γH2AX, pChk1, and Wip1 as Potential Markers of Persistent DNADamage Derived from Dibenzo[a,l]pyrene and PAH-ContainingExtracts from Contaminated SoilsMagdalena Niziolek-Kierecka,†,§ Kristian Dreij,*,†,§ Staffan Lundstedt,‡ and Ulla Stenius†

†Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden‡Department of Chemistry, Umea University, S-901 87 Umea, Sweden

ABSTRACT: Polycyclic aromatic hydrocarbons (PAHs) are formed during incompletecombustion of organic material and are ubiquitous environmental contaminants. Highlevels of PAHs are commonly found in soils at industrial sites, thereby constituting a riskfor humans and the environment. However, this risk is often difficult to estimate due tothe complexity of the contamination. In the present study, we investigated the cellularDNA damage response induced by extracts of PAH-contaminated soils collected atvarious industrial sites in Sweden. The results show that interactions of PAHs in the soilextracts caused activation of DNA damage signaling consistent with persistent DNAdamage. Signaling in HepG2 cells exposed to soil PAH extracts corresponding to 1 μMbenzo[a]pyrene was similar to that of 0.1 μM dibenzo[a,l]pyrene, a highly carcinogenicPAH known to produce persistent DNA damage. The response involved prolongedactivation of DNA damage marker (H2AX), check point kinase (Chk1), andphosphatases (Wip1). Furthermore, blocking DNA damage signaling using specificinhibitors and siRNA showed the important role of signaling through Chk1 for the levelof DNA damage. We conclude that the combination of prolonged Chk1 phosphorylation and induced expression of Wip1 mightserve as potential markers for persistent DNA damage induced by complex mixtures of environmental PAHs. Discrepanciesbetween mRNA and protein levels of Wip1 in response to soil extracts, in parallel with increased microRNA (miR)-16 levels,suggest a role of miR-16 in the regulation of DNA damage signaling in response to PAHs. Taken together, our data indicate thatPAH extracts induce irreparable DNA damage and that this is consistent with the prolonged activation of DNA damage signaling.

■ INTRODUCTIONPolycyclic aromatic hydrocarbons (PAHs) are a large group ofenvironmental pollutants formed during the incompletecombustion of organic materials. As a consequence, PAHs areemitted from a wide range of sources, including industrialprocesses, combustion engines, residential heating processes,and several natural processes. PAHs are of concern mainlybecause of their mutagenic and carcinogenic effects, and severalof them have thus been classified as possible or probable humancarcinogens by the International Agency of Research on Cancer(IARC).1 However, PAHs are seldom the only compounds ofconcern in PAH-contaminated environments. Often, a widerange of related compounds are also present.2 These may betoxic as well but have in general been much less studied ascompared to PAHs. One such related group of contaminantsthat may be of toxicological importance is the oxygenatedpolycyclic aromatic hydrocarbons (oxy-PAHs), which includepolycyclic aromatic quinones and ketones (Figure 1). Thesecompounds are frequently found at similar levels as PAHs insoil and urban air, for instance,3−5 and are also known to elicitvarious toxic effects. Oxy-PAHs are emitted from the samesources as PAHs but may also be formed through oxidation ofPAHs in the environment. This formation may be enhancedduring processes such as soil remediation, during which PAHdegradation is accelerated, which potentially may lead to

increased levels of oxy-PAHs and thereby increased toxicity,even if the original PAHs are degraded.6

PAHs usually occur at contaminated areas as complexmixtures. However, toxicity studies tend to be performed on asubstance by substance basis, a typical example beingbenzo[a]pyrene (BP). BP is classified as a human carcinogenby the IARC and serves in risk assessment as a referencecompound for mixtures of PAHs.7 However, previous data fromus as well as others clearly show that this approach or using“toxic equivalency factors” scales (TEF scales) will under-estimate the risk.7−9 For example, we previously showed thattoxicity equivalence quota (TEQ) values did not correlate withthe effect on DNA damage signaling induced by mixtures ofPAHs extracted from soil at several industrial areas in Sweden.In contrast to BP-induced transient DNA damage responses,low levels of PAH-containing soil extracts evoked persistentand nonrepairable damage.8

To counteract the effects of DNA-damaging agents, such asPAHs and oxy-PAHs, cells activate an intricate network ofsignal transduction pathways termed the DNA damageresponse (DDR).10 DDR coordinates cell cycle progressionwith DNA repair, helping to maintain genetic stability and

Received: October 7, 2011Published: March 12, 2012

Article

pubs.acs.org/crt

© 2012 American Chemical Society 862 dx.doi.org/10.1021/tx200436n | Chem. Res. Toxicol. 2012, 25, 862−872

preventing the formation of mutations and ultimately carcino-genesis. Key upstream components of DDR signaling includethe protein kinases ATM and ATR. The activation of ATM/ATR results in the phosphorylation of a diverse array ofdownstream targets, including checkpoint kinases Chk1 andChk2, and histone H2AX.11−13 In counteracting DDR, thewild-type p53-induced phosphatase 1 (Wip1) plays an extensiverole. Recent evidence indicates that Wip1 is a critical regulatorof DNA damage signaling pathways. Wip1 releases cells fromcell cycle arrest once DNA repair is completed bydephosphorylating many constituents of the DNA damagesignaling network.14

In the present study, we examined the effects of PAH- andoxy-PAH-containing soil extracts on DNA damage signaling inhuman hepatocellular carcinoma HepG2 cells to improve thescientific basis for risk assessment of PAH-contaminated soils.We employed several markers of DNA damage signaling,including checkpoint kinases and Wip1 phosphatase. We foundthat the combination of prolonged Chk1 phosphorylation andinduced expression of Wip1 might act as potential markers forpersistent DNA damage induced by complex environmentalPAH samples. Furthermore, our results suggest a role ofmicroRNA (miR-16) in the regulation of Wip1 activity inresponse to PAH-containing soil extracts.

■ MATERIAL AND METHODSCaution: PAHs are carcinogens, and thus, experimental handling must

be carried out under special safety conditions, for example, those outlined inthe NCI guidelines.Reagents. Wortmannin, BP, and all other reagents were purchased

from Sigma-Aldrich (Stockholm, Sweden) unless otherwise stated.Dibenzo[a,l]pyrene (DBP) was purchased from AccuStandard Inc.(New Haven, CT). Cell Signaling Technology (Beverly, MA) suppliedthe following: Chk1 siRNA, antibodies to total Chk1, Chk1phosphorylated at Ser317, Chk2 phosphorylated at Thr68, andH2AX phosphorylated at Ser139. Antibodies against Cdk2, secondaryrabbit and mouse antibodies, and control siRNA were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA). Wip1 antibody wasobtained from Calbiochem (Darmstadt, Germany). Gibco (Invitrogen,Paisley, United Kingdom) supplied growth media, fetal bovine serum,and cell culture reagents. Electrophoresis reagents were obtained fromBio-Rad Laboratories (Hercules, CA).Soil Extracts. The soil samples used originated from four

contaminated sites in Sweden, including one former gas work site(Husarviken) and three former wood preservation sites (Holmsund,Forsmo, and Hassleholm), further described in Mattson et al.8 Thesamples were pretreated, extracted, and fractionated as describedpreviously.5,8 Briefly, the fractions with particles less than 2 mm wereextracted by pressurized liquid extraction (PLE) using hexane/acetone(1:1, v/v) at 150 °C and 14 MPa. The resulting extracts were

fractionated on open silica gel columns using solvents of increasingpolarity to produce three fractions from each extracta prefractioncontaining aliphatic compounds, a nonpolar fraction containing PAHs,and a semipolar fraction containing oxygenated PAHs (oxy-PAHs).The prefraction was discarded, while the other two were evaporatedinto small volumes of DMSO in preparation of the subsequent cellculture exposures.

Cell Culture and Treatment. Human hepatocellular carcinomaHepG2 cells were obtained from the American Type CultureCollection (Rockville, MD). These cells are well suited for thisstudy because of their metabolic competence and ability to activatePAHs and other mutagens.15 Cells were cultured in minimal essentialmedium supplemented with 10% fetal bovine serum, sodium pyruvate(1 mM), nonessential amino acids (0.1 mM), penicillin (100 units/mL), and streptomycin (0.1 mg/mL) and maintained at 37 °C in 5%CO2. For each experiment, 0.6 × 106 HepG2 cells were seeded into 35mm dishes, grown for 72 h, and subsequently exposed to soil extractsor solvent control (0.1−0.2% DMSO) for up to 48 h. Cells wereexposed to extracts by using a dose equivalent to 1 μM BP. Whenmentioned, cells were preincubated with 100 nM wortmannin for 1 hand then exposed to soil extracts for indicated time.

Comet Assay. The alkaline version of the comet assay wasperformed as described previously.16 In brief, exposed cells wereresuspended in low melting point agarose (0.75% w/v) and spreadover precoated (0.3% agarose) slides. Cells were lysed in cold lysisbuffer (1% Triton X-100, 2.5 M NaCl, 10 mM Tris, and 0.1 M EDTA,pH 10) for 1 h on ice, washed in PBS for 5 min, and incubated in coldalkaline solution (0.3 M NaOH and 1 mM EDTA, pH > 13) for 40min on ice followed by electrophoresis using the same buffer at 29 V(1.15 V/cm) for 30 min. Slides were neutralized in 0.4 M Tris-HCl(pH 7.5), washed in H2O, dried overnight, and fixed in methanol for 5min. After the slides were stained with ethidium bromide, at least 50comets were scored per treatment performed in triplicates using aLeica DMLB fluorescent microscope and Comet Assay 3 (PerceptiveInstruments Ltd., Haverhill, United Kingdom).

Western Blotting. After incubation with soil extracts, cells werewashed once with ice-cold PBS and scraped into 0.1 mL of IPB-7buffer (20 mM triethanolamine−HCl, pH 7.8, 0.7 M NaCl, 0.5%Nonidet P-40, and 0.2% sodium deoxycholate) with proteaseinhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mg/mL leupeptin,1 mg/mL pepstatin, 1 mM sodium fluoride, 1 mM sodiummetavanadate, 0.1 mg/mL trypsin inhibitor, and 1 mg/mL aprotinin).Collected lysates were frozen in liquid nitrogen and stored at −20 °Cbefore analysis. In each lysate, the total protein content was measured,and samples were subjected to 5, 10, or 15% SDS-PAGE gel. Separatedproteins were transferred to a PVDF membrane, and their levels weredetected using specific antibodies and visualized with enhancedchemiluminescence (ECL or ECL plus, Amersham GE HealthcareBiosciences AB, Uppsala, Sweden). The results were analyzed withImage J version 1.34s.

siRNA Transfection. Twenty-four hours after seeding, HepG2cells were transfected with Lipofectamine 2000 reagent (Invitrogen)

Figure 1. Examples of some PAHs (A) and oxy-PAHs (B) present in the soil extracts used in this study.

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and 50 nM Chk1 or control siRNA for 48 h, according to themanufacturer's instructions.RNA Purification and Real-Time RT-PCR. Total RNA was

prepared using the RNeasy Mini Kit (Qiagen, Hilden, Germany) andfurther treated with TURBO DNA-f ree (Ambion, Austin, TX). cDNAwas generated using 1 μg of total RNA and the High Capacity cDNAReverse Transcription kit (Applied Biosystems, Foster City, CA)according to protocol. Subsequently, quantification of gene expressionwas performed in duplicate using Maxima SYBR Green qPCR MasterMix (Fermentas, St. Leon-Rot, Germany) with detection on anApplied Biosystems 7500 Real-Time PCR System (Applied Bio-systems). The reaction cycles used were 95 °C for 2 min and then 40cycles at 95 °C for 15 s and 60 °C for 1 min followed by melt curve

analysis. The primer sequences are shown in Table 1. Quantification ofmiR-16 was performed using a miRCURY LNA Universal RT miRcDNA synthesis kit, SYBR Green master mix, Universal RT, and LNAPCR primer set for miR-16 and normalized to U6 snRNA (all fromExiqon, Vedbaek, Denmark). Relative gene expression quantificationwas based on the comparative threshold cycle method (2−ΔΔCt).Statistical Analysis. All data are provided as means ± SEMs. Two-

tailed Student's and one-way ANOVA with Bonferroni's test were usedfor determining the significance of perceived differences betweenexperimental samples; p < 0.05 was considered to be statisticallysignificant.

■ RESULTSIn our earlier study, we showed that PAH extracts fromcontaminated soil induced DNA damage responses that couldnot be attributed to or explained by the BP content in thesamples.8 This unexpected signaling was most likely due to theformation of persistent DNA damage. Using immunocyto-chemical staining, we showed prolonged H2AX phosphor-ylation in response to 0.01−1 mg soil/mL medium.8

Phosphorylation of H2AX at Ser139 (γH2AX) reflects anoverall genotoxic insult in cells, resulting from diverse type ofDNA damage, and plays a central role in DNA repairpathways.17

Persistent Phosphorylation of H2AX and CYP1A1Activation in Response to PAH-Containing Soil Extracts.In our present work, we studied the effects of industrial soilextracts on DNA damage signaling. The levels of PAHs andoxy-PAHs in these extracts are presented in Table 2. The extentof γH2AX in HepG2 cells in response to PAHs extracts wasassessed by Western blotting. The results confirmed andextended previous results concerning the effects caused byvarious PAH- and oxy-PAH-containing extracts correspondingto 1 μM BP. As can be seen in Figure 2A, both PAH and oxy-PAH extracts from different contaminated sites induced a

stronger histone phosphorylation at 24 and 48 h than 1 μM BPalone. In general, PAH-containing extracts evoked strongerhistone phosphorylation than the corresponding oxy-PAHextract. Moreover, γH2AX levels increased for up to 48 h inresponse to PAH fractions from Holmsund and Hassleholmand the oxy-fraction from Forsmo. In contrast, both fractionsfrom Husarviken showed reduced levels of γH2AX at 48 h ascompared to 24 h. Collectively, these findings suggest thatmixtures of PAHs and oxy-PAHs from soil can induce theformation of persistent DNA damage.Many of the biological effects of PAHs such as oxidative

stress and formation of DNA damage are mediated throughactivation of the aryl hydrocarbon receptor (AhR) and thesubsequent induction of cytochrome P450 monooxygenases(CYP450s).18 We assayed the activation of the AhR bymeasuring the induction of CYP1A1 using real-time RT-PCR(RT2-PCR). The results showed that 1 μM BP caused 21-foldinduction of expression, whereas PAH and oxy-PAH soilfractions corresponding to 1 μM BP resulted in much strongerinduction of CYP1A1 (Figure 2B). Typically, PAH ligands forAhR activation are planar and unsubstituted,19,20 andaccordingly, it has been shown that oxy-PAHs induceCYP1A1 less strongly than PAHs.21 Surprisingly, the soilfractions containing the oxy-PAHs evoked strong induction ofCYP1A1, and notably, the oxy-PAH fraction from Hassleholminduced 1200-fold increased mRNA level of CYP1A1 (Figure2B). This observation suggests synergistic effects on CYP1A1activation resulting from the interactions of various compoundsin complex mixtures and may have important implications forDNA damage formation. Importantly, it has been emphasizedthat different interaction effects of PAHs, including activationof the AhR, have to be considered in the risk assessment of thecomplex mixtures,22 which is entirely in line with our findings.

PAH-Containing Soil Fractions Induce DNA DamageSignaling Pattern Similar to Persistent DNA Damage.On the basis of the on results shown in Figure 2, we carried outfurther studies with the PAH-containing fraction fromHassleholm to monitor DNA damage signaling over time.Because Chk1, Chk2, and H2AX are key signal transducers inDNA damage signaling, we assayed their phosphorylation.Effects of soil extracts were compared with 3 μM BP and 0.1μM DBP. We have previously shown that treatment of HepG2cells with these doses slightly elevate levels of cyclin-dependentkinase inhibitor 1A (p21) protein, suggesting cell cycle arrest orapoptosis.8,23 However, we observed no significant activation ofapoptosis as assessed by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage (results not shown).We observed a much stronger activation of Chk1 as

compared to Chk2 in response to all treatments (Figure 3A).

Table 1. Primer Sequences Used for RT2-PCR

gene sequence

CYP1A1 F: 5′-CACCATCCCCCACAGCAC-3′R: 5′-ACAAAGACACAACGCCCCTT-3

DDB2 F: 5′-TCATGCTCTGGAATTTTGGCA-3′R: 5′-ACTGGTTGGTATTGAGAGGGTTA-3′

P21 F: 5′-TGTCCGTCAGAACCCATGC-3′R: 5′-AAAGTCGAAGTTCCATCGCTC-3′

PCNA F: 5′-GCGCTAGTATTTGAAGCACCAA-3′R: 5′-CGATCTTGGGAGCCAAGTAGTA-3′

WIP1 F: 5′-TAGTGGTGCTCAGCCTGCAA-3′R: 5′-TCTGCGTCGCATGGTGAGT-3′

GADPH F: 5′-CGAGATCCCTCCAAAATCAA-3′R: 5′-TTCACACCCATGACGAACAT-3′

Table 2. Characteristics of the PAH-Containing Soil ExtractsCorresponding to 1 μM BPa

Holmsund Husarviken Hassleholm Forsmo

∑PAHs (μg) 49.1 9.5 19.4 25.6∑oxy-PAHs (μg) 7.9 1.4 1.9 3.8∑16 PAHsb (μg) 47.1 8.8 18.5 24.1∑cPAHsc (μg) 9.9 3.8 5.7 5.7TEQd 2.0 0.9 1.1 1.3

aPAH content recalculated from ref 5. bSixteen U.S. EPA priorityPAHs. cCarcinogenic PAHs according to IARC.1 dToxic equivalentsquota (TEQ) calculated using toxic equivalent factor (TEF) valuesfrom ref 60.

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In the presence of BP, pChk1 levels increased up to 16 hfollowed by a gradual decrease up to 48 h after treatment. Inresponse to DBP, the extent of pChk1 was more than 3-foldgreater than in response to BP at 16 h (∼29-fold relative tocontrol), and the high pChk1 levels were maintained up to 48h. In response to the Hassleholm PAH fraction, the amount ofpChk1 showed a time-dependent increase with about 12.5-foldhigher levels 48 h after treatment when compared to control(Figure 3B).The peak of γH2AX level in the presence of BP was observed

at 16 h and was followed by a gradual, however statisticallynonsignificant, decline with time, suggesting that damage isbeing repaired (Figure 3C). In contrast, the level of γH2AX inresponse to DBP 16 h after treatment was 6-fold in comparisonto control and stayed at this elevated level up to 48 h. Althoughthe γH2AX signal in response to the PAH fraction wasapproximately three times weaker as compared to DBP, itshowed a similar trend with sustained levels of activation.To confirm that the observed sustained DNA damage

signaling was consistent with persistent DNA damage, thelevels of DNA damage were measured using comet assay. Theresults showed sustained DNA damage levels at 48 h post-treatment as compared with 6 h in cells exposed to DBP andHassleholm PAH fraction. In contrast, we observed reducedlevels of DNA damage in cells treated with BP after 48 h(Figure 3D). Together, these results indicate that environ-mental PAH extracts, similar to DBP, induce more persistentDNA damage, which activates sustained activation of Chk1 andH2AX.DNA Damage Signaling through Chk1 Regulates the

Levels of DNA Damage. pChk1 and γH2AX participate inDNA damage recognition and repair, and the high levelsobserved in this study indicate activated DNA damagesignaling. To study the relationship between Chk1 and

γH2AX, we inhibited DNA damage signaling by wortmannin.Wortmannin is a sterol-like fungal metabolite that at lownanomolar concentrations irreversibly inhibits the kinaseactivities of ATM and ATR.24 Both kinases are cruciallyinvolved in DNA repair, and they activate several targetsproteins, including checkpoint kinases and H2AX. As shown inFigure 4A, PAH-induced pChk1 was also strongly diminishedand accompanied by enhancement of H2AX phosphorylation.Also, in the presence of wortmannin, the total amount of Chk1was decreased. In contrast to pChk1, wortmannin had a veryweak effect, if any, on phosphorylation of Chk2. Similarincreased levels of γH2AX were obtained by depletion of Chk1with siRNA and subsequent treatment of HepG2 cells withHassleholm PAH fraction (Figure 4B). Results in Figure 4clearly show that lack of active Chk1 corresponds to higherlevel of DNA damage induced by PAHs, as shown by increasedphosphorylation of H2AX.

Wip1 Expression Is Induced by BP, DBP, and PAH-Containing Soil Fractions. Phosphorylation status of keyproteins involved in DNA damage signaling has recently beenshown to be regulated by Wip1.14,25 Wip1 releases cells fromcell cycle arrest through dephosphorylation of proteins, such asH2AX, ATM, and Chk1.26−28 Exposing HepG2 cells to BP orDBP resulted in increased Wip1 protein levels around 16 hpost-treatment, followed by a decline. Exposure to HassleholmPAH fraction resulted in a comparable increase in Wip1 proteinlevels but at later time points (Figure 5A). Subsequently,mRNA levels of Wip1 in response to PAH treatment weremeasured by RT2-PCR. As can be seen in Figure 5B, 3 μM BPled to a significant but transient up-regulation at 12 h, whileexposure to 0.1 μM DBP resulted in significantly increased andsustained mRNA levels from 12 to 48 h. Interestingly, the soilextract displayed a time-dependent response similar to thecombination of BP and DBP (Figure 5C). An initial significant

Figure 2. Prolonged H2AX phosphorylation and CYP1A1 induction in HepG2 cells in response to soil PAH- and oxy-PAH extracts. (A) CellularγH2AX levels after exposing cells to BP (1 and 5 μM) or PAH extracts corresponding to 1 μM BP for 24 or 48 h. Cdk2 was used as a loadingcontrol. (B) CYP1A1 mRNA levels after exposing cells to BP (1 μM) or PAH extracts corresponding to 1 μM BP for 24 h. Abbreviations as follows:C, control; Fo, Forsmo; Hu, Husarviken; Ha, Hassleholm; and Ho, Holmsund. Results are presented as means ± SEMs, and experiments wereperformed at least twice in duplicate.

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but transient induction of Wip1 expression at 6 h was followedby a repression to background levels at 12 and 16 h and asecond significant induction at 24 and 48 h. These resultssuggest that induction of Wip1 might indicate the presence ofpersistent DNA damage.Chk1, Wip1, and miR-16 as Markers for Irreparable

DNA Damage. On the basis of our results, we investigatedwhether prolonged phosphorylation of Chk1, γH2AX, and

Wip1 expression is a general response to PAH-containing soilextracts. As shown in Figure 6A, PAH and oxy-PAH extractsfrom Holmsund and Husarviken caused similar effects on Chk1phosphorylation as the Hassleholm fraction. While bothfractions caused a time-dependent increase in phosphorylationof Chk1, the PAH-containing extracts induced higher levels ofpChk1 than the corresponding oxy-PAH fractions. Wip1protein levels showed a similar response as shown in Figure

Figure 3. Time-dependent induction of γH2AX. pChk1, pChk2, and DNA damage in HepG2 cells exposed to BP, DBP, or soil PAH extract. Cellswere treated with 3 μM BP, 0.1 μM DBP, or Hassleholm PAH extract corresponding to 1 μM BP for times indicated. (A) Cellular levels of Chk1,Chk2, and H2AX phosphorylation. Densitometry analysis of pChk1 and γH2AX is shown in B and C, respectively. Plotted values are means fromduplicate experiments, one of which is represented in A. Cdk2 was used as a loading control. *p < 0.05 as compared with 3 h samples by one-wayANOVA. (D) Levels of DNA damage as determined by Comet assay. Results are presented as means ± SEMs, and experiments were performed intriplicate. *p < 0.05 as compared to vehicle control by Student's t test.

Figure 4. DNA damage signaling through Chk1 regulates the levels of DNA damage. (A) HepG2 cells were treated with 100 nM wortmannin for 1 hand then exposed to soil extracts containing PAH or oxy-PAH fractions from Holmsund, Husarviken, and Hassleholm corresponding to 1 μM BP for24 h. (B) HepG2 cells were transfected with control (−) or Chk1 siRNA (+) and thereafter treated for 24 h with PAH extract from Hassleholm.Densitometry analysis is shown in B (right). Cdk2 was used as a loading control.

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Figure 5. Prolonged Wip1 expression is induced by DBP and PAH soil extracts. HepG2 cells were treated with 3 μM BP, 0.1 μM DBP, orHassleholm PAH extract corresponding to 1 μM BP for times indicated. Cellular Wip1 protein levels are shown in A, mRNA levels in response to BPand DBP are shown in B and in response to PAH extract in C. Cdk2 was used as a loading control. mRNA results are presented as means ± SEMs,and experiments were performed at least twice in duplicate. *p < 0.05 and ≥1.5-fold as compared to vehicle control by Student's t test.

Figure 6. Chk1, Wip1, and miR-16 as markers for irreparable DNA damage derived from PAH soil extracts. HepG2 cells were treated with soil PAHand oxy-PAH extracts corresponding to 1 μM BP. Levels of pChk1 at 24 and 48 h are shown in A, and plotted values for pChk1 (A, right) are meansfrom duplicate experiments, one of which is represented in A. *p < 0.01 as compared with 1 μM BP by one-way ANOVA. Wip1 protein levels at 24and 48 h are shown in B. Cdk2 was used as a loading control. Effects on Wip1 mRNA and miR-16 expression in response to PAH-enriched soilextracts are shown in C and D, respectively. Results are presented as means ± SEMs, and experiments were performed at least twice in duplicate. *p< 0.05 and ≥1.5-fold as compared to vehicle control by Student's t test.

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5A, with stronger and later induction of protein levels inresponse to PAH-containing soil extracts as compared to BP(Figure 6B). PAH and oxy-PAH fractions from Husarvikendisplayed reduced Wip1 protein levels at 48 h as compared to24 h. In accordance with DBP treatment (Figure 5B), Wip1mRNA expression levels were induced in response to PAH-containing soil extracts up to 48 h (Figure 6C). mRNA levelswere significantly induced in response to the PAH fractionsfrom Holmsund and Hassleholm at 24 and 48 h and the PAHfraction from Husarviken at 48 h. In response to oxy-PAHfractions, only the Hassleholm sample resulted in statisticallysignificant increased expression levels. These results furtherconfirm that complex PAH extracts from soil induce sustainedDNA damage signaling similar to DBP via Chk1 and Wip1.miRs are post-transcriptional regulators, and miR-16 has

recently been shown to be an important negative regulator ofWip1 in response to DNA damage, reducing the protein levelsas compared to mRNA expression level.29 To investigate ifWip1 protein levels are regulated by miR-16, we measured theexpression levels of the latter. As can be seen in Figure 6D,exposure to BP induced miR-16 expression at 24 h; however,this was not statistically significant (>1.5-fold, p = 0.19). At 48h, the expression level was similar to solvent control. Treatmentwith DBP resulted in an initial reduction in expression levels.All PAH soil fractions, except Husarviken samples, displayed aweaker expression pattern as compared to BP. In contrast,treatment with Husarviken PAH and oxy-PAH samples resultedin statistically significant increased expression of miR-16 at 24and 48 h, respectively.To further assess the effects on activation of DNA repair, the

effects of BP, DBP, and Hassleholm soil samples on the geneexpression levels of the DNA repair proteins damaged DNAbinding protein 2 (DDB2), proliferating cell nuclear antigen(PCNA), and p21 were assessed. Using RT2-PCR, we foundtime-dependent induction of all three genes in response to BPand DBP, while exposing cells to PAH- and oxy-PAH-containing extracts did not significantly affect the mRNA levelsof DDB2 and PCNA but significantly induced p21 mRNAlevels at 6 and 12 h (Figure 7A−C). These results suggest thatactivation of DNA repair at some level is either ineffective orinhibited in response to PAH soil extracts.

■ DISCUSSIONThe results presented here show that complex mixtures ofPAHs in soil extracts may cause DNA damage signalingconsistent with persistent DNA damage. The signaling inHepG2 cells exposed to PAH-containing soil extracts wassimilar to that of cells exposed to DBP, a highly carcinogenicPAH known to produce persistent DNA damage.30−32 Theresponse involved prolonged activation of H2AX, Chk1, andWip1 and is in agreement with results from comet assayshowing sustained DNA damage levels in response to DBP andsoil PAH extract. Furthermore, blocking the signaling usingspecific inhibitors and siRNA showed the important role ofsignaling through Chk1 for the level of DNA damage.Discrepancies between mRNA and protein levels of Wip1 inresponse to BP, in parallel with increased miR-16 levels, suggesta role of miR-16 in the regulation of DNA damage signaling inresponse to PAHs.All PAH-containing soil extracts tested (PAH and oxy-PAH

fractions normalized to 1 μM BP) induced γH2AX, and mostextracts displayed a more potent and persistent activation ofH2AX as compared to 1 μM BP. This is in agreement with our

earlier results showing that persistent γH2AX might indicateirreparable DNA damage.33 The measurement of phosphor-ylation of H2AX at Ser139 (γH2AX) has previously been usedas a biomarker for induction of DNA damage, including doubleand single strand breaks and the subsequent activation of DNArepair.34,35 However, it has been implied that γH2AX may act asa general biomarker of DNA damage rather than being specificfor strand breaks.36 Furthermore, it has recently been shownthat stable adducts derived from PAH diol epoxide metabolitesalso are capable of inducing γH2AX in the absence of strandbreaks.37 Exposure to PAHs causes a mixture of DNA damageincluding single and double strand breaks as well as stable bulkyDNA adducts.9 Penning and co-workers have recently shownthat PAHs can cause DNA strand breaks via oxidativemetabolism of PAHs by the human aldo-keto reductasesresulting in redox cycling amplifying the production of reactiveoxygen species.38,39 The γH2AX response pattern induced bysoil extracts was similar to that of DBP, possibly an effect frominteractions between the different PAHs present in the

Figure 7. Lack of induction of DDB2, PCNA, and p21 in response toPAH soil extracts. Cells were exposed for up to 48 h to 3 μM BP, 0.1μM DBP, or Hassleholm PAH fractions corresponding to 1 μM BPand PCNA (A), DDB2 (B), and p21 (C) mRNA levels were assayed.Results are presented as means ± SEMs, and experiments wereperformed at least twice in duplicate. *p < 0.05 and ≥1.5-fold ascompared to vehicle control by Student's t test.

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mixture.8 The similarity to DBP indicates a high carcinogenicpotential since DBP is the most mutagenic and carcinogenicPAH identified to date and is about 100 times more potent of acarcinogen as compared to BP.40 An explanation for its highbiological activity is the ability to cause persistent DNA damagethat escapes recognition and subsequent repair.30−32

Similar to the activation of H2AX, exposure to DBP andPAH soil extracts showed maintained or increasing levels ofChk1 phosphorylation over time, while BP caused a transientsignaling activation. These results were in agreement with DNAdamage levels assessed by comet assay. Activation of Chk1 byphosphorylation (pChk1) is mainly performed by ATR inresponse to single strand breaks41 and bulky adducts, such asthose derived from PAHs.42,43 Accordingly, our results suggestthat single strand breaks and stable DNA adducts are the mostformed DNA damage induced by PAH soil extracts; otherwise,we would expect a more evident activation of Chk2 through theaction of the more double strand break-specific ATM.41 Wefurther investigated the role of Chk1 for DNA damage signalingby pretreating cells with wortmannin. The results showedsignificantly lower levels of pChk1, confirming that ATM/ATRare responsible for the activation of Chk1. Additionally,pretreatment with wortmannin resulted in increased levels ofγH2AX. However, Yan et al. recently reported the same effectin HeLa cells exposed to BP and discussed that this might bedue to incomplete inhibition of ATM/ATR by wortmannin.44

The effect on γH2AX was further confirmed by siRNA of Chk1.Interestingly, it has previously been shown that activated Chk1can further phosphorylate and thereby activate Rad51 andBRCA2, proteins involved in homologous recombinationrepair, in response to camptothecin or UV exposure.45,46 Theresults presented here suggest a possible role for Chk1 in thecellular response to PAH exposure in activating and regulatingDNA repair. These findings warrant further investigation.These results indicate that environmental PAH mixtures induceDBP-like responses in Chk1 and H2AX activation, evoking atypical pattern of persistent or slowly repaired DNA damage.Chk1 and H2AX phosphorylation were then compared with

the levels of Wip1. Wip1 has been shown to dephosphorylateseveral key proteins involved in the checkpoint-signalingcascade in response to DNA damage, including H2AX,26,47,48

ATM,25,27 p53,28 and Chk1,25,28 thereby promoting checkpointinhibition and restoring chromatin structure once DNA damageis repaired. Consequently, premature activation of Wip1 mightinactivate the checkpoint possibly contributing to progressionthrough the cell cycle without proper DNA repair. Thesepotential oncogenic properties are further supported by theamplification and overexpression of PPM1D, the gene encodingWip1, in a number of human cancers including primary breasttumors.49,50

In response to BP, Wip1 mRNA levels were transiently up-regulated, while DBP caused a more sustained expression.Interestingly, mRNA levels in response to the Hassleholm PAHfraction displayed an expression pattern similar to thecombination of BP and DBP. This is in agreement with earlierresults in HepG2 cells in response to PAH treatment51 and ismost probably due to the regulation of p53, its maintranscriptional regulator, which has been shown to have apulsing activation pattern.52,53 PAH and oxy-PAH fractionsinduced high Wip1 expression levels at 24 and 48 h and againshowed a similar pattern to DBP. Surprisingly, the induction ofgene expression was not reflected in protein levels.Furthermore, Wip1 protein levels displayed a stronger and

later induction in response to soil samples as compared to BP.The delayed response is in agreement with more prolongedDNA damage signaling observed via γH2AX and pChk1 andthe sustained DNA damage levels as determined by cometassay. The observed discrepancy between mRNA and proteinlevels suggests that the activity and protein level of Wip1 mightbe regulated mainly at the level of translation.The role of translation was furthermore supported by the

results with miR-16. Zhang et al. recently showed that miR-16has an important role in regulating Wip1 in the DNA damageresponse specifically targeting the mRNA of Wip1 and therebyregulating the activity of Wip1.29 We found that BP inducedmiR-16 expression at 24 h, while treatment with DBP and soilextracts resulted in lower levels of miR-16. These resultscorrelated with Wip1 protein levels. The exception was thesamples from Husarviken, which contain relatively low levels ofPAHs (Table 2). Both PAH and oxy-PAH fractions fromHusarviken resulted in weaker DNA damage signalingcorrelating to rapid decline in phosphorylation of Chk1 andH2AX and decrease of Wip1 protein levels. Furthermore,parallel to induced Wip1 mRNA by the PAH fraction, miR-16was significantly induced. Taken together, our findingsdemonstrate for the first time that DNA damage signaling inresponse to PAH treatment induces gene expression of Wip1.Furthermore, the discrepancy between mRNA and proteinlevels in response to BP, in parallel with the increased miR-16levels, suggests a role of miR-16 in the regulation of DNAdamage signaling.The finding that PAH soil extracts caused persistent DNA

damage suggests that activation of DNA repair at some level iseither ineffective or inhibited. This was further confirmed bythe observed lack of induction of the two DNA repair genes,DDB2 and PCNA, in response to PAH extracts. In addition,expression levels of p21 were only transiently induced inresponse to the same treatment. DDB2 plays an important rolein the recognition of bulky DNA adducts54 and has been shownto be induced in response to PAH treatment.55 PCNA isinvolved in nucleotide and base excision repair, the two DNArepair mechanisms responsible for repairing DNA damagederived from PAHs.56,57 P21 is a regulator of the cell cycle, andits induction has been shown to correlate with DNA damageinduced by BP in HepG2 cells.58 Because all genes are p53-responsive, these results support our earlier findings showingthat 3 μM BP and 0.1 μM DBP activate p53 responses inHepG2 cells,23 whereas soil PAH fractions corresponding to 1μM BP to the same extent do not.8 The observed effects areprobably due to interactions and are in line with data showingthat complex mixtures are more potent carcinogens thanassumed, and the responsible mechanism warrants furtherinvestigation.Risk levels are often difficult to determine due to the

presence of mixtures of PAHs in contaminated soil. BP is oftenused as an indicator of carcinogenic PAHs in complex mixtures,although several studies have shown that this may under-estimate the cancer potency.7,59 As shown in Table 2, the TEQvalues varied between 0.9 and 2.0 for the different soil samples.Comparing these values to our results clearly demonstrates thatTEQ values do not correlate with the effect of the PAH-containing soil extracts on the studied DNA damage signalingproteins/genes. This is further confirmed by earlier studiesclearly showing that neither the BP concentration nor theavailable TEF scales are sufficient for predicting the risk ofcomplex PAH mixtures on DNA damage signaling.8,22 Our

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results demonstrate that complex PAH mixtures and oxy-PAHsthat have never been tested in animal experiments may causeunpredictable and persistent DNA damage signaling responseswith high potency. Hence, our study suggests that thedevelopment of new biotests reflecting the carcinogenicpotency is required. This study also underscores the need tounderstand how PAHs interact. The end points studied herecould be useful for evaluating the carcinogenic potency ofPAHs in contaminated soil and should be included in futurebiotests.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +46-8-52487796. Fax: +46-8-343849. E-mail: kristian.dreij@ki.se.

Author Contributions§These authors contributed equally to this manuscript.

FundingThis work was supported by Cancer och Allergifonden awardedto U.S., EU's European Regional Development Funds viaNorthern Sweden Soil Remediation Center (MCN) awarded toS.L., and European Commission Marie Curie IRG awarded toK.D.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe gratefully acknowledge Dr. Hanna Karlsson for skillfulassistance with performing the comet assay. We thank ProfessorBengt Jernstrom for valuable comments on the manuscriptsand helpful discussions.

■ ABBREVIATIONSAhR, aryl hydrocarbon receptor; BP, benzo[a]pyrene; Chk1,checkpoint kinase 1; Chk2, checkpoint kinase 2; DBP,dibenzo[a,l]pyrene; DDB2, damaged DNA binding protein 2;H2AX, histone H2AX; IARC, International Agency of Researchon Cancer; miR, microRNA; oxy-PAHs, oxygenated polycyclicaromatic hydrocarbons; P21, cyclin-dependent kinase inhibitor1A (CDKN1A); PAHs, polycyclic aromatic hydrocarbons;PCNA, proliferating cell nuclear antigen; TEF, toxicityequivalence factor; TEQ, toxicity equivalence quota; Wip1,wild-type p53-induced phosphatase 1

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