lower than in CF and 38 lowerthan in CF at HP (Figures 5(a) and 5(b))

The influence of replicative senescence represented bylow- and high-passage groups on antioxidant enzyme ratioswas calculated from the values of enzyme activities TheSODGPx ratio was 16-fold higher in T21F at LP than in CFas also occurred with the SODCAT and SOD(GPx+CAT)ratios This interenzymatic activity imbalance was accentu-ated with replicative senescence the SODGPx ratio was

23-fold higher in T21F than in CF at HP the SODCATratio 26 times higher in T21F than in CF at HP and theSOD(GPx+CAT) 24-fold higher in T21F than in CF at HP

35 Changes in SOD1 Expression and Activity in T21F inrelation to Replicative Senescence SOD1 activity (Figure 5)and mass (Figure 6) were higher in T21F at both LP (43mass increase 119875 lt 001) and HP (34 mass increase 119875 lt005) Considering that SOD1 accounts for approximately70ndash80 of total SOD activity [26] a correlation would beexpected between total SOD activity and SOD1 mass asdetermined by Western blot With replicative senescencewhile activities of SOD in the control group were almostidentical between LP and HP its expression analysis byWestern blot revealed a mass increase of 19 (119875 gt 005)(Figures 5 and 6) In T21F both activity andmasswere slightlyhigher at HP (14 and 12 resp 119875 gt 005)

On the other hand SOD2 expression was found to bedecreased with replicative senescence in both control fibrob-lasts (23 119875 gt 005) and T21F (51 119875 lt 001) (Figure 7(c))Moreover at HP SOD2 expression was significantly lower inT21F than in control fibroblasts (42 decrease 119875 lt 005)

36 mRNA Expression of SOD1 SOD2 and GPx in T21FGene expression of antioxidant enzymes SOD1 SOD2 andSe-dependent GPx was analyzed by real-time quantitativePCR to ascertain whether the increased production of ROSobserved at baseline in T21F was related tomRNA expressionof antioxidant enzymes The results show that the expressionof SOD1 was significantly higher in T21F at both HP and LP(Figure 7) it should be noted that the differences observedbetween controls and T21F decreased from 122 at LP (119875 lt0001) to 60 (119875 lt 005) at HP Analysis of SOD2 GPxand CAT expression yielded no differences between groups


Low passages High passagesCF T21F

120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

Low passages High passages

lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)

Figure 4 Protein carbonyls and malondialdehyde in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5) (a) Representativeprotein carbonyl Western blot of DNPH-derivatized cell lysates in MPER (b) Protein carbonyls were calculated from optical densities of thebands measured by an imaging technique Results were normalized to the band intensities measured in untreated fibroblasts at low passagesand expressed as mean plusmn SEM (c) MDA was determined in cell lysates by HPLC Values were corrected by the protein of the lysate andexpressed as mean plusmn SEM Statistical differences were analyzed by Studentrsquos 119905-test high versus low passages (119875 lt 005 119875 lt 001) T21Fversus CF (lowast119875 lt 005)

Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)

Figure 5 Antioxidant enzyme activities in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5) Enzymatic activities in celllysates were determined as described inMethods section Results at low (a) and high (b) passages are expressed as mean plusmn SEM of 12 separateexperiments performed in triplicate Statistical differences between T21F and CF were analyzed by Studentrsquos 119905-test lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast119875 lt 0001


Low passages High passagesCF T21F CF T21F

SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)

Figure 6 Effect of cell passages on SOD1 and SOD2 expression in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5) SOD1and SOD2 expression were analyzed by Western blot (a) and the bands were quantified by an imaging technique and normalized by 120573-actin(b and c) Results are normalized to the band intensities measured in untreated fibroblasts at low passages and expressed as mean plusmn SEMStatistical differences were analyzed by Studentrsquos 119905-test T21F versus CF (lowast119875 lt 005 lowastlowast119875 lt 001) high versus low passages (119875 lt 001)

Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)

Figure 7 Effect of cell passages on relative mRNA expression of SOD1 SOD2 GPx and catalase (CAT) in fibroblasts with trisomy 21 (T21F)(119899 = 5) and controls (CF) (119899 = 5) mRNA expression was analyzed by quantitative real-time PCR in fibroblasts at low and high passagesExpression data were normalized by the means of the double delta Ct method Results at low (a) and high passages (b) are expressed as meanplusmn SEM Statistical differences were analyzed by Studentrsquos 119905-test lowast119875 lt 005 lowastlowastlowast119875 lt 0001



p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (

)Low passages High passages

lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)

Figure 8 Effect of cell passages on p53 and p21 expression in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5) (a)Representative Western blot of p53 and p21 in cell lysates in M-PER (b) p53 and p21 expression was calculated from optical densities of thebands measured by an imaging technique and normalized by 120573-actin Results were normalized to the band intensities measured in untreatedfibroblasts at low passages and expressed as mean plusmn SEM (c) p53 and p21 were also quantified by ELISA in cell lysates of T21F and CF Resultswere normalized to the optical densitiesmeasured in untreated fibroblasts at low passages and expressed asmeanplusmn SEM Statistical differenceswere analyzed by Studentrsquos 119905-test T21F versus CF (lowast119875 lt 005) and high versus low passages (119875 lt 005 119875 lt 0001)

except for SOD2 at LP where a significant decrease wasobserved in T21F (43 119875 lt 005) It should be noted thatalthough not being statistically significant CAT expressionin T21F at HP was half with respect to CF

37 Replicative Senescence in T21F Control and T21F fibrob-lasts were seeded at LP and HP and expression of p21and p53 proteins was analyzed by Western blot to ascertainwhether in vitro replicative senescence of fibroblasts withtrisomy 21 is related to premature cell aging (Figure 8)TheWestern blot results indicated an increase in p53 protein

expression in T21F compared with controls (6 at LP and14 at HP) whereas p21 protein expression in T21F wassignificantly increased at both LP (18 119875 lt 005) andHP (26 119875 lt 005) with respect to CF (Figure 8(b))In vitro replicative aging led to a significant increase inp21 expression both in T21F (40 increase from EP 119875 lt0001) and in control fibroblasts (31 increase from EP 119875 lt005) Results of p53 and p21 expression quantified by ELISAshowed no statistically significant differences in p53 proteinexpression in T21F versus CF whereas p21 protein expressionsignificantly increased with in vitro replicative aging in T21F


CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)

Figure 9 Effect of in vitro replicative aging on SA-120573-Gal expression in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF) (119899 = 5)(a) Representative microphotographs of X-Gal staining in T21F and CF at low and high passages (b) Fold increase in positive cell counts forX-Gal from low to high passages in T21F and CF Cells were seeded in 12-well plates stained with X-Gal A minimum of 5 random fields werephotographed times100 with phase contrast for X-Gal-positive cell count Results are expressed as mean plusmn SEM Statistical differences betweenthe two passage groups were analyzed by Studentrsquos 119905-test lowast119875 lt 005

(30 119875 lt 005) and in control fibroblasts (35 119875 lt 005)(Figure 8(c))

Another method of cell aging assessment in vitrowas car-ried out using senescence-associated 120573-galactosidase activity(Figure 9(a)) SA-120573-Gal-positive cells were counted (mini-mum 5 random fields and 50ndash100 cellsfield) and the propor-tions of X-Gal-positive cells at LP and HP were calculatedfor each cell line (Figure 9(b)) While CF underwent atwofold increase in the number of X-Gal-positive cells at HPcompared with LP X-Gal-positive T21F at HP increased 25

times more than at LP which represented a 31 increase overcontrol values (119875 lt 005) It is noteworthy that given the greatinterindividual variability observed in the cell division rateand senescence process of both CF and T21F the number ofSA-Gal-positive cells was counted at low and high passagesand the differences were calculated in each cell line

To ascertain whether in vitro replicative aging is linked toan increase in lysosomal enzyme synthesis 120573-galactosidase(pH 4 and 6) and 120573-glucuronidase activities were determinedin T21F and CF at HP and LP (Table 2) Results indicate that


Table 2 Effect of replicative aging on lysosomal hydrolases

Low passages High passages120573-Gal (pH 6) 120573-Gal (pH 4) 120573-Gluc 120573-Gal (pH 6) 120573-Gal (pH 4) 120573-Gluc

CF 630 plusmn 04 2488 plusmn 46 422 plusmn 05 797 plusmn 07 2342 plusmn 22 654 plusmn 07a

T21F 742 plusmn 09 2368 plusmn 23 453 plusmn 07 819 plusmn 06 2105 plusmn 20 596 plusmn 07Activities of lysosomal enzymes 120573-galactosidase and 120573-glucuronidase in cell homogenates of control (119899 = 5) and trisomy 21 (119899 = 5) fibroblasts at low and highpassages Activities were corrected by the protein content of the cellular homogenates The 120573-galactosidase activity was determined at pH 40 and 60 Resultsare expressed as mean plusmn SEM Statistical differences between passages were analyzed with Studentrsquos 119905-test a119875 lt 005


Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)

Figure 10 Effect of cell passages on cytochrome c and caspase 9 expression in fibroblasts with trisomy 21 (T21F) (119899 = 5) and controls (CF)(119899 = 5) (a) Representative Western blot of cytochrome c and caspase 9 at low and high passages (b and c) Protein expression was calculatedfrom optical densities of the bands measured by an imaging technique and normalized by 120573-actin Results were normalized to the bandintensities measured in untreated fibroblasts at low passages and expressed as mean plusmn SEM Statistical differences were analyzed by Studentrsquos119905-test lowast119875 lt 005

in both T21F and CF replicative senescence was accompaniedby varying nonsignificant increases in 120573-galactosidase pH 6activity whereas greater and significant increases were foundin intracellular 120573-glucuronidase activity

38 Caspase-Dependent Apoptosis in T21F Since T21F over-produced ROS and it is known that ROS can also induceapoptosis cytochrome c and caspase 9 expressions wereanalyzed by Western blot to ascertain whether apoptosis wasactivated in T21F fibroblasts (Figure 10) Cytochrome c masswas found to be decreased in T21F both at LP (41 119875 lt 005)and at HP (27) with respect to CF Cytochrome c massremained unchanged in control fibroblasts from LP to HPwhereas there was a nonsignificant increase of 15 in T21FNo significant differences were found in caspase 9 expression

between both types of cells and between passages althoughexpression was slightly decreased in T21F with respect tocontrol fibroblasts

4 Discussion

In this study based on the hypothesis that fibroblasts withtrisomy of HSA21 from human fetuses due to a gene dosageeffect would have increased SOD1 expression with a subse-quent imbalance in enzymatic antioxidant system we soughtto delve into the consequences of such an imbalance whichwould involve increased ROS production causing oxidativemolecular damage possibly associated with premature cel-lular senescence Indeed the main objectives of this workwere to contribute to understanding of the pathophysiologic


mechanisms linking cellular metabolism with prematureaging and age-related pathologies in DS

We analyzed the degree of oxidative stress via intracellularROS levels which were elevated at baseline in T21F therebygenerating chronic oxidative stress furthermore the ROSproduction was always higher in prooxidant treated cellswhether at low or high passages Although we cannot explainwhy all cells produced less ROS at higher passages it mayhave been due to an increase in the expression of antioxidantenzymes The fluorescent probe CM-H

2DCFDA is often

considered to detect H2O2levels though it actually detects

cellular peroxides and not any specific free radical sincesensitivity of the probe varies according to the reactivespecies to which it binds thus the CM-H

2DCFDA assay

can be considered to quantify oxidative stress [27] As aconsequence of this continuous oxidative stress cells maysuffer molecular oxidative damage since these highly reactivespecies readily interact with macromolecules triggering achain of peroxidation affecting cellular membranes andorganelles Lipids are the molecules most prone to majoroxidative injury via a process known as lipoperoxidationwhich affects structures rich in polyunsaturated fatty acidssuch as cell membranes the susceptibility of which increasesas the number of double bonds rises and poses a constantthreat to cell integrity and function with ensuing tissuedamage In the present study MDA the main end-productof LPx was found to be raised significantly only in T21Fcells as replicative senescence progressed In fact LPx isan ongoing process which in normal physiology acts asa renovator of biological membranes although its excessiveactivation is linked with the pathogenesis of many diseasesand pathological processes thus LPx is considered a majorfactor in the ageing of aerobic cells [28]

Oxidative damage to proteins is one of the modificationsleading to severe failure of biological functions and celldeath protein carbonyl groups constitute the most impor-tant biomarker of protein oxidation and as they are morestable thanMDA their determination provides an additionaladvantage In this study carbonylated protein levels weredetected and quantified and a significant increase in T21Fwas observed compared with control cells Furthermorecarbonylated proteins increased in both cell types as replica-tive senescence progressed moreover a significant increasein oxidative injury to proteins was observed in fibroblastsfrom DS This noteworthy novel finding would point toa deleterious role of oxidatively modified proteins in earlycell senescence in T21F thereby linking protein oxidation topremature aging in DS

Replicative senescence of human fibroblasts has beenwidely studied and is considered to be a valid model forstudying aging Furthermore an accumulation of oxidizedproteins has been documented in senescent fibroblasts [29]The age-associated accumulation of oxidized proteins andlipids has been shown to disturb several normal cellularfunctions thereby contributing to the aging process byinhibiting the proteasome an important proteolytic systemunderlying age-dependent changes [30] A considerable bodyof evidence supports the involvement of free radicals in

the aging process and an accumulation of cellular com-ponents oxidatively damaged with age has been reportedproviding evidence that oxidant damage is one of severalfactors contributing to the aging process [31] The presenceof oxidative stress has long been associated with DS andmay be implicated in the development of chronic DS-relatedhealth complications [8 32] indeed OS has been shownto occur in the pathogenesis and progression of DS due toa dysregulation of geneprotein expression associated withthe trisomy characteristic of DS [33] The effects of OS inDS from embryonic life onwards have been documented astudy that evaluated OS biomarkers in the amniotic fluid ofwomen carrying DS fetuses indicated that oxidative damageis an early event in DS pathogenesis and might contribute tothe generation of deleterious DS phenotypes and abnormaldevelopment [34] Indeed neurons of DS patients havebeen found to exhibit a sharp increase in intracellular ROSaccompanied by elevated levels of LPx products [35] Theoccurrence of chronic oxidative injury in the brain as arisk factor for subsequent neurodegeneration in aged DSsubjects had been reported [36] In other neurodegenerativediseases and even during aging disturbances are producedin the intracellular pathways of oxidized protein degradationleading to a progressive accumulation of oxidativelymodifiedproteins with ensuing loss of cell functions particularlywith advanced age [37] Through SA-120573-Gal assay our resultsshowed cell aging to be more pronounced in DS embryonicfibroblasts possibly related to the increased ROS and subse-quent molecular and cellular oxidative damage

Oxidative stress and mitochondrial dysfunction areprominent features of DS and since mitochondria are themain source of ROS production their role is essential in age-related oxidative damage [15] Trisomy of HSA21 has alsobeen associated with mitochondrial dysfunction in cells andtissues from DS subjects which has led to the concept thatmitochondrial dysfunction contributes to the DS phenotypethat includes premature aging [35 38] In the present studybasal cellular ROS production at LP was found to be 29higher in T21F than in CF while in replicatively aged DSfibroblasts it was 48 higher than in normal fibroblaststhus indicating a significant rise in oxidative stress duringreplicative senescence not reported previously which from abroader viewpoint would support the accelerated senescencephenotype characteristic of Downrsquos syndrome IncreasedROS production has also been found to be accompaniedby mitochondrial dysfunction which occurs in DS cells asearly as in embryonic life [34] A study in DS fibroblastsfound a selective deficiency of complex I which contributes tomitochondrial ROS overproduction which further suggeststhat mitochondrial biogenesis is upregulated in DS [39]The relationship between oxidative stress and mitochondrialdysfunction is of special importance given the pivotal roleof mitochondria not only as a major site of productionbut also as a target of ROS Mitochondria constitute theorganelle where the ATP is generated and the continuousROS production could give rise to a drop in ATP synthesisa process that also occurs during cell aging We foundintracellular ATP content to be significantly decreased inT21F which could contribute to premature cell aging as


observed in DS fibroblasts of the present study Mitochon-drial function could be partially compromised in T21F sincethey exhibited increased production of ROS particularly themitochondrial radical superoxide and reduced ATP levelsThe involvement of possible abnormalities in mitochondrialenergy metabolism in DS pathogenesis has been studiedand a strong impairment of mitochondrial ATP synthesisdue to a reduction in the catalytic efficiency of certainproteins involved in mitochondrial ATP synthesis has beenfound [40] Although a significant decline in intracellularATP content and increased mitochondrial ROS productionwere detected in T21F which together would point to animpaired mitochondrial function a direct relationship couldnot be established as this study lacked direct measurementsof mitochondrial function

Most Downrsquos syndrome phenotypes would appear to berelated to alterations in gene expression derived from theextra copy of HSA21 and in line with the ldquogene dosage effectrdquohypothesis some DS features could result from the dosageimbalance of HSA21 genes [41 42] Considering that changesin gene expression and protein mass result in alterations inthe necessary synergic activity of the antioxidant enzymaticsystem the present study aimed to ascertain whether anantioxidant enzyme imbalance existed and if so whetherit persisted in successive passages Overexpression of somegenes encoded by HSA21 which include SOD1 the mostpotential inducer of OS since it plays a key role in theantioxidant defense system by catalyzing the dismutation ofsuperoxide radicals can disturb the activity ratio betweenSOD and GPx plus CAT giving rise to a prooxidant status intrisomic cells that may be of key importance in the pathogen-esis of DS In this study a marked increase in the mRNA ofSOD1 and significant increments in protein mass and activitywere found in T21F In a very recent study Gimeno et al [43]also found mRNA and protein levels of SOD1 to be increasedin DS fibroblasts However a point of interest of the presentwork is that we studied the antioxidant enzyme systemduringcell replicative senescence in our DS cell model and foundSOD1 activity and protein levels to be increased particularlyin older trisomic cells In living organisms trisomic cellstend to compensate for the excessive production of H

2O2

by increasing the enzymes that inactivate it However inthis in vitro study decreases (albeit with no significantdifferences) in expression and activities of GPx and CATwere observed thus the SODCAT+GPx activity ratio wassignificantly increased in T21F and the imbalance was morepronounced in aged cells The SODGPx SODCAT andSOD(GPx+CAT) ratios were higher in T21F than in CF atlow passages and this interenzymatic activity misbalance wasaccentuated with replicative senescence To our knowledgethese specific differences in the antioxidant enzyme defensesystem in relation to cell senescence have not been describedpreviously As a result of increased SOD1 activity without theconcomitant increase in complementary antioxidant defensemechanisms an excessive production of endogenous H

2O2

is generated thereby oxidizing biomolecules such as proteinsand lipids and damaging important cellular components aswe observed in this study In vivo the overproduction ofH2O2through the action of SOD1 has long been known to be

themain source of ROS inDS patients [8 32 44 45] poweredby an imbalance in SOD(GPx+CAT) ratio activities therebyinducing systemic oxidative stress [46] which appears to bea fundamental factor contributing to a senescent phenotypecharacteristic of DS However the free radical theory of agingis not the only theory proposed to explain the mechanism(s)involved in aging at molecular and cellular levels sinceother mechanisms such as telomere senescence genomicinstability and the mitochondrial hypothesis of aging alsoparticipate in cell senescence [47 48] To gain further insightinto themechanisms thatmight be implicated in the early lossof proliferative capacity and growth of T21F with replicativesenescence the expression of p21 and p53 proteins wasstudied in fibroblasts with trisomy 21 during the cell agingprocess in vitro

This study found that DS fibroblasts entered a state ofpremature senescence as shown by SA-120573-Gal assay and p21assessment it has been reported that in human fibroblastsprotein p21 levels rise through passages of primary cells [49]and that the overexpression of p21 leads to cell cycle arrest andthe appearance of cellular senescence markers such as SA-120573-Gal activation cell hypertrophy and flattening [50] and enzy-matic andmorphologic findings respectively of this study onembryonic DS cells The higher oxidative stress levels seen inDS fibroblasts in this work could increase the expression andactivation of p53 one of the main effectors of p21 [51] andinterestingly many of the neurodegenerative and immunedefects of DS patients correlate with an increased apoptosisrate linked to an increased expression of the proapoptotictumour suppressor p53 [52ndash54] However neither p53 northe proapoptotic proteins cytochrome c and caspase 9 werealtered in our DS cells although it should be noted thatWestern blots were performed with total cell lysates and thusit was not possible to ascertain whether cytochrome c wasbeing released from the mitochondria

While some reports have pointed to the activation ofthe apoptotic pathway as a pathophysiological mechanismbehind DS phenotypes the opposite might also be trueUnder certain conditions p21 functions as a potent anti-apoptotic factor acting at different levels of the cell deathcascade In fact p21 can promote resistance to apoptosisby inhibiting activation of caspase 9 and cytochrome crelease from mitochondria [51] Moreover although alteredapoptosis has been suggested as one of the mechanismsresponsible for different DS phenotypes most in vivo studieshave failed to find alterations in this process [55] Forinstance it has recently been reported that overexpression ofDyrk1a amember of the tyrosine-phosphorylation-regulatedprotein kinase family located at chromosome 21 [56] reducesthe magnitude of physiological apoptosis in the developingretina of trisomic Ts65Dn mice leading to a phenotypethat resembles that seen in DS individuals [57] ThereforeDyrk1a overexpression inDS fibroblasts could account for thediminished levels of caspase 9 and apoptosis seen in thesecells However as Liao and colleagues pointed out [58] thestory might not be this simple since some Down syndrome-associated proteins are also found to induce apoptosis in cellsThe results herein show that senescence but not apoptosisappears prematurely in cultured DS fibroblasts which could


account for the appearance of premature ageing signs seen inindividuals with Downrsquos syndrome

Abbreviations

AAPH 221015840-Azobis-2-methyl-propanimidamidedihydrochloride

DS Downrsquos syndromeHP High passagesLP Low passagesLPx LipoperoxidationOS Oxidative stressPCG Protein carbonyl groupsPD Population doublingsROS Reactive oxygen speciesT21F Fibroblasts with trisomy 21TBHP tert-Butyl hydroperoxide

Conflict of Interests

The authors declare that they have no conflict of interests

Authorsrsquo Contribution

Vıctor Rodrıguez-Sureda and Angel Vilches contributedequally to this work

Acknowledgments

Dr Rodrıguez-Sureda is supported by the CIBERER fromInstituto de Salud Carlos III This work was supported bygrants from the Fondo de Investigaciones Sanitarias (FIS1001605 and CIBERER Proyecto Intramural INTRA077191)of the Carlos III Institute of Health Spain The authors aregrateful to Miss C OrsquoHara for her help in the English versionof this paper

References

[1] AMegarbane A Ravel CMircher et al ldquoThe 50th anniversaryof the discovery of trisomy 21 the past present and futureof research and treatment of Down syndromerdquo Genetics inMedicine vol 11 no 9 pp 611ndash616 2009

[2] D L Nelson and R A Gibbs ldquoThe critical region in trisomy 21rdquoScience vol 306 no 5696 pp 619ndash621 2004

[3] H E Lockstone L W Harris J E Swatton M T Wayland AJ Holland and S Bahn ldquoGene expression profiling in the adultDown syndrome brainrdquo Genomics vol 90 no 6 pp 647ndash6602007

[4] M S Cheon M Dierssen S H Kim and G Lubec ldquoProteinexpression of BACE1 BACE2 and APP in down syndromebrainsrdquo Amino Acids vol 35 no 2 pp 339ndash343 2008

[5] A M W Coppus D Fekkes W M A Verhoeven S Tuinierand C M van Duijn ldquoPlasma levels of nitric oxide relatedamino acids in demented subjects with Down syndrome arerelated to neopterin concentrationsrdquo Amino Acids vol 38 no3 pp 923ndash928 2010

[6] P M Sinet ldquoMetabolism of oxygen derivatives in Downrsquossyndromerdquo Annals of the New York Academy of Sciences vol396 pp 83ndash94 1982

[7] B W Brooksbank and R Balazs ldquoSuperoxide dismutase glu-tathione peroxidase and lipoperoxidation in Downrsquos syndromefetal brainrdquo Brain Research vol 318 no 1 pp 37ndash44 1984

[8] F V Pallardo P Degan M drsquoischia et al ldquoMultiple evidencefor an early age pro-oxidant state in Down syndrome patientsrdquoBiogerontology vol 7 no 4 pp 211ndash220 2006

[9] J Muchova M Sustrova I Garaiova et al ldquoInfluence of ageon activities of antioxidant enzymes and lipid peroxidationproducts in erythrocytes and neutrophils of down syndromepatientsrdquo Free Radical Biology and Medicine vol 31 no 4 pp499ndash508 2001

[10] H D Teixeira R I Schumacher and R Meneghini ldquoLowerintracellular hydrogen peroxide levels in cells overexpress-ing CuZn-superoxide dismutaserdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 95 no14 pp 7872ndash7875 1998

[11] S I Liochev and I Fridovich ldquoThe role of119874∙2

minus in the productionof HO∙ in vitro and in vivordquo Free Radical Biology andMedicinevol 16 no 1 pp 29ndash33 1994

[12] CWWhite K BAvraham P F Shanley andYGroner ldquoTrans-genic mice with expression of elevated levels of copper-zincsuperoxide dismutase in the lungs are resistant to pulmonaryoxygen toxicityrdquoThe Journal of Clinical Investigation vol 87 no6 pp 2162ndash2168 1991

[13] N Nakao P Brundin K Funa O Lindvall and P OdinldquoTrophic and protective actions of brain-derived neurotrophicfactor on striatal DARPP-32-containing neurons in vitrordquoDevelopmental Brain Research vol 90 no 1-2 pp 92ndash101 1995

[14] B Halliwell and J M C Gutteridge ldquoThe definition and mea-surement of antioxidants in biological systemsrdquo Free RadicalBiology and Medicine vol 18 no 1 pp 125ndash126 1995

[15] P E Coskun and J Busciglio ldquoOxidative stress and mitochon-drial dysfunction in Downrsquos syndrome relevance to aging anddementiardquo Current Gerontology and Geriatrics Research vol2012 Article ID 383170 7 pages 2012

[16] Y Chen E McMillan-Ward J Kong S J Israels and S BGibson ldquoMitochondrial electron-transport-chain inhibitors ofcomplexes I and II induce autophagic cell death mediated byreactive oxygen speciesrdquo Journal of Cell Science vol 120 no 23pp 4155ndash4166 2007

[17] S Arbuzova T Hutchin and H Cuckle ldquoMitochondrial dys-function and Downrsquos syndromerdquo BioEssays vol 24 no 8 pp681ndash684 2002

[18] S P M Crouch R Kozlowski K J Slater and J Fletcher ldquoTheuse of ATP bioluminescence as a measure of cell proliferationand cytotoxicityrdquo Journal of Immunological Methods vol 160no 1 pp 81ndash88 1993

[19] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984

[20] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[21] P K Smith R I Krohn G T Hermanson et al ldquoMeasurementof protein using bicinchoninic acidrdquo Analytical Biochemistryvol 150 no 1 pp 76ndash85 1985

[22] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquo Nucleic acids research vol 29 no9 p e45 2001


[23] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoBiomarkers of diabetes-associated oxidativestress and antioxidant status in young diabetic patients with orwithout subclinical complicationsrdquo Free Radical Biology andMedicine vol 34 no 12 pp 1563ndash1574 2003

[24] P Martın-Gallan A Carrascosa M Gussinye and CDomınguez ldquoEstimation of lipoperoxidative damage and anti-oxidant status in diabetic children relationship with individualantioxidantsrdquo Free Radical Research vol 39 no 9 pp 933ndash9422005

[25] G P Dimri X Lee G Basile et al ldquoA biomarker that identifiessenescent human cells in culture and in aging skin in vivordquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 92 no 20 pp 9363ndash9367 1995

[26] M Lee D-H Hyun P Jenner and B Halliwell ldquoEffect ofoverexpression of wild-type and mutant CUZN-superoxidedismutases on oxidative damage and antioxidant defencesrelevance to Downrsquos syndrome and familial amyotrophic lateralsclerosisrdquo Journal of Neurochemistry vol 76 no 4 pp 957ndash9652001

[27] B Halliwell and M Whiteman ldquoMeasuring reactive speciesand oxidative damage in vivo and in cell culture how shouldyou do it and what do the results meanrdquo British Journal ofPharmacology vol 142 no 2 pp 231ndash255 2004

[28] A Sevanian and P Hochstein ldquoMechanisms and consequencesof lipid peroxidation in biological systemsrdquo Annual Review ofNutrition vol 5 pp 365ndash390 1985

[29] E K Ahmed C R Picot A-L Bulteau and B Friguet ldquoProteinoxidative modifications and replicative senescence of WI-38human embryonic fibroblastsrdquoAnnals of the New York Academyof Sciences vol 1119 no 1 pp 88ndash96 2007

[30] N Sitte M Huber T Grune et al ldquoProteasome inhibition bylipofuscinceroid during postmitotic aging of fibroblastsrdquo TheFASEB Journal vol 14 no 11 pp 1490ndash1498 2000

[31] R E Pacifici and K J A Davies ldquoProtein lipid and DNA repairsystems in oxidative stress the free-radical theory of agingrevisitedrdquo Gerontology vol 37 no 1ndash3 pp 166ndash180 1991

[32] J Kedziora and G Bartosz ldquoDownrsquos syndrome a pathologyinvolving the lack of balance of reactive oxygen speciesrdquo FreeRadical Biology and Medicine vol 4 no 5 pp 317ndash330 1988

[33] A Conti F Fabbrini P DrsquoAgostino et al ldquoAltered expressionof mitochondrial and extracellular matrix genes in the heart ofhuman fetuses with chromosome 21 trisomyrdquo BMC Genomicsvol 8 article 268 2007

[34] M Perluigi F di Domenico A Fiorini et al ldquoOxidative stressoccurs early in Down syndrome pregnancy a redox proteomicsanalysis of amniotic fluidrdquo ProteomicsmdashClinical Applicationsvol 5 no 3-4 pp 167ndash178 2011

[35] J Busciglio J Busciglio B A Yankner and B A YanknerldquoApoptosis and increased generation of reactive oxygen speciesinDownrsquos syndromeneurons in vitrordquoNature vol 378 no 6559pp 776ndash779 1995

[36] R C Iannello P J Crack J B De Haan and I Kola ldquoOxidativestress and neural dysfunction in Down syndromerdquo Journal ofNeural Transmission Supplement no 57 pp 257ndash267 1999

[37] P A Szweda B Friguet and L I Szweda ldquoProteolysis freeradicals and agingrdquo Free Radical Biology and Medicine vol 33no 1 pp 29ndash36 2002

[38] E Roat N Prada R Ferraresi et al ldquoMitochondrial alterationsand tendency to apoptosis in peripheral blood cells fromchildren with Down syndromerdquo FEBS Letters vol 581 no 3 pp521ndash525 2007

[39] D Valenti G A Manente L Moro E Marra and R A VaccaldquoDeficit of complex I activity in human skin fibroblasts withchromosome 21 trisomy and overproduction of reactive oxygenspecies by mitochondria involvement of the cAMPPKA sig-nalling pathwayrdquo Biochemical Journal vol 435 no 3 pp 679ndash688 2011

[40] D Valenti A Tullo M F Caratozzolo et al ldquoImpairment ofF1F0-ATPase adenine nucleotide translocator and adenylatekinase causes mitochondrial energy deficit in human skinfibroblasts with chromosome 21 trisomyrdquo Biochemical Journalvol 431 no 2 pp 299ndash310 2010

[41] M A Pritchard and I Kola ldquoThe lsquogene dosage effectrsquo hypothesisversus the lsquoamplified developmental instabilityrsquo hypothesis inDown syndromerdquo Journal of Neural Transmission Supplementno 57 pp 293ndash303 1999

[42] S E Antonarakis R Lyle E T Dermitzakis A Reymondand S Deutsch ldquoChromosome 21 and Down syndrome fromgenomics to pathophysiologyrdquo Nature Reviews Genetics vol 5no 10 pp 725ndash738 2004

[43] A Gimeno J L Garcıa-Gimenez L Audı et al ldquoDecreasedcell proliferation and higher oxidative stress in fibroblastsfrom down syndrome fetuses Preliminary studyrdquo Biochimica etBiophysica Acta vol 1842 no 1 pp 116ndash125 2014

[44] J B de Haan F Cristiano R Iannello C Bladier M JKelner and I Kola ldquoElevation in the ratio of CuZn-superoxidedismutase to glutathione peroxidase activity induces featuresof cellular senescence and this effect is mediated by hydrogenperoxiderdquoHumanMolecular Genetics vol 5 no 2 pp 283ndash2921996

[45] I T Lott ldquoAntioxidants in Down syndromerdquo Biochimica etBiophysica Acta vol 1822 no 5 pp 657ndash663 2012

[46] T R Garlet E B Parisotto G D S de Medeiros et alldquoSystemic oxidative stress in children and teenagers with Downsyndromerdquo Life Sciences vol 93 no 16 pp 558ndash563 2013

[47] F L Muller M S Lustgarten Y Jang A Richardson and HVan Remmen ldquoTrends in oxidative aging theoriesrdquo Free RadicalBiology and Medicine vol 43 no 4 pp 477ndash503 2007

[48] J F Passos G Saretzki S Ahmed et al ldquoMitochondrial dys-function accounts for the stochastic heterogeneity in telomere-dependent senescencerdquo PLoS Biology vol 5 no 5 article e1102007

[49] V S Romanov V A Pospelov and T V Pospelova ldquoCyclin-dependent kinase inhibitor p211198821198861198911 contemporary view on itsrole in senescence and oncogenesisrdquo Biochemistry vol 77 no 6pp 575ndash584 2012

[50] Y Y Sanders H Liu X Zhang et al ldquoHistone modificationsin senescence-associated resistance to apoptosis by oxidativestressrdquo Redox Biology vol 1 no 1 pp 8ndash16 2013

[51] R Mirzayans B Andrais A Scott and D Murray ldquoNewinsights into p53 signaling and cancer cell response to DNAdamage implications for cancer therapyrdquo Journal of Biomedicineand Biotechnology vol 2012 Article ID 170325 16 pages 2012

[52] M Salemi R A Condorelli C Romano et al ldquoCASP3 proteinexpression by flow cytometry in Downrsquos syndrome subjectsrdquoHuman Cell vol 27 no 1 pp 43ndash45 2014

[53] R Seidl S Fang-Kircher B Bidmon N Cairns and G LubecldquoApoptosis-associated proteins p53 and APO-1Fas (CD95) inbrains of adult patients with Down syndromerdquo NeuroscienceLetters vol 260 no 1 pp 9ndash12 1999

[54] A Sawa F Oyama N J Cairns N Amano and M MatsushitaldquoAberrant expression of bcl-2 gene family in Downrsquos syndromebrainsrdquoMolecular Brain Research vol 48 no 1 pp 53ndash59 1997


[55] N Rueda J Florez and C Martınez-Cue ldquoApoptosis in Downrsquossyndrome lessons from studies of human and mouse modelsrdquoApoptosis vol 18 no 2 pp 121ndash134 2013

[56] A Seifert L A Allan and P R Clarke ldquoDYRK1A phospho-rylates caspase 9 at an inhibitory site and is potently inhibitedin human cells by harminerdquo FEBS Journal vol 275 no 24 pp6268ndash6280 2008

[57] A Laguna M-J Barallobre M-A Marchena et al ldquoTrip-lication of DYRK1A causes retinal structural and functionalalterations inDown syndromerdquoHumanMolecular Genetics vol22 no 14 pp 2775ndash2784 2013

[58] J-M Liao X Zhou Y Zhang and H Lu ldquoMiR-1246 a new linkof the p53 family with cancer and Down syndromerdquo Cell Cyclevol 11 no 14 pp 2624ndash2630 2012

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


radicals to molecular oxygen and hydrogen peroxide whichin turn is converted by either glutathione peroxidase (GPxEC 11119) or catalase (CAT EC 11116) to water and oxygenthus providing a combined enzymatic action against oxygentoxicity An imbalance in the ratio of SOD to GPx and CATresults in the accumulation of H

2O2which may participate

in the Fenton reaction resulting in the formation of noxioushydroxyl radicals As the gene coding for the enzyme SOD1is localized to 21q221 almost all tissues of DS patientsvarious trisomy 21 cultured cells [6] and the brains of DSpatients [7] overexpress this gene Overexpression of SOD1as a consequence of gene dosage in trisomy 21 cells canlead to increased oxidative stress (OS) caused by an alteredSOD(GPx+CAT) activity ratio that may upset the oxidant-antioxidant balance thereby resulting in oxidative damageto molecules and vital cellular targets In fact evidence for amultiple prooxidant state in young DS patients would appearto support the hypothesis that ROS overgeneration couldpotentially contribute to some pathologic manifestations ofDS especially accelerated aging [8 9] However this hypoth-esis has never been unequivocally tested since cells overex-pressing SOD1 have been shown to produce less H

2O2[10]

which would lend support to the hypothesis proposed byLiochev and Fridovich [11] that more efficient superoxideanion dismutation in DS would prevent high H

2O2levels

since excess SOD1 can catalyze surrogate reactions therebygenerating hydroxyl radicals fromH

2O2 Furthermore trans-

genic mice designed to overexpress SOD1 turned out to beeven more resistant to oxygen toxicity than controls [12]Another study showed that SOD1 overexpression increasedthe survival of transplanted neurons [13]

The net intracellular oxidant activity depends on theoverall balance between ROS generation and the capacity ofcells to buffer these highly reactive species however whenthis capacity is exceeded oxidative stress is produced andconsequently ROS cause damage to macromolecules such asprotein lipids and DNA Oxidative stress is implicated in thepathogenesis of diverse chronic or acute diseases as well asin aging [14] and has been associated with DS and its majorphenotypic features such as premature aging [15]

Normal human fibroblasts cultured in vitro have limitedproliferation potential and eventually become senescent asa result of serial passages commonly known as replicativesenescence giving rise to the idea that cellular senescencemight be a determinant of human aging In vitro cellularsenescence is thus regarded as a useful model for elucidat-ing molecular mechanisms that underlie organismal agingfurthermore replicative cell senescence is continually beingshown to represent a validmodel of in vivo aging [16] In addi-tion to replicative senescence cellular senescence can alsobe induced by various forms of stress including oxidativestress thus cultured fibroblasts with trisomy 21 can serve asan excellent in vitro tool for aging research since they canbecome oxidative stress-induced senescent cells in whichfurthermore replicative senescence can be induced

The hypothesis of this study was that fibroblasts withtrisomy 21 (T21F) have high SOD expression and activitywhich lead to an imbalance in the antioxidant enzymatic sys-tems thereby provoking overgeneration of ROS and cellular

oxidative damage that could pathophysiologically underliepremature cell senescence in DS In addition to antioxidantenzyme activity alterations mitochondrial dysfunction [17]previously reported in DS would affect cell energy pro-duction and increase intracellular oxidant activity whichcombined could accelerate cellular senescence in T21F

This work aimed to assess intracellular oxidant activity ofcultured T21F and ascertain whether an imbalance exists inthe activities mRNA and protein expression of antioxidantenzymes SOD1 SOD2 GPx and CAT at low (3ndash7) and highpassages (8ndash12) during the cell replication process in vitro Inan attempt to determine whether oxidative stress is relatedto premature senescence in T21F senescence-associated 120573-galactosidase activity (SA-120573-Gal) and the expression of pro-teins p53 and p21 were analyzed at low and high passages dur-ing in vitro cell replication To our knowledge this is the firststudy relating ROS generation reduced ATP levels oxidativestress-induced molecular damage and cellular replicativesenescence in fibroblasts from DS the results of this studyshed new light on the complex relationship between oxidativestress and premature senescence in DS

2 Materials and Methods

21 Reagents and Materials Chemicals were of analyticalgrade and purchased fromSigma (St LouisMOUSA) unlessotherwise stated 221015840-azobis-2-methyl-propanimidamide di-hydrochloride (AAPH) 1-butanol 35-di-tert-4-butylhydrox-ytoluene 4-methylumbelliferone (4-MU) 4-MU-120573-D-galac-topyranoside 4-MU-120573-D-glucuronide 1133-tetraethox-ypropane 2-thiobarbituric acid adenosine 51015840-triphosphateassay mix acetonitrile diethylenetriaminepentaacetic acid(DTPA) dimethyl sulphoxide (DMSO) Dulbeccorsquos ModifiedEagle Medium (DMEM) Eaglersquos Minimal Essential Medium(MEM) fetal calf serum (FCS) Hankrsquos buffered salt solution(HBSS) sodium chloride sodium dodecyl sulfate (SDS)somatic cell ATP releasing reagent tert-butyl hydroperoxide(TBHP) TrissdotHCl Triton X-100 and Tween-20

Penicillin streptomycin 5-(and-6-)-chloromethyl-2101584071015840-dichlorodihydrofluorescein diacetate acetyl ester probe (CM-H2DCFDA) MitoSOX Red Mitochondrial Superoxide Indi-

cator MitoTracker Green FM and Hoechst 33342 werepurchased from Invitrogen (Carlsbad CA USA) Cell Prolif-eration Kit II (XTT) was purchased from Roche Applied Sci-ence (Basel Switzerland) 96-well and 12-well clear-bottomculture plates were from Corning (Corning NY USA) SODactivity determination kit (RANSOD) was from Randox(Crumlin UK) and GPx activity determination kit (cGPx-340) was from Bioxytech (Deltaclon Madrid Spain) Thefollowing antibodies were used for immunoblotting SOD1(sc-30080) and SOD2 (sc-11407) were purchased from SantaCruz Biotechnology (Santa Cruz CA USA) Dinitrophenyl-KLH (A-6430) was from Invitrogen p21 Waf1Cip 1 (2947)cytochrome c (4272) and cleaved caspase 9 (9505) werefrom Cell Signaling (Danvers MA USA) p53 (P8999) 120573-actin (A3854) rabbit IgG-HRP (A0545) and mouse IgG-HRP (A2304) were from Sigma-AldrichMammalian protein





2atmosphere







2DCFDA probe







2atmosphere





2PO4



2O2in CAT


2O2per minute










Low passages

0

2

4

6

8

10

Untreated AAPH TBHP

DCF

(RFU

)

T21FCF

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

2

4

6

8

10

DCF

(RFU

)

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)




3 Results










Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)


Low

pas

sage

sH

igh

pass

ages

(b)













Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2atmosphere







2DCFDA probe







2atmosphere





2PO4



2O2in CAT


2O2per minute










Low passages

0

2

4

6

8

10

Untreated AAPH TBHP

DCF

(RFU

)

T21FCF

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

2

4

6

8

10

DCF

(RFU

)

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)




3 Results










Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)


Low

pas

sage

sH

igh

pass

ages

(b)













Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















2atmosphere





2PO4



2O2in CAT


2O2per minute










Low passages

0

2

4

6

8

10

Untreated AAPH TBHP

DCF

(RFU

)

T21FCF

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

2

4

6

8

10

DCF

(RFU

)

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)




3 Results










Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)


Low

pas

sage

sH

igh

pass

ages

(b)













Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Low passages

0

2

4

6

8

10

Untreated AAPH TBHP

DCF

(RFU

)

T21FCF

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

2

4

6

8

10

DCF

(RFU

)

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)




3 Results










Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)


Low

pas

sage

sH

igh

pass

ages

(b)













Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Untreated TBHP

CF T21F CF T21F

Low

pas

sage

sH

igh

pass

ages

(a)


Low

pas

sage

sH

igh

pass

ages

(b)













Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Low passages

0

50

100

150AT

P (

CF) lowast

CFT21F

Untreated AAPH TBHP

lowastlowastlowast

lowastlowastlowast

(a)

High passages

0

50

100

150

ATP

( C

F)


CFT21F

Untreated AAPH TBHP

(b)











120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120573-actin

CFT21F

(a)

Protein carbonyls

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F


lowast

lowast

(b)

CFT21F

MDA

00

10

20

30

40


(120583m

olm

g pr

otei

n)

(c)


Low passages

000

003

006

40

60

80

SOD GPx CAT

Activ

ity (U

mg

prot

ein)

CFT21F

lowast

lowastlowast

(a)

CFT21F

High passages

000

003

006

40

60

80

Activ

ity (U

mg

prot

ein)

SOD GPx CAT

lowast

lowastlowast

lowastlowastlowast

(b)




SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















SOD1

SOD2

120573-actin

(a)

SOD1

00

05

10

15

20

CF at

low

pas

sage

s (

)


CFT21F

lowast

lowastlowast

(b)

CFT21F

SOD2

00

05

10

15

20

CF at

low

pas

sage

s (

)


lowast

(c)


Low passages

00

05

10

15

20

25

SOD1 GPx CATSOD2

mRN

A (r

elat

ive e

xpre

ssio

n)

CFT21F

lowast

lowastlowastlowast

(a)

CFT21F

High passages

00

05

10

15

20

25

mRN

A (r

elat

ive e

xpre

ssio

n)

SOD1 GPx CATSOD2

lowast

(b)




p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















p53

p21

120573-actin

(a)

p53 (Western blot)

00

05

10

15

20

CF at

low

pas

sage

s (

)


p21 (Western blot)

00

05

10

15

20

25

CF at

low

pas

sage

s (


lowast

lowast

(b)

p53 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)

CFT21F

CFT21F


p21 (ELISA)

00

05

10

15

20

CF at

low

pas

sage

s (

)


(c)






CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CF

T21F


(a)

0

100

200

300

Posit

ive c

ells

( o

f low

pas

sage

s)

CFT21F

SA-120573-Gal

lowast

(b)












Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Cytochrome c

Caspase 9

120573-actin

(a)

Cytochrome c

00

05

10

15

CF at

low

pas

sage

s (

)


CFT21F

lowast

(b)

CFT21F

Caspase 9

00

05

10

15

CF at

low

pas

sage

s (

)


(c)





4 Discussion





2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2DCFDA is often



2DCFDA assay









2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2O2


2O2







Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Abbreviations







Acknowledgments


References



































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article intracellular oxidant activity...

Documents