Free Radical Biology & Medicine 51 (2011) 1806–1814

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Original Contribution

Occult hepatitis C virus elicits mitochondrial oxidative stress in lymphocytes andtriggers PI3-kinase-mediated DNA damage response

Arpit Bhargava a,b, Gorantla V. Raghuram a,b, Neelam Pathak a, Subodh Varshney c, Suresh K. Jatawa a,Deepika Jain a,b, Pradyumna K. Mishra a,b,⁎a Research Wing, Bhopal Memorial Hospital and Research Centre, Bhopal, Indiab Division of Translational Research, Tata Memorial Centre, ACTREC, Navi Mumbai 410 210, Indiac Department of Surgical Gastroenterology, Bhopal Memorial Hospital and Research Centre, Bhopal, India

Abbreviations: 8-oxo-dG, 8-oxo-2′-deoxyguanosine;aldehyde-reactive probe; CM-H2DCFDA, 5-(and-6)-chdrofluorescein diacetate acetyl ester; DCF, 2′,7′-dichlorobreak; EDTA, ethylenediaminetetraacetic acid; ER, endorescein isothiocyanate; GR, glutathione reductase; HCV,hepatitis C infection; pATM, ataxia telangiectasia mutatataxia telangiectasia and Rad3-related phosphoserine-415; PE, phycoerythrin; PCR, polymerase chain reaction;kinase; ROS, reactive oxygen species; SOD, superoxide dlogical response.⁎ Corresponding author at: Division of Translational R

ACTREC, Navi Mumbai 410 210, India. Fax: +91 22 274E-mail address: pkm_8bh@yahoo.co.uk (P.K. Mishra

0891-5849/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.freeradbiomed.2011.08.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 May 2011Revised 11 August 2011Accepted 12 August 2011Available online 3 September 2011

Keywords:mtDNADNA repairApoptosisCirculating nucleosomesTranslational researchFree radicals

Occult hepatitis C viral infection (OHCI) is a newly reported pathological entity associated with increased riskof developing hepatocellular carcinoma and lymphoproliferative disorders. Although hepatocytes are theprimary sites of viral replication, hepatitis C virus is potentially lymphotropic, invading and propagating incells of the immune system. Lymphocytes, the extrahepatic viral reservoirs, are differentially implicated inthe occult and the active forms of the disease. This study aimed to elucidate the implications of mitochondrialoxidative stress on the immune pathophysiological mechanisms of OHCI. We herein report that OHCI inducesmitochondrial oxidative stress, leading to DNA double-strand breaks and elicitation of a phosphoinositol3-kinase-mediated cellular response in peripheral blood lymphocytes. Compared to controls, OHCI subjectsshowed higher accumulation of pATM, pATR, γH2AX, and p-p53, along with active recruitment of repair pro-teins (Mre11, Rad50, and Nbs1) and altered mitochondrial DNA content. Increased mitochondrial membranedepolarization and circulating nucleosome levels along with chromatid-type aberrations and decreased T-cellproliferative index observed in the OHCI group further indicated that this damagemight lead to Bax-triggeredmitochondria-mediated cellular apoptosis. Together our results provide themechanistic underpinnings of mi-tochondrial dysfunction in OHCI, a previously unknown paradigm, for explaining the immune pathogenesis ina redox-dependent manner.

ALT, alanine transferase; ARP,loromethyl-2′,7′-dichlorodihy-fluorescin; DSB, double-strandplasmic reticulum; FITC, fluo-hepatitis C virus; OHCI, occulted phosphoserine-1981; pATR,28; p-p53, p53 phosphoserine-PI3-kinase, phosphoinositol 3-ismutase; SVR, sustained viro-

esearch, Tata Memorial Centre,05061.).

rights reserved.

© 2011 Elsevier Inc. All rights reserved.

Hepatitis C virus (HCV) infection is prevalent in approximately 2% ofthe world's population and is one of the leading causes of cirrhosis, he-patocellular carcinoma, and lymphoproliferative disorders, includingmixed cryoglobulinema and non-Hodgkin B cell lymphoma [1]. HCV, asole member of the genus Hepacivirus (family Flaviridae), is a posi-tive-strand RNA virus that replicates by synthesizing a negative RNAstrand of 9600 bp [2]. The viral RNA encodes a large polyprotein of3100 amino acids, which is translated on the endoplasmic reticulum(ER) and posttranslationally processed by cellular and viral proteases

into 10 individual proteins. The structural proteins (core, E1, and E2)build up the virus particle, whereas the p7 polypeptide and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) arerequired for RNA replication and virion assembly [3]. The nonstructuralproteins orchestrate viral replication, forming a membrane-associatedreplication complex. All HCV proteins exposed to the cytosolic spaceare anchored to the ER membrane, except E1 and E2, which face theER lumen [4]. Because HCV has no known ability to integrate its nucle-otide sequence into the host genome and has too short a half-life, anuninterrupted HCV replication is essential to maintaining a continuousinfectious state.

Evidence derived from experimental systems illustrate the strongassociation of viral replication with mitochondrial oxidative stressand generation of reactive oxygen species (ROS) [5]. Oxidative stressimposed either directly by the virus or by the host immune responseis considered an important pathogenic mechanism in HCV infection.Viral proteins such as HCV core, E1, and NS3 are reported to be potentinducers of ROS [6]. These proteins inactivatemitochondrial respiratorychain enzymes and trigger blockade of the electron chain, dissipation ofthe mitochondrial transmembrane potential, and increased electronleakage causing hypergeneration of endogenous ROS [7]. Although thesynthesis and maturation of HCV proteins occur at the level of the ER,

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studies have shown that HCV proteins accumulate at the point of con-tact between the mitochondrial outer membrane and the ER. The viralproteins have been speculated tomigrate from the ER to the mitochon-dria by lateral diffusion via transient fusion of the membranous sub-compartments [8]. Therefore, direct interaction of HCV proteins withthe mitochondrial machinery in hepatic and extrahepatic sites is posit-ed, although themechanistic insights of this interaction remain obscure.

Despite the fact that hepatocytes are the primary sites of virus rep-lication, HCV is potentially lymphotropic. HCV invades and propagatesin cells of the immune system, albeit at a lower rate per cell thanhepatic tissue [9]. Indeed, HCV RNA positive and negative (replicative)strands as well as virus proteins have been identified in peripheralblood lymphocytes of chronic HCV patients [10,11]. HCV lymphotrop-ism is also supported by the presence of low levels of viral RNA incirculating lymphocytes of occult HCV patients, even after apparentresolution of the disease [12]. As lymphocytes, the viral reservoirs,are differentially implicated in the occult and the active forms of thedisease, the possible molecular repercussions of viral interaction atthe subcellular level need to be determined.

This work is an extension of our previous study in which weobserved sustained virological response (SVR) in 94.3% of genotype 3-infected patients after combination therapy [13]. SVR is defined asundetectable HCV RNA in serum, even after 24 weeks withdrawal ofstandard combination therapy (PEGylated interferon and ribavirin)[14]. The incidence of secondary occult HCV infections has been identi-fied in cases that achieved SVR. Further, we aimed to understand theimplications of viral interaction with the mitochondrial machinery ofthe host by evaluating downstream cellular effects. Oxidative stress,DNA damage repair response, mitochondrial DNA (mtDNA) contentand respiratory chain enzyme activity, T-cell proliferative index, andrelative expression of proapoptotic Bax along with cytogenetic analysisand apoptosis in the peripheral blood lymphocytes of occult and chronicHCV and control subjects were studied.

Materials and methods

Subject selection

A total of 68 chronic HCV patients were treated with standardcombination therapy of PEGylated interferon and ribavirin for 24 to48 weeks. Of these, 53 patients infected with genotype 3 achievedSVR [13]. The presence of HCV RNA was observed in peripheralblood lymphocytes in 10 (of 53) patients negative for serum HCVRNA but with normal liver function, thereby confirming secondaryoccult HCV infection. EDTA-conjugated blood samples were collectedfrom each subject by routine venipuncture and used for investigations.Diagnosis of chronic HCV (n=30) wasmade on the basis of a history ofmore than 6 months of liver disease together with a positive HCV anti-body test (second-generation enzyme immunoassay) and detectableHCV RNA by real-time PCR. The alanine transferase (ALT) levels ofthese patients were also raised on at least two determinationsperformed within 6 months and liver biopsy findings were consistentwith chronic HCVwithin 18 months before therapy. There was no sero-logic evidence of co-infection with other hepatotropic viruses. Controlsubjects (n=30) recruited for the study were without any clinical his-tory of hepatitis and considered healthy after routine laboratory analy-sis. Other possible associations of cellular injury, such as co-infection orconsumption of tobacco, alcohol, or drugs, were cause for exclusion. Allinvestigations were conducted as per institutional review boardguidelines.

Lymphocyte isolation and culture

Lymphocytes were isolated by density gradient centrifugation usingLymphosep (MP Biomedicals, Solon, OH, USA) and viability was

examined using the trypan blue dye exclusion test. Cells (1×106)were cultured andmitogenically stimulated as reported elsewhere [15].

Detection of HCV RNA

HCV RNA was isolated using the QIAamp viral RNA mini extractionkit (Qiagen, Hilden, Germany) following the protocol described previ-ously by Bhargava et al. [16]. Briefly, viral particles were lysed underhighly denaturing conditions followed by binding of viral RNA to a silicagel-based membrane and elution using RNase-free buffer. Detection ofHCV RNA was done using LightCycler 2.0 real-time PCR (Roche AppliedSciences, Mannheim, Germany) [13].

Evaluation of oxidative stress

Intracellular ROS generation was measured from the percent-age of 2′,7′-dichlorofluorescin (DCF) fluorescence using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetylester (CM-H2DCFDA) labeling (Molecular Probes, Invitrogen, Carlsbad,CA, USA) as described elsewhere [17]. Quantitative ELISA for theoxidative stress biomarker 8-oxo-2′-deoxyguanosine (8-oxo-dG) andantioxidant enzymes glutathione reductase (GR) and superoxide dismu-tase (SOD) (Trevigen, Gaithersburg, MD, USA) was performed as per themanufacturer's instructions and optical density was measured at 450and 340 nm [18].

Oxidative DNA damage quantification

Briefly, genomic DNA was isolated from the Wizard DNA purifica-tion kit (Promega) and dissolved in TE buffer at 100 μg/ml concentra-tion. Formation of aldehyde-reactive probe (ARP) sites in each samplewas quantified in accordance with the supplier's manual and absor-bance was measured at 650 nm on an ELISA reader [19].

Mitochondrial enzyme activity

Measurement of enzyme activity, expressed as nmol/min/mg lym-phocyte protein, of mitochondrial respiratory chain individual com-plexes was performed spectrophotometrically at 37 °C in a cuvettecontaining 1 ml ofmedium.We evaluated complex II (succinate ubiqui-none reductase), complex III (ubiquinol–cytochrome c reductase), andcomplex IV (cytochrome c oxidase) enzyme activities following themethod of Rustin et al. [20].

Relative quantitation of mitochondrial DNA/nuclear DNA ratio

Quantification of mtDNA in each subject was done through real-time PCR, with modifications [21]. In brief, two pairs of primers weredesigned and used in the two steps of relative quantification formtDNA content: one pair for the amplification of the mitochondrialNADH dehydrogenase ortholog 1 (MT-ND1) gene (ND1-F, 5′-CCCTAAAACCCGCCACATCT-3′; ND1-R, 5′-GAGCGATGGTGAGAGCTAAGGT-3′)in mtDNA and another for the amplification of the single-copy nucleargene human β-actin from the TIB MOLBIOL Universal Probe Library(Berlin, Germany). The data were expressed as the ratio of the meanmtDNA value of the triplicate measurements to the mean nDNAvalue (β-actin) of the triplicate measurements for a given sample(mtDNA/β-actin).

Assessment of DNA damage response

Immunofluorescence analysis was performed in all healthy controls(n=30), chronic patients (n=30), and patients with occult infections(n=10). Lymphocytes were fixed in 10% formaldehyde for 1 h, per-meabilized with 0.1% Triton X-100 for 30 min, blocked with 3% bovineserum albumin for 3 h, and incubated with primary antibodies against

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ataxia telangiectasia mutated phosphoserine-1981 (pATM), ataxiatelangiectasia and Rad3-related phosphoserine-428 (pATR), p53phosphoserine-15 (p-p53), and γH2AX (Abcam, Cambridge, UK,and Calbiochem, Nottingham, UK; dilution 1:1000). For qualitativemeasurement of pATM and γH2AX foci, cells were labeled withphycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugatedsecondary antibodies, respectively (dilution 1:200), for 1 h, and thenucleus was counterstained with 4,6-diamidinophenylindole (DAPI).Analysis was performed by a spectral bioimaging system using 4.0 soft-ware (Applied Spectral Imaging, Edingen, Neckarhausen, Germany)[22]. For quantitative measurements of pATM, pATR, and p-p53,the immunostained cells were acquired through a flow cytometer(FACSCalibur, BD IS, San Jose, CA, USA) [23].

Western blot of DNA repair proteins

Western blot analysis was performed to analyze the activation ofrepair proteins Mre11, Rad50, and Nbs1 using antibodies from SantaCruz Biotechnology (Santa Cruz, CA, USA) for all cases as describedby Raghuram et al. [19].

Analysis of apoptosis

Apoptosis was evaluated by staining cells with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide (JC-1) using aMitoscreen kit (BD Biosciences, San Diego, CA, USA). The assay wascarried out as per the supplier's instructions. The gate was applied inan FSC/SSC dot plot to restrict the analysis to lymphocytes only. Forthe gated cells, the ratio of FL1/FL2 was evaluated. In each case, a totalof 10,000 events were recorded in HI mode [24].

Relative expression analysis of bax by real-time PCR

For bax gene expression analysis, the method reported by Jain et al.[25] was followed. Briefly, total RNA was isolated using the Trizol re-agent (Invitrogen) and amplified using a one-step reverse transcriptasePCR kit through LightCycler 2.0 (Roche Applied Sciences). Primers weresynthesized from the TIB MOLBIOL Universal Probe Library. Primers forthe bax gene were forward sequence 5′-GGGTGGTTGCCCTTTTCTACT-3′and reverse sequence 5′-CCCGGAGGAAGTCCAGTGTC-3′, with corre-sponding housekeeping gene sequence (GAPDH) being forward 5′-GTATTGGGCGCCTGGTCACC-3′ and reverse 5′-CGCTCCTGGAAGATGGTGATGG-3′ [25].

Cell death detection ELISA

Measurement of circulating nucleosomes as the markers of apopto-tic cell deathwas done using a cell death detection ELISA Plus kit (RocheApplied Sciences) following all necessary instructions, and absorbancewas measured at 405 nm [26].

Table 1Clinical backgrounds of occult and chronic HCV patients compared with controls.

Control(n=30)

Occult(n=10)

Chronic(n=30)

Age 35±4 37±2 39±5Males/females 18/12 7/3 18/12ALT (U/L) 37.46±2.28 38.24±2.64 86.21±6.72*AST (U/L) 28.92±1.98 29.16±2.41 56.90±4.86*Bilurubin (g/dl) 0.76±0.18 0.81±0.14 2.98±0.26*Platelet counts (1×105/mm3) 3.40±0.71 3.01±0.62 1.32±0.19*Viral load (IU/ml) — 42.24±3.52 921.60±28.14

Data are expressed as means±SE; ALT, alanine aminotransferase; AST, aspartateaminotransferase.*p≤0.05.

Immune subset analysis

Immunophenotyping was performed using BD Tri Test TruCOUNTtubes following all the necessary instructions from the supplier(BD Biosciences). In brief, 50 μl of whole blood was added to theTruCOUNT tube, vortexed, and incubated for 15 min in the dark,followed by addition of 450 μl 1× FACS lysing solution and incubationfor 15 min. Quantitative estimation of the lymphocyte subset popula-tion was performed on a flow cytometry platform using the followingcombinations of antibodies: CD45+/CD3+/CD19+, CD45+/CD56+/CD3+, and CD3+/CD4+/CD8+[27].

BrdU lymphocyte proliferation

Cells were harvested and washed, and 5′-bromo-2′-deoxyuridine(BrdU) incorporation was studied by labeling cells with anti-BrdUFITC-conjugatedmonoclonal antibody (mAb; Santa Cruz Biotechnology).Dual labeling with anti-CD3 PE-conjugated mAb was done to restrictanalysis to T-cells only (Chemicon). Assay was performed on a flowcytometer platform using an FSC/SSC gating strategy to identify the clus-ter of T-cells and FL1/FL2 for measuring resultant fluorescence emittedfrom the FITC and PE channels [28].

Cytogenetic analysis

Briefly, the standard method of human peripheral blood lympho-cyte culturewas applied using samples of healthy controls and chronicand OHCI patients. Phytohemagglutinin-stimulated cultures were setup for 72 h. Metaphases were harvested after 3 h of colchicine (finalconcentration 10 μg/ml) exposure, hypotonic solution treatment,and fixing in methanol/acetic acid mixture followed by staining witha conventional Giemsa method. For control and occult and chronicHCV patients, a minimum of 50 metaphases each were selected ran-domly, photographed, and analyzed to determine chromosome aber-ration distribution and morphology [29,30].

Statistical analysis

The results are represented as themeans±SE. Statistical differencesbetween the studied groups were calculated using Student's t test oranalysis of variance. Analysis was performed using the Statistical Pack-age for Social Sciences software (SPSS, Inc., Chicago, IL, USA) and a pvalue of ≤0.05 established statistical significance.

Results

Demographic and clinical characteristics of patients

OHCI patientswere reported to be anti-HCVpositive, serumHCVRNAnegative, but positive for HCV RNA in peripheral lymphocytes with nor-mal ALT and aspartate aminotransferase levels. Their viral load rangedfrom 30 to 60 IU/ml. The chronic patients were positive for anti-HCVandhad serumHCVRNA levels ranging from800 to 1500 IU/ml. The clin-ical backgrounds of occult and chronic HCV patients compared withthose of the controls are shown in Table 1.

Oxidative stress

Intracellular ROS generationGeneration of intracellular ROS in terms of DCFH oxidation

(marked by DCF fluorescence) was measured as an initiator for oxida-tive stress. It was found that in comparison to controls, ROS genera-tion increased considerably during OHCI, contributing to oxidative

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damage. Mean percentage DCF fluorescence in occult and chronic HCVwas 28.85±4.86 and47.61±8.62%, respectively,whichwas significant-ly higher (p≤0.05) compared to controls (2.16±0.40%; Fig. 1A).

GR and SOD activitySimultaneously, measurement of the activity of the intracellular an-

tioxidant defense system enzymes GR and SOD showed depleted meanactivity levels in occult patients in comparison to controls. The reportedGR levels were significantly declined (p≤0.05) in occult (251.94±

Fig. 1. (A) Flow cytometric evaluation of ROS induction in peripheral blood lymphocytes labeled(upper left). Representative histograms fromhealthy controls (upper right) and occult (lower leand population of cells with elevated ROS (M2 zone). Data are presented asmeans±SE. (B) Gradepleted antioxidant defensemachinery enzymes, GR and SOD, in lymphocytes of occult and chconsidered statistically significant. (C) Levels of accumulated ARP sites in damaged nuclei of lymper 105 cells. Values indicate means±SE, and p≤0.05 was considered statistically significant.

36.93 mU/ml) and chronic HCV (189.16±28.72 mU/ml)-infected sub-jects, in comparison to controls (482.6±41.29 mU/ml). Mean SODlevels in occult patients were 232.89±32.4 mU/ml, whereas in chronicpatients and controls the levels were 124.98±28.19 and 345.87±24.61 mU/ml, respectively (Fig. 1B).

Oxidative stress8-Oxo-dG is a modified nucleoside base and is the most commonly

studied and detected by-product of DNA damage excreted upon DNA

with CM-H2DCFDA. FSC/SSC flowplot showing the gated population (R1) of lymphocytesft) and chronic HCVpatients (lower right) showing ratio between healthy cells (M1 zone)phical representation of oxidative stress (formation of 8-hydroxy-2′-deoxyguanosine) andronic patients compared with controls. Bars representmean±SE values, and p≤0.05wasphocytes of control and occult and chronic HCV patients. Data are presented as ARP sites

(D) Enzyme activity in complexes II, III, and IV.

Fig. 2. Comparative demonstration of mtDNA/nDNA ratio in lymphocytes of controlsand occult and chronic HCV patients. For each sample, mtDNA content was quantitatedas the ratio between mean mtDNA (ND1) and mean nuclear DNA value (β-actin) de-rived from triplicate measurements. Values are expressed as means±SE and p≤0.05was considered statistically significant.

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repair. In this study, the OHCI cohort presented a significantly higheraccumulation (p≤0.05) of 8-oxo-dG (98.83±16.45 ng/ml) in con-trast to controls (18.29±3.51 ng/ml), whereas in chronic cases itwas 169.71±31.28 ng/ml (Fig. 1B).

Oxidative DNA damageARP, a marker for oxidative DNA damage quantification, reacts spe-

cifically with an aldehyde group, which is the open-ring form of theapurinic/apyrimidinic sites on the damaged DNA. Lymphocytes isolated

Fig. 3. (A) Representative microphotographs (original magnification×400) showing immunfrom controls (n=30) and occult (n=10) and chronic HCV-infected subjects (n=30). Nuc(red). Scale bar, 5 μm. (B) Quantitative assessment of pATM, pATR, and p-p53 proteins byHCV patients (n=30). Columns represent means±SE showing percentage of positive (phoWestern blot analysis of DNA repair proteins Mre11 (74 kDa), Rad50 (180 kDa), and Nbs1(n=10) and chronic HCV (n=30) lymphocytes. (D) Histogram showing relative band denand chronic (n=30) subjects compared with controls (n=30). Bars display mean±SE valu

from OHCI patients reported higher accumulation (p≤0.05) of ARPsites (36.82) in the DNA per 105 cells in comparison to those of controls(3.21), whereas in chronic HCV cases it was 58.01 (Fig. 1C).

Mitochondrial enzyme activityMeasurement of individual enzymatic activities of complexes II, III,

and IV of the mitochondrial respiratory chain in lymphocytes of occultsubjects showed a significant decrease (p≤0.001) in values, 80.07±1.87, 124.19±2.22, and 107.97±1.30 nmol/min/mg protein versus87.54±1.77, 151.12±2.25, and 133.15±1.07 nmol/min/mg proteinin controls, respectively. However, complexes II (72.50±1.72 nmol/min/mg protein), III (108.24±1.73 nmol/min/mg protein), and IV(96.97±1.19 nmol/min/mg protein) recorded minimum activities inchronic HCV patients (Fig. 1D).

mtDNA/nDNA ratioA significantly lower mtDNA/nDNA ratio was observed in occult

and chronic HCV patients in comparison to the age- and gender-matched controls (p≤0.05). The mtDNA/nDNA ratio in occult andchronic patients was 0.38±0.04 and 0.31±0.08, respectively, andthe control value was 0.52±0.06 (Fig. 2).

DNA damage response

Qualitative immunofluorescence evaluation of ATM and γH2AX,primary proteins activateduponDNAdamage, displayedhigher accumu-lation of phosphorylated foci at Ser139 (γH2AX) and Ser1981 (pATM) in

ofluorescence analysis of pATM and γH2AX phosphorylation in nuclei of lymphocytesleus counterstained with DAPI (blue), γH2AX foci with FITC (green), and ATM with PEflow cytometer in lymphocytes of controls (n=30) and occult (n=10) and chronic

sphorylated) cells. p≤0.05 was considered statistically significant. (C) A representative(95 kDa), along with loading control β-actin (42 kDa) in controls (n=30) and occultsity (in percentage expression) for Mre11, Rad50, and Nbs1 proteins in occult (n=10)es, and p≤0.05 was considered statistically significant.

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OHCI patients in comparison to controls (Fig. 3A). In addition, quantita-tive analysis of these proteins also reported significantly higher levels(p≤0.05) of pATM (18.2±1.85%), pATR (21.6±1.92%), and p-p53(23.9±2.32%) in the OHCI cohort compared with age- and gender-matched controls (4.8±0.5, 5.4±0.8, and 6.4±0.87%; Fig. 3B). Theseobservations suggested that HCV, despite its occult state and scantyviral load (≤100 IU/ml), induces genotoxicity on the immune cells andelicits the PI3-kinase-mediated response pathway.

DNA repair proteins

Western blot analysis of the damage repair factors Mre11, Rad50,and Nbs1 revealed higher expression levels in OHCI patients in contrastto their controls, whereas in chronic infection expression of thesefactors was still higher, implying the extent of damage and genotoxicityinflicted by HCV on lymphocytes and thus disease severity (Fig. 3C).Software band analysis of these proteins showed a significant increase(p≤0.05) in their percentage expression, from 16.1±2.89 (Mre11),12.5±2.96 (Rad50), and 13.6±2.21% (Nbs1) in controls to 32.6±3.98, 32.7±4.92, and 29.3±4.87% in occult and 36.1±5.56, 35.2±6.82, and 36.8±4.32% in chronic subjects (Fig. 3D). Taken together,these results indicate that OHCI with low viral load (≤100 IU/ml) elicitsabrupt recruitment of repair proteins at the damage sites owing to anactive PI3-kinase-mediated response pathway.

Induction of apoptosis

JC-1 assay for mitochondrial membrane depolarizationThe membrane-permeative lipophilic cationic fluorochrome JC-1

was used as a probe for mitochondrial transmembrane potential(ΔΨ), a marker for apoptosis. JC-1 penetrates into cells, and its fluores-cence reflects ΔΨ. Compared with controls a significant increase(p≤0.05) in the cells with dissipated mitochondrial ΔΨ was recordedin OHCI. The mean percentage of cells with depolarized mitochondria

Fig. 4. Cytometric analysis of mitochondrial membrane potential in lymphocytes. Upper left:resentative dot plot from control illustrates ratio between healthy cells in FL2 (red—R2 zone)Lower right: chronic HCV. R3 zone represent population of cells with dissipated mitochond

in occult HCV patients was 46.92%, whereas in controls and chronicHCV, the levels were 0.88 and 68.33%, respectively (Fig. 4).

Relative expression analysis of baxQuantitative real-time PCR analysis of themitochondrial proapopto-

tic bax gene showed augmentation in expression levels (p≤0.05) foroccult and chronic subjects by 9.26±2.41- and 13.42±3.41-fold, re-spectively, compared to controls (1.8±0.09). These results implicatethe crucial role of bax in activation of subsequent apoptosis.

Raised circulating nucleosome levelIn comparison to controls, the observed increase (p≤0.05) in the

levels of circulating nucleosomes (markers of apoptotic cell death)in OHCI patients further strengthens our above observations thatOHCI exacerbates immune cells to cellular demise. The mean circulat-ing nucleosome levels in controls was 0.041 AU (arbitrary units), andin OHCI and chronic HCV patients the levels were 0.518 and 0.892 AU,respectively.

Immunophenotyping

Absolute numbers of T- and B-lymphocytes and NK cells recordeddifferences in control, chronic, and OHCI groups. However, valueswere statistically insignificant. Lower counts of the immune subsetsobserved in occult and chronic groups might attribute to the higherlymphocyte apoptotic index in contrast to controls (Fig. 5).

T-cell proliferative assay

The impact of HCV infection on T-cell proliferation was measuredin occult and chronic subjects. Interestingly, the OHCI cohort pre-sented a decline in BrdU-incorporated T-cell proliferating index(38.21±6.27%), and chronic infected patients showed a further de-creased index of proliferating T-cell population (35.73±4.12%);

FSC/SSC dot plot showing the gated population (R1) of lymphocytes. Upper right: rep-and cells with depolarized mitochondria FL1 (green—R3 zone). Lower left: occult HCV.rial membrane potential. Data are presented as means±SE.

Fig. 5. Cytometric analysis of lymphocyte subset enumeration for CD45+, CD19+, CD3+, CD4+, CD8+, and CD15+56+ cells in controls and occult and chronic HCV subjects. Barsrepresent means±SE of absolute counts, and p≤0.05 was considered statistically significant.

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however, the observed values were not statistically significant withrespect to controls (42.14±4.24%).

Cytogenetic analysis

Cytogenetic analysis showed chromatid-type aberrations in three ofthe OHCI patientswith open breaks on both chromatids of chromosome5 and deletion of a chromatid fragment on chromosome 11, whereasthe chronic HCV samples showed chromatid breaks, intrachromatidfusion, and endoreduplication as chromosomal aberrations, and controlsubjects presented a normal karyotype (Fig. 6). These results implicatethe clastogenic activity of HCV even under an occult scenario.

Discussion

Occult HCV infection is a newly reported pathological entity charac-terized by two different clinical situations: first, in anti-HCV-negativeand serum HCV RNA-negative patients with abnormal liver functiontests, and second, in anti-HCV-positive patients who have no detectableserum HCV RNA with normal liver enzymes. Because lymphoid tissuesare known to serve as reservoirs for HCV, this study aimed to evaluatethe involved pathophysiological mechanisms of OHCI in lymphocytes.We observed that OHCI induces mitochondrial oxidative stress, whichfurther leads to DNA damage and elicits a PI3-kinase-mediated re-sponse along with increased mitochondrial membrane depolarizationand apoptosis.

Alterations in the mitochondrial machinery are increasingly beingrecognized as key components in both acute and chronic allostaticstates, although the extent of their role in pathogenesis of such condi-tions is unknown. Chronic HCV infection is known to induce oxidativestress by affecting mitochondrial respiratory chain activity [8]. In vitrostudies have shown that viral proteins E1, core, and NS3 either directlyactivate ROSproduction or induce nitric oxide production,which causesinner mitochondrial membrane depolarization, resulting in massiveROS generation in the infected cells [5,31]. Evidence suggests that mod-ulation in the mitochondrial membrane potential is associated withthe activation of the Ca2+ uniporter, which leads to immoderate ROSgeneration and subsequent harmful effects to biomolecules, includingmtDNA located in the close proximity [32]. Although ROS are centrallyimplicated in chronic HCV-associated immune pathogenesis, precise un-derstanding of such phenomenon during OHCI remains inconspicuous.

Upon oxidative stress, abundance in the number ofmitochondria andmtDNA molecules is noticed, provided cells have efficient antioxidant

capacity and goodparentalmtDNA. Once the antioxidant defense systemis compromised, higher oxidative stress results in an increase in mito-chondria with defective machinery, thereby initiating a self-cascade ofROS generation and oxidative damage [33,34]. Our study showed thatOHCI leads to a significantly higher generation of ROS in peripheral lym-phocytes and depletion in the activity of the antioxidant defense en-zymes GR and SOD in comparison to controls (Figs. 1A and B). Theassociation of OHCI with oxidative stress was further revalidated byour study in OHCI patients that recorded incremented 8-oxo-dG forma-tion, an oxidative stress biomarker supposed to be produced through in-teraction of ROS with genomic DNA (Figs. 1B and C). The significantdecline observed in the mitochondrial respiratory chain enzymesamong OHCI subjects further supported the involvement of a perturbedmitochondrial machinery in the generation of oxidative stress (Fig. 1D).Once beyond threshold, oxidative stress significantly disturbs themtDNA integrity, which in turn could activate the nuclear DNA damageresponse, assuming that ROS and free radicals generated from the elec-tron transport chain are involved in signaling from the mitochondria tothe nucleus [35]. Our study reported a significantly low mtDNA/nDNAratio in lymphocytes of OHCI patients, which might be due to the closeproximity of mtDNA to ROS and lack of protective histones, chromatinstructure, introns, and proof-reading apparatus (Fig. 2).

Previous reports from chronic patients, along with in vitro observa-tions, have shown that HCV causes DNA double-strand breaks (DSBs)and enhances the mutation frequency of host cellular genes [36–38].Members of the PI3-kinase family, ATM and ATR, are believed to playa central role in the cellular response to genotoxic stress and are crucialfor maintaining the genome integrity [39]. Followed by their activationin response to damage, these kinases phosphorylate a number of down-stream proteins, including γH2AX and p53, and initiate cell signalingevents to induce cell cycle arrest or apoptosis [40]. The MRN complex,whose core contains Mre-11, Rad50, and Nbs1 proteins, is known tobe actively involved in the initial processing of DNA strand breaks up-stream and downstream of ATM in the DNA damage-response pathway[41]. Recently, it has been hypothesized that the HCV core protein bindsto the Nbs1 protein, thereby affecting the formation of the MRNcomplex and ATM activation and inhibition of repair enzymes tobind to DNA breaks [7]. Accumulation of DSBs also suggests thatan HCV-induced oxidative environment may overwhelm DNA re-pair mechanisms, leading to chromosomal abnormalities [42–45].Cytogenetic analysis in peripheral lymphocytes of occult subjectsdisplayed aberrations on both chromatids of chromosome 5 and de-letion of a chromatid fragment on chromosome 11, implicating the

Fig. 6. Cytogenetic analysis of lymphocytes of occult and chronic HCV. (a) Representative karyogram of control subjects showing normal karyotype. (b) Endoreduplication observedin representative karyotype of chronic HCV patients. (c, d) Chromatid break in chromosome 5 and deletion of chromosomal fragment of chromosome 11 in occult patients. (e, f)Intrachromosomal fusion in chromosome 10 and chromatid break in chromosome 12 in chronic patients.

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clastogenic activity of OHCI (Fig. 6). Notably, we also observed thatHCV, despite its occult state and low viral load, induces DNA damageand elicits a PI3-kinase-mediated response (Figs. 3A–D), ensuringthe mitochondria-mediated demise of lymphocytes. Such an effectmight also lead to an inevitable immunocompromised state in OHCI.

Depending on the severity of the generated ROS and the resultantmtDNA damage, mitochondria-mediated apoptosis is initiated by avariety of proapoptotic signals that cause an imbalance in the majorapoptosis regulators [46]. Bax, a proapoptotic member of the Bcl-2family, accumulates and triggers an increased permeability of theouter mitochondrial membrane. A recent in vitro study has reportedthat HCV induces a Bax-triggered apoptotic mechanism [6]. Congruent-ly, in this study, an apparent up-regulation of bax gene expressionlevels, along with significant dissipation of ΔΨ, suggested that OHCI in-duces the intrinsic mitochondria-mediated apoptotic pathway (Fig. 4).Additionally, we observed a decline in the proliferating T-cell index ofOHCI patients compared to controls. These observations imply that

the mitochondria-mediated oxidative damage in turn might contributeto OHCI-associated deregulated lymphocyte proliferative activity.

Collectively, our data indicate that OHCI, despite its low viral load, in-duces mitochondrial oxidative stress, eliciting a PI3-kinase-mediatednuclear DNA damage response and premature lymphocyte apoptosis.Our results provide the mechanistic underpinnings of mitochondrialdysfunction in OHCI, a previously unknown paradigm, for explainingthe immune pathogenesis in a redox-dependent manner. However, fur-ther patient follow-up would discern the clinical significance of thesemolecular repercussions in disease progression or recurrence.

Acknowledgments

This work was partially supported by the Intramural Research Pro-gramme of the Bhopal Memorial Hospital Research Centre, Bhopal,India, and the Department of Biotechnology, Government of India, NewDelhi.

1814 A. Bhargava et al. / Free Radical Biology & Medicine 51 (2011) 1806–1814

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