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Mutation Research 665 (2009) 51–60

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

journa l homepage: www.e lsev ier .com/ locate /molmutCommuni ty address : www.e lsev ier .com/ locate /mutres

ifferences in DNA damage and repair produced by systemic, hepatocarcinogenicnd sarcomagenic dibenzocarbazole derivatives in a model of rat liver progenitorells

uzana Valovicováa, Sona Marvanováb, Monika Mészárosováa, Annamária Srancíkováa,enka Trilecováb, Alena Milcovác, Helena Líbalovác, Jan Vondrácekb,d, Miroslav Machalab,an Topinkac, Alena Gábelováa,∗

Laboratory of Mutagenesis and Carcinogenesis, Cancer Reserach Institute, SAS, Vlárska 7, 833 91 Bratislava, SlovakiaDepartment of Chemistry and Toxicology, Veterinary Research Institute, 621 00 Brno, Czech RepublicLaboratory of Genetic Ecotoxicology, Institute of Experimental Medicine, AS CR, v.v.i. 142 20 Prague, Czech RepublicLaboratory of Cytokinetics, Institute of Biophysics, AS CR, 612 65 Brno, Czech Republic

r t i c l e i n f o

rticle history:eceived 29 December 2008eceived in revised form 17 February 2009ccepted 28 February 2009vailable online 13 March 2009

eywords:NA adductsNA strand breaksxidative stressinetics of DNA repair

a b s t r a c t

Liver progenitor (oval) cells are a potential target cell population for hepatocarcinogens.Our recent study showed that the liver carcinogens 7H-dibenzo[c,g]carbazole (DBC) and 5,9-dimethyldibenzo[c,g]carbazole (DiMeDBC), but not the sarcomagen N-methyldibenzo[c,g]carbazole(N-MeDBC), induced several cellular events associated with tumor promotion in WB-F344 cells, anin vitro model of liver oval cells [J. Vondracek, L. Svihalkova-Sindlerova, K. Pencikova, P. Krcmar, Z.Andrysik, K. Chramostova, S. Marvanova, Z. Valovicova, A. Kozubik, A. Gabelova, M. Machala, 7H-Dibenzo[c,g]carbazole and 5,9-dimethyldibenzo[c,g]carbazole exert multiple toxic events contributingto tumor promotion in rat liver epithelial ‘stem-like’ cells, Mutat. Res. Fundam. Mol. Mech. Mutagen.596 (2006) 43–56]. In this study, we focused on the genotoxic effects generated by these dibenzo-carbazoles in WB-F344 cells to better understand the cellular and molecular mechanisms involvedin hepatocarcinogenesis. Lower IC50 values determined for DBC and DiMeDBC, as compared withN-MeDBC, indicated a higher sensitivity of WB-F344 cells towards hepatocarcinogens. Accordingly,DBC produced a dose-dependent DNA-adduct formation resulting in substantial inhibition of DNAreplication and transcription. In contrast, DNA-adduct number detected in DiMeDBC-exposed cellswas almost negligible, whereas N-MeDBC produced a low level of DNA adducts. Although all diben-zocarbazoles significantly increased the level of strand breaks (p < 0.05) and micronuclei (p < 0.001)after 2-h treatment, differences in the kinetics of strand break rejoining were found. The strand breaklevel in DiMeDBC- and N-MeDBC-exposed cells returned to near the background level within 24 h aftertreatment, whereas a relatively high DNA damage level was detected in DBC-treated cells up to 48 h

after exposure. Additional breaks detected after incubation of DiMeDBC-exposed WB-F344 cells with arepair-specific endonuclease, along with a nearly 3-fold higher level of reactive oxygen species found inthese cells as compared with control, suggest a possible role of oxidative stress in DiMeDBC genotoxicity.We demonstrated qualitative differences in the DNA damage profiles produced by hepatocarcinogensDBC and DiMeDBC in WB-F344 cells. Different lesions may trigger distinct cellular pathways involved inhepatocarcinogenesis. The lowthe lack of hepatocarcinogenic

Abbreviations: AFB1, aflatoxin B1; B[a]P, benzo[a]pyrene; DBC, 7H-ibenzo[c,g]carbazole; DiMeDBC, 5,9-dimethyldibenzo[c,g]carbazole; Fpg,ormamidopyrimidine-DNA glycosylase/AP endonuclease; �H2AX, histone H2AXhosphorylated on Ser139; MI, mitotic index; MNi, micronuclei; MTT, methylhiazolyl blue tetrazolium bromide; N-MeDBC, N-methyldibenzo[c,g]carbazole;OS, reactive oxygen species; SCGE, single-cell gel electrophoresis.∗ Corresponding author.

E-mail address: alena.gabelova@savba.sk (A. Gábelová).

027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2009.02.014

amount of DNA damage, together with an efficient repair, may explainity of N-MeDBC.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

7H-Dibenzo[c,g]carbazole (DBC) (Fig. 1), a potential humancarcinogen (Group 2B) [1,2], is a ubiquitous environmental pol-

lutant. This agent has been found in various complex mixturesof organic compounds resulting from incomplete combustion oforganic materials such as soot and tars [3], diesel engine exhaust [4],synthetic fuel material [5], coal and oil processing [6], and tobaccosmoke [7]. DBC has been shown to be a potent multi-species and

52 Z. Valovicová et al. / Mutation Re

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Fig. 1. Chemical structure of 7H-dibenzo[c,g]carbazole.

ulti-site carcinogen, with local and systemic effects (reviewedn [8]). DBC carcinogenicity was demonstrated in different speciesmouse, rat, hamster, and dog) and tissues (skin, lung, forestomach,nd urinary bladder), with the liver being the prime target organfter DBC administration via different routes. In addition to hepa-ocellular adenoma and carcinoma, severe hepatocellular toxicity,ncluding biliary hyperplasia, has been observed in livers taken fromBC-treated mice [9–11].

The synthetic methyl derivative of DBC, 5,9-imethyldibenzo[c,g]carbazole (DiMeDBC), is one of the mostfficient strictly organ-specific carcinogens. This derivative isevoid of activity in the skin, but produces multiple malignant

iver tumors with lung metastases in 100% of animals after sub-utaneous injection or skin application [10,12,13]. In contrast toBC, sex dimorphism in response to DiMeDBC treatment wasbserved in mice; males were much less sensitive than femaleso this derivative [14]. The strict hepatocarcinogen DiMeDBC isemarkably less hepatotoxic than DBC; no histologically detectableoxicity has been found in the liver at lower (10 mg/kg) concentra-ion [14]. At high concentration (90 mg/kg), an early hepatocellularegeneration/necrosis was observed, mostly in centrilobular areas15].

In the liver, DBC and DiMeDBC produce tumors, DNA adductsnd gene mutations [13,16]. At equimolar concentrations, DiMeDBCnduces fewer mutations and DNA adducts than the parent com-ound [17], which is probably due to a reduced access to theH group for drug metabolizing enzymes, caused by a steric hin-rance of two methyl groups at the C5 and C9 positions [16].he heterocyclic nitrogen is supposed to have an important rolen liver carcinogenicity and strongly affects the biological activityf DBC. Substitution of methyl groups at the C6 and C8 posi-ions or directly at the heterocyclic nitrogen resulted in the lossf activity in the liver [16,18]. The synthetic methyl derivative N-ethyldibenzo[c,g]carbazole (N-MeDBC) induces sarcomas, respi-

atory tumors and papillomas, but lacks hepatocarcinogenic poten-ial [19,20]. Analogs of DBC bearing sulphur, oxygen or carbon inlace of nitrogen also do not demonstrate carcinogenic activity [21].

Cell lines established from a particular tissue are a valuable toolor better understanding of cellular and molecular mechanismshich may underlie the tissue specificity of chemical carcinogens.

he diploid rat epithelial cells WB-F344 are considered to be an initro model of liver oval cells [22]. The latter can proliferate whenegenerative hepatocyte proliferation is compromised, and can dif-erentiate into hepatocytes [23] and biliary epithelial cells [24].here is increased evidence that the small epithelial oval-shapedhepatic progenitor) cells are, in addition to hepatocytes, a possiblearget cell population for hepatocarcinogenesis [25,26]. These cellsave been observed in various models of rodent experimental car-inogenesis and human liver diseases associated with an increased

ncidence of hepatocellular carcinoma or cholangiocarcinoma [27].xperiments with polycyclic aromatic and heterocyclic aromaticydrocarbons have shown that the WB-F344 cell line is a usefulool for analysis of cellular and molecular mechanisms involved inoxic effects of environmental carcinogens [28–31].

search 665 (2009) 51–60

In our recent study, the liver carcinogens DBC and DiMeDBCsignificantly affected cellular pathways associated with tumor pro-motion in WB-F344 cells [32]. DBC was an efficient inhibitor ofgap-junctional intercellular communication (GJIC), and DiMeDBCmanifested the strongest aryl hydrocarbon receptor (AhR)-mediated activity. The tissue-specific sarcomagen N-MeDBC failedto substantially affect the cellular events associated with tumorpromotion. DBC and DiMeDBC, in contrast to N-MeDBC, inducedp53 phosphorylation, S-phase delay of cell cycle and apoptosis [32].The differences in cell response to DBC and DiMeDBC treatmentsuggested that these liver carcinogens may produce qualitatively(or at least quantitatively) different DNA lesions in WB-F344 cells.Because the genotoxic potential of DBC, DiMeDBC and N-MeDBChas not been studied in liver progenitor cells or in their mod-els such as WB-F344 cells, the primary objective of this workwas to assess the DNA damage profile generated by individualdibenzocarbazoles in this liver “stem-like” cell line. In additionto clastogenic effects (DNA adducts, DNA breakage, micronuclei),cellular responses (DNA repair kinetics, induction of oxidativestress, histone H2AX phosphorylation) were investigated to obtaina more comprehensive picture of the genotoxic effects generatedby the tissue-specific dibenzocarbazole derivatives in the WB-F344cell line.

2. Materials and methods

2.1. Chemicals

DBC (CAS No. 194-59-2), DiMeDBC and MeDBC were kindly provided by Dr.Francois Périn (Institute Curie, France). Benzo[a]pyrene (B[a]P, CAS No. 50-32-8),methyl thiazolyl blue tetrazolium bromide (MTT, CAS No. 298-93-1), and 4′ ,6-diamidino-2-phenylindole (DAPI, CAS No. 28718-90-30), anti-�-actin, Clone AC-15,anti-Mouse IgG, spleen phosphodiesterase, RNases A and T1, proteinase K, micro-coccal nuclease and nuclease P1 were purchased from Sigma–Aldrich (Deisenhofen,Germany). T4 polynucleotide kinase was from USB (Cleveland, OH, USA); �-32P-ATP (3000 Ci/mmol, 10 �Ci/�L) from GE Healthcare (Little Chalfont, UK); and0.1 mm polyethylene-imine cellulose thin-layer chromatography (TLC) plates fromMacherey-Nagel (Düren, Germany). P-H2AX (Ser 139) was from Upstate (Lake Placid,NY, USA), peroxidase-conjugated swine antirabbit immunoglobulin antisera wasfrom Sevapharma (Prague, Czech Republic), ECL Plus reagent was from GE Health-care (Little Chalfont, UK), and aflatoxin B1 (AFB1, CAS No. 1162-65-8) from Serva(BioTech, Slovakia). Stock solutions of DBC, MeDBC, DiMeDBC, B[a]P and AFB1 indimethyl sulfoxide (DMSO; 2 mM) were kept at −20 ◦C and diluted immediatelybefore use in DMSO. Media, fetal calf serum (FCS) and other chemicals used for cellcultivation were purchased from Gibco (KRD Limited, Slovakia). All other chemicalsand solvents were of high-performance liquid chromatography (HPLC) or analyticalgrade.

2.2. Cell culture

WB-F344 cells were kindly provided by Dr. J. E. Trosko (MSU, East Lansing, MI,USA). They were cultivated in modified Eagle’s minimum essential medium (MEM)with 50% increased concentrations of essential and non-essential amino acids, andsupplemented with sodium pyruvate (110 mg/L), 10 mM HEPES, 10% FCS and antibi-otics (penicillin 200 U/mL, streptomycin and kanamycin 100 �g/mL). Cells werecultured in a humidified atmosphere of 5% CO2 at 37 ◦C.

2.3. Treatment of cells

Exponentially growing WB-F344 cells were exposed to carcinogens for 2 h and24 h depending on the endpoint. Stock solutions (2 mM) of DBC, N-MeDBC, DiMeDBC,AFB1 and B[a]P in DMSO were further diluted to reach the final concentrations (DBC,MeDBC, DiMeDBC, B[a]P: 0.1–100 �M, and AFB1 0.001–1 �M). The final concentra-tion of vehicle (DMSO) never exceeded 0.5% (v/v) in any of the samples; controlcells (negative control) were therefore exposed to 0.5% DMSO. At treatment end,cells were washed twice with culture medium and then processed immediately, orincubated in fresh medium for different time intervals, and then processed.

2.4. MTT assay

Viability determination was carried out in plastic 96-well cell culture clusterplates at 5 × 103 cells/well and photometric evaluation (at 540 nm excitation and690 nm emission wavelengths) using the Multiskan Multisoft plate reader (Labsys-tems, Finland) and Genesis software provided by the producer. IC50 values were

tion Research 665 (2009) 51–60 53

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Table 1The IC50 values of DBC, DiMeDBC, N-MeDBC, B[a]P and AFB1 after 2 h and 24 h celltreatment. IC50 values were calculated using the CalcuSyn software.

Compounds

Cell exposure (h) 2 h [�M] 24 h [�M]

DBC 48 11DiMeDBC 30 9

and long-term (24 h) cell exposure. Equimolar concentrations rang-ing from 5 �M to 100 �M were used for dibenzocarbazoles andB[a]P, whereas 0.05–10 �M were chosen for AFB1. The param-eter IC50 was applied to compare the cytotoxicity of chemicals

Table 2The relative DNA adduct levels detected in WB-F344 cells exposed to DBC, N-MeDBC,DiMeDBC and B[a]P for 24 h.

Compounds Conc. [�M] DNA adducts/108 nucleotides

DBC 1 29.6 ± 10.610 56.3 ± 10.4

DiMeDBC 1 0.2 ± 0.410 0.5 ± 0.3

Z. Valovicová et al. / Muta

alculated from the dose–response curves using CalcuSyn software (Biosoft, Cam-ridge, UK).

.5. DNA isolation and 32P-postlabeling

DNA was isolated using RNases A and T1 and proteinase K treatment fol-owed by phenol/chloroform/isoamylalcohol extraction and ethanol precipitation33]. DNA concentrations were estimated spectrophotometrically by measuring UVbsorbance at 260 nm. DNA samples were stored at −80 ◦C until analysis.

32P-Postlabeling analyses were done as previously described [34]. Briefly, DNAamples (exact amount of DNA was 6 �g) were digested by a mixture of micrococcaluclease and spleen phosphodiesterase for 4 h at 37 ◦C. The nuclease P1 proce-ure was used for adduct enrichment. Adducted nucleotides were enzymatically

abelled using �-32P-ATP and T4 polynucleotide kinase, and separated by multidi-ectional polyethylenimine–cellulose thin-layer chromatography (TLC). The solventssed were: D1, 1 M sodium phosphate, pH 6.8; D2, 3.54 M lithium formate, 8.5 Mrea, pH 3.5; D3, 0.8 M lithium chloride, 0.5 M Tris, 8.5 M urea, pH 8.0; D4 = D1,ame direction as D3. After screen-enhanced autoradiography at −80 ◦C, the distinctNA adduct spots were cut out and evaluated by measuring 32P-radioactivity using

iquid scintillation spectroscopy. To determine the exact amount of DNA in each sam-le, aliquots of the enzymatic DNA digest were analyzed for nucleotide content byeverse-phase HPLC with UV detection, which simultaneously allowed for control-ing DNA purity. DNA adduct levels were expressed as adducts per 108 nucleotides.

BPDE-derived DNA adduct standard derived from the liver of rats treated withenzo[a]pyrene (100 mg, i.p.) was run in triplicate to control inter-assay variabilitynd to normalize calculated DNA adduct levels. Data are mean values of total DNAdducts derived from at least three biological replicates.

.6. Cumulative synthesis of macromolecules

Exponentially growing WB-F344 cells were exposed to dibenzocarbazoles1–10 �M) or positive controls for 2 h. After treatment, fresh medium con-aining [14C]thymidine (1 �Ci/mL) or [14C]uridine (1 �Ci/mL) or [14C]–L-leucine0.2 �M/mL) was added to all dishes. At different time intervals (30 min, 60 min,0 min, 120 min, 180 min, and 300 min), cells were rinsed with SSC buffer (0.15 Modium chloride, 0.015 M sodium citrate) and ice-cold 5% trichloroacetic acid (TCA)as added. The next day, cells were harvested and filtered through a membrane

0.45 �m pore), washed and dried. Radioactivity was measured on a liquid scintil-ation counter Beckman LS 1801.

.7. Single-cell gel electrophoresis (SCGE)

The procedure of Singh et al. [35], modified by Collins et al. [36] and Gabelovat al. [37] was followed. In brief, liver cells embedded in 0.75% LMP agarose andpread on a base layer of 1% NMP agarose in PBS buffer (Ca2+ and Mg2+ free)ere placed in a lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl,H 10 and 1% Triton X-100) at 4 ◦C for 1 h. In experiments focused on detectionf oxidative DNA damage, slides were washed three times for 5 min in endonucleaseuffer (40 mM HEPES–KOH, 0.1 M KCl, 0.5 mM EDTA, pH 8.0) and incubated withormamidopyrimidine–DNA glycosylase/AP nuclease (Fpg; 30 min) at 37 ◦C. Slidesere transferred to an electrophoretic box and immersed in an alkaline solution

300 mM NaOH, 1 mM Na2EDTA, pH > 13). After 40 min unwinding time, a voltage of5 V (300 mA) was applied for 30 min at 4 ◦C. Slides were neutralized with 3× 5 minashes with Tris–HCl (0.4 M, pH 7.4), and stained with 20 �L of ethidium bromide

EtBr, 10 �g/mL). EtBr-stained nucleoides were examined with an Olympus BX51uorescence microscope by image analysis using Komet 5.0 (Kinetic Imaging, Ltd.,iverpool, UK) software. The percentage of DNA in the tail (% tail DNA) was used asparameter for measurement of DNA damage (DNA strand breaks). One hundred

omets were scored per each sample in one electrophoretic run.

.8. Detection of reactive oxygen species (ROS)

Confluent WB-F344 cells were exposed to test compounds for 24 h. Hydro-en peroxide (exposure, 5 min) was the positive control. After exposure, cellsere washed twice with PBS, trypsinized, centrifuged, and resuspended withank’s balanced salt solution with 5% heat-inactivated fetal bovine serum

FBS). The cell suspension was incubated for 15 min with the fluorescent probe′ ,7′-dichlorofluorescein diacetate (DCFH-DA, 20 �M). Cells were washed again,entrifuged, and cooled on ice (except hydrogen peroxide-exposed cells). The flu-rescence of dichlorofluorescein (DCF) was analyzed on FACSCalibur (at 505 nmxcitation and 535 nm emission wavelengths) and analyzed with CellQuest softwareBecton Dickinson, San Jose, CA, USA).

.9. Micronucleus assay

Two sampling time intervals, 24 h and 48 h after treatment, were used to deter-ine the number of micronuclei (MNi) induced by individual dibenzocarbazole

erivatives after cell exposure for 2 h. WB-F344 cells were washed with 0.9% NaCl,ncubated in mild hypotonic solution (0.075 M KCl/0.9% NaCl, 1:19) for 10 min at7 ◦C, fixed with methanol–glacial acetic acid (3:1) for 15 min at 37 ◦C, rinsed with

N-MeDBC >100 >50B[a]P >100 15.8AFB1 2.2 0.2

distilled water and air dried. Fixed cells were stained with DAPI (2 �g/mL) for 30 minin the dark at room temperature, rinsed with McIlvaine’s buffer and distilled water,and dried and mounted with glycerol. MNi were identified based on the criteriaspecified by Miller et al. [38]. Proliferation status (mitotic index, MI) of WB-F344cells was measured according to Eckl and Raffelsberger [39]; cell death (apoptosisand necrosis) was determined using morphological criteria (fragmentation of nuclei)after Oberhammer et al. [40]. Two thousand cells per dish were analyzed using thefluorescence microscope Olympus BX51. Data are mean ± S.D. of at least two paralleldishes per one experiment from three independent experiments.

2.10. Western blotting

Confluent WB-F344 cells were exposed for 24 h to test compounds or to 0.1%DMSO as vehicle control. The effects of test compounds on histone H2AX phos-phorylation were determined in whole-cell lysates prepared by harvesting cells inlysis buffer (1% sodium dodecyl sulphate (SDS), TRIS, 10% glycerol, protease inhibitorcocktail). Total protein concentrations were determined with DC Protein Assay (Bio-Rad, Hercules, CA, USA). For Western blot analyses, equal amounts of total proteinwere subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS PAGE), electrotransferred onto polyvinylidene fluoride membrane Hybond-P(GE Healthcare), immunodetected using appropriate primary and secondary anti-bodies, and visualized by ECL Plus reagent according to manufacturer’s instructions.Determination of actin level was used to confirm equal protein loading. Densitomet-ric analyses using ImageJ 1.38e software (http://rsb.info.nih.gov/ij/) was employed tomeasure the intensity of bands corresponding to the phosphorylated H2AX (�H2AX)form. The fold of �H2AX expression was estimated semi-quantitatively as the ratioof induced to control level of band density.

2.11. Statistics

Data are given as means values ±S.D. The differences between treated sam-ples and untreated control were evaluated by the Student’s t-test. The threshold ofstatistical significance was set at p < 0.05.

3. Results

3.1. Cytotoxicity of DBC and its derivatives in the rat liverprogenitor WB-F344 cells

Cell viability after exposure to dibenzocarbazoles and the pos-itive controls B[a]P and AFB1 was measured after short-term (2 h)

N-MeDBC 1 0.8 ± 0.410 3.0 ± 1.8

B[a]P 1 27.6 ± 1.5

The mean ± S.D. from at least two independent experiments on each triplicate ofsheets.

54 Z. Valovicová et al. / Mutation Research 665 (2009) 51–60

F ithelia1 N-Mec and N

ua(t

ig. 2. Representative autoradiograms of 32P-labeled DNA isolated from rat liver ep0 �M DBC (C and D), 1 �M and 10 �M DiMeDBC (E and F), and 1 �M and 10 �Mompounds. Film exposure was as followed: control, B[a]P and DBC 24 h, DiMeDBC

nder study. Substantial differences in IC50 values between hep-tocarcinogenic and sarcomagenic carcinogens have been assessedTable 1). Considerable lower IC50 values were determined forhe liver carcinogens DBC and DiMeDBC, as well as the posi-

l WB-F344 cells incubated with vehicle (0.5% DMSO) (A), 1 �M B[a]P (B), 1 �M andDBC (G and H). Chromatographic conditions (see Section 2) were the same for all-MeDBC 72 h.

tive control AFB1 (IC50: 48 �M, 30 �M, and 2.2 �M, respectively)compared with B[a]P and tissue-specific sarcomagen N-MeDBC(IC50 > 100 �M for both agents) after 2 h exposure. Although long-term treatment (24 h) increased the cytotoxicity of all carcinogens

Z. Valovicová et al. / Mutation Research 665 (2009) 51–60 55

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detected in DBC-treated cells; a substantial number of strand breakswas observed even 48 h after treatment. The level of breaks pro-duced by N-MeDBC reached the background level within 24 h afterexposure.

ig. 3. The course of synthesis of DNA (A), RNA (B) and proteins (C) in WB-F344 celf synthesis of macromolecules was analyzed at several time intervals after treatme

nder study, N-MeDBC was less cytotoxic (IC50 > 50 �M) even after4 h treatment, as compared with DBC and DiMeDBC (IC50: 11 �Mnd 9 �M, respectively). These preliminary experiments suggestedhigher sensitivity of the liver progenitor cells towards both hepa-

ocarcinogens, DBC and DiMeDBC.

.2. Formation of DNA adducts by DBC and its derivatives

DNA adducts resulting from covalent binding of chemicals toNA are a critical event in the initiation of cancer [41]. Therefore

he ability of DBC, DiMeDBC and N-MeDBC to produce DNA adductsn the rat liver oval WB-F344 cells was evaluated at 1 �M and 10 �M.ased on our previous study [42], long-term (24 h) cell treatmentas applied to assess the level of stable DNA adducts in exposed

ells. Representative autoradiograms of DNA adducts formed inB-F344 cells treated with individual compounds under study are

hown in Fig. 2. Substantial differences in DNA adduct patterns wereetermined among individual dibenzocarbazoles. The DBC “finger-rint” was difficult to analyze due to close marked spots (Fig. 2Cnd D), while only two weak (but distinct) spots were found in cellsxposed to the organ-specific hepatocarcinogen DiMeDBC (Fig. 2End F). In contrast, four clear spots were detected in N-MeDBC chro-atograms (Fig. 2G and H). B[a]P, the positive control, produced

ne dominant spot representing a well-known BPDE-DNA adductFig. 2B) and no DNA adducts were found in control cells (Fig. 2A).t equimolar (1 �M) concentrations, DBC induced a comparable

evels of DNA adducts as the reference compound B[a]P, whereashe DNA-adduct level generated by DiMeDBC at 1 �M and 10 �Mas very low, close to the detection limit (Table 2). The DNA adduct

evel produced by N-MeDBC was also very low, nearly 40-fold lowerhan that of DBC. To elucidate and verify the inability of DiMeDBCo produce DNA adducts in WB-F344 cells, a time-course studyas undertaken. DiMeDBC-treated cells were harvested at 2 h time

ntervals during exposure for 24 h, and DNA adducts measured by2P-postlabeling. No DiMeDBC-related adducts were found at earlyime points, suggesting that the lack of DNA adducts in DiMeDBC-xposed cells was not due to rapid elimination of DNA bulky adductsuring 24-h treatment (data not shown). Because the cytotoxicffects of DiMeDBC were comparable with those of DBC, furtherxperiments were undertaken to clarify the mechanism(s) whichay underlie the toxic activity of DiMeDBC in WB-F344 cells.

.3. The effect of DBC and its derivatives on the replication andranscription of DNA and proteosynthesis

Based on our previous study [42] and MTT assay results (Table 1),he short-term (2 h) treatment interval was chosen to evaluate theenotoxic effects of dibenzocarbazoles induced in WB-F344 cells.he effect of dibenzocarbazoles on replication (DNA synthesis),ranscription (RNA synthesis) and proteosynthesis was assessed

r treatment with DBC. Cells were exposed to DBC at 1–10 �M for 2 h. The intensitych point represents the mean from at least two independent experiments.

at concentrations 1–10 �M after 2 h cell exposure. DBC caused asubstantial dose-dependent delay of DNA and mainly RNA synthe-ses during 5 h post-cultivation of WB-F344 cells in fresh medium(Fig. 3A and B), while proteosynthesis was less affected (Fig. 3C).In contrast, neither DiMeDBC nor N-MeDBC, as well as the posi-tive controls B[a]P and AFB1, significantly influenced the course ofsynthesis of macromolecules under identical treatment conditions(data not shown).

3.4. DNA strand breaks and the kinetics of DNA repair

DNA strand breaks are readily detected as the cellular responseto exposure; therefore DNA strand break formation has been pro-posed to be a standard biomarker of DNA damage. The capacity ofdibenzocarbazoles to generate DNA breaks was measured withinthe concentration range 0.1–20 �M (Fig. 4). All dibenzocarbazoles,regardless of their tissue specificities, induced significant levels ofstrand breaks in exposed cells (p < 0.05 to p < 0.001). Although DBCappeared to be the most efficient producer of DNA strand breaks,the level of breaks induced by this agent was not significantly differ-ent from DiMeDBC or N-MeDBC. In contrast, substantial differencesin the kinetics of DNA strand break rejoining were determined incells exposed to dibenzocarbazoles (Fig. 5). Relatively fast removalof strand breaks was observed in DiMeDBC-treated cells; the levelof strand breaks reached the steady-state level within 16 h aftertreatment. A significant delay in DNA strand break rejoining was

Fig. 4. Detection of DNA damage in WB-F344 cells exposed to DBC, DiMeDBC andN-MeDBC for 2 h by the alkaline SCGE. Cells were treated with equimolar concentra-tions 0.1–20 �M and DNA breakage was analyzed immediately after treatment. Thebars represent the mean ± S.D. from three independent experiments on each tripli-cate of slides. The data were analyzed statistically by Student’s t-test. Significantlydifferent from the control, *p < 0.05; **p < 0.01; ***p < 0.001.

56 Z. Valovicová et al. / Mutation Research 665 (2009) 51–60

Table 3The frequency of micronuclei induced by DBC, N-MeDBC, DiMeDBC, AFB1 and B[a]P in WB-F344 cells after 2 h treatment and the mitotic index of exposed cells determined24 h and 48 h after cell exposure.

Compound Conc. [�M] Micronuclei (%) Mitotic index (%)

24 h 48 h 24 h 48 h

Control 0 2.7 ± 0.3 3.8 ± 0.7 4.1 ± 1.4 4.4 ± 1.0

DBC 0.5 6.3 ± 1.8*** 8.2 ± 1.1*** 4.1 ± 1.4 6.1 ± 2.01 8.0 ± 1.5*** 8.8 ± 0.9*** 5.8 ± 1.5 7.0 ± 2.02.5 8.4 ± 1.1*** 11.0 ± 1.1*** 5.4 ± 1.8 7.7 ± 1.3

DiMeDBC 0.5 6.6 ± 1.4*** 8.3 ± 1.4*** 6.4 ± 1.6 5.9 ± 1.11 6.2 ± 1.1*** 8.8 ± 1.0*** 5.6 ± 0.2 8.0 ± 1.02.5 6.9 ± 1.7*** 8.5 ± 1.5*** 6.2 ± 2.2 7.0 ± 2.0

N-MeDBC 0.5 6.1 ± 1.2*** 7.3 ± 2.0** 6.0 ± 2.0 5.7 ± 1.81 7.7 ± 1.1*** 9.3 ± 1.7*** 6.9 ± 1.5 7.9 ± 2.32.5 6.5 ± 1.3*** 8.2 ± 1.4*** 5.6 ± 1.4 6.6 ± 1.2

B[a]P 0.5 10 ± 0.9*** 9.3 ± 1.1*** 7.0 ± 0.8 4.2 ± 1.91 10.7 ± 1.3*** 9.2 ± 1.3*** 6.4 ± 1.4 5.1 ± 1.72.5 13.1 ± 0.9*** 11.3 ± 1.9*** 6.0 ± 2.0 5.4 ± 1.3

AFB1 0.1 4.4 ± 0.7*** 6.0 ± 0.8*** 5.1 ± 1.3 4.5 ± 1.160.5 5.0 ± 1.0*** 6.0 ± 0.6*** 4.8 ± 1.1 4.9 ± 2.21 5.3 ± 0.5*** 6.6 ± 0.5*** 5.3 ± 1.7 5.9 ± 1.6

D

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*** Significantly different from control group at p < 0.001.

.5. Clastogenic activity of DBC and its derivatives

Micronucleus formation has become an important endpoint inenotoxicity studies because a positive correlation exists betweenarcinogenicity and clastogenicity of chemical agents [43]. Thelastogenic activities of dibenzocarbazoles and the reference com-ound B[a]P were assessed in concentration range 0.5–2.5 �M,hereas concentrations 0.1–1 �M were used for AFB1. WB-F344

ells were exposed to dibenzocarbazoles and positive controls forh. The frequency of MNi and MI were assessed in WB-F344 cellst 24 h and 48 h after treatment (Table 3). In agreement withxperiments focusing on the strand break formation, all diben-ocarbazoles induced a significant level of MNi compared withontrol cells. A dose-dependent increase of MNi was detected inBC-treated cells at both sampling times (r = 0.802 and 0.880,

espectively), but the rise in MNi generated by the strict hepato-arcinogen DiMeDBC and the tissue-specific sarcomagen N-MeDBC

ere less significant (DiMeDBC: r = 0.687 and 0.624; N-MeDBC:

= 0.576 and 0.629, respectively). B[a]P, the positive control, washe most potent inducer of MNi in WB-F344 cells. Under thesereatment conditions, no substantial variation in the frequency of

ig. 5. The kinetics of DNA strand break rejoining in WB-F344 cells exposed toquimolar (1 �M) DBC, DiMeDBC and N-MeDBC for 2 h. DNA strand break levelas measured at several time intervals after treatment. The columns represent theean ± S.D. from three independent experiments on each of triplicate of slides. Dataere analyzed by Student’s t-test. Significantly different from the control, *p < 0.05;

**p < 0.001.

apoptotic and necrotic cells was determined in WB-F344 cells dueto exposure to chemicals under study (data not shown). A trendtowards increased MI that did not reach a statistical significancewas found in exposed cells (Table 3).

3.6. Oxidative DNA damage and oxidative stress

It is generally accepted that accumulation of oxidative dam-age to DNA is implicated in various human diseases, includingcancer and aging [44]. In chemical carcinogenesis, highly redox-active molecules produced during biotransformation of organiccompounds can contribute to oxidative stress generation result-ing in oxidative damage to DNA. To evaluate the mechanism bywhich the strict hepatocarcinogen DiMeDBC could mediate geno-toxic effects (strand breaks, MNi) in WB-F344 cells, the modifiedSCGE assay was utilized [36]. This modification involves incuba-tion of DNA with specific DNA repair endonuclease, the Fpg protein,which cleaves DNA in the site of base modifications, i.e., fragmented(FAPY-adducts) and oxidized (8-oxodG) purines. A slight but sta-tistically significant level of Fpg-sensitive sites was determined inDiMeDBC-treated cells (Fig. 6A). Neither DBC nor N-MeDBC pro-duced a significant level of base modifications in exposed cells. Thekinetics of Fpg-sensitive sites removal produced by DiMeDBC wasrelatively slow; a detectable level of DNA breaks was determinedup to 14 h after treatment (Fig. 6B). To verify the capacity of diben-zocarbazoles to induce oxidative stress and generate ROS whichmay cause oxidative DNA lesions, flow cytometric analysis of ROSproduction was carried out (see Section 2). WB-F344 cells wereexposed to dibenzocarbazoles for 24 h; hydrogen peroxide (5 min,on ice) was the positive control in these experiments. At equimolarconcentration (1 �M), DiMeDBC was the most potent producer ofROS compared with DBC and N-MeDBC (Table 4). Although the ROSlevel generated by DiMeDBC was lower than that produced by thepositive control, it might still be sufficient to induce oxidative DNAdamage in exposed cells.

3.7. Phosphorylation of histone H2AX by DBC and its derivatives

Phosphorylation of histone H2AX (termed �H2AX) representsone of early events occurring in cells due to genotoxic stress [45].

Z. Valovicová et al. / Mutation Research 665 (2009) 51–60 57

Fig. 6. The level of oxidative DNA damage detected in WB-F344 cells exposed to equimolar (1 �M) DBC, DiMeDBC and N-MeDBC concentration for 2 h (A) and the kineticsof oxidative DNA damage repair (B). Opened portions of the bars represent DNA strand breaks (strand breaks and alkali-labile sites) detected immediately after treatment inthe absence of Fpg endonuclease, the filled portion of the bar represent additional DNA strand breaks detected in the presence of Fpg endonuclease (Fpg-sensitive sites). Thecolumns represent the mean ± S.D. from at least two independent experiments on eachin WB-F344 cells was analyzed at several time intervals after treatment. Points represenslides. Data were analyzed statistically by Student’s t-test. Significantly different from thein the absence of Fpg endonuclease; ap < 0.01.

Table 4The relative level of reactive oxygen species (ROS) generated by equimolar (1 �M)DBC, DiMeDBC and N-MeDBC concentration in WB-F344 cells after 24 h treatment.Hydrogen peroxide (250 �M, 5 min treatment) was used as the positive control.

Compound Treatment [%]

DMSO 24 h 100DBC 24 h 166 ± 12.0*,a

DiMeDBC 24 h 295 ± 61.7*

N-MeDBC 24 h 205 ± 32.3*

H2O2 5 min 707 ± 77.3*,a

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ata represent the mean ± S.D. of three independent experiments.a Significantly different from DiMeDBC at p < 0.05.* Significantly different from control (DMSO) at p < 0.05.

H2AX accumulation was measured after 24 h exposure to dibenzo-arbazoles at 0.1–10 �M (Fig. 7). Dibenzo[a,l]pyrene, the strongestnown genotoxin among polycyclic aromatic hydrocarbons (PAHs)as the positive control in these experiments. Exposure of WB-

ig. 7. Histone H2AX phosphorylation (�H2AX) detected in WB-F344 cells exposedo equimolar (0.1 �M, 1 �M and 10 �M) DBC, DiMeDBC, N-MeDBC, 0.1 �M DB[a,l]Ppositive control), and 0.1% DMSO (negative control) for 24 h. For Western blot anal-ses, equal amounts of total protein were subjected to 10% SDS PAGE mini gel. Actinas used as loading control. For immunodetection, appropriate primary and sec-ndary antibodies were utilized. RI is the relative band intensity, the ratio of inducedo control level of band density. All RI values were checked and corrected relative toctin levels.

triplicate of slides. The kinetics of Fpg-sensitive site removal induced by DiMeDBCt the mean ± S.D. from at least two independent experiments on each triplicate of

control, *p < 0.05; ***p < 0.001; significantly different from DiMeDBC-treated cells

F344 cells to the liver carcinogens DBC and DiMeDBC resulted ina significant induction of histone H2AX phosphorylation; up tofivefold increase of �H2AX was determined at the highest concen-tration (Fig. 7). Only a weak increase in �H2AX level was detectedin cells exposed to the tissue-specific sarcomagen N-MeDBC.

4. Discussion

This study is a follow-up of a previous investigation whichsuggested that the “stem-like” rat liver epithelial cells may bea potential target cell population for the liver carcinogens DBCand DiMeDBC [32]. Variability in cellular processes triggered bythese agents in WB-F344 cells suggested differences in DNA lesionsproduced by DBC and DiMeDBC. Because the genotoxic effects ofdibenzocarbazole derivatives have not been studied in this typeof liver cells, this work was initiated as part of ongoing researchdevoted to understanding the cellular and molecular mechanismsinvolved in chemical hepatocarcinogenesis.

A higher susceptibility of WB-F344 cells towards the liver car-cinogens, as compared with sarcomagens, was observed after 2 htreatment (Table 1). The 24-h exposure to the systemic carcino-gen DBC resulted in a significant, dose-dependent increase of DNAadducts (Fig. 2C and D). At equimolar (1 �M) concentration, the DNAadduct level was comparable with that of B[a]P (Table 2). The DNAadduct pattern produced by DBC in WB-F344 cells was similar tothat found in human hepatoma HepG2 cells [46], but distinct fromthe one detected in mouse liver cells in vivo [8,16,18]. These obser-vations seem to imply that additional drug-metabolizing enzymesexpressed in the liver may be involved in DBC metabolism. Based onthe differences in tissue distribution patterns of DBC-DNA-adductsdetected in vivo [16,18] and in vitro [18,47,48], two metabolic path-ways have been proposed for DBC: (i) activation involving thering-carbon atoms, as in the case of PAHs, and (ii) metabolismat the pyrrolic NH group [8,19,49]. Although stable DNA adductsare supposed to be major DNA lesions responsible for the carcino-genicity of this agent in vivo [50], DBC, which has a relatively lowionization potential [51], can also form unstable, depurinating DNA

adducts through one-electron oxidation mediated by radical cationpathway [52]. Aldo–keto reductases (AKR), which compete withcytochrome P450 (CYP) enzymes, can play a significant part in DBCmetabolism [53,54]. Phenols are the major DBC metabolites foundin vitro using mouse and rat liver microsomes [55–57] as well as

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uman CYP1 enzymes [58]. The proximate carcinogens 3-OH-/4-H-DBC [59] may be further activated to yield DBC-o-quinonesia the AKR pathway, which can form stable and unstable DNAdducts. o-Quinones were also identified as a minor componentf DBC metabolites in vitro [55]. DBC-3,4-dione is supposed to behe ultimate DBC metabolite [53]. DNA lesions produced by DBCesulted in a substantial dose-dependent inhibition of DNA and RNAyntheses (Fig. 3), caused DNA breakage (Fig. 4) and micronucleusormation (Table 3). DBC-bulky adducts generate serious distortionn DNA, resulting in a considerable delay in DNA damage removalFig. 5); even 48 h after exposure, a significant level of DNA strandreaks was detected in DBC-treated cells. Significant phosphoryla-ion of histone H2AX (Fig. 7) and p53 protein [32] were determinedn DBC-treated cells.

Surprisingly, the strict hepatocarcinogen DiMeDBC producedn almost negligible level of stable DNA adducts under identicalreatment conditions (Fig. 2E and F; Table 2). A time-course studyimed at the kinetics of DNA-adduct formation in cells exposedo DiMeDBC excluded a loss of bulky adducts already during cellreatment owing to rapid DNA repair. No specific DNA adduct spotsere detected after the exposure of WB-F344 cells by DiMeDBC

or 2 h, 4 h, 6 h, and 12 h. DBC and DiMeDBC are potent liverarcinogens, but quantitative and qualitative differences in theiriological effects were detected in vivo. At equimolar concentra-ions, DiMeDBC induced fewer mutations and DNA adducts evenith different chromatographic mobility than the parent com-ound DBC [10,13,16,18]. DBC hepatocarcinogenicity is associatedith strong hepatotoxicity whereas, under identical treatment con-itions, no histologically detectable toxicity has been found in the

iver of DiMeDBC-treated mice [14].Despite the lack of stable DNA adducts, a significant level of

trand breaks and MNi were ascertained in DiMeDBC-exposed cellsFig. 4, Table 3). In contrast with DBC, the increase in strand breaksas not concentration dependent. One of the reasons of such phe-omenon might be a rapid repair of DNA damage at these lowoncentrations as suggested, e.g., by Doak et al. [60]. Indeed theesults depicted in Fig. 5 seem to suggest that the repair of breaksnduced by DiMeDBC is more rapid, when compared with DBC; noNA strand breaks were determined in cells 16 h after exposure.n explanation might be induction of oxidative stress. It is sup-osed that ROS generated by various agents are eliminated to someegree by inherent cellular defence mechanisms as presented, e.g.,y Jenkins and colleagues [61]. DiMeDBC (Table 4) induced a sig-ificant level of ROS, as compared with both control and DBC.he modified comet assay was used to analyze the DNA-damagerofiles induced by individual dibenzocarbazoles to evaluate theechanism of DiMeDBC genotoxicity in WB-F344 cells. A signifi-

ant rise in strand breaks due to incubation of DiMeDBC-exposedells with Fpg protein, a repair-specific endonuclease, suggestedhat base modifications such as oxidized damage (e.g., 8-oxodG) oring-opened DNA adducts (Fapy-DNA adducts) may be responsi-le for DiMeDBC genotoxicity in these cells (Fig. 6A). In addition,imilarity in the kinetics of DNA damage removal in the presencer absence of Fpg protein was found in DiMeDBC-exposed cellsFigs. 5 and 6B). No DiMeDBC-DNA adducts were detected also in79MZh1A2 cells stably expressing human CYP1A2 despite a sig-ificant level of mutations and micronuclei detected in exposedells [48]. In agreement with our present study, further experimentsevealed possible role of oxidative DNA damage or unstable DNAdducts in DiMeDBC genotoxicity in V79MZh1A2 cells.

DiMeDBC is a potent aryl hydrocarbon receptor (AhR)-agonist

nd accordingly increased significantly the expression of AhR-ediated genes in WB-F344 cells, mainly CYP1A1 [32], although

t is poorly metabolized by this cytochrome P450. No mutations,NA strand breaks, micronuclei or DNA adducts were detected in79MZh1A1 cells stably expressing CYP1A1 [48,62–64]. Based on

search 665 (2009) 51–60

these data, we hypothesized that DiMeDBC might cause a releaseof ROS due to uncoupling of the catalytic cycle of CYP1A1 similarlyas the planar halogenated hydrocarbons. Both events, oxidativedamage and CYP1A1 induction were detected in cells exposed todioxin (TCDD) or polychlorinated biphenyl (PCB) congeners, whichare AhR-agonists but are, at the same time, poorly metabolized byCYP1A1 [65–67]. On the other hand, AhR activation alone may alsocause a shift in the cellular redox balance, resulting in cellular oxida-tive stress [68,69]. Oxidative DNA damage produced by DiMeDBCmay lead to a replication fork collision resulting in histone H2AXphosphorylation (Fig. 7). H2AX, a variant of histone H2A, is a crit-ical factor for cellular protection [70]. It is rapidly phosphorylatedat serine 139 in response to DNA double strand breaks [71], repli-cation arrest [72], apoptosis [73], transcription inhibition [74], andoxidative stress [75].

Although the level of stable adducts determined in N-MeDBC-treated WB-F344 cells was very low (Fig. 2G and H, Table 2), itwas probably sufficient to produce DNA strand breaks and MNi(Fig. 4, Table 3). DNA strand break formation has been proposedto be a standard biomarker of DNA damage; however, this parame-ter is probably not specific enough to identify differences in tissuespecificity of chemical compounds. DNA strand breaks are formedas a consequence of various events; they are induced directly bythe agent, produced due to DNA damage removal in the process ofDNA repair or can result spontaneously by release of unstable DNAadducts leading in alkali labile sites. DNA lesions generated by N-MeDBC were efficiently repaired; no DNA strand breaks were foundin N-MeDBC-treated cells 24 h after exposure (Fig. 5). Efficient elim-ination of N-MeDBC metabolites via conjugation reactions cannotbe excluded, as, e.g., Perin et al. [49] showed that high N-MeDBCmutagenicity in vitro due to activation by pure microsomes wassignificantly reduced in the presence of cytosolic fraction. Despitethe lack of biological activity of this tissue-specific sarcomagen inthe liver [76]; a low level of DNA adducts (a level ∼300-fold lowerthan that produced by DBC) was detected in mouse liver exposedto N-MeDBC [20]. Contrary to DBC and DiMeDBC, N-MeDBC didnot exhibit cytotoxicity (Table 1), cell-cycle arrest or apoptosis inWB-F344 cells [32]. No significant histone H2AX (Fig. 7) and p53phosphorylation was found in N-MeDBC-treated WB-F344 cells[32].

In conclusion, the present study clearly demonstrated thatdifferent mechanisms may underlie genotoxic effects of liver car-cinogens DBC and DiMeDBC in the WB-F344 cell line, a promising invitro model of rat liver oval cells. DNA damage induced by the tissue-specific sarcomagen N-MeDBC may result in DNA strand breaksand MNi. The extent of these genotoxic effects was insufficient toproduce additional cellular events related to hepatocarcinogene-sis. Stable DNA adducts may mediate DBC genotoxicity in WB-F344cells, but oxidative stress resulting in oxidative damage to DNAis likely to be the causal factor in DiMeDBC genotoxicity. Differ-ent DNA lesions may trigger distinct cellular pathways, resultingin diverse cell responses detected in WB-F344 cells. The previousstudy also showed that the strict hepatocarcinogen DiMeDBC is arelatively potent agonist of AhR, which is an important player incarcinogenesis. High levels of apparently active AhR characterizevarious tumors, even in the absence of exogenous ligands [77–79].AhR can interact via molecular cross-talk with multiple signallingpathways involved in cellular growth, differentiation, and regula-tion of cell adhesion and migration [80,81]. Future studies shouldestablish the significance of oxidative damage induced by DiMeDBCunder in vivo conditions, as well as its relevance to hepatocarcino-

genesis.

Conflict of interest

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cknowledgements

The authors wish to thank Professor F. Périn, Department ofenotoxicity and Carcinogenicity, Institute Curie, France, who pro-ided the dibenzocarbazole derivatives; and Professor J. E. Trosko,SU, East Lansing, MI, USA, who kindly offered the rat liver ovalB-F344 cells. The authors express their appreciation to Mrs. A.

okáliková for excellent technical assistance.This study was supported by the grants awarded by the Sci-

ntific Grant Agency of SAS (No. 2/6063/26), Czech Ministryf Education (No.2B08005), and Czech Ministry of AgricultureMZE0002716201). Zuzana Valovicová, M.Sc. was a fellow of theuropean Social Fund Project (13120200038).

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