Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

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ARTICLE IN PRESSG ModelUT 11334 1–11

Mutation Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

j ourna l h om epage: www.elsev ier .com/ l ocate /molmutComm uni t y ad dress : www.elsev ier .com/ locate /mutres

onhomologous DNA end joining and chromosome aberrations inuman embryonic lung fibroblasts treated with environmentalollutants

avel Rossner Jr. ∗, Andrea Rossnerova, Olena Beskid, Nana Tabashidze, Helena Libalova,aterina Uhlirova, Jan Topinka, Radim J. Sram

epartment of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, Prague, Czech Republic

r t i c l e i n f o

rticle history:eceived 18 January 2014eceived in revised form 25 February 2014ccepted 7 March 2014vailable online xxx

eywords:enzo[a]pyrenehromosome aberrationsouble-strand DNA breaksxtractable organic matteronhomologous DNA end joining repair

a b s t r a c t

In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA inhuman embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P)and extractable organic matter (EOM) from ambient air particles <2.5 �m (PM2.5) on nonhomologousDNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected inwinter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cellswere treated for 24 h with the following concentrations of tested chemicals: B[a]P: 1 �M, 10 �M, 25 �M;EOMs: 1 �g/ml, 10 �g/ml, 25 �g/ml. We tested several endpoints representing key steps leading fromDSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells andthe frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and17 using fluorescence in situ hybridization. Our results show that 25 �M of B[a]P and most of the tested

doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompaniedby induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequentlyaffected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligationactivity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatmentof human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs.

© 2014 Published by Elsevier B.V.

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. Introduction

Combustion of organic material generates a complex mixture

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f chemicals and represents an important source of fine particlePM2.5) air pollution [1]. PM2.5 is deposited in lung alveoli wheret causes inflammation. Chemical compounds bound to PM2.5 may

Abbreviations: B[a]P, benzo[a]pyrene; CA, chromosome aberration; c-PAH,arcinogenic polycyclic aromatic hydrocarbon; DSB, double strand DNA break;MSO, dimethylsulfoxide; EOM, extractable organic matter; FG/100, genomic fre-uency of translocations per 100 cells; FISH, fluorescence in situ hybridization;-H2AX, phosphorylated H2AX; HEL12469, human embryonic lung fibroblasts; NEX,uclear extract; NHEJ, nonhomologous end joining repair; PAH, polycyclic aromaticydrocarbon; PM, particulate matter; PM2.5, particulate matter of aerodynamiciameter < 2.5 �m; ROS, reactive oxygen species.∗ Corresponding author at: Videnska 1083, 14220 Prague, Czech Republic.el.: +420 241062763; fax: +420 241062785.

E-mail address: [email protected] (P. Rossner Jr.).

ttp://dx.doi.org/10.1016/j.mrfmmm.2014.03.006027-5107/© 2014 Published by Elsevier B.V.

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then enter the bloodstream and cause damage to both lympho-cytes and cells of distant organs. Recently, the International Agencyfor Research on Cancer (IARC) classified outdoor air pollution andparticulate matter from outdoor air pollution as carcinogenic tohumans [2]. Mechanisms of carcinogenicity include direct DNAdamage (chromosomal aberrations, bulky DNA adduct formation,oxidative stress), as well as changes in gene expression involvedin DNA damage and repair pathways, inflammation, immune andoxidative stress response [2]. Carcinogenic polycyclic aromatichydrocarbons (c-PAHs) are responsible for most of the genotoxicactivity of organic extracts from particulate matter (EOM) resultingin the formation of bulky DNA adducts [3]. Benzo[a]pyrene (B[a]P),a model c-PAH, is classified as carcinogenic to humans [4].

There are three major pathways of detoxification/metabolic

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

activation of PAHs: the pathway via dihydrodiol epoxides employ-ing CYP450 enzymes, the pathway via radical cations by oneelectron oxidation, and the pathway catalyzed by dihydrodioldehydrogenases resulting in the formation of ortho-quinones [5].

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ulky DNA adducts represent a predominant form of DNA dam-ge following insult by c-PAHs. They are repaired by the action ofucleotide excision repair. However, PAH-o-quinones generated byihydrodiol dehydrogenases are redox active and may contribute toeactive oxygen species (ROS) formation and subsequent oxidativetress [6]. ROS may attack DNA and cause DNA single- or double-trand breaks. If repaired by nonhomologous end joining (NHEJ) oromologous repair, double strand DNA breaks (DSBs) may result intable chromosomal aberrations, as translocations and reciprocalranslocations. Apart from this mechanism, DSBs may be induceds an indirect result of bulky DNA adduct formation. The presencef adducts may block DNA synthesis and uncoupling of the othertrand thus resulting in DSB formation [7]. The induction of unsta-le chromosomal aberrations by c-PAHs or their metabolites wasepeatedly observed in various cell lines [8–14]. Although chromo-omal translocations are more serious than unstable aberrationsecause they may be fixed in the genome potentially leading toearrangements of regulatory elements of genes including onco-enes [15], very few studies analyzing the effect of c-PAHs on modelell lines in vitro have been published [16,17].

In the present study, we aimed to analyze processes associ-ted with the treatment of human embryonic lung fibroblastsHEL12469 cells) with EOMs and B[a]P. EOMs were prepared byrganic extraction from PM2.5 collected in two Czech cities (Prague,strava) differing in sources and concentrations of c-PAHs. Weoncentrated on the induction of DSBs (measured as levels of phos-horylated histone H2AX), expression of selected NHEJ proteinsKu70, Ku80, XRCC4), ligation activity of HEL12469 nuclear extractsnd the frequency of stable aberrations of chromosomes 1, 2, 4, 5, 7nd 17. We hypothesized that ROS generation after EOM and B[a]Preatment will result in the induction of DSBs, followed by expres-ion of NHEJ proteins and increased ligation activity of nuclearxtracts which in turn will increase the frequency of chromosomeranslocations.

. Materials and methods

.1. PM2.5 collection, preparation of EOM and chemical analysis

Sample collection, EOM preparation and chemical characteriza-ion were described in detail in our previous study [18]. Briefly,M2.5 was collected by a HiVol 3000 air sampler (model ECO-VS3000, Ecotech, Australia) on Pallflex filters T60A20 in two

ocations in the Czech Republic: Prague (a sampler was located out-ide the city center, with moderate traffic intensity) and Ostrava (aampler was located in a heavily polluted industrial area). The sam-les were collected in the winter and summer seasons of 2011, for4 h each day for 4–5 weeks. The total number of filters obtainedor individual location and sampling season ranged from 28 to 31.ach filter was extracted with a mixture of dichloromethane andyclohexane, the extracts (EOMs) were pooled and aliquots used forhemical analysis and cell treatment. Quantitative chemical anal-sis of PAHs was performed by HPLC with fluorimetric detection.or the in vitro experiments, EOM samples were evaporated to dry-ess under a stream of nitrogen and the residue redissolved inimethylsulfoxide (DMSO). The stock solution of each EOM sam-le contained 50 mg of EOM/ml DMSO. EOMs were kept in thereezer at −80 ◦C. The extraction of PM2.5 and chemical analysesere performed in the laboratories of the certified company ALSzech Republic, Prague (EN ISO CSN IEC 17025).

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.2. Cell cultivation and treatment

The growth characteristics of the HEL12469 cells (ECACC, Salis-ury, UK) used in the present study and cytotoxicity of the tested

PRESSsearch xxx (2014) xxx–xxx

chemicals were described in detail previously [18]. The cells weregrown at 37 ◦C in a humidified atmosphere containing 5% CO2 inminimal essential medium EMEM supplemented with 10% fetalbovine serum (FBS), 2 mM glutamine, 1% non-essential amino acids,0.2% sodium bicarbonate, 50 U/ml penicillin and 50 �g/ml strepto-mycin. After reaching confluency, the cells were treated with 0.25%trypsin/0.02% EDTA in PBS and used for individual applications.We tested the following concentrations of B[a]P (Sigma–Aldrich, St.Louis, MO, USA) and EOM: B[a]P: 1, 10 and 25 �M; EOMs: 1, 10 and25 �g/ml. We assessed the cytotoxicity of the selected concentra-tions of the tested compounds using the cell proliferation reagent,WST-1 (Roche, Mannheim, Germany), following the manufacturer’sinstructions (see [18] for details).

2.3. Expression of �-H2AX, Ku70, Ku80 and XRCC4 proteins

The cells (1 × 106) were seeded in 75 cm2 tissue culture flasks(TPP, Trasadingen, Switzerland) in 15 ml culture medium sup-plemented with 10% FBS and incubated for 2–3 days at 37 ◦C.Treatment involved replacing the culture medium with 10 ml freshmedium containing the test chemicals. The cells were treated for24 h, harvested by scraping and stored at −80 ◦C until furtherprocessing.

For Western blotting analysis, the cells were lysed in extractionbuffer (1% SDS, 10% glycerol, 100 mM Tris, pH 7.4) supplementedwith protease and phosphatase inhibitors, and the protein con-centration was estimated using the BCA reagent (Sigma, St. Louis,MO, USA). Proteins (12 �g/sample) were separated by SDS-PAGEin 4–12.5% gels (0.75 mm) and transferred onto a Hybond-P PVDFmembrane (GE Healthcare, Piscataway, NJ, USA). The membranewas blocked for 2 h at room temperature in TBS buffer (10 mMTris, 150 mM NaCl, pH 7.4) containing 2% ECL Prime BlockingAgent (GE Healthcare, Piscataway, NJ, USA). The proteins weredetected by incubation of the membrane with primary antibod-ies against �-H2AX (ab22551; 0.1 �g/ml), Ku70 + Ku80 (ab53126;dilution 1:2000) and XRCC4 (ab97351; dilution 1:500) from Abcam(Cambridge, UK) at 10 ◦C overnight and a secondary antibodyconjugated with horseradish peroxidase (for �-H2AX: NA931, GEHealthcare, Piscataway, NJ, USA, dilution 1:10,000; for Ku70 + Ku80and XRCC4: NA934, GE Healthcare, Piscataway, NJ, USA; dilution1:100,000 and 1:10,000 for Ku70 + Ku80 and XRCC4, respectively)at room temperature for 1 h. The antibodies were diluted in TBS-T buffer (TBS + 0.1% Tween 20) containing 2% ECL Prime BlockingAgent. To develop the chemiluminiscence signal the membranewas incubated with ECL Prime WB Detection Reagent (GE Health-care, Piscataway, NJ, USA) for 5 min at room temperature in the darkand exposed to Amersham Hyperfilm (GE Healthcare, Piscataway,NJ, USA). The intensity of the bands on the scanned films was ana-lyzed using ImageJ software (http://rsbweb.nih.gov/ij/index.html).The results were expressed as a percentage of the band intensityof the control sample normalized to a loading control [the mem-brane was stained with amido black solution (0.1% Amido Black10B, Sigma–Aldrich, St. Louis, MO, USA; 45% methanol; 10% aceticacid)].

2.4. NHEJ repair activity

The activity of NHEJ was measured in nuclear extracts ofHEL12469 cells essentially as described in [19,20], with some mod-ifications. The cells (4 × 105) were seeded in 25 cm2 tissue cultureflasks (TPP, Trasadingen, Switzerland) and cultivated in 10 ml cul-ture medium supplemented with 10% FBS for 2–3 days. Before

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

treatment, the medium was replaced with 5 ml fresh mediumcontaining the test chemicals. After 24 h treatment, the cellswere harvested and immediately used for nuclear extract (NEX)preparation. NEXs were prepared using the NE-PER Nuclear and

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ytoplasmic Extraction Reagents Kit (Thermo Scientific, Waltham,A, USA) with protease inhibitors (Thermo Scientific, Waltham,A, USA) added. The volume of the reagents depended on the num-

er of cells harvested. Five microliters of NEX were used for analysisf protein concentration using the BCA Kit (Sigma, St. Louis, MO,SA) and the remaining volume of NEX was mixed with glycerol

final concentration 20% v/v) and stored at −80 ◦C until analysis.Plasmid pUC18 (Invitrogen, Eugene, OR, USA) linearized by

igestion with EcoRI (Thermo Scientific, Waltham, MA, USA)as used as a substrate for the ligation reaction. NEX contain-

ng 4 �g of proteins in a total volume of 6 �l was mixed with0 ng (concentration 40 ng/�l) of linearized pUC18, 1 �l of end-

oining mix (containing 200 mM HEPES, 800 mM KCl, 100 mMgCl2), 1 mg/ml bovine serum albumin, 1 mM dithiothreitol and

mM adenosine triphosphate. The reaction mixture (total vol-me of 20 �l) was incubated for 2 h at 25 ◦C. The reaction wastopped by adding RNase (final concentration 1 mg/ml) and pro-einase K (final concentration 1 mg/ml). DNA fragments wereeparated by electrophoresis in 1% agarose gel in TBE bufferunning at 5 V/cm for 1 h. Gels were stained with SYBR® SafeInvitrogen, Eugene, OR, USA) diluted 1:10,000× and visualizedsing the GelDoc-ItTM Imaging System (UVP, Cambridge, UK). The

ntensity of the DNA fragments was analyzed using ImageJ soft-are (http://rsbweb.nih.gov/ij/index.html). The repair activity was

xpressed as a percentage of ligation of the linearized substratealculated as (intensity of ligation products/substrate) × 100. Aegative control (linearized pUC18 without NEX) was run in eachxperiment. To verify the method before running the test samples aeries of optimization experiments was conducted using differentmounts of NEX proteins and pUC18 in a reaction mixture as wells different incubation periods.

.5. Fluorescence in situ hybridization (FISH)

The cells (1 × 106) were seeded in 75 cm2 tissue culture flasksTPP, Trasadingen, Switzerland) in 15 ml culture medium sup-lemented with 10% FBS and incubated for 2–3 days at 37 ◦C.reatment involved replacing the culture medium with 10 ml freshedium containing the test chemicals. The cells were treated for

4 h. Colchicine (Sigma–Aldrich, St. Louis, MO, USA) was added at final concentration of 0.5 �g/ml three hours before the end of thencubation. After treatment, the cells were trypsinized, collectedy centrifugation, resuspended in a hypotonic 0.075 M KCl solution,xed with methanol/acetic acid and stored at −20 ◦C until droppingn slides. The FISH staining protocol of chromosomes 1, 2, 4, 5, 7 and7 was adapted from the manufacturer’s instructions (MetaSys-ems, Altlussheim, Germany). FISH analysis was performed usingwo sets of Customized XCP-Mix probes: the first set containingrobes for chromosome 1 (red), 4 (green) and 17 (red and green,onverted as yellow); the second set with the same color labels butpecific for chromosomes 2, 5, and 7. The slides were first stainedith the first set, analyzed, destained and re-hybridized with the

econd set.Analyses of FISH slides counterstained with DAPI mixed with

ectashield mounting medium (Vector Laboratories, Burlingame,A, USA) were performed using a Zeiss Axioscope and Olympus BX1 microscopes (final magnification: 1000×) equipped with a triplelter to visualize simultaneously the DAPI (blue), FITC (green) andy3 (red) signals. Color images with CAs were collected and ana-

yzed using ISIS software, version 5.0 (MetaSystems, Altlussheim,ermany). The number of scored metaphases per tested chemicalnd concentration was 1500–3000 for each set of painted chromo-

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omes.All aberrant cells were classified according to the Proto-

ol for Aberration Identification and Nomenclature (PAINT) [21].he genomic frequencies of translocations (FG) were calculated

PRESSearch xxx (2014) xxx–xxx 3

as suggested by Lucas et al. [22]. Exchange (translocations)frequencies obtained from each chromosome were calculated forthe whole genome by dividing the observed frequencies by thefactor of 2.05fp(1 − fp), where fp is the fraction of painted DNAconverted by individual chromosomes. The genomic frequencyof translocations obtained from the three chromosomes together(including the exchanges between painted chromosomes) wereestimated by dividing the observed frequencies by the factor2.05{(fr + fg + fy)[1 − (fr + fg + fy)] + frfg + fgfy + frfy} [23], where fr, fgand fy are the fractions of the genome [24] converted by chromo-somes 1, 4 and 17, and 2, 5, 7, for the first and second painted setof chromosomes, respectively. Genomic frequencies of transloca-tions (FG) were recalculated per 100 cells and expressed as genomicfrequencies of translocations per 100 cells (FG/100). Conversion ofpainted metaphase cells to whole-genome equivalents (defined ascell equivalents) was calculated using equations appropriate forthree-color paints [25]. Cell equivalents ranged from 463 to 927 forpainted chromosomes 1, 4 and 17 and from 509 to 1017 for paintedchromosomes 2, 5 and 7.

2.6. Statistical analysis

Differences between individual groups in the mean values ofNHEJ repair activity were tested using the two-tailed Student’st-test; a p-value of 0.05 or less indicated a statistically signifi-cant difference between the compared groups. The calculationswere performed using IBM SPSS (Chicago, IL, USA) v. 20 statisti-cal software. Observed frequencies of chromosomal translocationsin individual chromosomes were compared with expected valuesas described by Johnson et al. [26].

3. Results

3.1. Air sampling and chemical analysis of EOMs

Information on air sampling and chemical analysis of EOMswas reported in detail in our previous study [18]. Here we pro-vide a brief summary of the results. The PM2.5 samples werecollected in two cities (Prague, Ostrava) in two seasons (win-ter, summer) of 2011. The concentrations of PM2.5 (�g/m3)in the ambient air were as follows: 34.7, 49.2, 9.2 and 16.6,for Prague-winter, Ostrava-winter, Prague-summer and Ostrava-summer, respectively. The concentrations of B[a]P followed asimilar trend. Although for cell treatment we used equal con-centrations of EOMs (1, 10 and 25 �g/ml), the concentrations ofanalyzed PAHs in individual EOMs differed (Table 1). Total PAHconcentration was significantly higher in the Ostrava-winter thanin the Prague-winter EOM; the same was true for EOMs collectedin the summer season. It should be noted that the PAH concen-trations in EOMs used for cell treatment were several orders ofmagnitude lower than the concentrations of B[a]P tested in ourexperiments.

3.2. DSBs and expression of NHEJ proteins

We first examined the ability of B[a]P and EOMs to induceDSBs which are a prerequisite for the formation of chromosomeaberrations. B[a]P at the highest tested dose (25 �M) increased �-H2AX levels, and the doses of 1 �M and 10 �M seemed to haveno effect under the tested conditions (Fig. 1). Most EOMs werepotent inducers of DSBs. Although the effect of the EOM col-lected in the winter season in Prague was similar to B[a]P (2.5-fold

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

increase in �-H2AX levels after treatment with the highest dose),other EOMs induced H2AX phosphorylation ranging from 16- to84-fold above the control level (Fig. 2). Moreover, particularlyfor the Ostrava-winter EOM, we observed a clear dose–response

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Table 1PAH content in pooled extractable organic matter (EOM) from individual samplings.

Analyzed PAHs Concentrations of PAHs in EOMs from individual sampling locations (ng/�g EOM)

Prague-winter* Ostrava-winter Prague-summer* Ostrava-summer

Benz[a]anthracene 0.22 0.41 0.01 0.11Benzo[a]pyrene 0.20 0.25 0.01 0.14Benzo[b]fluoranthene 0.30 0.41 0.03 0.34Benzo[g.h.i]perylene 0.16 0.16 0.02 0.17Benzo[k]fluoranthene 0.13 0.15 0.01 0.13Chrysene 0.34 0.38 0.02 0.16Dibenz[a.h]anthracene 0.02 0.03 0.01 0.02Indeno[1.2.3.cd]pyrene 0.15 0.14 0.02 0.13

Total PAHs 1.51 1.93 0.13 1.20

* gue-suS 8 to 3

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Significant differences (p < 0.001) in PAH concentrations between EOMs from Pratudent’s t-test was used to compare PAHs concentrations in EOMs obtained from 2

elationship between the tested concentrations of the EOM and

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nduction of DSBs.The effect of most tested compounds on Ku70 and Ku80 expres-

ion was weak (Figs. 1 and 2). A dose-dependent increase in Ku70

ig. 1. Induction of DSBs and expression of NHEJ proteins in HEL12469 cells treated with

f the respective bands. (B) Protein expression normalized per sample loading (assessed

mmer vs. Ostrava-summer and Prague-winter vs. Ostrava-winter. The two-tailed1 individual filters before pooling.

expression was observed after treatment with the Ostrava-summer

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

EOM (Fig. 2). We further observed inconsistent effects of otherEOMs on the expression of this protein. For the Ku80 protein, anincrease was noted only after treatment with the lowest dose of

B[a]P; a representative result of three independent western blot analyses. (A) Scansby Amido Black staining).

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F ed wia ample

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ig. 2. Induction of DSBs and expression of NHEJ proteins in HEL12469 cells treatnalyses. (A) Scans of the respective bands. (B) Protein expression normalized per s

[a]P (Fig. 1). In contrast, XRCC4 expression was induced by both[a]P (Fig. 1) and all tested EOMs (Fig. 2). The effect of B[a]P seemedo be more pronounced; the levels of expression ranged from a

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.60- to 11.1-fold increase when compared with the expressionf this protein in the control sample. All the tested EOMs affectedRCC4 expression to a similar extent (2- to 3-fold increase), with

he exception of the highest dose of the Ostrava-winter EOM which

th four different EOMs; a representative result of three independent western blot loading (assessed by Amido Black staining).

caused more than five-fold induction of the protein when comparedwith the control sample.

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

3.3. NHEJ repair activity

The activity of non-homologous DNA end-joining measured asthe ability of nuclear extracts to ligate a pUC18 plasmid substrate

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F f HEL1t

dw5t

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ig. 3. Activity of NHEJ repair measured as ligation efficiency of nuclear extracts ohree independent experiments.

igested with EcoRI was not significantly affected by treatment

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ith the tested samples (Fig. 3). The control NEX converted about0% of the substrate to end-joining products during 2 h incuba-ion. The NEX from the treated samples had a similar or about 10%

2469 cells treated with B[a]P and EOMs. The figure shows mean values ± SD from

lower ligation activity, but the differences were not statistically

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

significant. The NEX obtained from the Ostrava-summer samplesconsistently increased the repair activity to about 60%, but theresults were not significant.

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00 ce

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Fig. 4. Genomic frequency of translocations per 1

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.4. Stable chromosome aberrations

Genomic frequency of translocations per 100 cells calculatedased on the analysis of translocations of six chromosomes (1, 2,

lls. HEL12469 cells treated with B[a]P and EOMs.

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

4, 5, 7 and 17) was elevated above the control level (FG/100 = 0.19)after treatment with all tested samples (Fig. 4). However, we didnot find distinct differences between the compounds. To furthercharacterize the effect of B[a]P and EOMs on DNA damage we

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ig. 5. Frequency of chromosome translocations for individual chromosomes expcharts in the right column). Results shown for the effect of (A) B[a]P + EOMs; (B) B[

alculated the frequency of translocations for individual chromo-

Please cite this article in press as: P. Rossner Jr., et al., Nonhohuman embryonic lung fibroblasts treated with environmental phttp://dx.doi.org/10.1016/j.mrfmmm.2014.03.006

omes for all compounds and for B[a]P and EOMs separately. Wexpressed the results both as the number of translocations per000 cells and the number of translocations/1000 cells/1 giga basef a respective chromosome. The latter value takes into account

per 1000 cells (charts in the left column) and normalized per chromosome sizely; (C) EOMs only. Numbers below horizontal axes indicate chromosome number.

the chromosome size. The results reported in Fig. 5 indicate that

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

chromosome size does not significantly affect the frequency ofchromosome translocations. The pattern of frequencies of chromo-some translocations for B[a]P- and EOM-treated cells was different.EOMs preferentially induced DNA damage to chromosomes 7 and

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7, followed by chromosome 4. Frequencies of aberrations around.002/1000 cells/Gb were observed for chromosomes 5, 2 and 1.requencies of translocations caused by B[a]P were similar forhromosomes 7 and 4, followed by chromosome 2; frequenciesf translocations observed for chromosomes 5, 17 and 1 wereow (around 0.001/1000 cells/Gb). To further characterize theseesults we compared the observed frequencies of translocationsith expected values. For B[a]P treatment, the frequency of translo-

ations was 2.3-fold and 1.90-fold higher than the expected valueor chromosome 7 and 4, respectively. For EOM treatment, dam-ge to chromosomes 7 and 17 was similar and reached about-fold higher than expected frequency. For other chromosomes thebserved frequency was equal or lower than the expected valuedata not shown).

. Discussion

In the present study we conducted a series of experiments withhe aim of elucidating mechanisms associated with the treatmentf a human non-tumor lung cell line with carcinogenic PAHs, both aodel compound (B[a]P) and a complex mixture containing c-PAHs

btained from organic extracts from environments with differentources of air pollution. We analyzed a set of parameters related tonduction and repair of DSBs that should affect a final endpoint –he frequency of stable chromosome aberrations.

Double-strand DNA breaks are considered the most lethal formf DNA damage. It is estimated that in dividing cells about 10 DSBser cell per day are formed [27]. In general, DSBs can be repairedy two major mechanisms: homologous recombination (HR) andon-homologous end-joining. However, HR is limited only to the- and G2-phase of the cell cycle and requires sufficient homol-gy between repaired DNA strands. Therefore, NHEJ is regardeds the most important pathway of DSBs repair [28]. The repairrocess is started by binding of the Ku70/Ku80 heterodimer toNA ends. The Ku proteins are among the most abundant cel-

ular proteins and they exhibit a very high affinity to DNA ends28]. The DNA–Ku complex then attracts the catalytic subunitf the DNA-dependent protein kinase (DNA–PKcs) which causeshe phosphorylation of other proteins participating in the repairathway, including XRCC4. This protein stabilizes ligase IV whichltimately causes ligation of DNA strands (for details, see [28]).

Detection of phosphorylated histone H2AX is used as a reliablearker of DSBs [29]. It has been demonstrated that B[a]P treatment

nduces H2AX phosphorylation [30–32], although the intensityepends on the cell type used reflecting the metabolic activityf the tested model [33]. Similarly, extracts of soils contaminatedith PAH mixtures caused dsDNA damage in HepG2 cells [34]. As

DSB is a prerequisite for the formation of stable chromosomalberrations, we first determined whether the tested compoundsnduced phosphorylation of H2AX. We detected increased �-H2AXevels after treatment with all tested compounds, with the excep-ion of lower concentrations (1 and 10 �M) of B[a]P. As these dosesnduced chromosome translocations, this observation should note interpreted as the inability of lower concentrations of B[a]Po cause DSBs. The result rather suggests that at the time of cellollection DSBs had already been repaired. We also observed pro-ounced differences in the ability of individual EOMs to induce-H2AX. The Prague-summer EOM was the most potent inducer ofSBs, while the weakest effect (comparable with B[a]P treatment)as detected after the Prague-winter EOM treatment. Because the

Please cite this article in press as: P. Rossner Jr., et al., Nonhohuman embryonic lung fibroblasts treated with environmental phttp://dx.doi.org/10.1016/j.mrfmmm.2014.03.006

rague-summer EOM contained the lowest concentrations of B[a]Pnd other PAHs, we conclude that the ability of EOMs to induceSBs cannot by assigned exclusively to PAHs, but rather to otheronstituents that were not identified in the chemical analyses of

PRESSearch xxx (2014) xxx–xxx 9

the EOMs (e.g. other PAHs and their derivatives, n-alkanes, sterolsand various industrial contaminants [35]).

To the best of our knowledge, the effects of PAHs on the expres-sion of Ku70/80 and XRCC4 proteins in model cell lines has not yetbeen studied. The studies in cells exposed to X-rays [36] and etopo-side [37] suggested that expression of Ku70 and Ku80 proteins isnot induced by these factors. This may be explained by the fact thatKu proteins are one of the most abundant cellular proteins [28] andtheir levels are sufficient to perform their functions. In line withthis theory is our observation that the effect of B[a]P on Ku70/80expression is weak. However, EOMs, which in general were morepotent inducers of DSBs, exhibited some effect on the induction ofKu70 expression, which was mostly limited to the highest testeddoses of EOMs. Unlike Ku proteins, the expression of XRCC4 wasconsistently induced by treatment with the tested compounds and,particularly for EOMs, its expression levels were in agreement withlevels of phosphorylated H2AX.

Although protein expression analyses give some indication ofthe effect of tested compounds on the model organism, functionalassays should provide the ultimate information on repair and otherprocesses taking place in the cell. The in vitro NHEJ assays havebeen used in various set ups for more than 20 years (reviewedin [38]). The method we applied in our study is based on theprotocol of Perrault et al. [19] which employed pUC18 digestedwith EcoRI incubated with nuclear extracts of the treated cells.We hypothesized that treatment of the cells with the tested com-pounds would result in induction of NHEJ activity. In fact, althoughnumerous authors used the in vitro NHEJ assay for various appli-cations [38,39], none of them focused on inducibility of the NHEJreaction. Unexpectedly, we observed that the tested compoundshad a very small effect on NHEJ activity: the Ostrava summer EOMwas the only one to cause a consistent increase in the repair activ-ity, although the results were not statistically significant. Theremay be several reasons for the lack of a positive effect. First, lin-earized plasmid may not be an optimal substrate to mimic DNAdamage caused by the tested compounds, where ROS probablyplay a major role. It has been reported that naturally occurringoxidatively induced DSBs are far more complex than DNA breaksproduced by restriction enzymes and require further nucleolyticprocessing [39]. Thus, some enzymatic activities required for therepair of DSBs induced by PAHs and other compounds present inEOMs may not be detected by the in vitro NHEJ reaction. The effi-ciency of the NHEJ reaction is further dependent on the structure ofthe DNA break [40], thus we cannot exclude the possibility that theNHEJ reaction using the plasmid linearized by another restrictionenzyme would give different results. Finally, some authors advo-cate using whole-cell rather than nuclear extracts in the NHEJ assay[40] given the fact that some NHEJ proteins (e.g. Ku) are present inthe cytoplasm and transported to nuclei only after DNA damage[41]. However, this report was contradicted by another study [36]in which Ku proteins in human fibroblasts were found to be local-ized exclusively in the nucleus and only a minor fraction of XRCC4was detected in the cytoplasm. Moreover, the distribution of theproteins was not altered by irradiation of the cells [36]. The authorsof this study also measured DSB repair activity, but used a differentapproach (the pulldown assay) which allowed analysis of the activ-ity of the DNA–PK complex. They did not observe any induction ofthe repair activity upon X-ray irradiation of the fibroblasts.

To analyze the effects of the tested compounds on stable chro-mosome translocations we selected 6 chromosomes: 1, 2, 4, 5,7, and 17. The rationale for this selection was the fact that mostof these chromosomes represent the largest chromosomes thus

mologous DNA end joining and chromosome aberrations inollutants, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2014),

giving us an opportunity to analyze a significant proportion ofthe genome (35.8% of the human genome). Chromosome 17 wasincluded because the p53 gene, which is of particular interest withregard to human cancers, is located on its short arm. The number

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f chromosomes analyzed for stable CAs and the fact that a model,on-tumor cell line was used make our study unique. Most stud-

es have been performed in human peripheral blood lymphocytes42]. The fraction of exchanges detected using our set of probesas 0.59 which is comparable with the results obtained for human

ubjects after painting six chromosomes [42]. Our data indicate thatll tested compounds induced stable chromosome translocations.lthough the ability of EOMs to induce chromosome aberrationsas not been reported so far, B[a]P was shown to cause abnormal-

ties of chromosome 8 and 5 in human mammary epithelial cells16]. Another study found that B[a]P diol epoxide, an active metabo-ite of B[a]P, caused instability of chromosomes 2, 3 and 16 in auman lung cancer cell line [17].

We further concentrated on the identification of chromosomeshich are preferentially damaged after treatment with B[a]P and

OMs. The sensitivity of the chromosomes does not seem to dependn their size: chromosome 7 was most commonly affected regard-ess of the tested compound. It has been reported that aberrationsf chromosomes 1–6 assessed in lymphocytes of human subjectsxposed to PAHs were nonrandomly distributed and that chro-osome 6 was most frequently damaged. The authors observed

ncreased aberration frequencies as chromosome size decreased43]. They argued that PAHs may be preferentially targeting specifichromosomes. Although this observation was made on human sub-ects exposed to air pollutants and thus is not directly comparable

ith our data, it suggests that there may be common principleshich affect damage to chromosomes. The position of chromo-

omes in the nucleus may be one of them. It has been shownhat in human diploid fibroblasts, smaller chromosomes, inde-endently of gene density, are located closer to the center of theucleus, while larger chromosomes are found closer to the nuclearim [44]. Thus, chromosomes located near the nucleus peripheryay be at increased risk of damage. This observation may explain

he higher frequency of chromosome 7 translocations. However,t is still unclear why chromosome 1 was the least damaged of allhe studied chromosomes. Interestingly, chromosome 1 codes theighest number of proteins. Some authors argue that in certain cellypes gene-rich chromosomes were preferentially located towardhe center of the nucleus (e.g. [45]), which again may decrease theisk of DNA damage. Thus, at present a definite conclusion can-ot be drawn. The fact that there are differences in the patternf translocation frequencies between B[a]P and EOMs suggest thatther unidentified mechanisms and EOM components play a role.

. Conclusions

In our study we demonstrated the ability of B[a]P and EOMso induce DSBs, activate the expression of NHEJ proteins and causehromosome translocations. Components of EOMs other than PAHseem to be more important in the induction of DNA damage whichs in agreement with the mechanisms of DSBs formation in whichOS play an important role. The tested compounds preferentiallyffected chromosome 7; however, different mechanisms of actionf B[a]P and EOMs are underscored by the high frequency of damageo chromosome 17 after EOMs treatment. Compounds responsi-le for these differences remain to be identified. Our data indicatehat HEL12469, the non-tumor human lung cell line is suitable forn vitro testing of compounds whose mechanism of action involvesSBs and induction of chromosome aberrations.

Please cite this article in press as: P. Rossner Jr., et al., Nonhohuman embryonic lung fibroblasts treated with environmental phttp://dx.doi.org/10.1016/j.mrfmmm.2014.03.006

onflict of interest statement

The authors declare that there are no conflicts of interest.[

PRESSsearch xxx (2014) xxx–xxx

Acknowledgement

This work was supported by the Grant Agency of the Czech

Republic (P503/11/0084).

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