The effect of dibenzo[a,l]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction...

Mutation Research 471 (2000) 57–70

The effect of dibenzo[a,l]pyrene and benzo[a]pyrene onhuman diploid lung fibroblasts: the induction of DNA adducts,

expression of p53 and p21WAF1 proteins and cell cycle distribution

Blanka Binková∗, Yves Giguère, Pavel Rössner Jr., Miroslav Dostál, Radim J. ŠrámLaboratory of Genetic Ecotoxicology, Regional Institute of Hygiene of Central Bohemia, c/o Institute of Experimental Medicine,

Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic

Received 23 May 2000; received in revised form 8 August 2000; accepted 9 August 2000

Abstract

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but thedetailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene(DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lungfibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by32P-postlabelling, the expression ofp53 and p21WAF1 proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNAadduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA bindingwas detectable even at the lowest concentration used (24 h exposure, 0.01mM for both PAHs). The highest DNA adduct levelswere observed after 24 h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced byDB[a,l]P was∼ 8–10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at1mM and for DB[a,l]P at 0.1mM: 237± 107 and 2360± 798 adducts/108 nucleotides, respectively). The induction of p53and p21WAF1 protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53(R = 0.832,P < 0.001 andR = 0.859,P < 0.001, for DB[a,l]P and B[a]P, respectively) and p21WAF1 levels (R = 0.808,P < 0.001 andR = 0.797,P = 0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and thephase of cell growth. The results showed that a detectable increase of p53 and p21WAF1 proteins (≥1.5-fold as comparedwith controls) requires a minimal DNA adduct level of∼ 200–250 adducts/108 nucleotides for both PAHs tested and suggestthat the level of adducts rather than their structure triggers the p53 and p21WAF1 responses. The cell cycle was altered after12–16 h of treatment, and after 24 h of exposure to 0.1mM DB[a,l]P in growing cells, there was∼ 24% increase in S phasecells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0mM B[a]P resulted in moresubtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53and p21WAF1 without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed inour study is beneficial for the survival of the cells remains to be further established. © 2000 Elsevier Science B.V. All rightsreserved.

Abbreviations:B[a]P, benzo[a]pyrene; BgChDE, benzo[g]chrysene-11,12-dihydrodiol-13,14-epoxide;BPDE, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[±]; DB[a,l]P, dibenzo[a,l]pyrene; E-MEM, Eagle’s-minimal essential medium;

∗Corresponding author. Tel.:+4202-475-2675; fax:+4202-475-2785.E-mail address:[email protected] (B. Binkova).

1383-5718/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S1383-5718(00)00111-X

58 B. Binkova et al. / Mutation Research 471 (2000) 57–70

EROD, 7-etoxyresorufin O-deethylase; G2/M, G2/mitosis; HEL, human diploid lung fibroblasts; LDH, lactate dehydrogenase; PAHs,polycyclic aromatic hydrocarbons; PEI, polyethylene imine; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline solution; TBS-T,TBS-Tween; TLC, thin layer chromatography

Keywords:PAHs; Human lung fibroblasts; DNA adducts; p53 and p21WAF1 proteins; Western blotting; Flow cytometry

1. Introduction

A ubiquitous class of organic pollutants adsorbedto respirable air particles (aerodynamic diameter<

2.5mm), such as polycyclic aromatic hydrocarbonsand their derivatives, may be considered to pose ahuman health risk as a consequence of continuouslow-level exposure to such complex mixtures [1–3].These organic compounds are products of the incom-plete combustion of gasoline, diesel fuel, coal and oil,as well as products of chemical and photochemical re-actions in the environment, and they have both muta-genic and carcinogenic activities [4–6]. Many singlePAHs are carcinogenic in rodent bioassays, and theyare suspected carcinogens in humans [7,8]. PAHs ex-hibit their biological effects through metabolic acti-vation by cytochrome P450s enzymes to electrophilicspecies capable of reacting with nucleophilic sites ofDNA to form adducts. The formation and persistenceof carcinogen-DNA adducts are believed to be criti-cal events for the initiation of neoplasia in target cells.It is assumed that the risk of mutations arising fromDNA adducts does not depend only on the adduct levelbut also on their mutagenic potential as well as on theDNA sequence of neighboring adducts [9,10].

Nowadays, the modulation of cell survival and celldeath pathways is believed to be a central processin carcinogenesis [11]. The DNA damage response iscomplex and occurs in concert with transcriptional in-duction of many proteins. The tumour suppressor genep53, known as the most common target for mutationsin human cancer [12,13], is believed to be critical formaintaining genomic integrity. Strong evidence sup-ports the notion that the sequence of events mediatedby p53 protein follows by two major pathways: cell cy-cle arrest and apoptosis [14,15]. The cyclin-dependentkinase inhibitor p21WAF1 gene product [16–18] is animportant mediator of the p53-induced cell cycle ar-rest to delay transit from G1 to S phase and/or from G2to M phase, thus preventing the effect of DNA dam-age on the gene functions. If DNA repair is not suc-

cessfully accomplished, p53 may promote the deathof affected cells by triggering apoptosis.

The role of p53 and p21WAF1 proteins in responseto environmentally relevant chemicals and their com-plex mixtures has not been thoroughly investigated.Attention has mostly been directed towards agentsused clinically in cancer therapy [11]. Only a few re-cent studies have dealt with the DNA damage causedby single PAHs or their DNA-reactive metabolites andthe induction of p53 and/or p21WAF1 proteins. Thestudy of Bjelogrlic et al. [19] was the first to show thatDNA adducts formed in vivo in mouse skin after topi-cal treatment with benzo[a]pyrene are associated withincreased levels of p53 protein. Similarly, Ramet et al.[20] found a correlation between p53 expression andDNA adducts following B[a]P-treatment of breast car-cinoma MCF-7 and lung carcinoma A-549 cell lines.Venkatachalam et al. [21] showed that p53 proteinlevels were increased in a dose- and time-dependentmanner after B[a]P-diol epoxide (BPDE) treatment ofvarious repair-proficient and repair-deficient humancells, but the level of DNA damage was not measuredsimultaneously. Stierum et al. [22] also observed p53accumulation in in vitro stimulated human peripheralblood lymphocytes after treatment with anti-BPDE.The induction of p53 protein in cell cultures was alsoproposed as a new approach for genotoxicity testingof potential chemical carcinogens [23,24].

The results obtained so far for the induction ofthe cyclin-dependent kinase inhibitor p21WAF1 aresomewhat contradictory. Dipple et al. [10] proposeda so-called ‘stealth’ property of PAHs according tothe results they obtained with mammary carcinomaMCF-7 cells exposed to several PAHs’ metabolitesthat showed accumulation of p53 without any induc-tion of p21WAF1 [25]. Kaspin and Baird [26] and Luchet al. [27], using the same carcinoma cell line MCF-7,showed that exposure of cells to reactive metabo-lites of B[a]P or DB[a,l]P induced p53 protein levelsthat correlated with DNA adduct levels. In contrastwith previous studies, they also detected an increase

B. Binkova et al. / Mutation Research 471 (2000) 57–70 59

of p21WAF1 protein levels. The contradictory resultsobserved may reflect the various experimental ap-proaches as well as the sensitivity of the assays used.

The response of cells to genotoxic damage medi-ated by p53 expression appears to be also dependenton the cell types investigated [28]. Therefore, themain objective of the present study was to assess thecellular response of human lung diploid fibroblasts toexposure to two main representatives of carcinogenicPAHs — B[a]P and DB[a,l]P. B[a]P is one of thebest-characterised members of the family of PAHspresent in environmental samples. DB[a,l]P, the mostpotent carcinogen ever tested [29,30], has been alsofound in soil and sediment samples [31]. We usednormal (non-mutated) human diploid cells that arecapable of activating parent PAHs to reactive metabo-lites to better understand the mechanism of actionthat may be responsible for the carcinogenic potentialof these compounds in humans. We simultaneouslyassessed multiple endpoints such as DNA adductsby 32P-postlabelling, p53 and p21WAF1 proteins bywestern blotting and cell cycle distribution by flowcytometry. The cellular response to both B[a]P andDB[a,l]P was followed in a dose- and time-dependentmanner in growing cells as well as at near-confluence.

2. Materials and methods

2.1. Chemicals

Spleen phosphodiesterase was purchased fromBoehringer-Mannheim; ribonuclease A and T1, pro-teinase K, micrococcal nuclease, nuclease P1, pro-pidium iodide, actinomycin D and protein assay kit(No.5656) from Sigma; T4 polynucleotide kinase fromUSB Co; polyethylene-imine cellulose TLC plates(0.1 mm) from Macherey-Nagel; B[a]P (99% pure)from Supelco, Inc; DB[a,l]P from Midwest ResearchInstitute (Kansas City, MO); primary antibodies Ab-6(clone DO-1) and Ab-1 (clone EA10) and p53 westernblotting standard from Oncogene Research Products(Cambridge, MA, USA); secondary anti-mouse IgG(NA931), ECL protein molecular weight markers(RPN2107), streptavidin-horseradish peroxidase con-jugate (RPN1231), HybondTM C-pure membranes,chemiluminiscence detection reagents andg-32P-ATP(3000 Ci/mmol, 10mCi/ml) were purchased fromAmersham Pharmacia Biotech; colchicine from Fluka

and phosphate buffered saline (CellWASH) fromBecton–Dickinson. All other chemicals and solventswere of HPLC or analytical grade.

2.2. Cell cultures and their treatments

Human embryonic lung diploid fibroblasts (HEL;passage levels 20–25, Sevapharma, a.s., Prague) weregrown in minimal essential medium E-MEM (Sevap-harma, a.s., Prague) supplemented with 10% bovineserum and 100 U/ml penicillin. Cells were seeded inplastic cell culture flasks (75 cm2 for protein and DNAadduct analysis; 25 cm2 for flow cytometry) at an ini-tial concentration of∼17 000 cells/cm2 and incubatedat 37◦C in 5% CO2. The medium was replaced withfresh medium before treating cells with B[a]P andDB[a,l]P, both of them dissolved in DMSO. The finalconcentration of DMSO did not exceed 0.1% of totalincubation volume. Control samples included in eachincubation set were treated with DMSO alone. Fordose-response experiments the cells were exposed for24 h to different concentrations of B[a]P or DB[a,l]Pat the 5th or 7th days after seeding. For time-responseexperiments the cells were exposed to a final concen-tration of either 1.0mM B[a]P or 0.1mM DB[a,l]P. Allincubation sets were repeated at least three times withduplicate samples. At the end of treatment the cellswere always examined microscopically for morpho-logical changes, then harvested by trypsinization. Thecells from 75 cm2 culture flasks for DNA adduct andprotein analysis were washed three times in PBS, re-suspended in 3 ml PBS and divided into two aliquots(2 ml for DNA isolation; 1 ml for preparation of sam-ples for western blotting). The cell pellets were storedfrozen at−20◦C until processing the following day.The handling of cells for flow cytometry is further de-scribed below.

The viability of the cells treated with both B[a]Pand DB[a,l]P was >95% at the time of harvesting,as determined by a trypan blue exclusion assay. Thedoses used did not show any cytotoxicity as measuredby lactate dehydrogenase activity assay (ELISA kit,Boehringer Mannheim).

2.3. DNA isolation and32P-postlabelling

The cell pellets were homogenised in a solutionof 10 mM Tris-HCl, 100 mM EDTA and 0.5% SDS,


pH 8.0. DNA was isolated using RNAses A andT1 and proteinase K treatment followed by phe-nol/chloroform/isoamyl alcohol extraction [32]. DNAconcentration was estimated spectrophotometricallyby measuring the UV absorbance at 260 nm. DNAsamples were kept at−80◦C until analysis.

32P-postlabelling analysis was performed as previ-ously described [2,33]. Briefly, DNA samples (6mg)were digested by a mixture of micrococcal endonu-clease and spleen phosphodiesterase for 4 h at 37◦C.The nuclease P1 was used for adduct enrichmentaccording to an interlaboratory trial standardised pro-tocol [34]. The labelled DNA adducts were resolvedby two-directional thin layer chromatography on10 cm× 10 cm PEI-cellulose plates. Solvent systemsused for TLC were the following: D-1: 1 M sodiumphosphate, pH 6.8; D-2: 3.8 M lithium formate, 8.5 Murea, pH 3.5; D-3: 0.8 M lithium chloride, 0.5 M Tris,8.5 M urea, pH 8.0. Autoradiography was carried out at−80◦C for 1–24 h. The radioactivity of distinct adductspots was measured by liquid scintillation counting.To determine the exact amount of DNA in each sam-ple, aliquots of the DNA enzymatic digest (0.5mgof DNA hydrolysate) were analysed for nucleotidecontent by reverse-phase HPLC with UV detection,which simultaneously allowed for controlling the pu-rity of the DNA. DNA adduct levels were expressedas adducts per 108 nucleotides. A B[a]P-derived DNAadduct standard (4± 0.6 adducts/108 nucleotides)was run in triplicate in each postlabelling experimentto control for interassay variability and to normalisethe calculated DNA adduct levels.

2.4. Western blotting of p53 and p21WAF1 proteins

The cell pellets were lysed in an appropriate vol-ume of sample buffer (0.063 M Tris-HCl pH 6.8,2% SDS, 5% 2-mercaptoethanol, 10% glycerol) con-taining the protease inhibitors leupeptin (2.5mg/ml),aprotinin and pepstatin (both of them 1.25mg/ml) andphenylmethylsulfonyl fluoride (PMSF, 2.5mg/ml).The samples were heated to 100◦C for 5 min andcentrifuged at 5000 rpm. for 10 min. The proteinconcentration was determined by the Peterson mod-ification of the micro-Lowry method using Folin& Ciocalteau’s phenol reagent (Sigma kit). Thealiquots of these samples were stored at−80◦C untilanalysis.

Prior to western blotting, the samples were di-luted with an appropriate volume of sample buffer(coloured with bromophenol blue) to contain 15mgprotein/12ml and heated to 100◦C for 5 min. The pro-teins were separated by 12.5% SDS-polyacrylamidegel electrophoresis on 0.75 mm mini-gels and trans-ferred to HybondTM C-pure membrane (BIORADMini-Protean and Trans-Blot systems). The mem-branes were blocked for 1 h at room temperaturewith Tris-buffered saline (TBS; 10 mM Tris, 150 mMNaCl, pH 7.4) supplemented with 5% non-fat dry milkpowder. After repeated washing with TBS-Tween so-lution (TBS-T; 0.1% Tween-20) they were incubatedovernight in a cold room at 8◦C with primary anti-bodies for p53 (Ab-6, clone DO-1; 0.025mg/ml) orfor p21WAF1 (Ab-1, clone EA10; 0.25mg/ml). Bothof the primary antibodies were diluted in TBS-T sup-plemented with 1% non-fat dry milk powder. Afterincubation with the primary antibodies, the blots wererepeatedly washed with TBS-T and then incubated for1 h at room temperature with the secondary antibody(anti-mouse IgG conjugated to horseradish peroxi-dase) diluted 1:2000 in TBS-T supplemented with 1%non-fat dry milk powder. The membranes were thenrepeatedly washed with TBS-T, and the proteins weredetected using an enhanced chemiluminiscence tech-nique (Amersham) and several exposures of BioMaxMR-2 films (Kodak). p53 western blotting standard(Oncogene) was used as a positive control for p53protein detection and ECL protein molecular weightmarkers (Amersham) for p21WAF1. The intensity ofbands was quantified with an image acquisition andanalysis system (GDS-8000 Chemi System, UVP,Inc., CA, USA).

2.5. Flow cytometry

HEL cells were grown and exposed to B[a]P andDB[a,l]P under the same conditions as for DNA adductanalyses. Treatment with 1.0 nM actinomycin D (usedas a positive control for G1 arrest) was performed totest the capacity of HEL cells to arrest in G1 phasein response to DNA damage. After cell treatments,the cells (total amount∼1 − 2 × 106) were washedtwice with PBS and harvested with 0.05% trypsin in0.15% Na2EDTA. The cells were centrifuged, washedtwice in PBS and resuspended in 0.3 ml of 50% inac-tivated fetal calf serum in PBS. The cells were then


fixed in ice-cold 70% ethanol and stored overnightat 4◦C. The fixed cells were washed twice with PBSand incubated with propidium iodide (50mg/ml) andRNase A (10mg/ml) for 60 min at 37◦C. Data acquisi-tion was performed using an argon laser fluorescenceactivated cell analyser (FACSort, Becton-Dickinson)and the CELLQuest software version 1.2.2 providedby the manufacturer. Cell cycle analysis was done inModFitTM 2.0 (Verity Software House, ME). For eachsample, 16 000 events were acquired and, accordingto the guidelines for the analysis of DNA content,the coefficient of variance was<8.0%, the reducedchi-squares of analysis were between 0.9 and 5.0, andthe % of background diploid aggregates and debriswas<20.0% [35].

3. Results

3.1. 32P-postlabelling of DNA adducts

Representative autoradiographs of DNA adductsformed in HEL cells after treatment with B[a]P andDB[a,l]P for all of the incubation conditions used areshown in Fig. 1. B[a]P induced only one major adductspot identical with anti-BPDE-derived DNA adduct,as was tentatively confirmed by HPLC analysis us-ing a procedure previously described [33] (data notshown). Exposure of cells to DB[a,l]P induced threemajor spots under the TLC chromatographic condi-tions used, but HPLC analysis indicated two major

Fig. 1. Autoradiographs of32P-postllabelled DNA adducts formed in human lung fibroblasts after 24 h exposure to (A) benzo[a]pyrene(1mM), (B) dibenzo[a,l]pyrene (0.1mM) and (C) DMSO alone (control sample). Adducts were32P-labelled and separated as described inSection 2.3. Exposure was carried out for 1 h (B) or for 5 h (A, C) at−80◦C.

radioactive peaks for spots 1 and 3, and three majorpeaks for spot 2. Quantitative analysis was performedon each spot. The total DB[a,l]P-DNA adduct levelspresented here are the sum of all these ‘DNA adductspots’.

The DNA adduct levels detected after 24 h expo-sure of cells to both PAHs at different days afterseeding are shown in Table 1. The highest DNAadduct levels were observed after 24 h exposure ofcells at near-confluence (7th day after cell seeding).The total DB[a,l]P-DNA adduct levels were approx-imately one order of magnitude higher as comparedwith B[a]P-derived DNA adducts. For time- anddose-response experiments, the fifth day (the cells arestill in the growth stage), and seventh day after cellseeding (>90% of cells at G0/G1 phase) were chosenas a compromise between DNA adducts, protein ana-lysis (sufficient number of cells for DNA and proteinisolation) and cell cycle measurements.

DNA adduct formation was investigated in relationto the time of cell treatment with both PAHs, at con-centrations of 1.0mM B[a]P or 0.1mM DB[a,l]P. Thedata are shown in Fig. 2. The DNA adduct levels exhi-bited a similar time-dependent increase for both com-pounds and both days of application after cell seeding,indicating that the DNA adduct formation proceededat similar rates during the whole incubation time re-gardless of the day of application.

The concentration dependence of DNA adduct for-mation was studied in detail for both compounds inconcentration ranges from 0.01 to 40mM (B[a]P) and


Table 1Formation of DNA adducts in human diploid lung fibroblasts treated with B[a]P and DB[a,l]P for 24 h at different days after seedinga

Day of 24 h exposure after cell seeding DNA adducts/108 nucleotidesBenzo[a]pyrene (1mM) Dibenzo[a,l]pyrene (0.1mM)

3rd 73.8 123.84th 124.1 747.85th 157.4 1071.46th 191.3 1563.87th 223.1 2145.0

a 3th–6th day after cell seeding: growth stage of cells. 7th day after cell seeding: near-confluent cells, e.g. more than 90% of cells atG0/G1 phase. Data represent the mean of two independent incubations.

from 0.01 to 1mM (DB[a,l]P) at near-confluence andfor 24 h cell treatment. The data are shown in Fig. 3.DNA binding was detectable even at the lowest con-centration used, e.g. at 0.01mM for both compounds(1.8 and 183 adducts/108 nucleotides, for B[a]P andDB[a,l]P, respectively). B[a]P-DNA adduct levels in-creased linearly at concentrations between 0.01–1mMand reached a maximal value at 1mM B[a]P (237adducts/108 nucleotides). Increasing the B[a]P con-centration over 4mM decreased B[a]P-DNA adductlevels even if no morphological changes were ob-served or no cytotoxicity was detected by LDH assay(data not shown). A similar dose-response curve wasobserved for DB[a,l]P, but in a concentration rangeone order of magnitude lower (maximal value of 2360

Fig. 2. Time-response of human lung fibroblasts exposed to 1mM B[a]P and 0.1mM DB[a,l]P during the growth stage (5th day afterseeding) or/at near-confluence (7th day after seeding). Mean± S.D. represent values from three independent incubations with duplicatesamples. Each DNA sample was analysed at least in two independent32P-poslabelling experiments.

adducts/108 nucleotides at 0.1mM). The same courseof dose dependence was also observed in growing cells(5th day after cell seeding; data not shown).

3.2. Western blotting of p53 and p21WAF1 proteins

A representative western blot analysis of p53 andp21WAF1 proteins in HEL cells exposed for 24 h toDB[a,l]P at concentrations ranging from 0.01 to 1mMat near-confluence or in the growth stage is shownin Fig. 4a,b. Quantification of the western blot re-sults obtained for the samples of different incubationsets showed an apparent increase in p53 protein le-vels as compared with control levels after 16 h of ex-posure of the cells to 0.1mM DB[a,l]P regardless of


Fig. 3. Dose-response of human lung fibroblasts exposed for 24 h to B[a]P and DB[a,l]P at near-confluence (7th day after seeding).Mean± S.D. represent values from at least three independent incubations and two independent32P-poslabelling experiments.

the day of application. The induction of p53 proteinwas followed by an increase in p21WAF1 protein lev-els. Detailed western blot analysis of cells exposedto DB[a,l]P for 24 h in the concentration range of0.01–1mM at near-confluence as well as in the growthstage indicated a significant relationship between thelevels of DNA adduct formation and the induction ofboth p53 and p21WAF1 proteins as shown in Fig. 5.The western blot analysis performed with the samplesof the cells exposed to B[a]P showed a detectable andreproducible increase of both proteins over the con-trol levels (>1.5-fold) only for the samples in whichB[a]P-DNA adduct levels reached maximal values un-der the incubation conditions used (Fig. 6).

3.3. Cell cycle distribution by flow cytometry

We investigated the effect of DB[a,l]P and B[a]P onthe cell cycle of HEL cells after exposing them to thesame conditions as for the DNA adduct analysis andthe expression of p53 and p21WAF1 proteins. The cellswere grown for 24 h in the presence of both PAHs atthe concentrations of maximal DNA adduct formation,i.e. 0.1mM DB[a,l]P and 1.0mM B[a]P. As a positivecontrol for G1 arrest, exposure to 1.0 nM actinomycinD was used. The exposure of growing cells for 24 hresulted in an increased proportion of cells in G0/G1as compared to control cells treated with DMSO aloneand a decreased proportion of cells in S phase (datanot shown). However, 24 h exposure to DB[a,l]P did

not result in G0/G1 arrest, but rather in an increase inthe proportion of cells in S phase compared with con-trols, while both G0/G1 and G2/M phases decreasedin the cells exposed to DB[a,l]P. Although the alter-ations were not as strong, 24 h treatment with B[a]Presulted in an increase in the proportion of cells inS phase as compared with controls. The same trendswere observed for the cells at near-confluence (7th dayafter seeding), but the differences were smaller thanfor growing cells because more than 90% of the cellswere in G0/G1, i.e. less cells were cycling (data notshown).

To further establish if HEL cells were arrested inS phase in the presence of DB[a,l]P or B[a]P, wecarried out experiments with colchicin (0.5mg/ml; anantimitotic agent). The colchicin was added duringthe last 4 h of the 24 h treatment. The proportion ofcontrol cells exposed to colchicin alone showed asignificant increase in G2/M as compared with con-trols. In contrast, the cells co-exposed to DB[a,l]P andcolchicin did not show any increase in G2/M, whileco-incubation with B[a]P resulted in a partial increasein G2/M as compared with controls (data not shown).These results indicated that, similarly to what wasfound for some other PAHs [10], exposure to DB[a,l]Pand B[a]P does not induce a G1 arrest but rather adelay or an arrest in S phase.

We also investigated the time- and dose-responseof the cell cycle exposed to both PAHs. The differ-ences between exposed and control cells treated in


Fig. 4. p53 and p21WAF1 protein levels in human lung fibroblasts exposed to DB[a,l]P for 24 h in the concentration range from 0.01 to1mM at near-confluence (a) and in the growth stage (b). The cells were harvested and processed as described in Section 2. 15mg of proteinin 12ml of sample buffer were resolved by SDS-PAGE on a 12.5% mini-gel. p53 western blotting standard from Oncogene (Cat.#WB21)was used as a positive control for p53 protein and ECL protein molecular weight markers (Amersham) for p21WAF1. CON: control sampletreated with DMSO alone.

a time-dependent manner for all phases of the cellcycle are shown in Fig. 7. During the first 12 h fol-lowing treatments, control samples and cells exposedto 0.1mM DB[a,l]P had a similar distribution of cellcycle phases. Thereafter, major differences were ob-served after 24 h of treatment (Fig. 7a). There wasan approximately 24% increase in the proportion ofcells in S phase after DB[a,l]P treatment as comparedwith controls, while G0/G1 and G2/M decreased.The exposure to 1.0mM B[a]P also resulted in thesame trends after 12 h of treatment (Fig. 7b), butsmaller differences were found compared with ex-posure to DB[a,l]P. Again, in near-confluent cells,

similar trends were noted, but the differences weresmaller compared with growing cells (data notshown).

Finally, we studied the dose-response of the cell cy-cle by treating growing cells for 24 h with both PAHsat the concentrations of maximal DNA adduct forma-tion, as well as on each side of their concentrationpeaks. Compared to controls cell distribution betweenthe three phases of the cell cycle was significantly al-tered even after exposure to 0.02mM DB[a,l]P, sug-gesting an S phase delay as shown in Table 2. This wasnot unexpected since even at this low concentrationof DB[a,l]P, DNA adducts were highly induced (>500


Fig. 5. Relationship between the induction of p53 and p21WAF1 proteins and DNA adduct levels in human lung fibroblasts exposed toDB[a,l]P in a dose- and time-dependent manner at near-confluence (7th day after seeding) or during the growth stage (5th day afterseeding). The induction of proteins was evaluated as the relation of the protein level in the exposed cells to the protein level of control cellstreated with DMSO alone. The data points represent individual western blotting data obtained for the samples from different incubationsets (R = Spearman rank correlation coefficient,P = significance level).

adducts/108 nucleotides, Fig. 3). After the exposureof cells to concentrations between 0.10 to 10.0mMB[a]P the differences in the distribution of exposedand control cells between the three phases of the cell

Fig. 6. Relationship between the induction of p53 and p21WAF1 proteins and DNA adduct levels in human lung fibroblasts exposed toB[a]P in a dose- and time-dependent manner at near-confluence (7th day after seeding) or during the growth stage (5th day after seeding).The induction of proteins was evaluated as the relation of the protein level in the exposed cells to the protein level of control cells treatedwith DMSO alone. The data points represent induvidual western blotting data obtained for the samples from different incubation sets(R = Spearman rank correlation coefficient,P = significance level).

cycle were also significant (Table 2). However, we ob-served more pronounced alterations of the cell cycle at1.0mM B[a]P, similar to what was found for maximalDNA adduct formation (Fig. 3).


Fig. 7. Differences in the distribution of the cell cycle betweencontrol (DMSO) and (a) DB[a,l]P or (b) B[a]P after various timesof exposure. After 5 days of growth, the cells were washed,fresh medium was added, and the cells were treated for 2, 4, 8,12, 16, or 24 h in the presence of 0.1mM DB[a,l]P or 1.0mMB[a]P. After exposure, the cells were harvested by trypsynisation,RNA was digested by RNase A, and DNA was stained withpropidium iodide. DNA content analysis was performed by flowcytometry as described in Section 2. For each phase of the cellcycle, the values represent the mean difference of the proportionbetween control (DMSO) and PAH-exposed samples (Exposedminus control samples) from three independent incubations. Errorbars are S.D.

4. Discussion

A few recent papers have dealt with the role ofp53 and/or p21WAF1 proteins in the cellular responseto DNA damage caused by some parent PAHs ortheir DNA-reactive metabolites. The majority of thesestudies used the breast carcinoma cell line MCF-7[10,20,25–27], while others used human [21] ormouse [23,24] fibroblasts and stimulated peripheralblood lymphocytes [22]. The induction of p53 pro-tein in mouse fibroblasts was also proposed as a newapproach for the genotoxicity testing of potential che-

Table 2The cell cycle distribution of growing human diploid lung fibrob-lasts after 24 h exposure (at 5th day after seeding) to differentdoses of DB[a,l]P and B[a]Pa

Chemical (mM) Cell cycle phaseG0/G1 S G2/M

Dibenzo[a,l]pyreneControl 84.0± 2.4 9.5± 0.4 6.5± 2.10.02∗ 70.5 ± 5.3 29.5± 5.3 N.D.0.10∗ 76.0 ± 3.5 24.0± 3.5 N.D.1.00∗ 71.5 ± 5.6 27.0± 4.5 1.5± 1.0

Benzo[a]pyreneControl 88.0± 1.2 8.0± 0.7 4.0± 1.10.10∗ 84.5 ± 1.9 12.0± 1.3 3.5± 1.01.00∗ 82.0 ± 3.7 13.5± 2.7 4.5± 1.04.00∗ 83.5 ± 1.4 12.5± 1.7 4.0± 0.8

10.0∗ 85.5 ± 2.1 10.5± 1.6 4.0± 0.7

a The data represent the mean±S.D of duplicate samples fromat least three independent incubations. N.D.: Not detectable.

∗ P < 0.001 (x2 statistic) compared with control for the celldistribution between the three cell cycle phases.

mical carcinogens, but the levels of DNA damage andp21WAF1 protein were not simultaneously measured[22,23]. So far, the study of the cyclin-dependentkinase inhibitor p21WAF1 produced contradictoryresults.

The principal aim of this study was to investigatesimultaneously DNA adduct formation, induction ofp53 and p21WAF1 proteins and the cell cycle distribu-tion in human diploid lung fibroblasts after exposureto B[a]P and DB[a,l]P. We used normal human cellsbecause they are closer to in vivo conditions and be-cause tumour cell lines are likely to contain multiplemutations that may affect DNA damage response andthe cell cycle [21,36]. DNA adduct analysis showedthat these cells are capable of activating parent PAHsto their reactive metabolites. DNA binding was de-tectable even at the lowest concentration used, e.g.at 0.01mM for both compounds. Both PAHs exhib-ited a time- and dose-dependent increase of DNAadduct levels regardless of the exposure either ingrowing or nearly-confluent cells. The highest adductlevels were found in confluent cells at the concen-trations of 1mM for B[a]P and 0.1mM for DB[a,l]P,but the DNA damage induced by DB[a,l]P was oneorder of magnitude higher as compared with B[a]P(237 and 2360 adducts/108 nucleotides, respectively).The dose-response curves showed a deflection from


linearity at higher concentrations indicating a satu-ration of metabolic activation enzymes [37] or theirinactivation, possibly due to the PAH’s reactivemetabolites (formation of protein adducts). This lastsuggestion supports the study of Till et al. [38] whomeasured 7-ethoxyresorufin O-deethylase (EROD)activity in rat hepatocytes after treatment with sev-eral PAHs. They observed a similar course of thedose-response curve for EROD activities induced byB[a]P (DB[a,l]P was not studied) as we found forB[a]P-DNA adducts in HEL cells.

DNA damage induced in HEL cells by DB[a,l]P in-creased the cellular content of both p53 and p21WAF1

proteins as compared to control cells treated under thesame conditions with vehicle alone (up to∼10-fold in-crease of p53 and up to∼6-fold increase of p21WAF1),regardless of whether the cells were still growing orwere nearly-confluent (over 90% of cells in G0/G1phase). The effect of DB[a,l]P on the cellular levelsof both proteins was similar for various times anddoses of exposure that gave similar DNA adduct lev-els. The results from time- and dose-responses showedthat there were significant correlations between DNAadduct levels and the levels of both proteins (p53:R = 0.832,P < 0.001 andR = 0.859,P < 0.001;p21WAF1: R = 0.808, P < 0.001 andR = 0.797,P = 0.001, for DB[a,l]P and B[a]P, respectively). Theresults showed that a detectable increase of p53 andp21WAF1 proteins (≥1.5-fold increase as comparedwith controls) required a minimal DNA adduct level of∼ 200–250 adducts/108 nucleotides and suggest thatthe level of adducts rather than their structure triggersthe p53 and p21WAF1 responses. Similar conclusionresulted from the experiments of Luch et al. [27] withMCF-7 cells treated withsyn- and anti-dihydrodiolepoxides of DB[a,l]P.

Our results of time-dependent induction of p53protein levels after exposure of cells are consistentwith the studies of Yang et al. [23] who measuredp53 induction following treatment of mouse fibrob-lasts with direct- and indirect-acting carcinogens.The indirect-acting genotoxic carcinogens caused aninduction of p53 with the peak increase appearing be-tween 16 to 24 h of treatment (probably correspondingto the DNA damage required to activate expressionof p53).

Dose- and time-dependent increases of p53 andp21WAF1 protein levels in the human carcinoma cell

line MCF-7 were observed by Kaspin et al. [26] afterexposure to BPDE and also by Luch et al. [27] afterexposure to DB[a,l]P-dihydrodiol epoxides, even ifthey used different experimental designs. Therefore,they suggested that a p53-dependent transductionpathway might result in a cell cycle arrest at G1or G2/M checkpoints allowing cells to repair DNAdamage prior to continuing into the S phase or toundergoing mitosis. However, neither of these studiesinvestigated the effect of exposure on the cell cycle.

In contrast, Khan et al. [25] did not observeany induction of p21WAF1 protein in human mam-mary carcinoma MCF-7 cells following exposure tobenzo[g]chrysene-11,12-dihydrodiol-13,14-epoxide(BgChDE). In their study, they simultaneously mea-sured p53 and p21WAF1 protein levels and cell cycledistribution, while the levels of DNA damage werenot determined. They showed that cell treatment withthis carcinogen led to an increase in p53 levels with-out any induction of p21WAF1, and they demonstrateda lack of G1 arrest but instead a delay of cells inthe S phase. Therefore, they suggested that a lack ofG1 arrest is consistent with the absence of p21WAF1

cyclin-dependent kinase inhibitor induction. Accord-ing to further investigations with two other dihydro-diol epoxide metabolites of PAHs, Dipple et al. [10]proposed that the so-called ‘stealth’ property may bea fairly general response to hydrocarbon carcinogenactive metabolites.

In our study, despite a significant increase of bothp53 and p21WAF1 protein levels after treatment ofHEL cells with DB[a,l]P, we did not observe G1 ar-rest but instead a delay of cells in the S phase. Af-ter 16–24 h of cell treatment with 0.1mM DB[a,l]P(the concentration corresponding to the highest DNAadduct levels detected), the distribution of exposedcells between the three phases of cell cycle was sig-nificantly altered compared with control. We observeda remarkable increase in the proportion of cells in Sphase, while the G2-M phase was almost completelydepleted, along with a decrease in G1 phase cells, i.e.the cells were blocked in S phase. Similarly, an S phasedelay was observed by Khan et al. [25] after expo-sure of MCF-7 cells to BgChDE but without induc-tion of p21WAF1. Black et al. [39] also detected an Sphase block in human lymphoblasts exposed to BPDEor N-acetoxy-2-acetylaminofluorene. They found thatthe perturbation of the cell cycle was preceded by a


reduction of cell viability and was associated with aninhibition of population growth. Therefore, they sug-gested that an accumulation of cells in S phase leadingto cell death is typical for compounds inhibiting DNAsynthesis. However, cell viability was not affected un-der the experimental conditions we used. Consistentwith our results, Shenberger et al. [40] also observed Sphase growth arrest with increased expression of p53and p21WAF1/CIP1 proteins induced by oxidative DNAdamage (free oxygen radicals) in human bronchialsmooth muscle cells. They suggested that activationof p53andWAF1genes may prevent DNA replicationwithout inducing apoptosis to allow for repair of DNAdamage.

The mechanism behind the absence of a G1 ar-rest but rather an S phase delay, even if both thep53 and p21WAF1 levels are elevated, is unclear.The DB[a,l]P-reactive metabolites may interfere withthe binding of p21WAF1 to cyclin-dependent kinasesor p21WAF1 may be inefficient due to a qualitativedeficiency (protein adducts formed with reactivemetabolites). In an attempt to avoid the propagationof genomic modifications, the cells may be arrestedin S phase to repair DNA damage before completereplication and mitosis occurs [41–43]. Alternatively,the cells may be irreversibly blocked in S phase be-cause they cannot progress further due to extensiveDNA damage. Since p53 and p21WAF1 were increasedin human fibroblasts exposed to DB[a,l]P, along withan increase in the proportion of cells in S phase anda decrease in G2/M, the possibility of an S phasearrest as a downstream defense mechanism to repairdamaged DNA after escape from the G1 checkpointcannot be excluded.

There is a growing body of evidence thatsome genotoxic carcinogens may induce cell cy-cle defense mechanisms through p53-dependent orp53-independent pathways, with or without p21WAF1

induction. The measurement of p53 protein induc-tion is helpful but not sufficient to evaluate whethercells recognise and turn on (or not) the mechanismsresponsible for molecular defense and integrity, andultimately how the cell cycle is altered by exposure.It is becoming clear that a p53 increase does not al-ways induce G1 arrest and that the absence of p53induction does not necessarily mean the absence ofDNA damage and of G1 arrest. It seems that data onDNA damage, the proteins involved in the regulation

of the cell cycle as well as on cell cycle alterationsare necessary for a better understanding of the effectand consequences of the action of genotoxic agents.

In conclusion, the present study clearly demon-strated that exposure of human diploid lung fibrob-lasts to both PAHs, DB[a,l]P and B[a]P, resulted intime- and dose-dependent DNA damage accompaniedby increased levels of p53 and p21WAF1 proteins.Surprisingly, there was no G1 arrest but instead adelay of cells in the S phase, nowadays-called the‘S phase checkpoint’. There is a common perceptionthat the S phase checkpoint is equivalent to the G1checkpoint because both response pathways delay orinhibit the initiation of DNA synthesis. Whether theS phase delay observed in our study after exposure ofhuman diploid fibroblasts to selected PAHs is benefi-cial for the survival of the cells remains to be furtherestablished.

Acknowledgements

The authors would like to thank Miss RadkaBoráková for excellent technical help and Dr. JamesDutt for correction of English language. The CzechMinistry of the Environment (grants VaV/ 340/1/1997,VaV/340/2/00) supported this study.

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