Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients

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w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Protein

carbonyl

content:

a

novel

biomarker

for

aging

in

HIV/AIDS

patients

Vaishali

Kolgiri

∗

,

Vinayak

Wamanrao

Patil

GrantGovernmentMedicalCollegeandSirJJGroupofHospitals,DepartmentofBiochemistry,Mumbai,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received24May2016 Accepted21September2016 Availableonline4November2016

Keywords:

Carbonylcontent DNAdamage

Humanimmunodeﬁciencyvirus (HIV)infection

8-OH-dG ROS

a

b

s

t

r

a

c

t

Background:Themajorcomplicationsof“treated”HumanImmunodeﬁciencyVirus(HIV) infectionarecardiovasculardisease,malignancy,renaldisease,liverdisease,bonedisease, andperhapsneurologicalcomplications,whicharephenomenaofthenormalaging pro-cessoccurringatanearlierageintheHIV-infectedpopulation.Thepresentstudyisaimed toexploreproteincarbonylcontentasabiomarkerfordetectingoxidativeDNAdamage inducedARTtoxicityand/oracceleratedaginginHIV/AIDSpatients.

Objective:Toinvestigatethepotentialofcarbonylcontentasabiomarkerfordetecting oxida-tiveDeoxyribonucleicacid(DNA)damageinducedAntiretroviralTheraphy(ART)toxicity and/oracceleratedaginginHIV/AIDSpatients.

Methods:Inthiscase–controlstudyatotal600subjectswereincluded.Allsubjectswere ran-domlyselectedandgroupedasHIV-negative(controlgroup)(n=300),HIV-infectedARTnaive (n=100),HIV-infectedonﬁrstlineART(n=100),andHIV-infectedonsecondlineART(n=100). Seronegativecontrolsubjectswereage-andsex-matchedwiththeARTnaivepatientsand thetwoothergroups.CarbonylproteinwasdeterminedbythemethoddescribedinLevine etal.DNAdamagemarker8-OH-dGwasdeterminedusing8-hydroxy-2-deoxyGuanosine StressXpressELAKitbyStressMarqBiosciences.

Results:ProteincarbonylcontentlevelsandoxidativeDNAdamageweresigniﬁcantlyhigher (p<0.05)inHIV-infectedpatientsonsecondlineARTandHIV-infectedpatientsonﬁrstline ARTthanARTnaivepatientsandcontrols.Inalinearregressionanalysis,increasedprotein carbonylcontentwaspositivelyassociatedwithincreasedDNAdamage(OR:0.356;95%CI: 0.287–0.426)p<0.05.

Conclusions: CarbonylcontentmayhasaroleasabiomarkerfordetectingoxidativeDNA damageinducedARTtoxicityand/oracceleratedaginginHIV/AIDSpatients.Largerstudies arewarrantedtoelucidatetheroleofcarbonylcontentasabiomarkerforprematureaging inHIV/AIDSpatients.

Abbreviations:ART,antiretroviraltherapy;ELISA,EnzymeLinkedImmunosorbentAssay;HIV,humanimmunodeﬁciencyvirus;8-OHdG, 8-hydroxydeoxyguanosine;ROS,reactiveoxygenspecies.

∗ _{Corresponding}_author.

E-mailaddress:vaishali.h@gmail.com(V.Kolgiri). http://dx.doi.org/10.1016/j.bjid.2016.09.007

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Introduction

The AIDS epidemic continues to spread in the South-East Asia (SEA)Region. TheSouth-EastAsiaisthe second most affectedWHOregionintheworld,aftersub-SaharanAfrica. Todate,closeto40millionpeoplethroughouttheworldhave beeninfectedwithhumanimmunodeﬁciencyvirus(HIV).Of these, almost 6.4million are in the SEA Region. Over99% ofcaseshavebeenreportedfromfourcountries–Thailand, India, Indonesia and Myanmar.1 _More _than ₂₅ antiretrovi-raldrugsfromsixtherapeuticclassesarenowavailablefor the managementofHIV infection.Most patientswho take medication achievedurable andperhapslifelong viral sup-pression,sotheclassicAIDSrelatedconditionsarebecoming less common.However,treated patientsdo nothave com-pletelyrestored health.Comparedwithpeoplewithout HIV infection,patients withthe infectionwho are treated with antiretrovirals have increased risk for several “non-AIDS” complications,manyofwhicharecommonlyassociatedwith aging.2

Themajorcomplicationsof“treated”HIVdiseaseare car-diovasculardisease,malignancy,renaldisease,liverdisease, bonedisease,andperhapsneurologicalcomplications,which arephenomenaofthenormalagingprocess,occuratan ear-lierageintheHIVinfectedpopulation.3

AcriticalresearchquestioniswhetherHIVisaccelerating agingitselfthroughpathwaysandmechanismscommonto theagingprocessor,alternatively,HIVmaysimplybean addi-tionalriskfactorforawidenumberofchronicconditionsthus accentuatingtheprevalenceofdiseaseateveryage.This situ-ationisfurthercomplicatedbyconcernthattoxicitiesofART, notHIVitself,maycontributetoagingandage-relatedillness. SpeciﬁcbiomarkersofagingarelimitedinHIV-infectedhosts andimpactedbyantiretroviraltherapy,andahighrateof mod-iﬁablelifestyleconfounders(e.g.,smoking,substanceabuse, alcohol)andco-infections(e.g.,viralhepatitis)inHIV-infected participants.Thereisaneedforvalidatedbiomarkersofaging inthecontextofHIVandART.4–7

ToshowthatHIVinfectionisassociatedwithaccelerated normalaging,oneﬁrstneedstounderstandwhatismeantby normalagingandtoﬁndawayofmeasuringit.

Aging,alsodescribedasorganismalsenescence,isdeﬁned asastatewithdecreasedabilitytorespondtostress,decreased homeostaticbalance,andanincreasedriskofdeveloping age-relateddiseases.8

Oxidativestressleadstoanelevatedoxidationof macro-molecules, such as DNA, lipids, and proteins. It was postulated that age-relatedaccumulation ofdamaged, oxi-dized,andaggregatedproteinsmightcontributetotheaging process.8,9

Oxidation of proteins leads to a partial unfolding and, therefore, to aggregation. Protein aggregates impair the activity ofcellular proteolytic systems (proteasomes, lyso-somes),resultinginfurtheraccumulationofoxidizedproteins. In addition, the accumulation of highly cross linked pro-teinaggregatesleadstofurtheroxidantformation,damage to macromolecules and ﬁnally, to apoptotic cell death. Furthermore, protein oxidation seems to play a role in the development of various age related diseases, such as

neurodegenerativediseases,type2diabetes,cancer,and car-diovasculardisease.2

Two ofthe most commonlymeasured oxidativeprotein modiﬁcations are protein carbonyls and 3-NT, which are usedtoassess thecellularstatusofproteinoxidation. Pro-teincarbonylsaregeneratedduetotheoxidationofproline, arginine, lysine, threonine, and other amino-acid residues and due to the oxidation of the protein backbone. More-over, they might be the result of secondary reactions of amino acids (cysteine, histidine, and lysine) with reactive carbonylcompounds(ketones,aldehydes),emergedinlipid peroxidationorglycation/glycoxidationreactions.9 _One_way tomeasureproteinoxidationistodeterminelevelsofprotein carbonyls.10

Under normal physiological conditions in all aerobic organisms, thereisabalancemaintained between endoge-nousoxidantsandnumerousenzymaticandnon-enzymatic antioxidant defences.When animbalanceoccurs,oxidants produceextensiveoxidativedamagetoDNA,which,inturn, contributestoaging,malignanttumors,andother degenera-tivediseases.11,12_Oxidative_injury_is_integral_to_HIV/AIDS_as_a potentinducerofviralactivation,viralreplication,andDNA damageininfectedcells.13,14

AlthoughmanydamagedDNAlesions havebeen identi-ﬁed,wehavechosen8-hydroxy-2-deoxyguanosine(8-OHdG) as our biomarker ofoxidative damage. The importanceof this lesion stems from the fact that it is both abundant in DNA and it is mutagenic. Current evidence suggests that8-OHdGlesionspresentinDNAduringcellular replica-tion results insomatic mutation, the driving force behind carcinogenesis.13,15 _{Determination} _and _analysis _of _8-OHdG can beperformed inanimal organs and inhumansample (urine,humanorgans,leukocyteDNA)asabiomarkerof oxida-tivestress,aging,andcarcinogenesis.16,17_Many_studies_in_the past20yearsandimprovementsinthequantitative estima-tionof8-OHdGbyvariousanalyticaltechniquesinbloodcells orinurinehaveestablisheditasaveryimportantbiomarker notonlyforcarcinogenesisbutalsoforaginganddegenerative diseases.18–21

ThesearchofsuitablebiomarkersforaginginHIV/AIDS patientsisongoing.Thereisneedofbiomarkerforpremature agingthatcouldalertthecliniciantofurtherinvestigatenon AIDSrelateddiseasesinHIV/AIDSpatients.Suchatoolshould be inexpensive and facilities for its determination easily available.

Inthisstudywedeterminedproteinoxidationmarker car-bonyl content and correlated it with DNA damagemarker 8-OHdGinHIV-infectedpatientsonART,ARTnaïve,and HIV-negativecontrols.

Methods

Subjectselection

A case–control study was carried out on HIV-1 infected patientsattheoutpatientinfectiousdisease unit(OPD)and ARTcentreoftheSirJJHospital&GrantGovernment Medi-calCollege,Mumbaioveraperiodofoneyear,fromFebruary 2014 to March 2015. We have selected 300 subjects from

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OPDafterevaluationoftheirmedicalrecords(negativeserial ELISA/WesternblottestforHIVbeforethreemonthsof sam-plecollection)asHIV-negativecontrolsand300HIV-infected subjectsdetectedbyserialELISA/Westernblotmethodfrom ARTcentre.Allsubjectswererandomlyselectedandgrouped as HIV-negative (control group) (n=300), HIV-infected ART naive (n=100), HIV-infected on first line ART (n=100) and HIV-infectedonsecondlineART(n=100).Seronegative con-trolsubjectswereage-andsex-matchedwiththeARTnaive patientsandthetwoothergroups.Weusedpoweranalysis methodforsampleselectionat5%significancelevelfor95% confidenceinterval.

Wedonot havedata regardingbaselineviral load,CD4 nadir,andviralsuppressionafterstartingARTforthestudy population, as these are not included in routine tests for HIV/AIDSpatientsatARTcentreoftheSirJJHospital&Grant Government Medical College, Mumbai. As this was a self-fundedstudywecouldnotperformthesetestsduetolimited funds.

Ethicalapproval

Thestudyprotocolwasapprovedbytheinstitutionalethics committee(No.IEC/Pharm/902/2013)andNationalAIDS Con-trolOrganizationDelhi,India(T-11020/67/2011-NACO).

Inclusioncriteria

All participantswere 20 years ofage or older HIV-infected patientsdetectedbyserialELISA/Westernblot methodand wereincludedinthisstudyaftergivingtheirinformed con-sent.Wehaveselected300subjectsfromOPDafterevaluation oftheirmedicalrecords(negativeserialELISA/Westernblot testforHIVbeforethreemonthsofsamplecollection)as HIV-negativecontrols(n=300).

Exclusioncriteria

Exclusion criteria includedpregnant women, patients with chronicdiseases like hepatitis,diabetes, renal impairment, cardiovascular co-morbidities, neurological psychiatric dis-orders, various malignancies, as well as heavy smokers, alcoholics,tobacco-chewers,andHIV-infectedpatientswith withdrawal of combination ART. The demographic details were collected from each patient and entered into the pro-forma. Subsequent to this, a detailed history was taken.

Samplecollection

Venous blood sampleswere collected inplain and lithium heparin vacutainers as an anticoagulant. Blood was cen-trifuged (4000g, 10min, 4◦C) to separate the plasma. The collectedplasmawas storedat−70◦Cwithaseptic precau-tions.Plainbloodsamples2haftercollectionwerecentrifuged at3000rpmfor5min;serumwasseparatedandcollectedin steriletubes.

DifferenttreatmentregimensasperNACOguidelines

ThelistofantiretroviraltherapyadministeredtoIndian HIV-infectedpatientswasasfollows:

Firstlinetherapy Secondlinetherapy

Tenofovir Tenofovir

Lamivudine Lamivudine

Efavirenz Ritonavir

Atazanavir

Biochemical

methods

Determinationofproteincarbonylcontent

Protein carbonyl proteins were assayed by

2,4-dinitrophenylhydrazine reaction.22 _The _assay _is _based _on the spectrophotometric detection of the reaction between 2,4-dinitrophenylhydrazine(DNPH)withproteincarbonylto formproteinhydrazone.Carbonylcontentwasdeterminedas nmolmg−1protein.Thetotalproteincontentwasmeasured usingcolorimetrickitbasedonbiuretmethod.

DeterminationofDNAdamagemarker 8-hydroxy-2-deoxyguanosine(8-OHdG)

Plasma of all the cases and control sampleswas used for themeasurementof8-OHdGlevelsusingcompetitiveinvitro

Enzyme-Linked Immunosorbent Assay (ELISA) kit obtained fromStressXpressELAKitbyStressMarqBiosciences.8-OHdG measurementswereperformedusingmicrotiterELISAplate pre-coatedwithanti-mouseIgG.50␮Lsample,50␮L8-OHdG AChE(acetylcholinesterase)tracerand 50␮L8-OHdG mono-clonal antibodywere added toeach well and incubated at 4◦Cfor18h.Afterthewellswerewashedﬁvetimes,200␮L Ellman’s reagent was added to each well. The wells were incubatedatroomtemperatureinthedarkfor100min.The absorbancewasread atwavelengthof420nm.ELISA assay displaysIC50(50%B/B0)andIC80(80%B/B0)valuesof

approxi-mately100and30pgmL−1,respectively.

Statisticalmethods

Datawere expressedasthemean±SD.Student’sttestwas performed to assess differences between two means. Sta-tisticalanalysis wasperformed usingEPI-INFO 7statistical softwareformedicalresearchstudies.Differencesinmeans between groups were tested using independent-sample

t-tests. p<0.05 was considered as statistically significant. Mann–Whitneytestfornonparametriccontinuousvariables, Chi-squareorFisher’sexacttestforcategoricalvariableswere usedtotestforstatisticalsignificance.Weusedlinear regres-sionmethodforcorrelationofproteincarbonylcontentand DNAdamagemarker8-OH-Dg,andweusedlogistic regres-sionmethodforcomparisonbetweentwoagegroupsandART second-line,ARTfirst-line,andARTnaïve.

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Table1–Patientscharacteristics,CD4count,plasmaDNAdamagemarker8-OHdG,andserumcarbonylcontent.

Group Subgroup Male/female Age(mean) CD4(mean) Timeonanti-retroviral therapy(mean)

8-OHdG Carbonylcontent count years cells/␮L months ng/mL nmol/mgofprotein HIV-negative controls 20–40yrs 87/63 34.8±5.1 NA NA 1.07±0.25 0.64±0.08 40–60yrs 92/58 47.3±5.4 NA NA 1.30±0.32 0.66±0.10 HIV-infectedART naive 20–40yrs 28/22 31.6±4.2b ₆₂₄_±₂₀₂b _NA _1.81_±_0.66a _0.76_±_0.13a 40–60yrs 33/17 45.2±6.1b ₆₀₂_±₁₃₄b _NA _1.95_±_0.56a _0.77_±_0.18a HIV-infectedon ﬁrstlineART 20–40yrs 30/20 34.3±5.2b ₅₀₂_±₂₃₃b _63.6a _2.50_±_0.91a _1.06_±_0.31a 40–60yrs 41/9 48.3±4.8b ₄₆₅_±₁₆₅b _65.7a _3.13_±_0.92a _1.08_±_0.37a HIV-infectedon

secondlineART

20–40yrs 36/14 35.6±4.3b ₄₀₀_±₁₄₄b _74.3a _2.79_±_0.95a _1.20_±_0.36a

40–60yrs 43/7 47.5±5.3b ₃₇₆_±₁₈₃b _76.8a _3.27_±_0.99a _1.22_±_0.36a

Valuesaremean±SEMofsubjects.Thedatawereanalyzedbyone-wayANOVA.

a _p_<_0.01_signiﬁcant_when_compared_to_control.

b _p_<_0.05_signiﬁcant_when_compared_to_control.

Results

Atotalof600(300HIV-infectedand300HIVnegative)subjects wereincludedinthisstudy.Allthesubjectsweredividedin twoagegroups(20–40yrs)and(40–60yrs).AsseeninTable1, the mean carbonylcontentlevels were significantlyhigher than thatof thecontrol groupforthe ART-naïve(p<0.001) group,thoseonfirst-lineART(p<0.001)andonsecond-line ART(p<0.001)inbothagegroups.DNAdamagemarkerlevels werealsosignificantlyhigherthanthatofthecontrolgroup fortheARTnaïve(p=0.001),thoseonfirst-lineART(p<0.001) andonsecond-lineART(p<0.001)inbothagegroups.Carbonyl contentandDNAdamagemarkerlevelscorrelatedpositively witheachotherforthetotalpatientsample(OR:0.356;95%CI: 0.287–0.426)[p<0.001].Theseresultssupportedthenotionof carbonylcontentasthebettermarkerofinflammatory activ-ityandareinagreementwiththeconceptofARTtoxicityas aconditionwithincreasedinflammatoryactivity withDNA damage.

CarbonylcontentwassigniﬁcantlyhigherinHIV-infected patientsonARTthanHIV-infectedARTnaïvesubjectsandHIV negativecontrols.ThemeancarbonylcontentforHIV-infected ARTnaivepatientsinagegroup20–40yrswas0.76nmol/mg ofproteinandinagegroup40–60yrswas0.74nmol/mgof pro-tein.ThemeancarbonylcontentforHIV-infectedinbothage groups,20–40yrsand40–60yrsonART(ﬁrstline)were1.06and 1.21nmol/mgofproteinand1.06and1.22nmol/mgofprotein forthoseonsecondline.

Weconsiderthenormalvalueforcarbonylcontentisupto 0.75nmol/mgofproteinandmorethan0.75nmol/mgof pro-teinisconsidered asabnormalvalueofcarbonylcontent.22 Carbonylcontentwasabnormalin14(28%)ARTnaive HIV-infectedpatientsintheagegroup20–40yrsandin17(34%) intheagegroup40–60yrs.Thecarbonylcontentwas signif-icantlymoreabnormalinHIV-infectedpatientsinbothage groups(20–40yrsand40–60yrs)onART:31(62%)onﬁrstline and33(66%)onsecondline,and35(70%)and38(76%), respec-tively.Theseresultsshowthatcarbonylcontentwasincreased inHIV-infectedpatientsonART.Inalogisticregression analy-siscarbonylcontentwassigniﬁcantlydifferentbetweenthe age groups of 20–40yrs and 40–60yrs (OR: 0.609; 95% CI:

0.4502–0.8258–p<0.001),betweensecond-lineandfirst-line ART(OR:0.7959;95%CI:0.5183–1.222–p<0.296),andbetween naïve patients and those on first-line (OR: 5.717; 95% CI: 3.762–8.672–p<0.001).Themean8-OH-DGforHIV-infected patientswashigherthanthatforHIVnegativesubjects.The mean 8-OH-DG forHIV-infected ART naivepatients in age group 20–40yrswas 1.81ng/mL and inage group 40–60yrs was1.95ng/mL.Themean8-OH-DGforHIV-infectedinboth agegroups(20–40yrsand40–60yrs)onART(firstline)were 2.50ng/mLand3.13ng/mL,and(secondline)2.79ng/mLand 3.27ng/mL,respectively.ARTacceleratesDNAdamagein HIV-infectedpatients.TheseresultsshowincreasedDNAdamage inHIV-infectedpatientsonART.Inthisstudyweusedlinear regressionmethodtocorrelateproteincarbonylcontentwith DNA damage.Weobservedthat increasedproteincarbonyl contentlevelswerepositivelyassociatedwithincreasedDNA damage(OR:0.356;95%CI:0.287–0.426–p<0.001).Increased meancarbonylcontentvalueswereobservedinHIV-infected patientsonfirstlineandsecondlineARTofbothagegroups (20–40yrsand 40–60yrs)than ARTnaiveand negative con-trols;similarpatternwasobservedwithDNAdamagemarker 8-OH-DGinHIV-infectedpatientsonfirstlineandsecondline ARTofbothagegroups(20–40yrsand40–60yrs),asshownin Table1.

Discussion

Infectionwiththehumanimmunodeﬁciencyvirus-1(HIV)is associated withclinical symptoms ofacceleratedaging, as evidencedbyincreasedincidenceanddiversityofage-related illnessesatrelativelyyoungages.Cardiovasculardisease, dia-betes,andseveralotherconditionsaremoreprevalentatall agesinthosewithHIV,suggestingthereisanextra“hit”by HIVand/orART–thatis,accentuatedaging.4–7_There_is con-siderable evidence suggesting that oxidative stress playsa critical role inbothin vitrosenescence andin vivoaging.23 Telomerescaptheendsofchromosomesandconsistof hex-americTTAGGGrepeatsandtheprotective‘shelterin’protein complex.Telomeraseinhibitionhasbeenconsidereda possi-blemechanismbywhichARTinHIV/AIDScausesaccelerated aging.24

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Averyimportantmarkerofoxidativestressisprotein car-bonylation, measured through estimating protein carbonyl groupscontent in serum.Measuring proteincarbonyl con-tentisadvantageousoverotherbiomarkersofoxidativestress duetotheirearlyformationanddetectablestabilityarising from proteinside chains(Pro,Arg,Lys, and Thr).25 _Protein carbonylationisatypeofproteinoxidationthatcanbe pro-motedbyreactiveoxygenspecies.Itusuallyreferstoaprocess thatformsreactiveketonesoraldehydesthatcanbereacted by2,4-dinitrophenylhydrazine(DNPH)toformhydrazones.26 Oxidative modification of proteins isknown to affect pro-tein function. Protein carbonyls and protein nitrotyrosine arewidelyusedandchemicallystablebiomarkersofprotein oxidation. Theproteincarbonyls (PC)group are formed by eitherdirectoxidationofcertain aminoacid residues, par-ticularly lysine, arginine, threonine, proline, and histidine or secondarily reaction with product of lipid peroxidation (e.g.,HNE)orglycoxidationreactionwithlysinegroup.27 Pro-teincarbonylsarebetterstudiedthanproteinnitration.The concentrationofPCwasstable,yieldedquantitativeresults, and appearedtoreflect disease endpoints ina biologically significantway.Carbonylatedproteinsarerelativelydifficult toinducecomparedwiththe SHoxidationofcysteine and methionine,andelevatedlevelsofcarbonylationarethought tobeasignnotonlyofoxidativestress,butalsoof disease-derivedproteindysfunction.Therefore,carbonylatedproteins can be used as oxidative stress biomarkers according to theirspecialpropertiesincludingirreversibility, unrepairabil-ity, stability inphysicalcondition andinduction ofprotein aggregation.Proteinsaremajorbio-moleculesthatdrive cel-lularactivities.Growingevidencesuggeststhatonco-proteins, tumorsuppressorproteinsand oxidativestress-related pro-teins play significant roles in carcinogenesis.27 _Aging _is accompaniedbyaccumulationofdamagedDNA,misfolded proteins, and oxidized proteins. A multitude of data indi-catethatproteinoxidation,accumulationofoxidizedproteins, and protein aggregation as well as the impairment of the proteosomal system play a major role in the aging pro-cessandinthedevelopmentofsome,ifnotall,age-related diseases.28

Freeradicalsmayalsobringabouttheoxidativedamages ofDNA thatare manifestedbythedevelopmentofvarious complications.TheoxidativelyinducedDNAdamagetypically associatedwithROSareapurinic/apyrimidinic(AP)DNAsites, oxidized purines and pyrimidines, single strand and dou-blestrandDNAbreaks.TheaccumulationofoxidativeDNA damagemay belinkedwithage-associated neurodegenera-tivedisorders Alzheimer’sdisease,Parkinson’s disease and amyotrophiclateral sclerosis.ROSgenerate8-oxodG,which can be used as an indicator of oxidative DNA damage in relation to oxidative stress-driven diseases. Kryston et al. reviewed the role of oxidative stress and DNA damage in humanmalignancies,andsuggestedtheideaofusingseveral oxidatively generated DNA lesions like 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxodG), also known as 8-hydroxydG (8-OHdG),thymineglycol,APsitesandoxidativelygenerated clusteredDNAlesions(OCDLs)asnovelbiomarkersof oxida-tivestress.16_The_most_commonly_used_marker_of_oxidatively modiﬁed DNA is 8-hydroxy-2-deoxyguanosine (8-OHdG), a productofoxidativelymodiﬁedDNAbaseguanine.DNAcanbe

oxidizedtoproducemanyoxidativeproducts;however, oxida-tionoftheC-8ofguanineisoneofthemostcommonoxidative events and results in a mutagenic lesion that produces predominantly G-to-Ttransversionmutations.8-OHdG was foundtobeincreasedinanormalhumanLECcultureafter induced oxidative stress.17 _Biological _materials_most _often used tomeasurelevels of8-OHdGinclude,serum, plasma, urineandtissues.Inrecentyears8-hydroxydeoxyguanosine (8-OHdG)hasemergedasamarkerofoxidativedamageinthe patho-physiologicalprocessesofcancer.18,19_Levels_of_8-OHdG invarious biologicalsampleshasbeencorrelated with dis-easeactivity,thus8-OHdGisausefulmarkerforstudyofDNA damagecausedbyfreeradicals.Thereisample circumstan-tialevidencethatoxidativestressleadstotheaccumulationof DNAdamageduringaging.20_These_data_suggest_that_carbonyl contentand8-OHdGlevelsarepotentiallyusefulbiomarkers ofaginginHIV/AIDSpatients.Itwillbeimportanttoaccount fordifferentialexposuretoriskfactors(e.g.,smoking,alcohol, non-HIVsexuallytransmitteddiseases)betweenHIV-infected and HIV-uninfected populations that are likelyto resultin residualconfoundingwhenassessingassociationsofHIVwith increasedriskofage-relatedillness.Therefore,wehave cho-senthesubjectpopulationandcontrolpopulationwithsimilar economical background and similar confounders (smoking andalcohol).

All experimentsdescribed inthisstudy were performed blindly withrespecttosubjectstatus(ARTnaïve,ART first-line, ART second-line HIV patients, and control subjects). In thepresent study thevalueofcarbonylcontentas gen-eral marker of the inflammatory status was examined by comparing carbonyl content levels to that of other recog-nizedbiomarkerofinflammation,i.e.,DNAdamagemarker 8-OHdG. Asseen inFig. 1, themean carbonylcontent lev-els weresignificantlyhigherthan thatofthecontrolgroup fortheART-naïvepatients(p<0.001),thoseonfirst-lineART (p<0.001),andthoseonsecond-lineART(p<0.001).8-OHdG levelswerealsosignificantlyhigheramongARTnaïvepatients (p=0.001), those on first-line ART (p<0.001), and those on second-line ART (p<0.001)than those ofthe controlgroup (Fig.2).

These results supportedthe notionofcarbonyl content asabettermarkerofinflammatoryactivity andare inline withtheconceptofadvancedHIV/AIDSasaconditionwith increased inflammatory activity. Protein carbonyl content and 8-OHdG levels are significantlyincreased afterART in HIV/AIDS patients of both age groups (Figs. 1 and 2). Sig-nificant increase in protein carbonyl content and 8-OHdG levelswereobservedinHIV-infectedpatientsintheagegroup 40–60yearswhencomparedtothoseintheagegroup20–40 years.Infact,proteincarbonylcontentlevelsshowastrong andsignificantcorrelationwithDNAdamage.Thuswehave observedacloseassociationbetweencarbonylcontent and DNA damage inHIV/AIDS withART (Fig. 3). This suggests thatthe accumulationofcarbonylcontentmaybeanearly indicationofincreasedDNAdamagemarker8-OHdGlevels whichmayindicatesacceleratedaginginHIV/AIDSpatients onART.

Inﬂammationandactivationofcoagulationpathwaysare central to the pathophysiology leading to morbidity and mortalityinHIV-infectedpatients,asdemonstratedbydata

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1.40 Carbonyl content 1.20 1.00 0.80 0.60 0.40 0.00 0.20

ART first line ART second line

Antiretroviral theraphy

A

v

er

age carbon

yl content (nmol/mg of protein)

ART yet to start Control

Group_20_40 yrs

Group_40_60 yrs

Group_20_40 yrs

Group_40_60 yrs

Group_20_40 yrs _{Group_40_60 yrs}

Group_20_40 yrs Group_40_60 yrs

Fig.1–Averagecarbonylcontentamongsubjects.

Group_20_40 yrs

Group_40_60 yrs

Group_20_40 yrs

Group_40_60 yrs

Group_20_40 yrs Group_40_60 yrs

Group_20_40 yrs Group_40_60 yrs ART first line ART second line

Antiretroviral theraphy status

ART yet to start Control 3.50 3.00 2.50 2.00 1.50 1.00 0.00 0.50 Plasma 8-OH-DG [ng/ml]

DNA damage marker

Fig.2–AverageDNAdamagemarkeramongsubjects.

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 ART First Line ART Second Line ART Yet to Start

Control ART First Line ART Second Line ART Yet to Start Control Group_40_60 Yrs Group_20_40 Yrs

Average carbonyl content[nmol/mg]

&8-OH-DG[ng/ml]

Co-relation of carbonyl content& dna damage marker

Fig.3–Co-relationofcarbonylcontent&DNAdamage marker.

from theStrategiesforManagement ofAntiretroviral Ther-apystudy,whichshowedthatIL-6andD-dimerwerestrongly related tonon-AIDS –deﬁning comorbiditiesand all-cause mortalityinpatientsonART.InHIVinfection,theheightened inﬂammationandimmunedysfunctionrelatedtoaccelerated aginghasstimulatedassessmentofseveralrelevant biomark-ersincludingC-reactiveprotein,IL-6,andD-dimerandtheir associations withage-relatedmorbidityand HIVinfection.4 Aweaknessofthepresentstudywasthatthosebiomarkers couldnotincluded.

We do not yet know if antioxidant therapy is use-ful to prevent premature aging in HIV/AIDS patients. This studydemonstratedtheextentofoxidativeproteindamage and its association with DNA damage. Hence determina-tion of protein carbonyl content can serve as a simple biomarker formonitoring the magnitudeofoxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDSpatients.Furtherstudiesareneededtoclarifythis issue.

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Conclusion

ThisstudyinHIV/AIDSpatientsshowedcarbonylcontentasa betterindicatoroftheprematureagingduetoeffectofART.It isclearthattherapeuticside-effectsandeffectsofHIV-1 infec-tiontogethermustbeconsideredinthepathogenesisofaging intheHIV/AIDSpopulation,asantiretroviraltherapyislinked directlytotheinfection.Thereisanabundanceofevidence implicatingROSasonesourceofDNAdamageassociatedwith aging,cancer,stresssignaling,andother conditions.Inthe presentstudyweobservedthatcarbonylcontentispositively correlatedtoDNAdamagemarker8-OHdGlevelsinHIV/AIDS patients.Overall,ourresultsdemonstratethatthecarbonyl contentmayhavearoleasabiomarkerfordetectingoxidative DNAdamageinducedARTtoxicityand/oracceleratedagingin HIV/AIDSpatients.Determinationofcarbonylcontentisavery simpleandcost-effectivemethodhencewesuggestthat car-bonylcontentmaybeusedasabiomarkerfordetermination ofARTtoxicityand/orprematureaginginHIV/AIDSpatients. Infuture,managementofprematureagingandDNAdamage inHIV/AIDSpatientsshouldbeprimeconcernforhealthcare physicians.

Authors

contribution

VKcarriedoutthesampleanddatacollection.VKcarriedout allexperimentalwork,dataanalysisanddraftthemanuscript. VPassistedVKinexperimentalworkanddraftdesigning.VP helpedinsubjectselectionandsampleselection.Allauthors readandapprovedtheﬁnalmanuscript.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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1. VajpayeeM,MohanT.Currentpracticesinlaboratory monitoringofHIVinfection.IndianJMedRes. 2011;134:801–22.

2. WendelkenL,ValcourV.ImpactofHIVandagingon neuropsychologicalfunction.JNeurovirol.2012;18:256–63. 3. DeeksSG.HIVinfection,inﬂammation,immunosenescence,

andaging.AnnuRevMed.2011;62:141–55.

4. PathaiS,BajillanH,LandayAL,HighKP.IsHIVamodelof acceleratedoraccentuatedaging?JGerontolA:BiolSciMed Sci.2014;69:833–42.

5. HorvathS,LevineAJ.HIV-1infectionacceleratesage accordingtotheepigeneticclock.JInfectDis. 2015;212:1563–73.

6. PerrinS,CremerJ,RollP.HIV-1infectionandﬁrstlineART induceddifferentialresponsesinmitochondriafromblood lymphocytesandmonocytes:theANRSEP45“aging”study. PLoSONE.2012;7:e41129.

7.RickabaughTM,BaxterRM,SehlM.Accelerationof

age-associatedmethylationpatternsinHIV-1-infectedadults. PLoSONE.2015;10:e0119201.

8.HöhnA,KonigJ,GruneT.Proteinoxidationinagingandthe removalofoxidizedproteins.JProteomics.2013;92:132–59. 9.StadtmanER,BerlettBS.Reactiveoxygen-mediatedprotein

oxidationinaginganddisease.ChemResToxicol. 1997;10:485–94.

10.Dalle-DonneI,RossiR,GiustariniD.Proteincarbonylgroups asbiomarkersofoxidativestress.ClinChimActa.

2003;329:23–38.

11.SmithCD,CarneyJM,Starke-ReedPE.Excessbrainprotein oxidationandenzymedysfunctioninnormalAgingandin Alzheimerdisease.ProcNatlAcadSciUSA.1991;88: 10540–3.

12.Starke-ReedPE,OliverCN.Proteinoxidationandproteolysis duringagingandoxidativestress.ArchBiochemBiophys. 1989;275:559–67.

13.StadtmanER.Proteinoxidationandaging.Science. 1992;257:1220–4.

14.KrystonTB,GeorgievAB,PissisP.Roleofoxidativestressand DNAdamageinhumancarcinogenesis.MutatRes.

2011;711:193–201.

15.AukrustP,LunaL,UelandT,Johansen.Impairedbaseexcision repairandaccumulationofoxidativebaselesionsinCD4+_T

cellsofHIV-infectedpatients.Blood.2005;105:4730–5. 16.ValavanidisA,VlachogianniT,FiotakisC.

8-Hydroxy-2-deoxyguanosine(8-OHdG):acriticalbiomarker ofoxidativestressandcarcinogenesis.JEnvironSciHealthC. 2009;27:120–39.

17.Olinski,Rozalski,Gackowski.Urinarymeasurementof 8-oxodg,8-oxogua,and5HMUra:anoninvasiveassessment ofoxidativedamagetoDNA.AntioxidRedoxSignal. 2006;8:1011–9.

18.YermilovV,Rubio,Becchi,Friesen,Pignatelli.Formationof 8-nitroguaninebythereactionofguaninewithperoxynitrite invitro.Carcinogenesis.1995;16:2045–50.

19.Rodriguez,Song,Crouse.Invivobypassof8-oxodG.PLoS Genet.2013;9:e1003682.

20.Melis,vanSteeg,LuijtenM.OxidativeDNAdamageand nucleotideexcisionrepair.AntioxidRedoxSignal. 2013;18:2409–19.

21.Hiraku,Kawanishi,Ichinose.TheroleofiNOS-mediatedDNA damageininfection-andasbestos-inducedcarcinogenesis. AnnNYAcadSci.2010;1203:15–22.

22.LevineRL,GarlandD,OliverCN,etal.Determinationof carbonylcontentinoxidativelymodiﬁedproteins.Methods Enzymol.1990;186:464–78.

23.TorresRA,LewisW.AgingandHIV/AIDS:pathogeneticroleof therapeuticsideeffects.LabInvest.2014;94:120–8.

24.PandeyG,ShihabudeenMS,DavidHP.Associationbetween hyperleptinemiaandoxidativestressinobesediabetic subjects.JDiabetesMetabDisord.2015;14:24.

25.SuzukiYJ,CariniM,ButterﬁeldAD.Proteincarbonylation. AntioxidRedoxSignal.2010;12:323–5.

26.ReegS,GruneT.Proteinoxidationinaging:doesitplayarole inagingprogression?AntioxidRedoxSignal.2015;23: 239–55.

27.ThananR,OikawaS,HirakuY.Oxidativestressandits signiﬁcantrolesinneurodegenerativediseasesandcancer. IntJMolSci.2015;16:193–217.

28.SedelnikovaaOA,RedonaCE.RoleofoxidativelyinducedDNA lesionsinhumanpathogenesis.MutatRes.2010;704:152–9.