Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM2.5) in human lung epithelial A549 cells

Available online at www.sciencedirect.com

Cancer Letters 270 (2008) 144–155 www.elsevier.com/locate/canlet

Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM2.5) in human lung epithelial A549 cells Sylvain Billet a,b, Imane Abbas a, Je´re´mie Le Goff b, Anthony Verdin a, Ve´ronique Andre´ b, Paul-Eric Lafargue c, Adam Hachimi c, Fabrice Cazier d, Francßois Sichel b, Pirouz Shirali a,*, Guillaume Garcßon a a

LCE EA 2598, Toxicologie Industrielle et Environnementale, Universite´ du Littoral – Coˆte d’Opale, Maison de la Recherche en Environnement Industriel de Dunkerque 2, 189A, Avenue Maurice Schumann, 59140 Dunkerque, France b UPRES-EA 1772 – IFR 146 ICORE, Groupe Re´gional d’Etudes sur le CANcer, Universite´ de Caen Basse-Normandie et Centre Re´gional de Lutte Contre le Cancer Francßois Baclesse, Avenue du Ge´ne´ral Harris, 14000 Caen, France c MicroPolluants Technologie S.A., 5, Impasse des Anciens Hauts Fourneaux, Z.I. du Gassion, BP 80293, 57108 Thionville Cedex, France d Centre Commun de Mesures, Universite´ du Littoral – Coˆte d’Opale, Maison de la Recherche en Environnement Industriel de Dunkerque 1, 145, Avenue Maurice Schumann, 59140 Dunkerque, France Received 29 January 2008; received in revised form 29 April 2008; accepted 29 April 2008

Abstract To improve the knowledge of the underlying mechanisms of action involved in air pollution Particulate Matter (PM)induced toxicity in human lungs, with a particular interest of the crucial role played by coated-organic chemicals, we were interested in the metabolic activation of Polycyclic Aromatic Hydrocarbons (PAH)-coated onto air pollution PM, and, thereafter, the formation of PAH–DNA adducts in a human lung epithelial cell model (A549 cell line). Cells were exposed to Dunkerque city’s PM2.5 at its Lethal Concentrations at 10% and 50% (i.e. LC10 = 23.72 lg/mL or 6.33 lg/cm2, and LC50 = 118.60 lg/mL or 31.63 lg/cm2), and the study of Cytochrome P450 (CYP) 1A1 gene expression (i.e. RT-PCR) and protein activity (i.e. EROD activity), and the formation of PAH–DNA adducts (i.e. 32P-postlabeling), were investigated after 24, 48, and/or 72 h. PAH, PolyChlorinated Dibenzo-p-Dioxins and -Furans (PCDD/F), Dioxin-Like PolyChlorinated Biphenyls (DLPCB), and PolyChlorinated Biphenyls (PCB)-coated onto collected PM were determined (i.e. GC/MS and HRGC/HRMS, respectively), Negative (i.e. TiO2 or desorbed PM, dPM; EqLC10 = 19.42 lg/mL or 5.18 lg/cm2, and EqLC50 = 97.13 lg/mL or 25.90 lg/cm2), and positive (i.e. benzo(a)pyrene; 1 lM) controls were included in the experimental design. Statistically significant increases of CYP1A1 gene expression and protein activity were observed in A549 cells, 24, 48 and 72 h after their exposure to dPM, suggesting thereby that the employed outgassing method was not efficient enough to remove total PAH. Both the CYP1A1 gene expression and EROD activity were highly induced 24, 48 and 72 h after cell exposure to PM. However, only very low levels of PAH–DNA adducts, also not reliably quantifiable, were reported 72 h after cell exposure to dPM, and, particularly, PM. The relatively low levels of PAH together with the presence of PCDD/F, DLPCB, and PCB-coated onto Dunkerque City’s PM2.5 could notably contribute to explain the

*

Corresponding author. Tel.: +33 3 28237610; fax: +33 3 28237171. E-mail address: [email protected] (P. Shirali).

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.04.044

S. Billet et al. / Cancer Letters 270 (2008) 144–155

145

borderline detection of PAH–DNA adducts in dPM and/or PM-exposed A549 cells. Hence, remaining very low doses of PAH in dPM or relatively low doses of PAH-coated onto PM were involved in enzymatic induction, a key feature in PAHtoxicity, but failed to show a clear genotoxicity in this in vitro study. We also concluded that, in the human lung epithelial cell model we used, and in the experimental conditions we chose, bulky-DNA adduct formation was apparently not a major factor involved in the Dunkerque City’s PM2.5-induced toxicity. Ó 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: A549 cells; Air pollution Particulate Matter; Polycyclic Aromatic Hydrocarbons; Lung toxicity; Cytochrome P450 1A1; 32 P-postlabeling; PAH–DNA adducts

1. Introduction Long-term exposure to fine Particulate Matter (PM) air pollution is associated with increased incidence of mortality [1–3]. Among the 58 million deaths worldwide identified in 2005, 1.3 million were from lung cancer [4]. Air pollution caused about 5% of trachea, bronchus and lung cancer (i.e. 71,000 deaths) [2,4–6]. The adjusted relative risk associated with a 10 lg/m3 increase in annual average PM2.5 for the 1999–2000 period led to a 13% increase in lung cancer mortality [1]. Even if tobacco use takes a major part in etiology of lung cancer, other explanation like genetic and lifestyle factors, and occupational or environmental exposure to carcinogens have also to be considered. Indeed, one of the most important risk factor that have to be take into account is the relatively low exposure to airborne carcinogens, like Polycyclic Aromatic Hydrocarbons (PAH), generally occurring close to point emissions, such as coal-, woodand fuel-power plants, steel and petrochemical factories, and/or near mobile sources, such as motor vehicle exhaust [7,8]. The exposure level to PAH emitted from these sources is relatively low as compared to other, as such as diet, occupation, or tobacco smoke [9]. The half-life of airborne PAH is of the order of days but can be longer when they are coated onto ambient PM [9]. Indeed, air pollution PM is a very complex and heterogeneous mixture of chemicals (i.e. metals; salts; carbonaceous material; Volatile Organic Compounds, VOC; PAH; etc.) and/or biological (i.e. bacteria, endotoxins, fungi, etc.) elements, which can in fact be attached to a carbonaceous core being use as a condensation nuclei [10,11]. After their absorption, PAH are distributed into lung cells and/or tissues, where they can be biotransformed. The metabolic activation of PAH occurs via two classes of enzymes: phase I (i.e. oxidation, reduction, hydrolysis) and phase II (i.e. con-

jugation) enzymes. Cytochrome P450s (CYPs) are essential heme-containing enzymes that play critical functions in the conversion of organic chemicals into water soluble metabolites, thereby helping their excretion. Accordingly, in human lungs, PAH, which require metabolic activation to biologically reactive intermediates to elicit their adverse health effects, are metabolized by the CYP superfamily member CYP1A1 [2]. Certain chemically reactive intermediates arising from PAH metabolic activation in lungs could thereafter interact with DNA target sites to produce adducts, thereby giving rise to mutation, and eventually, tumor initiation [2]. The gene expression of CYP enzymes can be modulate in response to the activation of key transcription factors by specific substrates; in particular, in lung cells, the activation of Aryl hydrocarbon Receptor (AhR) by PAH induces CYP1A1 mRNA transcription [2,12]. Mutagenic activities of outdoor air pollution from anthropogenic combustion-related sources or its main components have already been shown in Salmonella assays, as recently reviewed [13]. Such data have revealed that the PAH present in almost all combustion-related complex mixtures constitute a significant source of genotoxicity among at least 500 mutagenic components of varying chemical classes [13,14]. However, other factors, often neglected, such as PM size, component interactions, secondary chemical reactions in the atmosphere, and/or sampling season are known to affect the genotoxicity of air pollutants [15–18]. The photodegradation and chemical transformation of PAH emissions in the atmosphere during warmer months might also induce the formation of highly biologically reactive products, such as B(a)P-7,8-Dihydrodiol-9,10-Epoxide (BPDE), and quinones, able to form DNA adducts [19,20]. Ultimately, in humans, hereditary and acquired susceptibilities have been reported to influence the formation of aromatic DNA adducts [21].

146

S. Billet et al. / Cancer Letters 270 (2008) 144–155

The most frequently used biological markers to monitor PAH exposure are their metabolites (e.g. 1-hydroxypyrene), and PAH–DNA and/or –protein adducts. Since PAH–protein adducts are not correlated with low concentrations of PAH exposure, they do not appear as valid markers to assess environmental exposure [15,22,23]. Conversely, PAH–DNA adduct levels are particularly relevant to monitor it since they are mechanistically linked to the induction of cancer [24–28]. A review in molecular epidemiology concluded that DNA adducts measured by the 32P-postlabeling method had become a biological marker of choice for PAH monitoring in both the cases of occupational and/or environmental exposure [29]. Consensus protocols, standards, and quality assurance methods for 32P-postlabeling have been developed through a working group and inter-laboratory trial exercise [28,30]. Hence, to improve the knowledge of the underlying mechanisms of action involved in air pollution PM-induced toxicity in human lungs, with a particular interest of the crucial role played by coatedorganic chemicals, a PM sampling was realized in Dunkerque, a French seaside City characterized not only by the proximity of lots of industrial activities, but also by a heavy motor vehicle traffic. The most toxicologically relevant physical and chemical characteristics of collected PM were determined, and its potential role in the induction of PAH and/or VOC-metabolizing enzymes were carried out in a human lung epithelial cell model (A549 cell line) [31]. Hence, in this work, we focused our attention on the ability of collected PM to induce CYP1A1 gene expression and protein activity and, thereafter, PAH–DNA adduct formation in A549 cells in culture. We were closely interested in the presence of collected PM-coated inducible-CYP1A1 enzyme substrates, and, therefore, in the required distinction between PAH, which are able to be converted into reactive metabolites that directly react with DNA to form bulky-DNA adducts, on the one hand, and PolyChlorinated Dibenzo-p-Dioxins (PCDD) and -Furans (PCDF), Dioxin-Like PolyChlorinated Biphenyls (DLPCB) and PolyChlorinated Biphenyls (PCB), which are not able to form bulky-DNA adducts detectable by postlabeling method [32]. To better elucidate the role of coated PAH, TiO2 particles and PM having undergone a thermal desorption (i.e. desorbed PM, dPM) were included as negative controls in the experimental design of this study.

2. Materials and methods 2.1. Chemicals Cell culture reagents were from Invitrogen (Cergy Pontoise, France). Titanium (IV) oxide powder (anatase; purity: 99%; primary particle size: 0.2 lm; surface not coated) was from Acros Organics (Noisy Le Grand, France). Pesticide grade/high purity solvents (i.e. toluene, hexane, methylene chloride) and reagents (i.e. silica, Silicagel, 60, 63–200 lm; basic alumina, Brockman I, 90, 63– 200 lm); sodium sulfate, anhydrous, for analyses, ASC, ISO) were from LGC Promochem (Molsheim, France) and WWR (Darmstadt, Germany), respectively. Benzo(a)pyrene, proteinase K, ribonuclease A and T1, micrococcal nuclease, 20 -deoxyadenosine-30 -monophosphate (dAp), nuclease P1, urea, formic acid, lithium hydroxide, bicinchoninic acid kit, and reagents to study EthoxyResorufin O-deethylase (EROD) activity were from Sigma– Aldrich (St-Quentin Fallavier, France). RIPA lysis buffer was from Santa Cruz Biotechnology (Heidelberg, Germany). Polymerase Chain Reaction (PCR) primer pairs were from Proligo France SAS (Paris, France), and all other reagents for Reverse Transcription (RT) and PCR were provided by Roche Diagnostics (Meylan, France). Carrier-free [c-32P]ATP (3000 Ci/mmol) was from GE Healthcare Europe GmbH (Orsay, France). T4 polynucleotide kinase was from Q Biogen (MP Biomedicals, llkirch, France); Phosphodiesterase Type II (calf spleen) was from Calbiochem (VWR International France, Fontenay-Sous-Bois, France). PolyEthyleneImine (PEI)-cellulose F plastic sheets were from Macherey-Nagel (Hoerdt, France). 2.2. PM sampling, physical and chemical characteristics, and outgassing 2.2.1. PM sampling PM was collected in Dunkerque (51°040 N; 2°380 E), a French sea-side City on the southern coast of the North-Sea, using high volume cascade impactor [33]. 2.2.2. PM physical and chemical characteristics [31] PM size distribution, as carried out by Scanning Electron Microscopy (SEM), showed size ranging from 0.33 to 5.0 lm, with a peak at 0.45 lm. The highest number of PM (i.e. 92.15%) was detected in size classes including PM with a size 6 2.5 lm: 0–0.5 lm (33.63%), 0.5–1.0 lm (30.61%), 1.0–1.5 lm (14.33%), 1.5–2.0 lm (8.69%), and 2.0–2.5 lm (4.89%). Adsorption data from the Brunauer Emmett Teller (BET) method provided a specific surface area of 1 m2/g for PM, and 50 m2/g for dPM. Inductively coupled plasma-atomic emission spectrometry showed that Fe (7.84%), Al (5.83%), Ca (4.95%), Na (1.88%), K (0.97%), Mg (0.81%), Pb (0.80%), and Ti (0.51%) were the most abundant inorganic elements. Gas

S. Billet et al. / Cancer Letters 270 (2008) 144–155

Chromatography/Mass Spectrometry (GC/MS), after thermal desorption at 500 °C and cold trapping, or soxhlet extraction with dichloromethane, allowed notably to identify lots of PAH-coated onto PM [31]. PCDD/F and DLPCB were analyzed by High-Resolution Gas Chromatography/High-Resolution Mass Spectrometry (HRGC/HRMS). PM sample (i.e. 20 mg) was spiked with 12 isotopically labeled PCDD and PCDF (i.e. 480 pg for the tetra-, penta-, and hexa-chlorinated congeners, and 960 pg for the hepta- and octa-chlorinated congeners), and 12 isotopically labeled PCB (i.e. 1000 ng for each) then extracted with toluene by using ASE 200 pressurized liquid extractor (Dionex, Voisins Le Bretonneux, France). The extract was cleaned-up according to the liquid chromatographic procedures described in the USEPA Methods 1613 and 1668. A silica column: (from top to bottom, 1 g of anhydrous sodium sulfate, 1 g of activated silica, 8 g of 30% sulfuric acid impregnated silica, 1 g of activated silica, 4 g of 23% sodium hydroxide impregnated silica) allowed to isolate PCDD/F by elution with n-hexane. A basic alumina column (from top to bottom, 1 g of anhydrous sodium sulfate, 15 g of activated basic alumina, activity I) allowed to concentrate DLPCB and PCB after elution with toluene, and PCDD/F after elution with n-hexane/methylene chloride (1:1, v:v). DLPCB and PCB, and PCDD/F fractions were reconstituted by adding 10 lL of a standard solution containing either 13C PCB 70, 13C PCB 111 and 13C PCB 170, or 13C 1,2,3,4 TCDD and 13C 1,2,3,7,8,9 HxCDD, respectively, to monitor recoveries. The levels of PCDD/F and DLPCB, and PCB in the dusts were determined using HRGC/HRMS on an Autoconcept, which is a high-resolution double focussing mass spectrometer of EB geometry produced by Mass Spectrometry International (MSI), Manchester, UK. The system is directly coupled to a HRGC (HP6890, Agilent Technologies, Massy, France) fitted with a split/splitless injector. The capillary column for GC separation was J&W DB 5 MS, 60 m length, 0.25 mm internal diameter and 0.25 lm film thickness. The mass spectrometer was operated at a resolution of 10,000. The source was operated at a temperature of 250 °C with an electron voltage of 30 eV at a trap current of 300 lA. The identification criteria specified in U.S.EPA Methods 1613 and 1668 respect to the GC column performance and MS performance were fully satisfied by the data obtained in this study (separations, resolution and sensitivity capabilities). Laboratory blanks were analyzed with the samples, and showed no contamination. The recoveries of labeled compounds were ranging from 77% to 97%. 2.2.3. PM outgassing dPM, i.e. PM having undergone a thermal desorption at 400 °C under a secondary vacuum and having thereby kept inorganic structures and lost most of organic chemicals, were used as negative controls [31,33].

147

2.3. Cell line and culture conditions We used the A549 cell line that derived from a type II-like human alveolar epithelial carcinoma (ATCC no CCL-185). Adherent A549 cells were cultured in sterile plastic flasks (Corning; Thermo Fisher Scientific, Courtaboeuf, France), in Minimum Essential Medium (MEM) with Earle’s salts, containing: 5% (v/v) Fetal Bovine Serum (FBS), 1% (v/v) L-glutamin (200 mM), 1% (v/v) penicillin (10,000 IU/mL), and 1 % (v/v) streptomycin (10,000 lg/mL) (Invitrogen). Exponentially growing cells were maintained at 37 °C, in a humidified atmosphere containing 5% CO2. All the A549 cells we used in the study of the gene expression and catalytic activity of CYP1A1, on the one hand, and the study of DNA adduct pattern, on the other hand, derived from the same initial cell culture. 2.4. Cell exposure and sampling 2.4.1. Cell exposure Depending on the incubation time, A549 cells were seeded at different density (i.e. 6  106, 3  106 or 1.5  106 cells/20 mL, respectively) in culture plastic flasks and incubated at 37 °C, in a humidified atmosphere containing 5% CO2, for 24, 48 or 72 h. After 3 h, culture supernatants were removed to eliminate non-adherent cells. Only living cells were incubated in the continuous presence of PM at its Lethal Concentration at 10% (i.e. LC10 = 23.72 lg PM/mL; or 6.33 lg PM/cm2) or 50% (i.e. LC50 = 118.60 lg PM/mL; or 31.63 lg PM/cm2) for 24, 48 or 72 h, without renewing the culture media. Non-exposed cells served as controls, and TiO2- and dPM-exposed cells served as negative controls. Cells were exposed to TiO2 or to dPM at equivalent concentrations to inorganic LC10 (i.e. EqLC10 = 19.42 lg/mL; or 5.18 lg/cm2) or LC50 (i.e. EqLC50 = 97.13 lg/mL; or 25.90 lg/cm2), integrating thereby mass losses arising from the thermal desorption of the organic fraction. Benzo(a)pyrene (B(a)P; 1 lM)-exposed cells served as positive controls [34,35]. For each incubation time (i.e. 24, 48 or 72 h), 8 culture flasks were chosen at random as controls, 4 culture flasks per TiO2 or dPM concentration as negative controls, 4 culture flasks per PM concentration as exposed cells, and 4 culture flasks per B(a)P concentration as positive controls. 2.4.2. Cell sampling After 24, 48 or 72 h of incubation, adherent cells were removed and centrifuged (500g; 10 min; 4 °C). Cell pellets were washed twice with 5-mL aliquots of cold phosphatebuffered saline (0.01 M; pH 7.2), and aliquots were quickly frozen at 80 °C to the further determination of gene expression and catalytic activity of CYP1A1, on the one hand, and PAH–DNA adduct formation after 72 h of incubation, on the other hand.

148

S. Billet et al. / Cancer Letters 270 (2008) 144–155

2.5. Study of gene expression and catalytic activity of CYP1A1 2.5.1. Gene expression of CYP1A1 Total RNA were extracted from cell-aliquots using RNeasy Mini Kit (Qiagen). Conditions for the RT were as published elsewhere [34]. Highly specific primer pair (F: 50 -TCT TTC TCT TCC TGG CTA TC-30 ; R: 50 -CTG TCT CTT CCC TTC ACT CT-30 ) and PCR thermocycling conditions were designed as reported by Iwanari et al. [36]. 2.5.2. Preparation of microsomes Microsomes were prepared as described by Guengerich [37]. In brief, cells (i.e. 6  106 cells) were lysed with 500 lL RIPA Lysis buffer (10 mM Tris–HCl, 150 mM NaCl, pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail), 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 20,000g for 10 min at 4 °C, supernatants were centrifuged at 110,000g for 1 h at 4 °C. Microsomal pellets were suspended in 100 mM phosphate buffer, pH 7.4, containing 1 mM EDTA, quickly frozen in liquid nitrogen, and stored at 80 °C. Total microsomal protein contents were determined with bicinchoninic acid reagent [38]. 2.5.3. Catalytic activity of CYP1A1 EROD activities were determined according to Donato et al. [39]. Briefly, catalytic activities of CYP1A1 were carried out by incubating microsomes (i.e. 100 lg total microsomal proteins) for 15 min at 37 °C in a 125 lL final volume of 100 mM phosphate buffer, pH 7.4, containing NADPH regenerating system (5 mM MgCl2, 1 mM NADP, 10 mM glucose-6-phosphate, and 0.3 U/mL glucose-6-phosphate dehydrogenase), and 10 lM ethoxy-res orufin as corresponding substrate. Reactions were stopped by adding 125 lL of methanol and the resorufin formed was determined fluorimetrically (excitation wavelength: 544 nm; emission wavelength: 590 nm) by using a computerized microplate reader (software: Ascent v2.6; hardware: Fluoroskan Ascent; Thermo Labsystems). 2.6. Study of DNA adduct pattern

Enrichment of DNA adducts was obtained by treatment with nuclease P1 for 30 min at 37 °C. Adduct labeling step was carried out during 30 min at 37 °C in the presence of T4 polynucleotide kinase and 30 lCi 32P-cATP. Separation of adducts was achieved by multidirectional PEI-cellulose Thin Layer Chromatography (TLC) (12 cm  12 cm). Four migration solvents were successively used: (i) 1 M sodium phosphate, pH 6.8 (overnight); (ii) 3 M lithium formate, 7 M urea, pH 3.5; (iii) 0.7 M lithium chloride, 0.45 M Tris–HCl, 7.7 M urea, pH 8.0; and (iv) 1.7 M sodium phosphate, pH 5.0 (overnight). Autoradiograms were Kodak XOmat film exposed to TLC-plates for 72 h at 80 °C with intensifying screens. Every sample was processed at least in triplicate. A positive control (i.e. calf thymus DNA exposed to BPDE) and a negative control (i.e. without any DNA) were included. Two samples with 3 pmol of dAp were labeled and diluted to 1 mL final. Using each of them, three deposits of 4 lL were separated on TLC sheets. After counting, dAp mean values served to estimate the labeling of 5 lg of dNp. Spot areas on TLCplates were excised and the radioactivity was quantified using a scintillation counter (Cerenkov mode). Results were expressed as Relative Adduct Levels (RAL): RAL = cpmadducts/[(cpmdAp/0.012)  3240  5] = 1.22  106 cpmadducts/cpmdAp, with: 0.012 pmol of dAp/deposit, 3240 = pmol of dNp in 1 lg DNA, and 5 = lg of DNA by sample [41]. The above-described protocol confer to the 32 P-postlabeling method a sensitivity of about 1 adduct/ 109 nucleotides, which will, therefore, be sufficiently sensitive for its application to a wide range of cell and/or tissue samples from human populations exposed to ambient air pollution. 2.7. Statistical analysis Treatment attribution at random has been made following an 11 element-permutation table. Results are expressed as mean values and standard deviations. For each incubation times, data from cell cultures exposed to PM, negative or positive controls were compared with those from nonexposed cell cultures. Thereafter, we looked for correlation between the different markers under study. Statistical analyses were performed by the Mann–Whitney U test and the non-parametric Spearman test (Software: SPSS for Windows, v12.0.1; 2003; Paris, France). Statistically significant differences were reported with p values < 0.05.

2.6.1. Genomic DNA extraction After RNA and protein elimination by enzymatic treatment with RNAses A and T1, followed by proteinase K, genomic DNA was extracted from A549 cell pellets using the Nucleospin Tissue Kit (Macherey-Nagel Eurl, Hoerdt, France).

3. Results

2.6.2. Postlabeling method This method was adapted from Randerath and Reddy [40,41]. Briefly, 5 lg of DNA were digested by micrococcal nuclease and spleen phosphodiesterase during 3.5 h.

The concentrations of PAH (lg/g), PCDD/F (pg/g), DLPCB (pg/g), and marker PCB (pg/g)-coated onto PM are shown in Tables 1–3, respectively. Among the PAH family members, 14 compounds were detected in the

3.1. PAH, PCDD/F, DLPCB, and marker PCB-coated onto PM

S. Billet et al. / Cancer Letters 270 (2008) 144–155

PM sample, and their concentrations ranged from 4.7 lg/ g (i.e. fluoranthene or pyrene) to 141.9 lg/g (i.e; methylnaphthalene) (Data from Billet et al. [31]). With other respects, a complex mixture of tetra-, penta-, hexa-, hepta-, and octa-chlorinated congeners of PCDD/F were observed in collected PM. Their concentrations ranged from < 5 pg/g (i.e. 2,3,7,8-TCDD) to 10,918.02 pg/g (i.e. OCDD) for PCDD, and from 69.94 pg/g (i.e. 2,3,7,8TCDF) to 1645.36 pg/g (i.e. 1,2,3,4,6,7,8 HpCDF) for PCDF. Both DLPCB and marker PCB were coated onto collected PM. Their concentrations ranged from <50 pg/g (i.e. PCB 123, PCB 114, or PCB 169) to 16,277 pg/g (i.e. PCB 118) for DLPCB, and from 16,576 pg/g (i.e. PCB 101) to 51,907 lg/g (i.e. PCB 28) for marker PCB. 3.2. Effects of PM on gene expression and protein activity of CYP1A1 As shown in Table 4, a statistically significant induction of CYP1A1 gene transcript was observed in A549 cells, 24, 48 and 72 h after their exposure to dPM at its EqLC10 (p < 0.05) and EqLC50 (p < 0.01), to PM at its LC10 (p < 0.01) and LC50 (p < 0.01), and to B(a)P (p < 0.01), compared to controls. In contrast, no significant variation of CYP1A1 gene transcript was reported after A549 cell exposure to TiO2 neither at its Eq LC10 nor at its Eq LC50, versus controls. Table 5 showed that significant increases in CYP1A1 catalytic activity was observed in A549 cells 24, 48 and 72 h after their exposure to PM at its LC10 (p < 0.05) and LC50 (p < 0.05), versus controls. Whatever the considered incubation time, A549 cell exposure to dPM at its LC10 and LC50, on the one hand, and to B(a)P, on the other hand, induced statisTable 1 PAH detected in collected PM Compounds

Concentration (lg/g)

Naphthalene Methylnaphthalene Dimethylnaphthalene Anthracene Phenanthrene Dibutylphthalate Fluoranthene Benzo(b + k)fluoranthene Benzo(a)pyrene Bis(ethyl.hexyl)phthalate Acenaphthene Fluorene Pyrene Benzo(a)anthracene or chrysene

38.1 141.9 90.2 47.1 28.3 110.1 4.7 6.6 7.9 41.4 11.1 6.3 4.7 4.9

Concentrations (lg/g) of the Polycyclic Aromatic Hydrocarbons (PAH) detected in the air pollution Particulate Matter (PM) as carried out by thermal desorption or by solvent extraction prior to analysis with gas chromatography–mass spectrometry. (Adapted from Billet et al. [31].)

149

Table 2 PCDD/F detected in collected PM Congener

Concentration (pg/g)

I-TEQ (pg I-TEQ/g)

2,3,7,8 TCDD 1,2,3,7,8 PeCDD 1,2,3,4,7,8 HxCDD 1,2,3,6,7,8 HxCDD 1,2,3,7,8,9 HxCDD 1,2,3,4,6,7,8 HpCDD OCDD P PCDD

<5 115.28 139.51 378.39 384.43 2644.62 10,918.02 14,580.27

5.00 57.64 13.95 37.84 38.44 26.45 10.92 190.24

2,3,7,8 TCDF 1,2,3,7,8 PeCDF 2,3,4,7,8 PeCDF 1,2,3,4,7,8 HxCDF 1,2,3,6,7,8 HxCDF 2,3,4,6,7,8 HxCDF 1,2,3,7,8,9 HxCDF 1,2,3,4,6,7,8 HpCDF 1,2,3,4,7,8,9 HpCDF OCDF P PCDF P PCDD/F

69.94 153.32 329.33 367.05 395.15 556.23 70.11 1645.36 269.62 1058.59 4914.74

6.99 7.67 164.67 36.71 39.52 55.62 7.01 16.45 2.70 1.06 338.39

19,495.02

528.63

Concentrations (pg/g) and International-Toxic Equivalent Quantities (I-TEQ; pg I-TEQ/g) of the PolyChlorinated Dibenzop-Dioxins (PCDD) and -Furans (PCDF) detected in the air pollution Particulate Matter (PM) as carried out by solvent extraction prior to analysis with high-resolution gas chromatography– P P high-resolution mass spectrometry. PCDD, PCDF, and P PCDD/F were calculated as sum of Tera- (T), Penta- (Pe), Hexa- (Hx), Hepta- (Hp), and Octa (O)-chlorinated PCDD, PCDF, or PCDD/F, respectively. I-TEQ were calculated using International-Toxic Equivalent Factors (I-TEF) defined by NATO/CCMS (1988) [54].

tically significant increases of EROD activity as compared to controls (p < 0.05). In return, no significant change of EROD activity was reported 24, 48 and 72 h after A549 cell exposure to TiO2, versus controls. Accordingly, a statistically significant correlation was observed between the gene transcripts of CYP1A1 and the EROD activities of CYP1A1 (Spearman’s rho = 0.722, p < 0.001).

3.3. Effects of PM on DNA adduct formation As shown by Fig. 1 , 32P-postlabeling method showed the presence of several DNA adduct spots (I, II, and III) on the autoradiograms of TLC sheets from A549 cells 72 h after their exposure to dPM at its Eq LC50, to PM at its LC50, and to B(a)P (1 lM). Since DNA adduct spot I was detected in controls, it could not be related to dPM, PM or BaP exposure. This spot will not be considered further. In contrast, Spot II appeared only after dPM, PM and B(a)P exposure, at a weak, comparable, but not reliably quantifiable, intensity in the three patterns. Three

150

S. Billet et al. / Cancer Letters 270 (2008) 144–155

Table 3 DLPCB and marker PCB detected in collected PM

4. Discussion

Congener

Lung cancer was significantly associated with elevated exposure to air pollution PM [1,42]. An hypothesis currently under investigation is that the organic chemicals-coated onto airborne PM could be closely involved in its pulmonary toxicity [31,43]. Hence, to improve the current knowledge, we were also interested in the ability of Dunkerque City’s PM2.5 to induce the gene expression and protein activity of CYP1A1 and, thereafter, bulky PAH–DNA adduct formation in A549 cells in culture. The physical and chemical characterization of collected PM showed not only PAH family members but also PCDD/F, DLPCB, and marker PCB associated to PM either through adherence to the carbonaceous core particle or as integral components [31]. It is also very interesting to report that the PAH concentrations measured in Dunkerque City’s PM2.5 were equal or smaller than those observed in PM samples collected in two different locations in Paris [44]. With regards to PCDD/F, the concentrations found in Dunkerque City’s PM2.5 under study were somewhat similar to those reported in airborne PM collected in two sites located at 1.1 and 2.1 km downwind from a municipal incinerator, respectively [45]. The presence of relatively low doses of PAH and other related compounds were also closely related to the anthropogenic emission sources located near the sampling site; apart the heavy motor vehicle traffic, iron, steel, and aluminum industries, oil refinery, and basic chemistry could be cited. Collected PM consisted also in a complex and heterogeneous mixture of pollutants, generally present at relatively low doses,

PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB PCB P

81 77 123 118 114 105 126 167 156 157 169 189

PCB PCB PCB PCB PCB PCB P

28 52 101 138 153 180

DLPCB

PCB

Concentrations (pg/g) <100 1447 <50 16,277 <50 9937 <50 2624 5015 1544 <50 912 37,757 51,907 50,616 16,576 32,815 23,628 21,645 197,187

Concentrations (pg/g) of Dioxin-Like PolyChlorinated Biphenyls (DLPCB) and marker PolyChlorinated Biphenyls (PCB) detected in the air pollution Particulate Matter (PM) as carried out by solvent extraction prior to analysis with high-resolution gas P chromatography–high-resolution mass spectrometry. DLPCB P and PCB were calculated as sum of DLPCB and PCB, respectively.

distinct DNA adduct spots, also included together in the area III, were observed with high intensity 72 h after A549 cell exposure to B(a)P (1 lM). The mean calculated RAL for this area III was 89.5  108 with a standard deviation of 24.1  108. Moreover, two of the three spots observed in the area III were also detected in the BPDE control electrophoresis pattern (see Fig. 1).

Fig. 1. PAH–DNA adduct patterns. Fig. 1 shows DNA adduct patterns observed by 32P-postlabeling method in A549 cells 72 h after their exposure. Non-exposed A549 cells served as Controls, and A549 cells exposed to desorbed Particulate Matter (i.e. dPM) at concentrations equivalent to its Lethal Concentration at 50% (i.e. Eq LC50 = 97.13 lg PM/mL or 25.90 lg PM/cm2), integrating weight losses due to the organic desorption, were used as negative controls. A549 cells were exposed to Particulate Matter (i.e. PM) at its Lethal Concentration at 50% (i.e. LC50 = 118.60 lg PM/mL or 31.63 lg PM/cm2). A549 cells exposed to Benzo(a)Pyrene (B(a)P), and calf thymus DNA exposed to B(a)P-7,8-Dihydrodiol-9,10-Epoxide (BPDE Control) served as positive controls.

Table 4 Gene expression of CYP1A1 in PM-exposed A549 cells Exposure

24 h 48 h 72 h

C

TiO2 EqLC10

TiO2 EqLC50

dPM EqLC10

dPM EqLC50

PM LC10

PM LC50

B(a)P 1 lM

1 ± 0.340 1 ± 0.231 1 ± 0.269

0.922 NS ± 0.350 0.894NS ± 0.264 0.869NS ± 0.232

1.205NS ± 0.502 0.824NS ± 0.053 1.018NS ± 0.278

1.620* ± 0.342 1.342** ± 0.254 1.578* ± 0.541

2.159** ± 0.671 1.683** ± 0.406 1.916** ± 0.545

2.889** ± 0.309 2.341** ± 0.203 2.419** ± 0.719

3.382** ± 1.054 2.669** ± 0.679 2.893** ± 1.020

3.235** ± 0.849 2.165** ± 0.334 2.65** ± 0.251

Table 5 Catalytic activity of CYP1A1 in PM-exposed A549 cells Exposure C 24 h 48 h 72 h

195 ± 16 115 ± 53 124 ± 50

TiO2 EqLC10 NS

177 ± 9 136NS ± 19 90NS ± 22

TiO2 EqLC50 NS

192 ± 9 649NS ± 21 240NS ± 32

dPM EqLC10

dPM EqLC50

*

*

20,166 ± 949 19,271* ± 3233 13,306* ± 2978

20,493 ± 3336 29,186* ± 4909 14,480* ± 1243

PM LC10 *

25,455 ± 2248 23,331* ± 7023 13,689* ± 3721

PM LC50 *

49,420 ± 2453 31,817* ± 7528 31,347* ± 3255

B(a)P 1 lM

S. Billet et al. / Cancer Letters 270 (2008) 144–155

Gene expression of cytochrome P450 1A1 (CYP1A1) (fold-induction versus controls) in A549 cells 24, 48 or 72 h after their exposure. Non-exposed A549 cells served as Controls (C), and A549 cells exposed to TiO2 or desorbed Particulate Matter (dPM) at concentrations equivalent to PM’s Lethal Concentrations at 10% (i.e. Eq LC10 = 19.42 lg PM/mL or 5.18 lg PM/cm2) and 50% (i.e. Eq LC50 = 97.13 lg PM/mL or 25.90 lg PM/cm2), integrating weight losses due to the organic desorption, were used as negative controls. In contrast, A549 cells exposed to Benzo(a)Pyrene (B(a)P) served as positive controls for the induction of the gene expression of CYP1A1. A549 cells were exposed to Particulate Matter (PM) at its Lethal Concentrations at 10% (i.e. LC10 = 23.72 lg PM/mL or 6.33 lg PM/cm2) or 50% (i.e. LC50 = 118.60 lg PM/mL or 31.63 lg PM/cm2). Gene expression of CYP1A1 are described by their mean ± standard deviation of 8 replicates for controls (C), and 4 replicates for negative controls, PM exposures, and positive controls (Mann–Whitney U-test; versus controls; NS: not significant; *p < 0.05; and **p < 0.01).

16,337* ± 988 42,960* ± 3938 35,727* ± 505

Catalytic activity (i.e. EthoxyResorufin O-Deethylase, EROD) of Cytochrome P450 1A1 (CYP1A1) (expressed in pmol/min/mg protein) in A549 cells 24, 48 or 72 h after their exposure. Non-exposed A549 cells served as Controls (C), and A549 cells exposed to TiO2 or desorbed Particulate Matter (dPM) at concentrations equivalent to PM’s Lethal Concentrations at 10% (i.e. Eq LC10 = 19.42 lg PM/mL or 5.18 lg PM/cm2) and 50% (i.e. Eq LC50 = 97.13 lg PM/mL or 25.90 lg PM/cm2), integrating weight losses due to the organic desorption, were used as negative controls. In contrast, A549 cells exposed to Benzo(a)Pyrene (B(a)P) served as positive controls for the induction of the organic metabolic activation. A549 cells were exposed to Particulate Matter (PM) at its Lethal Concentrations at 10% (i.e. LC10 = 23.72 lg PM/mL or 6.33 lg PM/cm2) or 50% (i.e. LC50 = 118.60 lg PM/mL or 31.63 lg PM/cm2). EROD activities are described by their mean ± standard deviation of 8 replicates for controls (C), and 4 replicates for negative controls, PM exposures, and positive controls (Mann–Whitney U-test; versus controls; NS: not significant; *p < 0.05).

151

152

S. Billet et al. / Cancer Letters 270 (2008) 144–155

thereby rendering very difficult the share of the toxicity ascribable to each fraction (inorganic vs organic, notably). However, as shown by Billet et al. [31], the role of physical carrier played by PM could enhance both the penetration and the retention of coated-organic chemicals into the lung, thereby enabling them to exert a longer toxicity [31]. Some of organic chemicals present in collected PM require tissue-specific activation to become biologically reactive. Xenobiotic-metabolizing enzymes including CYP1A1 are also present in the human lung and lung-derived cell lines, possibly contributing to in situ activation and inactivation of PAH. We also showed that the PAH and related compounds-coated onto Dunkerque City’s PM2.5 significantly induced both the gene expression and the catalytic activity of CYP1A1 in A549 cells. There was a statistically significant correlation between the gene transcripts and the catalytic activities of CYP1A1. This finding suggested that the relatively low doses of PAH-coated onto collected PM, ranging from 4.7 lg/g for fluoranthene to 141.9 lg/g for methylnaphthalene, and including pyrene (4.7 lg/g), benzo(b + k)fluoranthene (6.6 lg/g), B(a)P (7.9 lg/g), phenanthrene (28.3 lg/ g), and anthracene (47.1 lg/g), activated the transcription of AhR and induced PAH metabolic activation [2,12]. Other related compounds (i.e. PCDD/ F, DLPCB, and marker PCB), present at very variable concentrations in the airborne PM under study, could also be involved in the reported induction of both the gene expression and the catalytic activity of CYP1A1 in dPM and particularly PM-exposed A549 cells. Hence, the statistically significant increase of both the gene transcription and the EROD activity of CYP1A1 in dPM-exposed A549 cells, clearly showed that the employed outgassing method was not efficient enough to remove total PAH, PCDD/F, DLPCB, and marker PCB, as confirmed elsewhere [33]. Although the metabolic activation involved protective enzymes, in the case of PAH, it could contribute to the production of highly chemically reactive metabolites that could interact with DNA target sites, and produce DNA adducts, giving rise to mutation and, eventually, tumor initiation. There are a lot of frequently used biological markers of PAH exposure. Among them, PAH– DNA adducts are tentatively correlated to cancer risk because they represent the integrated consequence of exposure, absorption, distribution, metabolic activation, and DNA adverse interaction, and

the kinetic of DNA repair [24–28,46]. Generally, P-postlabeling has sensitivity ranging from 1 adduct/108 to 1 adduct/1010 nucleotides, thereby being sensitive enough for an application to a wide range of human environmental exposures [47,48]. In this study, quantifiable PAH–DNA adduct spots were seen in B(a)P-exposed A549 cells, thereby contributing to validate the protocol designed to detect bulky PAH–DNA adducts. Dunkerque City’s PM2.5 also induced faint DNA adducts 72 h after A549 cell exposure to dPM and PM. However, a comparable spot was seen after A549 cell exposure to B(a)P at 1 lM, which is a concentration about 250-fold higher than this resulting from PM exposure. It could also be speculated that, if the spot II corresponded to a B(a)P–DNA adduct, it would be greatly more intense in A549 cells exposed to B(a)P than in those exposed to PM. Conversely, if this spot would be closely linked to another PAH, it should be absent in A549 cells exposed to B(a)P. Consequently, we considered that this spot could not be definitively ascribed to PM exposure. Taken together, the results reported in dPM- and PM-exposed A549 cells revealed an apparent discrepancy between the induction of the gene expression and catalytic activity of CYP1A1, one the one hand, and the low level of PAH–DNA adducts, on the other hand. Some explanations might be considered. Firstly, it should be emphasized that the B(a)P concentration in A549 cells exposed to PM at their LC50, assuming the whole B(a)P-coated onto PM to be released, would be about 250-fold lower than in A549 cells exposed to B(a)P at 1 lM. Secondly, in other studies assessing the genotoxicity of pollutant mixtures, adduct spots formed by single genotoxic chemicals are too discrete, and only Diagonal Radioactive Zones (DRZ) can be used [17]. However, such DRZ did not appear in our work despite the presence of a lot of PAHcoated onto PM. Thirdly, one must keep in mind that other authors, carefully studying the ability of various air pollution PM to induce PAH–DNA adducts in different mammalian cell models in culture, confirmed their genotoxic potentials [17,18,49]. However, these results were reported following mammalian cell models exposure to Extractable Organic Matter (EOM) arising from air pollution PM, and also not to ambient PM such as collected Dunkerque City’s PM2.5. There could, therefore, be a great difference between the relatively high doses of PAH present in EOM, on the

32

S. Billet et al. / Cancer Letters 270 (2008) 144–155

one hand, and the relatively low doses of PAHcoated onto ambient PM, on the other hand. The major discrepancy between the EOM tested in the three studies vs the collected PM tested in the present work rendered very difficult any comparison between the effective doses of PAH in contact to target cell models. However, taking into account the characteristics of the PM sampling we used, an estimate, just as an indication, could be calculated. For example, the effective doses used by Sevastyanova et al. to compare the genotoxic potential of OEM from urban air PM were significantly higher (i.e. from 100- to 1000-fold) than those we used in this work [17]. Fourthly, the time of exposure we chose (i.e. 72 h) could allow the activation of mechanisms of DNA repair; the choice of shorter exposure times could perhaps minimize them. Fifthly, chemical reactions occurring in the atmosphere are known to affect the genotoxicity of PM [8,13,15–18,50– 53]. Sixthly, several co-contaminants of PAH, like PCDD/F, DLPCB, and PCB are often detected on airborne PM. While these chemicals are powerful AhR agonists and CYP1A1 gene expression and EROD activity inducers at low doses, they do not form bulky-DNA adducts [32]. One of the two overarching goals of Wu et al. [32] was to determine the effect of TCDD treatment on B(a)P-induced DNAadduct formation in the liver of BALB and CBA female mice, that have the AhRb2 genotype in common. Therefore, B(a)P was administered to mice 24 h after vehicle (control) or TCDD treatment. Reduced formation of B(a)P–DNA adducts was observed at the lowest dose of B(a)P tested (50 mg kg1) following TCDD exposure. At the higher B(a)P dose (200 mg kg1) following TCDD exposure, there was not a significant strain difference in B(a)P–DNA adduct formation. In agreement with the interesting results reported by Wu et al. [32], the relatively low levels of PAH together with the presence of other related compounds (i.e. PCDD/F, DLPCB, and PCB)-coated onto Dunkerque City’s PM2.5 could contribute to explain the very low, and therefore not reliably quantifiable, levels of PAH–DNA adducts detected in dPM and/or PM-exposed A549 cells. In conclusion, relatively low doses of PAHcoated onto air pollution PM2.5 were able to significantly induced both the gene expression and the catalytic activity of CYP1A1, but, in the experimental conditions chosen in this work, did not generate reliably quantifiable PAH–DNA adducts in A549 cells. We also concluded that, in the human lung

153

epithelial cell model and in the experimental conditions we chose, bulky-DNA adduct formation was apparently not a major factor involved in the Dunkerque City’s PM2.5-induced toxicity. Nevertheless, the research of the underlying mechanisms responsible for the toxicity of air pollution PM still stays one of the major challenges in further studies; in future work, the genotoxic potential of collected PM will notably be achieve by either classical bacterial assay (Ames test) or the Functional Analysis of Separated Alleles of p53 in Yeast (FASAY) after A549 cell exposure. Acknowledgements The Laboratoire de Recherche en Toxicologie Industrielle et Environnementale (LCE EA2598) participates in the Institut de Recherche en ENvironnement Industriel (IRENI), which is financed by the Re´gion Nord-Pas de Calais, the Ministe`re de l’Enseignement Supe´rieur et de la Recherche, and European Funds (FEDER). The research described in this article benefited from grants from the Agence Francßaise de Se´curite´ Sanitaire de l’Environnement et du Travail (AFSSET; Convention n°EST-2007-48), the Ministe`re de l’Enseignement Supe´rieur et de la Recherche (Convention n°16848-2005), and the Re´gion Nord-Pas de Calais (Convention n° 08070005) [54]. References [1] C.A. Pope III, R.T. Burnett, M.J. Thun, E.E. Calle, D. Krewski, K. Ito, et al., Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution, JAMA 287 (2002) 1132–1141. [2] A.G. Schwartz, G.M. Prysak, C.H. Bock, M.L. Cote, The molecular epidemiology of lung cancer, Carcinogenesis 28 (2007) 507–518. [3] P. Vineis, K. Husgafvel-Pursiainen, Air pollution and cancer: biomarker studies in human populations, Carcinogenesis 26 (2005) 1846–1855. [4] World Health Organization, The World Health Report – Reducing Risks to Health, Promoting Healthy Life, Report, 2002, ISBN-13 9789241562072. [5] K. Hemminki, G. Pershagen, Cancer risk of air pollution: epidemiological evidence, Environ. Health Perspect. 102 (Suppl 4) (1994) 187–192. [6] U.S.EPA, Air Quality Criteria for Particulate Matter (vol. I, EPA/600/P-99/002aF and vol. II, EPA/600/P-99/002bF), Report, 2004. [7] A. Kibble, R. Harrison, Point sources of air pollution, Occup. Med. (Lond.) 55 (2005) 425–431. [8] M. Peluso, P. Srivatanakul, A. Munnia, A. Jedpiyawongse, A. Meunier, S. Sangrajrang, et al., DNA adduct formation

154

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24]

S. Billet et al. / Cancer Letters 270 (2008) 144–155 among workers in a Thai industrial estate and nearby residents, Sci. Total Environ. 389 (2008) 283–288. M.M. Mumtaz, J.D. George, K.W. Gold, W. Cibulas, C.T. DeRosa, ATSDR evaluation of health effects of chemicals. IV. Polycyclic aromatic hydrocarbons (PAHs): understanding a complex problem, Toxicol. Ind. Health 12 (1996) 742– 971. R.M. Harrison, J. Yin, Particulate matter in the atmosphere: which particle properties are important for its effects on health?, Sci Total Environ. 249 (2000) 85–101. T.M. de Kok, H.A. Driece, J.G. Hogervorst, J.J. Briede, Toxicological assessment of ambient and traffic-related particulate matter: a review of recent studies, Mutat. Res. 613 (2006) 103–122. L.M. Tompkins, A.D. Wallace, Mechanisms of cytochrome P450 induction, J. Biochem. Mol. Toxicol. 21 (2007) 176– 181. L.D. Claxton, P.P. Matthews, S.H. Warren, The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity, Mutat. Res. 567 (2004) 347–399. M. Sorensen, H. Autrup, O. Hertel, H. Wallin, L.E. Knudsen, S. Loft, Personal exposure to PM2.5 and biomarkers of DNA damage, Cancer Epidemiol. Biomarkers Prev. 12 (2003) 191–196. G. Castano-Vinyals, A. D’Errico, N. Malats, M. Kogevinas, Biomarkers of exposure to polycyclic aromatic hydrocarbons from environmental air pollution, Occup. Environ. Med. 61 (2004) e12. B. Binkova, D. Vesely, D. Vesela, R. Jelinek, R.J. Sram, Genotoxicity and embryotoxicity of urban air particulate matter collected during winter and summer period in two different districts of the Czech Republic, Mutat. Res. 440 (1999) 45–58. O. Sevastyanova, Z. Novakova, K. Hanzalova, B. Binkova, R.J. Sram, J. Topinka, Temporal variation in the genotoxic potential of urban air particulate matter, Mutat. Res. 649 (2008) 179–186. J. Topinka, L.R. Schwarz, F.J. Wiebel, M. Cerna, T. Wolff, Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter, Mutat. Res. 469 (2000) 83–93. D. Palli, A. Russo, G. Masala, C. Saieva, S. Guarrera, S. Carturan, et al., DNA adduct levels and DNA repair polymorphisms in traffic-exposed workers and a general population sample, Int. J. Cancer 94 (2001) 121–127. J. Lewtas, Air pollution combustion emissions: characterization of causative agents and mechanisms associated with cancer, reproductive, and cardiovascular effects, Mutat. Res. 636 (2007) 95–133. S. Kato, E.D. Bowman, A.M. Harrington, B. Blomeke, P.G. Shields, Human lung carcinogen–DNA adduct levels mediated by genetic polymorphisms in vivo, J. Natl. Cancer Inst. 87 (1995) 902–907. H. Autrup, B. Daneshvar, L.O. Dragsted, M. Gamborg, M. Hansen, S. Loft, et al., Biomarkers for exposure to ambient air pollution – comparison of carcinogen–DNA adduct levels with other exposure markers and markers for oxidative stress, Environ. Health Perspect. 107 (1999) 233–238. P.B. Farmer, R. Singh, B. Kaur, R.J. Sram, B. Binkova, I. Kalina, et al., Mutat. Res. 544 (2003) 397–402. J. Lewtas, D. Walsh, R. Williams, L. Dobias, Air pollution exposure–DNA adduct dosimetry in humans and rodents:

[25]

[26]

[27] [28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

evidence for non-linearity at high doses, Mutat. Res. 378 (1997) 51–63. F.A. Beland, M.C. Poirier, Significance of DNA adduct studies in animal models for cancer molecular dosimetry and risk assessment, Environ. Health Perspect. 99 (1993) 5–10. M.C. Poirier, A. Weston, B. Schoket, H. Shamkhani, C.F. Pan, M.A. McDiarmid, et al., Biomonitoring of United States Army soldiers serving in Kuwait in 1991, Cancer Epidemiol. Biomarkers Prev. 7 (1998) 545–551. D.H. Phillips, DNA adducts as markers of exposure and risk, Mutat. Res. 577 (2005) 284–292. M.C. Poirier, A. Weston, Human DNA adduct measurements: state of the art, Environ. Health Perspect. 104 (Suppl 5) (1996) 883–893. P.B. Farmer, D.E. Shuker, What is the significance of increases in background levels of carcinogen-derived protein and DNA adducts? Some considerations for incremental risk assessment, Mutat. Res. 424 (1999) 275–286. D.H. Phillips, M. Castegnaro, Standardization and validation of DNA adduct postlabelling methods: report of interlaboratory trials and production of recommended protocols, Mutagenesis 14 (1999) 301–315. S. Billet, G. Garcon, Z. Dagher, A. Verdin, F. Ledoux, F. Cazier, et al., Ambient particulate matter (PM2.5): physicochemical characterization and metabolic activation of the organic fraction in human lung epithelial cells (A549), Environ. Res. 105 (2007) 212–223. Q. Wu, J.S. Suzuki, H. Zaha, T.M. Lin, R.E. Peterson, C. Tohyama, et al., Differences in gene expression and benzo[a]pyrene-induced DNA adduct formation in the liver of three strains of female mice with identical AhR(b2) genotype treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin and/or benzo[a]pyrene, J. Appl. Toxicol. (2008). S. Billet, F. Ledoux, Z. Dagher, D. Courcot, G. Garcon, F. Cazier, et al., Stability of organic compounds in atmospheric particulate matter and its relation with textural properties, Chem. Eng. Trans. 10 (2006) 101–107. G. Garcon, P. Gosset, F. Zerimech, B. Grave-Descampiaux, P. Shirali, Effect of Fe(2)O(3) on the capacity of benzo(a)pyrene to induce polycyclic aromatic hydrocarbon-metabolizing enzymes in the respiratory tract of Sprague–Dawley rats, Toxicol. Lett. 150 (2004) 179–189. M.W. Powley, G.P. Carlson, Species comparison of hepatic and pulmonary metabolism of benzene, Toxicology 139 (1999) 207–217. M. Iwanari, M. Nakajima, R. Kizu, K. Hayakawa, T. Yokoi, Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences, Arch. Toxicol. 76 (2002) 287–298. F.P. Guengerich, Separation and purification of multiple forms of microsomal cytochrome P-450. Partial characterization of three apparently homogeneous cytochromes P-450 prepared from livers of phenobarbital- and 3-methylcholanthrene-treated rats, J. Biol. Chem. 253 (1978) 7931–7939. P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, et al., Measurement of protein using bicinchoninic acid, Anal. Biochem. 150 (1985) 76–85. M.T. Donato, J.V. Castell, M.J. Gomez-Lechon, Characterization of drug metabolizing activities in pig hepato-

S. Billet et al. / Cancer Letters 270 (2008) 144–155

[40]

[41]

[42]

[43]

[44]

[45]

[46]

cytes for use in bioartificial liver devices: comparison with other hepatic cellular models, J. Hepatol. 31 (1999) 542– 549. K. Randerath, M.V. Reddy, R.C. Gupta, 32P-labeling test for DNA damage, Proc. Natl. Acad. Sci. USA 78 (1981) 6126–6129. R.C. Gupta, M.V. Reddy, K. Randerath, 32P-postlabeling analysis of non-radioactive aromatic carcinogen–DNA adducts, Carcinogenesis 3 (1982) 1081–1092. D.E. Abbey, P.K. Mills, F.F. Petersen, W.L. Beeson, Longterm ambient concentrations of total suspended particulates and oxidants as related to incidence of chronic disease in California Seventh-Day Adventists, Environ. Health Perspect. 94 (1991) 43–50. J. Liden, A. Ek, L. Palmberg, S. Okret, K. Larsson, Organic dust activates NF-kappaB in lung epithelial cells, Respir. Med. 97 (2003) 882–892. A. Baulig, J.J. Poirault, P. Ausset, R. Schins, T. Shi, D. Baralle, et al., Physicochemical characteristics and biological activities of seasonal atmospheric particulate matter sampling in two locations of Paris, Environ. Sci. Technol. 38 (2004) 5985–5992. M.R. Chao, C.W. Hu, H.W. Ma, G.P. Chang-Chien, W.J. Lee, L.W. Chang, et al., Size distribution of particle-bound polychlorinated dibenzo-p-dioxins and dibenzofurans in the ambient air of a municipal incinerator, Atmos. Environ. 37 (2003) 4945–4954. R.L. Melnick, Carcinogenicity and mechanistic insights on the behavior of epoxides and epoxide-forming chemicals, Ann. NY Acad. Sci. 982 (2002) 177–189.

155

[47] G. Boysen, S.S. Hecht, Analysis of DNA and protein adducts of benzo[a]pyrene in human tissues using structurespecific methods, Mutat. Res. 543 (2003) 17–30. [48] D.H. Phillips, Detection of DNA modifications by the 32Ppostlabelling assay, Mutat. Res. 378 (1997) 1–12. [49] A.K. Sharma, K.A. Jensen, J. Rank, P.A. White, S. Lundstedt, R. Gagne, et al., Genotoxicity, inflammation and physico-chemical properties of fine particle samples from an incineration energy plant and urban air, Mutat. Res. 633 (2007) 95–111. [50] L.D. Claxton, G.M. Woodall Jr., A review of the mutagenicity and rodent carcinogenicity of ambient air, Mutat. Res. 636 (2007) 36–94. [51] J. Topinka, O. Sevastyanova, B. Binkova, I. Chvatalova, A. Milcova, Z. Lnenickova, et al., Biomarkers of air pollution exposure – a study of policemen in Prague, Mutat. Res. 624 (2007) 9–17. [52] F. Perera, D. Brenner, A. Jeffrey, J. Mayer, D. Tang, D. Warburton, et al., DNA adducts and related biomarkers in populations exposed to environmental carcinogens, Environ. Health Perspect. 98 (1992) 133–137. [53] E. Grzybowska, K. Hemminki, M. Chorazy, Seasonal variations in levels of DNA adducts and X-spots in human populations living in different parts of Poland, Environ. Health Perspect. 99 (1993) 77–81. [54] NATO/CCMS, International toxicity equivalency factors (ITEF) method of risk assessments for complex mixtures of dioxins and related compounds (North Atlantic Treaty Organization/Committee on The Challenges of Modern Society), Report, vol. 176, 1988.