Role of air pollution Particulate Matter (PM2.5) in the occurrence of loss of heterozygosity in multiple critical regions of 3p chromosome in human epithelial lung cells (L132)

Toxicology Letters 187 (2009) 172–179

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Role of air pollution Particulate Matter (PM2.5 ) in the occurrence of loss of heterozygosity in multiple critical regions of 3p chromosome in human epithelial lung cells (L132) Franc¸oise Saint-Georges a , Guillaume Garc¸on b , Fabienne Escande c , Imane Abbas b , Anthony Verdin b , Pierre Gosset d , Philippe Mulliez a , Pirouz Shirali a,∗ a

Service de Pneumologie, Hôpital Saint-Philibert, Groupement Hospitalier de l’Institut Catholique-Faculté Libre de Médecine de Lille, Rue du Grand But, BP 249, 59462 Lomme Cedex, France b LCE-EA2598, Toxicologie Industrielle et Environnementale, Université du Littoral - Côte d’Opale, Maison de la Recherche en Environnement Industriel de Dunkerque 2, Avenue Maurice Schumann, 59140 Dunkerque, France c Laboratoire d’Oncologie et Génétique Moléculaires, Centre de Biologie Pathologie, Boulevard du Pr J. Leclercq, Centre Hospitalier Régional et Universitaire, 59037 Lille Cedex, France d Laboratoire d’Anatomie et de Cytologie Pathologique, Hôpital Saint-Vincent, Groupement Hospitalier de l’Institut Catholique-Faculté Libre de Médecine de Lille, Rue du Port, 59046 Lille Cedex, France

a r t i c l e

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Article history: Received 26 January 2009 Received in revised form 19 February 2009 Accepted 23 February 2009 Available online 9 March 2009 Keywords: L132 cells Air pollution Particulate Matter 3p chromosome multiple critical regions Loss of heterozygosity Microsatellite instability

a b s t r a c t Lung cancer still remains the most frequent type of cancer all around the world and the leading cause of cancer-related death. Even if tobacco use takes a major part in etiology of lung cancer, other explanations like genetic and lifestyle factors, and occupational and/or environmental exposure to carcinogens have to be considered. Hence, in this study, we were interested in the ability of in vitro short-term exposure to air pollution Particulate Matter (PM) to induce genomic alterations in Dunkerque City’s PM2.5 -exposed human epithelial lung cells (L132). The occurrence of MicroSatellite (MS) alterations in 3p multiple critical regions (i.e. 3p14.1, 3p14.2, 3p14.3, 3p21.1, 3p21.31, and 3p21.32) identified as showing frequent allelic losses in benign or malignant lung diseases, was also studied in Dunkerque City’s PM2.5 -exposed L132 cells. Negative (i.e. TiO2 ; desorbed PM, dPM), and positive (i.e. benzo[a]pyrene, B[a]P) controls were also included in the experimental design. Loss Of Heterozygosity (LOH) and/or MicroSatellite Instability (MSI) were reported 72 h after L132 cell exposure to dPM (i.e. 61.71 ␮g dPM/mL or 12.34 ␮g dPM/cm2 ), PM (i.e. 75.36 ␮g PM/mL or 15.07 ␮g PM/cm2 ), or B[a]P (i.e. 1 ␮M). In agreement with the current literature, such MS alterations might rely on the ability of dPM, PM or B[a]P to induce oxidative stress conditions, thereby altering DNA polymerase enzymes, enhancing DNA recombination rates, and inhibiting DNA repair enzymes. Hence, we concluded that the occurrence of dramatic MS alterations in 3p chromosome multiple critical regions could be a crucial underlying mechanism, which proceeded the lung toxicity in air pollution PM-exposed target L132 cells. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Air pollution Particulate Matter (PM) is an important environmental health risk factor for respiratory and cardiovascular morbidity and/or mortality (Maier et al., 2008; Schwartz et al., 2007; Vineis and Husgafvel-Pursiainen, 2005). Accordingly, longterm exposure to high concentrations of air pollution PM can increase the risk of lung cancer, Chronic Obstructive Respiratory

∗ Corresponding author at: LCE EA2598, Toxicologie Industrielle et Environnementale, MREI 2, ULCO, 189A, Avenue Maurice Schumann, 59140 Dunkerque, France. Tel.: +33 3 28237610; fax: +33 3 28237171. E-mail address: [email protected] (P. Shirali). 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.02.016

Diseases (COPD), and arteriosclerosis, whereas short-term exposure can cause exacerbation of several forms of other respiratory diseases, including bronchitis and asthma, as well as changes in heart rate variability (Brunekreef and Holgate, 2002; Dominici et al., 2007; Elliott et al., 2007; Hales and Howden-Chapman, 2007; Pope, 2004; Samet et al., 2000; Sorensen et al., 2003). Nowadays, lung cancer still remains the most frequent type of cancer all around the world and the leading cause of cancer-related death, not only in males but also in females (Black et al., 1997; Boyle et al., 2007; Carpagnano et al., 2005; Okutan et al., 2004; Schottenfeld, 2000). Even if tobacco use takes a major part in etiology of lung cancer, other explanations like genetic and lifestyle factors, and occupational and/or environmental exposure to various carcinogens have also to be considered (De Kok et al., 2006; Dominici et al., 2007;

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Elliott et al., 2007; Hales and Howden-Chapman, 2007; Peluso et al., 2008). Besides reduction of chemical emission, and cessation and/or prevention of human exposure, early detection of lung diseases is clearly the only way to counteract the lung disease epidemic by improving the overall survival (Brambilla et al., 2003). A recent approach involved locating molecular markers of lung diseases (Carpagnano et al., 2005; Pan et al., 2005; Samara et al., 2006). Molecular markers were firstly studied through their application in the early diagnosis of lung cancer or tumor progression, potentially permitting the individualization of early cell damage and non-invasive staging of the disease (Carpagnano et al., 2005). Human cancers are now widely accepted as genetic diseases characterized by the accumulation of molecular genetic and epigenetic abnormalities in critical target specific genes (Ellegren, 2000; Hancock, 1999; Schlotterer and Wiehe, 1999). Hence, genetic and epigenetic abnormalities (i.e. gene mutation, genetic instability, promoter methylation, and overexpression, etc.) in the genes closely involved in cell cycle, senescence, apoptosis, repair, differentiation and cell migration control have been reported in different biological samples of patients with lung cancer (Brambilla et al., 2003; Carpagnano et al., 2005; Pan et al., 2005). One of the major challenges in cancer diagnosis is the use of cancer-specific markers for the early detection of sporadic cancer (Brambilla et al., 2003). Although the analysis of genetic alterations of critical target specific genes provide an accurate molecular basis for assessment of the cancer stages, most of these alterations could only be detected at an advanced stage and could be a laborious and expensive undertaking with a low return in treating the disease (Pan et al., 2005; Samara et al., 2006; Vineis and Husgafvel-Pursiainen, 2005). However, recent insights into the molecular basis of cancer have recognized the occurrence of some triggering molecular events likely to result in the development of lung cancer, including MicroSatellite (MS) alterations (Carpagnano et al., 2005; De la Chapelle, 2003; Zhivotovsky and Kroemer, 2004). Some studies have also confirmed that MS alterations (i.e. Loss Of Heterozigosity, LOH; MicroSatellite Instability, MSI) could be used as molecular markers for genetic alterations such as deletion of Tumor Suppressor Genes (TSG), and could manifest susceptibility to cancer in apparent phenotypically normal preneoplastic cells that may eventually progress to become cancer (Zhou et al., 2000). Moreover, molecular markers, and notably MS alterations, were also recently studied through their application in the genetic predisposition to benign lung diseases as such as COPD, asthma, sarcoidosis, and idiopathic pulmonary fibrosis [for review, see Samara et al., 2006). Hence, allelic loss and cytogenetic studies have pointed to several candidate regions in different chromosomes of the genome, as such as 1p, 3p, 5q, 8p, 9p, 11q, 13q, 17p, 18q, and X, for the involvement of TSG in lung cancer susceptibility, and 3p, 5q, 6p, 8p, 9p, 9q, 11q, 13q, 14q, and 17q, in other lung disease susceptibility (i.e. COPD, asthma, sarcoidosis, and idiopathic pulmonary fibrosis) (Brambilla et al., 2003; Carpagnano et al., 2005; Musti et al., 2006; Pan et al., 2005; Samara et al., 2006). In particular, it was shown several years ago, that deletion of the short arm of chromosome 3 is very frequent. Very early in the evolution of preinvasive disease, localized specific MS alterations in 3p chromosome occur, even in phenotypically normal or hyperplastic epithelium (Kerr, 2001). Multiple critical regions identified as showing frequent allelic losses in non-malignant lung diseases or primary lung cancer have been identified as 3p12, 3p14.2, 3p21, and 3p25 on chromosome 3p (Boyle et al., 2001; Greenberg et al., 2002; Hirao et al., 2001; Koy et al., 2008; Marsit et al., 2004; Musti et al., 2006; Pan et al., 2005; Samara et al., 2006; Sanz-Ortega et al., 2001; Trimeche et al., 2008). In order to contribute to a better knowledge of the underlying mechanisms of action involved in air pollution PM2.5 -induced lung toxicity, we have undertaken an extensive in vitro research.

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Briefly, after the collection of air pollution PM2.5 in Dunkerque, a French highly industrialized seaside city, the most toxicologically relevant physical and chemical characteristics have been carried out (Billet et al., 2007, 2008). We have also already shown that in vitro short-term exposure to Dunkerque City’s PM2.5 induced doseand time-dependent oxidative damage, activation of nuclear factorkappa B/inhibitory kappa B complex, inflammatory response, and apoptotic events in cells originated from the normal lung tissue of a human embryo in culture (L132 cell line) (Dagher et al., 2005, 2006, 2007; Garc¸on et al., 2006, 2007). Moreover, Abbas et al. (2009) have recently reported that the metabolic activation of the very low doses of Volatile Organic Compounds (VOC) and/or Polycyclic aromatic Hydrocarbons (PAH)-coated onto Dunkerque City’s PM2.5 constituted one of the underlying mechanisms of action closely involved in its cytotoxicity in L132 cells. Considering this in vitro short-term exposure system, we were therefore interested in the key role potentially played by Dunkerque City’s PM2.5 in the occurrence of genetic alterations, through the study of MS alterations using well defined human polymorphic markers in 3p multiple critical regions (i.e. 3p14.1: D3S1261; 3p14.2: D3S1300 and D3S1312; 3p14.3: D3S3719; 3p21.1: D3S3561; 3p21.31: D3S3629; 3p21.32: D3S3624). 2. Materials and methods 2.1. Chemicals Minimum Essential Medium (MEM) with Earle’s salts, Fetal Bovine Serum (FBS), l-Glutamin, Penicillin/Streptomycin solution, Trypsin, 0.05% with EthyleneDiamine-Tetraacetic Acid (EDTA), and sterile Phosphate-Buffered Solution (PBS) were from InVitrogen SARL (Cergy Pontoise, France). Titanium (IV) oxide powder (anatase; purity: 99%; primary particle size: 0.2 ␮m; surface not coated) was from Acros Organics (Noisy Le Grand, France). Benzo[a]Pyrene (B[a]P) was from Sigma–Aldrich (St-Quentin Fallavier, France). Qiagen (Courtaboeuf, France) supplied QIAamp DNA Mini kit and Qiagen Multiplex PCR kit. Fluorescent dye-labeled forward primers and unlabeled reverse primers were from Applied Biosystems France SA (Paris, France). 2.2. Methods 2.2.1. PM sampling, physical and chemical characteristics, and outgassing PM sampling: PM was collected in Dunkerque City (51◦ 04 N; 2◦ 38 E; France), a French seaside city located on the southern coast of the North-Sea, using a high volume cascade impactor (Billet et al., 2007). Briefly, no back-up filter was used to maintain a constant aspiration flow rate, and the lowest stage was doubled to increase the efficiency of smallest particle sampling. Meteorological data (i.e. wind speed, wind direction, temperature, etc.) were obtained from Météo France (Villeneuve d’Ascq, France). With regards to the direction of the prevailing winds, Dunkerque City is located below the atmospheric emission arising from the nearby industrial activities (i.e. iron and steel industry, aluminum industry, oil refinery, basic chemistry, pharmaceutical industry, plant health production, food industry, etc.) and motor vehicle traffic. Physical and chemical characteristics: PM size distribution, as carried out by scanning electron microscopy, showed that the highest number of PM (i.e. 92.15%) was detected in size classes including PM with a size ≤2.5 ␮m: 0–0.5 ␮m (33.63%), 0.5–1.0 ␮m (30.61%), 1.0–1.5 ␮m (14.33%), 1.5–2.0 ␮m (8.69%), and 2.0–2.5 ␮m (4.89%) (Billet et al., 2007). Adsorption data from the Brunauer–Emmett–Teller method provided a specific surface area of 1 m2 /g for PM, and 49.9 m2 /g for dPM (Billet et al., 2007). Inductively coupled plasma-atomic emission spectrometry showed that Fe (78.4 mg/g), Al (58.3 mg/g), Ca (49.5 mg/g), Na (18.8 mg/g), K (9.7 mg/g), Mg (8.1 mg/g), Pb (8.0 mg/g), and Ti (5.1 mg/g) were the most abundant inorganic elements. Gas chromatography/mass spectrometry, after thermal desorption at 500 ◦ C and cold trapping, or soxhlet extraction with dichloromethane, revealed the presence of VOC (e.g. benzene: 106.5 ␮g/g; diethylbenzene: 170.3 ␮g/g; tetramethylbenzene: 89.5 ␮g/g; toluene: 42.2 ␮g/g; xylene: 65.4 ␮g/g; etc.) and PAH (e.g. anthracene: 47.1 ␮g/g; phenanthrene: 28.3 ␮g/g; pyrene: 4.7 ␮g/g; benzo(a)pyrene: 7.8 ␮g/g; benzo(a)anthracene: 4.9 ␮g/g)-coated onto collected PM, respectively (Billet et al., 2007). High resolution gas chromatography/high resolution mass spectrometry after solvent extraction indicated the coating of PolyChlorinated Dibenzo-p-Dioxins (PCDD: (14,580 pg/g or 190 pg I-TEQ/g), and PolyChlorinated DibenzoFurans (PCDF: 4,914 pg/g or 338 pg I-TEQ/g), Dioxin-Like PolyChlorinated Biphenyls (DL-PCB: 37,757 pg/g), and marker PolyChlorinated Biphenyls (PCB: 197,187 pg/g) onto collected PM (Billet et al., 2008). PM outgassing: Desorbed PM (dPM), i.e. PM having undergone a thermal desorption at 400 ◦ C under a secondary vacuum and having thereby kept inorganic

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structures and lost most of organic chemicals, was used as negative control (Billet et al., 2007). 2.2.2. Cell line, culture conditions, and cell exposure Cell line: The cell line we used originates from the normal lung tissue of a human embryo, and is deposited under the designation L132 in the American Type Culture Collection (ATCC; ATCC number: CCL-5). The morphology of these cells is epithelial, and they exhibit typical features of pneumocytes (Erfle and Mellert, 1996). Culture conditions: L132 cells were cultured in sterile plastic flasks (Corning; Fisher Scientific Labosi SAS, Elancourt, France), in MEM with Earle’s salts, containing: 1% (v/v) l-Glutamin, 10% (v/v) FBS, 1% (v/v) penicillin (10,000 IU/mL), and 1% (v/v) streptomycin (10,000 ␮g/mL). Exponentially growing cells were maintained at 37 ◦ C, in a humidified atmosphere containing 5% CO2 . All the L132 cells we used in this study derived from the same initial cell culture. Cell exposure: L132 cells were seeded in sterile 25 cm2 plastic flasks (i.e. 500 × 103 cells/5 mL MEM with Earle’s salts containing 1% (v/v) l-Glutamin; 10% (v/v) FBS, 1% (v/v) penicillin (10,000 IU/mL), and 1% (v/v) streptomycin (10,000 ␮g/mL)), and incubated at 37 ◦ C, in a humidified atmosphere containing 5% CO2 , for 24 h. Thereafter, cell culture supernatants were carefully removed to eliminate nonadherent cells. Only living L132 cells were also incubated in the continuous presence of PM at its calculated Lethal Concentration (LC) at 50% (i.e. LC50 = 75.36 ␮g PM/mL or 15.07 ␮g PM/cm2 ) for 72 h of incubation, without renewing the cell culture media. Garc¸on et al. (2006) have already calculated LC values from Dunkerque City’s PM2.5 under study in L132 cells using the Colony Forming Method. L132 cells incubated only with cell culture media were used as controls, and TiO2 or dPM-exposed L132 cells as negative controls. L132 cells were exposed to TiO2 or dPM at their Equivalent Lethal Concentration (Eq LC) to inorganic (i.e. Eq LC50 = 61.71 ␮g/mL or 12.34 ␮g/cm2 ), integrating thereby mass losses arising from the thermal desorption of the PM-coated organic fraction. B[a]P (1 ␮M)-exposed cells served as positive control (Abbas et al., 2009). Hence, 2 culture flasks were chosen at random as nonexposed cells, 2 culture flasks as negative controls, 1 culture flask as exposed cells, and 1 culture flasks as positive control. 2.2.3. L132 cloning by limit-detection method Just after their 72-h incubation as controls, negative controls, PM-exposed cells, or positive control, L132 cells were harvested by trypsinisation (trypsin-EDTA, 0.05% (v/v)) and cloned by the limit-dilution method. Briefly, L132 cells were seeded in sterile 96-well culture plates (Costar; Fisher Scientific Labosi SAS) at a density of 1 cell/200 ␮l MEM with Earle’s salts containing 1% (v/v) l-Glutamin; 10% (v/v) FBS, 1% (v/v) penicillin (10,000 IU/mL), and 1% (v/v) streptomycin (10,000 ␮g/mL)), and incubated at 37 ◦ C, in a humidified atmosphere containing 5% CO2 , for 24 h. Thereafter, the effective seeding of each well was checked by optical microscopy, and cell culture supernatants were carefully removed to eliminate non-adherent cells. Limit-dilution method was also used to select and clone L132 cells, and ensure their exponentially growing until their number was sufficient to allow a reliable DNA extraction (i.e. 21 days). 2.2.4. Microsatellite markers and loss of heterozygosity analysis Microsatellite markers: The MS markers were chosen to reflect the multiple critical DNA regions frequently lost in the short arm of the chromosome 3 in benign and malignant lung diseases (Pan et al., 2005; Park et al., 1999; Samara et al., 2006; Sanz-Ortega et al., 2001; Wistuba et al., 2000). A panel of 7 MS markers spanning in 3p multiple critical regions (i.e. 3p14.1: D3S1261; 3p14.2: D3S1300 and D3S1312; 3p14.3: D3S3719; 3p21.1: D3S3561; 3p21.31: D3S3629; 3p21.32: D3S3624) were used (Fig. 1). Details of the investigated MS markers, their chromosomal location, and their heterozygosity frequency, cited from Ensembl Genome Browser (http://www.ensembl.org/index.html) and GeneLoc Home Page (http://genecards.weizmann.ac.il/geneloc/index.shtml), are provided in Table 1. LOH analysis: Genomic DNA was isolated from L132 cells using QIAamp DNA Mini Kit, according to the manufacturer’s recommendations. Thereafter, MS fragment analysis was carried out after fluorescent-labeled multiplex Polymerase Chain Reaction (PCR) amplification using fluorescent dye-labeled forward primer (i.e. 6FAM, NED, or VIC) and unlabeled reverse primer (Applied Biosystems France SA). To facilitate multiplexing, the expected product sizes of the amplified MS markers and the annealing temperatures were considered; the sequences of the fluorescent dye-labeled forward primers and unlabeled reverse primers we used are provided in Table 1. Multiplex PCR amplifications were realized in 25 ␮L reaction volumes containing 100 ng genomic DNA, using the Qiagen Multiplex PCR kit, according to the manufacturer’s instructions. The cycling conditions we applied on a Applied Biosystems 7500 Fast Real-Time PCR System (Applera France SA, Courtaboeuf, France) were as follow: initial activation step (15 min, 95 ◦ C), 40 repetitions of the 3-step cycling (i.e. denaturation: 30 s, 94 ◦ C; annealing: 90 s, 60 ◦ C; extension: 60 s, 72 ◦ C), and final extension step (i.e. 30 min, 60 ◦ C). Thereafter, capillary electrophoresis was performed using an 3130XL Genetic analyzer DNA sequencer (Applied Biosystems France SA), and the results were analyzed using the GeneScan software (Applied Biosystems France SA). MS fragments present as a single fragment in non-exposed L132 cells DNA represented homozygous alleles and were considered non-informative. On comparison of non-exposed L132 cell DNA and exposed L132 cell DNA, presence of a new DNA band in exposed L132 cells was defined as MSI, whereas LOH was scored by the visual detection of the complete

Fig. 1. Localization of the well-defined human polymorphic markers under study in 3p multiple critical regions (i.e. 3p14.1: D3S1261; 3p14.2: D3S1300 and D3S1312; 3p14.3: D3S3719; 3p21.1: D3S3561; 3p21.31: D3S3629; 3p21.32: D3S3624) (MutL Homologue 1, MLH1; Fragile Histidine Triad, FHIT).

absence of a band in the exposed L132 cell DNA from a constitutively heterozygous sample. 2.2.5. Statistical analysis MS status were provided as the fragment size (bp) and the frequency (%) of 10 replicates for controls (i.e. non-exposed cells), negative controls (i.e. TiO2 , dPM), PM-exposed cells, and positive control (i.e. B[a]P). Results from negative controls (i.e. TiO2 , dPM), PM-exposed cells, or positive control (i.e. B[a]P), were compared with those from controls (i.e. non-exposed cells). Statistical analysis was performed by the Chi-square test with Yates correction (Software: SPSS for Windows, v10.05, 2000; Paris, France). Statistically significant differences were reported with p values <0.05.

3. Results 3.1. LOH analysis at 3p chromosome multiple critical regions in PM-exposed L132 cells The MS markers we used to study the effects of air pollution PM on genetic alterations in L132 cells in culture were chosen to reflect the multiple critical DNA regions frequently lost in the short arm of the chromosome 3 in benign and malignant lung diseases. The MS markers under study were also located on well defined human polymorphic markers in 3p multiple critical regions (i.e. 3p14.1: D3S1261; 3p14.2: D3S1300 and D3S1312; 3p14.3: D3S3719; 3p21.1: D3S3561; 3p21.31: D3S3629; 3p21.32: D3S3624). Before to study the ability of air pollution PM to induce MS alterations in 3p multiple critical regions in L132 cells, the heterozygous status of the MS under study was checked in non-exposed L132 cells. As shown in Tables 1 and 2, despite their relative high frequency of heterozygosity provided by the Geneloc Home Page (http://genecards.weizmann.ac.il/geneloc/index.shtml) three (i.e.

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Table 1 Characteristics of the polymorphic loci at the well-defined human polymorphic markers in 3p multiple critical regions. Accession number

Locus name

Chromosome localization, genetic map (bp)

Product size (bp)

Labeled forward primer/unlabeled reverse primer

Fluorescent dye

Heterozygosity frequency

Z16488

D3S1261

3p14.1 68,785,036–68,785,220

185

5 -TGGGTGAAATGGGAGC-3 5 -GGCAAGTTAGGTGGCAGAAG-3

6-FAM

0.84

Z17006

D3S1300

3p14.2 60,484,947–60,485,189

243

5 -AGCTCACATTCTAGTCAGCCT-3 5 -GCCAATTCCCCAGATG-3

VIC

0.83

Z17154

D3S1312

3p14.2 62,381,450–62,381,672

223

5 -GGCTCCCCAGGGTAAG-3 5 -TGGGTTCTGCCTCCAA-3

NED

0.75

Z51863

D3S3719

3p14.3 55,425,928–55,426,196

269

5 -TCATGCAGAATCTCATGGGT-3 5 - CGCCCTGACTGGCTCTTA-3

6-FAM

0.82

Z52269

D3S3561

3p21.1 52,316,909–52,317,131

223

5 -TCCTGGGGACTGTGATG-3 5 -GGTGACTGGAGGTTCAAG-3

6-FAM

0.73

Z53408

D3S3629

3p21.31 49,558,179–49,558,426

248

5 -CAAGCAGATGTTGCTGCC-3 5 -GGAATTACCTGGATTGCCC-3

6-FAM

0.78

Z53367

D3S3624

3p21.32 44,588,821–44,588,972

152

5 -GGGATATGACTGCCCAAC-3 5 -GCCTCAAAATGCGAATG-3

6-FAM

0.72

Characteristics of the polymorphic loci at the well-defined human polymorphic markers in 3p multiple critical regions were provided by Ensembl Genome Browser (http://www.ensembl.org/index.html) and GeneLoc Home Page (http://genecards.weizmann.ac.il/geneloc/index.shtml).

D3S1300, D3S3561, D3S3629) of the seven MS we chose have homozygous (uninformative) status in non-exposed lung target L132 cells in culture. Therefore, only the four other MS (i.e. D3S1261; D3S1312; D3S3719; D3S3624) we included in this study have constitutional heterozygous (informative) status in the non-exposed target lung cell model we used and were also considered thereafter to study the appearance of MS alterations in 3p multiple critical regions in PM-exposed L132 cells (Tables 1 and 2). As shown in Table 3, no change in the heterozygous status of the four MS was observed in TiO2 -exposed L132 cells, versus controls (i.e. non-exposed cells). In contrast, statistically significant alterations in the constitutional heterozygous status of the four MS were seen in dPM or PM-exposed L132 cells, versus controls (i.e. nonexposed cells; p < 0.01), thereby inducing a LOH. As shown in Fig. 2, at D3S1312, there was also a loss of one (i.e. 211) of the two alleles (i.e. 211/221) resulting in a LOH in dPM or PM-exposed L132 cells, versus controls (i.e. non-exposed cells; p < 0.01). At DS1261 and D3S3624, a loss of both the alleles (i.e. 183/193 and 141/143, respectively) and the occurrence of one longer size allele (i.e. 206 and 145,

Table 2 Status of the polymorphic loci at the well-defined human polymorphic markers in 3p multiple critical regions under study in non-exposed L132 cells. Accession number

Locus name

Chromosome localization, genetic map (bp)

Microsatellite status

Z16488

D3S1261

3p14.1 68,785,036–68,785,220

Heterozygous

Z17006

D3S1300

3p14.2 60,484,947–60,485,189

Homozygous

Z17154

D3S1312

3p14.2 62,381,450–62,381,672

Heterozygous

Z51863

D3S3719

3p14.3 55,425,928–55,426,196

Heterozygous

Z52269

D3S3561

3p21.1 52,316,909–52,317,131

Homozygous

Z53408

D3S3629

3p21.31 49,558,179–49,558,426

Homozygous

Z53367

D3S3624

3p21.32 44,588,821–44,588,972

Heterozygous

Characteristics of the polymorphic loci at the well-defined human polymorphic markers in 3p multiple critical regions were provided by Ensembl Genome Browser (http://www.ensembl.org/index.html) and GeneLoc Home Page (http://genecards.weizmann.ac.il/geneloc/index.shtml).

respectively) were reported in dPM or PM-exposed L132 cells as compared to controls (i.e. non-exposed cells; p < 0.05 or p < 0.01). In contrast, at D3S3719, there were a loss of both the alleles (i.e. 265/272) and the occurrence of one shorter size allele (260) in dPM or PM-exposed L132 cells, versus controls (i.e. non-exposed cells; p < 0.01). In B[a]P-exposed L132 cells, the same heterozygous status alterations as those previously reported in dPM- or PM-exposed L132 cells were observed at the four MS under study, but their frequency was statistically significant only at D3S1261, versus controls (i.e. non-exposed cells; p < 0.01).

4. Discussion Recent insights into the molecular basis of various lung diseases have recognized the occurrence of some triggering molecular events, including MS alterations (Carpagnano et al., 2005; De la Chapelle, 2003; Samara et al., 2006; Thomas et al., 2006; Yohena et al., 2007; Zhivotovsky and Kroemer, 2004). However, their specific association with individual patterns of exposure to tobacco or other lung xenobiotics has been less well-described. Hence, in this work, we were interested in the key role potentially played by Dunkerque City’s PM2.5 in the occurrence of LOH and/or MSI, using well-defined human polymorphic markers in 3p chromosome multiple critical regions. One of the main findings of this work was, therefore, that in vitro short-term exposure of L132 cells to Dunkerque City’s PM2.5 induced statistically significant alterations in the constitutional heterozygous status of the four MS under study, versus controls. Two different MS alterations were reported in the target lung cell model we used, versus the controls (i.e. non-exposed cells): (i) at D3S1312, there was a loss of one (i.e. 211) of the two alleles (i.e. 211/221), (ii) at D3S1261, D3S3624 and D2S3719, a loss of both the alleles (i.e. 183/193, 141/143, and 265/272, respectively) and the occurrence of one new-size allele (i.e. 206, 145, and 260, respectively). Accordingly, in Dunkerque City’s PM2.5-exposed L132 cells, the subsistence of only one of the two parental alleles observed at D3S1312 could typically arise from the occurrence of a LOH (Brambilla et al., 2003). Moreover, the disappearance of both the parental alleles, on the one hand, and the occurrence of a single longer size allele at D3S1261 and D3S3624, or a shorter size allele at D3S3719, on the other hand, might rely on the existence of a LOH followed by a MSI on the residual allele, in the homogeneous cell population arising from the cloning of one L132 cell

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Table 3 Status of the polymorphic loci at the well-defined human polymorphic markers in 3p multiple critical regions under study in exposed L132 cells. Exposure

Control dPM PM B[a]P

Microsatellite status D3S1261

D3S1312

D3S3719

D3S3624

183/193 (100%) 206 (30%)a 183/193 (70%)a 183/193 (60%)a 206 (40%)a 183/193 (60%)a 206 (40%)a

211/221 (100%) 211/221 (60%)a 221 (40%)a 211/221 (60%)a 221 (40%)a 211/221 (80%)c 221 (20%)c

265/272 (100%) 265/272 (60%)a 260 (40%)a 265/272 (60%)a 260 (40%)a 265/272 (80%)c 260 (20%)c

141/143 (100%) 141/143 (60%)a 145 (40%)a 141/143 (60%)a 145 (40%)a 141/143 (80%)c 145 (20%)c

Status of the polymorphic loci at the well-defined human polymorphic markers in 3p critical regions under study (i.e. D3S1261, D3S1312, D3S3719, and D3S3624) 72 h after the incubation of L132 cells either as (i) controls (i.e. incubated only with cell culture media), (ii) negative controls (i.e. incubated in the continuous presence of TiO2 particles or desorbed Particle Matter (dPM) at their Equivalent Lethal Concentration (Eq LC) at 50%; Eq LC50 = 61.71 ␮g/mL or 12.34 ␮g/cm2 ), (iii) Particulate Matter (PM)-exposed cells (i.e. incubated in the continuous presence of PM at its calculated Lethal Concentration at 50%; LC50 = 75.36 ␮g PM/mL or 15.07 ␮g PM/cm2 ), or (iv) positive control (i.e. incubated in the continuous presence of Benzo[a]Pyrene, B[a]P; 1 ␮M). Limit-dilution method was also used to select and clone L132 cells after their exposure, and ensure their exponentially growing until their number was sufficient to allow a reliable DNA extraction (i.e. 21 days). Microsatellite status is provided as the fragment size (bp) and the frequency (%) of 10 replicates for controls, negative controls, PM-exposed cells, or positive control (Chi-square test with Yates correction; vs controls). a p < 0.01. b p < 0.05. c Not significant.

by limit-detection method (Samara et al., 2006). Taken together, these results supported the occurrence of dramatic LOH, followed by MSI, in a target in vitro cell model exposed to air pollution PM, directly collected in the human near-environment. It is now wellknown that, depending upon their natural and/or anthropogenic emission sources, air pollution PM is a very complex and heterogeneous mixture of chemical and/or biological elements (e.g. metals, salts, carbonaceous material, VOC, PAH, PCDD/F, DL-PCB, PCB, endotoxins), which can in fact interact and be coated onto PM carbonaceous cores (Alfaro-Moreno et al., 2002; Billet et al., 2007;

Elder et al., 2000; Englert, 2004; Knaapen et al., 2002; Soukup and Becker, 2001). Hence, Billet et al. (2008), determining the most toxicologically relevant physical and chemical characteristics of Dunkerque City’s PM2.5 , revealed the presence of such very complex and heterogeneous inorganic and organic fractions. Negative (i.e. TiO2 and dPM) and positive (i.e. B[a]P) controls were therefore included in the experimental design of this work in order to help us to discriminate between the accurate role potentially played by both these fractions (i.e. particulate, inorganic versus organic).

Fig. 2. Representative examples of MicroSatellite (MS) analysis at D3S1261, D3S1312, D3S3719, and D3S3624 in non-exposed L132 cells and in Particulate Matter (PM)-exposed L132 cells at its Lethal Concentration (LC) at 50% (i.e. LC50 = 75.36 ␮g PM/mL or 15.07 ␮g PM/cm2 ). There was a constitutional heterozygous status at the four MS under study in non-exposed L132 cells. In contrast, in PM-exposed L132 cells, there were: (i) at D3S1312, a loss of one (i.e. 211) of the two alleles (i.e. 211/221), (ii) at D3S1261 and D3S3624, a loss of both the alleles (i.e. 183/193 and 141/143, respectively) and the occurrence of one longer size allele (i.e. 206 and 145, respectively), and (iii) at D3S3719, a loss of both the alleles (i.e. 265/272) and the occurrence of one shorter size allele (i.e. 260).

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With regards to the negative controls we used, no alteration of the MS heterozygous status was reported in TiO2 -exposed L132 cells, whereas statistically significant LOH and/or LOH followed by MSI, similar as those previously described in PM-exposed L132 cells, were seen in the four MS under study in dPM-exposed cells, versus controls (i.e. non-exposed cells). The apparent discrepancy between the results arising from the exposure to both these negative controls could easily be explain by role of TiO2 only as a carrier particle, on the one hand, and the role of dPM as only representative of the inorganic fraction of PM, on the other hand (Billet et al., 2007). Taken together, these results could confer the responsibility for the induction of MS alterations in PM-exposed L132 cells to its inorganic fraction. However, after the consideration of the results arising from thermogravimetric and differential thermal analysis, Billet et al. (2007) indicated that the remove of the organic fraction-coated onto PM using this outgassing method was not sufficient to eliminate the totality of the coated-PAH. Moreover, Abbas et al. (2009), studying the gene expression of PAH-metabolizing enzymes, reported significant increases of CYP1A1 transcripts in L132 cells 24, 48 and 72 h after their exposure to dPM, thereby confirming that the employed outgassing method was not enough efficient to remove total PAH. Hence, in the experimental design of this work, B[a]P was, therefore, chosen as positive control of the key role potentially played by the organic fraction, which can in fact be attached-onto the carbonaceous cores being use as condensation nuclei within air pollution PM. In vitro short-term exposure of L132 cells to B[a]P caused various alterations in the constitutional heterozygous status of the four MS under study in exposed L132 cells, as compared to controls (i.e. nonexposed cells). Accordingly, in human lungs, PAH, which require metabolic activation to biologically reactive intermediates to elicit their adverse health effects, are metabolized by the Cytochrome P450 (CYP) superfamily member CYP1A1 into chemically reactive intermediates which could thereafter interact with DNA target sites to produce adducts, thereby giving rise to mutation, and eventually, tumor initiation (Miller and Ramos, 2001; Omiecinski et al., 1999; Schwartz et al., 2007; Whitlock, 1999). Yohena et al. (2007) revealed the occurrence of a LOH at D2S132, but not at the eight other MS under study, in B[a]P-exposed A549 cells. However, A549 cells derived from a type II-like human alveolar epithelial carcinoma and some genetic alterations and/or abnormalities have already occurred (Castell et al., 2005). All these statements were in agreement with the B[a]P-induced LOH and/or LOH followed by MSI at the four MS under study we reported in L132 cells. To the current knowledge, this is the first time that in vitro short-term exposure to air pollution PM is related to the induction of MS alterations in 3p chromosome multiple critical regions at MS well-described as being altered in benign and malignant lung diseases. However, these results asked some underlying questions in terms of toxicological relevance. Firstly, Wistuba et al. (2000) also consider that the LOH of the short arm of chromosome 3 is a universal lesion in the pathogenesis of lung cancer, whereas other authors considered that the LOH of short arms of certain chromosomes, and in particular 3p, housed at certain well-defined loci many TSG (i.e. MutL Homologue 1, MLH1; Fragile Histidine Triad, FHIT; see also Fig. 1) closely involved in the early stages of lung cancer (Brambilla et al., 2003; Powell et al., 2003; Schayek et al., 2006; Yohena et al., 2007). Liloglou et al. (2001) indicated that the LOH only revealed the chronic exposure to certain carcinogens (e.g. PAH, derivatives of nicotine alkaloids and relatives) but not necessarily the inevitable dramatic evolution of the damaged cell to initiated cell. Secondly, another particularity of the results provided by the present work was, at D3S1261 and D3S3624, on the one hand, and D3S3719, on the other hand, the complete disappearance of the two parental alleles and, in contrast, the occurrence of one different-size allele. Although there is no underlying mecha-

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nism well-described to explain the occurrence of a new allele with a different-size versus parental alleles, man could suggest the successive occurrence of a LOH and MSI at the loci of these three MS in homogeneous cell populations arising from the cloning of one L132 cell by limit-detection method. MS are, because of their characteristic repetitive sequence structures, particularly vulnerable to Replication Errors (RER), reflecting a malfunction during cell replication. RER or MSI phenotype is caused by biallelic inactivation of DNA Mismatch Repair (MMR) gene (i.e. predominantly MLH1), resulting of allelic loss and/or hypermethylation of its promoter region. Such events are common in human cancers. However, the notion of MSI in the bronchopulmonary cancer or the neck cancer have been studied only recently (Koy et al., 2008; Malhotra et al., 2004; Trimeche et al., 2008). MLH1 gene is mapped to 3p22.2, near the critical multiple 3p region loss in the exposed L132 cells (Fig. 1). To confirm the eventual contribution of MLH1 in the occurrence of new alleles observed in this study, it will be interesting to confirm the MSI phenotype by using a set of five mononucleotide markers (i.e. BAT25, BAT26, NR21, NR22, NR24) as recommended by the National Cancer Institute, and to study the methylation status and protein expression of MLH1 (Boland et al., 1998). Several authors have already highlighted the simultaneous induction of LOH and MSI within the same tumor tissue; however, their observations concerned different loci (i.e. 3p14, 3p21) and MS markers (Liloglou et al., 2001; Malhotra et al., 2004; Schayek et al., 2006; Trimeche et al., 2008). Moreover, Sanz-Ortega et al. (2001) have already observed the existence of MSI at 9p21 locus in tumors, and the occurrence of LOH at the same locus but in normal mucosa adjacent to these tumors. Thirdly, the results provided by this work could be explained in terms of underlying mechanisms of action, closely involved in such MS alterations. Indeed, while the underlying molecular causes of LOH and/or MSI still remain unclear, Zienolddiny et al. (2000) evoked the key role of oxidative stress conditions; they closely supported the hypothesis that MS alterations would be directly caused by Reactive Oxygen Species (ROS), notably arising from inflammatory response, which results in altered DNA polymerase enzymes, high rates of recombination of DNA with inhibition of DNA repair enzymes. Accordingly, Garc¸on et al. (2006) showed that the oxidative stress response proceeded cytotoxicity in Dunkerque City’s PM2.5 -exposed L132 cells, and, thereafter, Dagher et al. (2006, 2007) indicated that Dunkerque City’s PM2.5 are closely involved in the oxidative stress-induced activation of the NF-␬B/I␬B complex, and, therefore, in the gene expression and protein secretion of various inflammatory mediators in Dunkerque City’s PM2.5 -exposed L132 cells. In particular, they reported that, in Dunkerque City’s PM2.5 -exposed L132 cells, there were concentration- and/or timedependent changes not only in lipid peroxidation and superoxide dismutase activity, but also in 8-hydroxy-2 -deoxyguanosine formation and poly(ADP-ribosyl)ation, on the one hand, and in gene expression and/or protein secretion of various pro-inflammatory mediators (e.g. tumor necrosis factor-alpha, interleukin-1 beta, interleukin-8, transforming growth factor-beta, nitric oxide), on the other hand. In agreement with the current literature, several lines of evidence revealed that some of the main covalent metals detected in Dunkerque City’s PM2.5 (e.g. Fe: 78.4 mg/g, Al: 58.3 mg/g, Pb: 8.0 mg/g, Mn: 3.5 mg/g, Zn: 1.84 mg/g) could be closely involved in redox systems, which can lead to the initiation of radical reactions, notably through the Fenton reaction and/or the Haber-Weiss cycle (Billet et al., 2007; Knaapen et al., 2002; Soukup and Becker, 2001). Moreover, it is now well-known that the metabolic activation by enzyme-catalyzed reactions of the VOC and/or the PAH-coated onto PM could result in the excessive production of ROS capable of interfering with cell homeostasis (Miller and Ramos, 2001; Omiecinski et al., 1999; Whitlock, 1999). In addition, the phagocytosis of air pollution PM by several types of the lung cells is generally

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well-described as another underlying mechanism from which ROS production could generally arise, thereby leading to dramatic oxidative stress conditions (De Kok et al., 2006). However, before concluding, caution is necessary when tackling the question of the helpfulness of in vitro models to improve the extrapolation to the experiments in animals and/or the situations in humans. For example, in this work, we showed the occurrence of genetic alterations (i.e. LOH and/or MSI) in 3p chromosome multiple critical regions in L132 cells 72 h after their exposure to Dunkerque City’s PM2.5 at its LC50 (i.e. LC50 = 75.36 ␮g PM/mL or 15.07 ␮g PM/cm2 ). Hence, any extrapolation from the artificial acute exposure conditions we used in the human lung target cell model under study to the realistic chronic exposure conditions of the humans operating in their general occupational and/or general environments would be very helpful for a better assessment and/or management of the human health risk. Further consideration of this key question indicated, however, a general lack of information about the potential uses of data generated from in vitro systems in the risk assessment process. Scientific statements have notably highlight the potential discrepancies between the mechanisms of action occurring in in vitro systems and those appearing in animal systems, and whether the in vitro observations have any real relevance to the mechanisms of PM-induced health affects (Devlin et al., 2005). Conversely, other scientific statements have indicated that many cellular pathways are activated similarly both in vitro and in vivo and carefully conducted in vitro experiments can be performed using relevant target cells and exposure scenarii (Castell et al., 2005). The major limitation of in vitro studies is that cells have been removed from their normal environment: there are also no neighboring cells or tissues to interact with, yet intercellular signaling is central to tissue and organ homeostasis, and cell exposure in a manner that mimics in vivo exposure can be very difficult. Hence, one should be careful and reserved with extrapolation of in vitro observations to the in vivo situation (Carere et al., 2002; Devlin et al., 2005; Maier et al., 2008). For example, it is often very difficult to extrapolate from the PM dose applied to individual cells in culture to the inhaled doses that reach the epithelial cells in animals and/or humans. Indeed, it is likely that the one time application of PM to a target cell model is remarkably different than short-term or long-term inhalation exposures of animals and/or humans. Another example deals with both the deposition and the clearance kinetics of inhaled particles throughout the respiratory tract. Indeed, the deposition is governed essentially by particle size, ventilator parameters, as well as airway characteristics, whereas the clearance of the particulate fraction, once deposited, is dependent on its physical and chemical characteristics. Knowledge of the deposition and the clearance of inhaled particles is also important for the extrapolation of results from in vitro observations to the in vivo situation, in particular when applied to toxicological investigations. However, despite these unavoidable limitations, in vitro experiments continue to be a prerequisite for a preliminary understanding of mechanism of action that, in general, is currently obtainable only through animal and/or human studies. The observations reported in in vitro approaches require also further validation through experiments in animals and studies in humans before to be validated (Carere et al., 2002; Devlin et al., 2005; Maier et al., 2008). Keeping in mind all the above remarks, the present findings suggested that both the oxidative stress and inflammatory responses proceeded cytotoxicity in PM-exposed L132 cells, and could thereby be closely involved in the occurrence of genetic alterations in 3p chromosome multiple critical regions in Dunkerque City’s PM2.5 exposed L132 cells. The research of the MSI phenotype and the methylation status and protein expression of MLH1will therefore be one of the major challenges in a further in vitro work.

Conflict of interest statement All the authors of the present manuscript declare that there are no conflicts of interest. Acknowledgements The Laboratoire de Recherche en Toxicologie Industrielle et Environnementale (LCE EA2598) participates in the Institut de Recherches en ENvironnement Industriel (IRENI), which is financed by the Région Nord-Pas de Calais, the Ministère de l’Enseignement Supérieur et de la Recherche, and European Funds (FEDER). We also thank the “Plateau Commun de Biologie Moléculaire du Centre de Biologie-Pathologie” of the Centre Hospitalier et Universitaire (CHR&U) of Lille. The research described in this article benefited from grants from the Agence Franc¸aise de Sécurité Sanitaire de l’Environnement et du Travail (AFSSET; Convention n◦ EST2007-48), the Ministère de l’Enseignement Supérieur et de la Recherche (Convention n◦ 16848-2005), and the Région Nord-Pas de Calais (Convention no. 08070005). All the authors of the present manuscript declare no one of the above-cited sponsors had any involvement in the study design, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. References Abbas, I., Saint-Georges, F., Billet, S., Verdin, A., Mulliez, P., Shirali, P., Garc¸on, G., 2009. Air pollution particulate matter (PM2.5 )-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbonmetabolizing enzymes in an in-vitro coculture lung model. Toxicol. In Vitro 23, 37–46. Alfaro-Moreno, E., Martinez, L., García-Cuellar, C., Bonner, J.C., Murray, J.C., Rosas, I., Ponce-de-Leon Rosales, S., Miranda, J., Osornio-Vargas, A.R., 2002. Biological effects induced in vitro by PM10 from three different zones of Mexico City. Environ. Health Perspect. 110, 715–720. Billet, S., Garc¸on, G., Dagher, Z., Verdin, A., Ledoux, F., Courcot, D., Aboukais, A., Shirali, P., 2007. Ambient particulate matter (PM2.5 ): physicochemical characterization and metabolic activation of the organic fraction in human lung epithelial cells (A549). Environ. Res. 105, 212–223. Billet, S., Abbas, I., Le Goff, J., Verdin, A., Andre, V., Lafargue, P.E., Hachimi, A., Cazier, F., Sichel, F., Shirali, P., Garc¸on, G., 2008. Genotoxic potential of polycyclic aromatic hydrocarbons-coated onto airborne particulate matter (PM2.5 ) in human lung epithelial A549 cells. Can. Lett. 270, 144–155. Black, R.J., Bray, F., Ferlay, J., Parkin, D.M., 1997. Cancer incidence and mortality in the European Union: cancer registry data and estimates of national incidence for 1990. Eur. J. Cancer 33, 1075–1107. Boland, C.R., Thibodeau, S.N., Hamilton, S.R., Sidransky, D., Eshleman, J.R., Burt, R.W., Meltzer, S.J., Rodriguez-Bigas, M.A., Fodde, R., Ranzani, G.N., Srivastava, S., 1998. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 58, 52448–52457. Boyle, J.O., Lonardo, F., Chang, J.H., Klimstra, D., Rusch, V., Dmitrovsky, E., 2001. Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations. Clin. Cancer Res. 7, 259–266. Boyle, P., Gandini, S., Gray, N., 2007. Epidemiology of lung cancer: a century of great success and ignominious failure. In: Hansen, H.H. (Ed.), Textbook of Lung Cancer. Martin Dunitz, London, UK, pp. 13–25. Brambilla, C., Fievet, F., Jeanmart, M., de Fraipont, F., Lantuejoul, S., Frappat, V., Ferretti, G., Brichon, P.Y., Moro-Sibilot, D., 2003. Early detection of lung cancer: role of biomarkers. Eur. Respir. J. 21, 36S–44S. Brunekreef, B., Holgate, S., 2002. Air pollution and health. Lancet 360, 1233–1242. Carpagnano, G.E., Foschino-Barbaro, M.P., Mulé, G., Resta, O., Tommasi, S., Mangia, A., Carpagnano, F., Stea, G., Susca, A., Di Gioia, G., De Lena, M., Paradiso, A., 2005. 3p microsatellite alterations in exhaled breath condensate from patients with non-small cell lung cancer. Am. J. Respir. Crit. Care Med. 172, 738–744. Carere, A., Stammati, A., Zucco, F., 2002. In vitro toxicology methods: impact on regulation from technical and scientific advancements. Toxicol. Lett. 127, 153–160. Castell, J.V., Donato, M.T., Gomez-Lechon, M.J., 2005. Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach. Exp. Toxicol. Pathol. 57, 189–204. Dagher, Z., Garc¸on, G., Gosset, P., Ledoux, F., Surpateanu, G., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2005. Pro-inflammatory effects of Dunkerque city air pollution particulate matter 2.5 in human epithelial lung cells (L132) in culture. J. Appl. Toxicol. 25, 166–175.

F. Saint-Georges et al. / Toxicology Letters 187 (2009) 172–179 Dagher, Z., Garc¸on, G., Billet, S., Gosset, P., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2006. Activation of different pathways of apoptosis by Dunkerque city air pollution particulate matter (PM2.5 ) in human epithelial lung cells (L132) in culture. Toxicology 225, 12–24. Dagher, Z., Garc¸on, G., Billet, S., Verdin, A., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2007. Role of nuclear factor-kappa B activation in the adverse effects induced by air pollution particulate matter (PM2.5 ) in human epithelial lung cells (L132) in culture. J. Appl. Toxicol. 27, 284–290. De Kok, T.M.C.M., Driece, H.A.L., Hogervorst, J.G.F., Briede, J.J., 2006. Toxicological assessment of ambient and traffic-related particulate matter: a review of recent studies. Mutat. Res. 613, 103–122. De la Chapelle, R., 2003. Microsatellite instability. N. Engl. J. Med. 349, 209–210. Devlin, R.B., Frampton, M.L., Ghio, A.J., 2005. In vitro studies: what is their role in toxicology? Exp. Toxicol. Pathol. 57, 183–188. Dominici, F., Peng, R.D., Zeger, S.L., White, R.H., Samet, J.M., 2007. Particulate air pollution and mortality in the United States: did the risks change from 1987 to 2000? Am. J. Epidemiol. 166, 880–888. Elder, A.C.P., Gelein, R., Finkelstein, J.N., Cox, C., Oberdörster, G., 2000. Endotoxin priming affects the lung response to ultrafine particles and ozone in young and old rats. Inhal. Toxicol. 12, 85–98. Ellegren, H., 2000. Microsatellite mutations in the germline: implications for evolutionary inference. Trends Genet. 16, 551–558. Elliott, P., Shaddick, G., Wakefield, J.C., de Hoogh, C., Briggs, D.J., 2007. Long-term associations of outdoor air pollution with mortality in Great Britain. Thorax 62, 1088–1094. Englert, N., 2004. Fine particle and human health—a review of epidemiological studies. Toxicol. Lett. 149, 235–242. Erfle, V., Mellert, W., 1996. Human cell line LC5 and its use US Patent 5582967 dated December 10, 1996. Garc¸on, G., Dagher, Z., Zerimech, F., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., Shirali, P., 2006. Dunkerque city air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol. In Vitro 20, 519–528. Garc¸on, G., Billet, S., Dagher, Z., Gosset, P., Verdin, A., Courcot, D., Shirali, P., 2007. Toxicological mechanisms underlying the adverse effects of ambient particulate matter (PM2.5 ) in the lung: contribution of an in-vitro approach. In: Bodine, C.G. (Ed.), Air Pollution Research Advances. Nova Science Publishers, New York, pp. 77–124. Greenberg, A.K., Yee, H., Rom, W.N., 2002. Preneoplastic lesions of the lung. Respir. Res. 3, 20–29. Hales, S., Howden-Chapman, P., 2007. Effects of air pollution on health. BMJ 335, 314–315. Hancock, J.M., 1999. Microsatellites and other simple sequences: genomic context and mutational mechanisms. In: Golstein, D.B., Schlotterer, C. (Eds.), Microsatellites: Evolutions and Applications. Oxford University Press, pp. 1–9. Hirao, M., Gushiken, T., Imokawa, H., Kawai, S., Inaba, H., Tsukuda, M., 2001. Solitary neurofibroma of the nasal cavity: resection with endoscopic surgery. J. Laryngol. Otol. 115, 1012–1014. Kerr, K.M., 2001. Pulmonary preinvasive neoplasia. J. Clin. Pathol. 54, 257–271. Knaapen, A.M., Shi, T., Borm, P.J., Schins, R.P., 2002. Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter. Mol. Cell. Biochem. 234/235, 317–326. Koy, S., Plaschke, J., Luksch, H., Friedrich, K., Kuhlisch, E., Eckelt, U., Martinez, R., 2008. Microsatellite instability and loss of heterozygosity in squamous cell carcinoma of the head and neck. Head Neck 30, 1105–1113. Liloglou, T., Maloney, P., Xinarianos, G., Hulbert, M., Walshaw, M.J., Gosney, J.R., Turnbull, L., Field, J.K., 2001. Cancer-specific genomic instability in bronchial lavage: a molecular tool for lung cancer detection. Cancer Res. 61, 1624–1628. Maier, K.L., Alessandrini, F., Beck-Speier, I., Hofer, T.P.J., Diabaté, S., Bitterle, E., Stöger, T., Jakob, T., Behrend, H., Horsch, M., Beckers, J., Ziesenis, A., Hültner, L., Frankenberger, M., Krauss-Etschmann, S., Schulz, H., 2008. Health effects of ambient particulate matter—biological mechanisms and inflammatory responses to in vitroand in vivo particle exposures. Inhal. Toxicol. 20, 319–337. Malhotra, P., Behera, D., Srinivasan, R., Majumdar, S., Wali, A., Mir, S., Kaur, R., 2004. Detection of microsatellite alterations in bronchial washings in squamous cell lung cancer: the first study from India. Jpn J. Clin. Oncol. 34, 439–444. Marsit, C.J., Hasegawa, M., Hirao, T., Kim, D.H., Aldape, K., Hinds, P.W., Wiencke, J.K., Nelson, H.H., Kelsey, K.T., 2004. Loss of heterozygosity of chromosome 3p21 is associated with mutant TP53 and better patient survival in non-small-cell lung cancer. Cancer Res. 64, 8702–8707. Miller, K.P., Ramos, K.S., 2001. Impact of cellular metabolism on the biological effects of benzo(a)pyrene and related hydrocarbons. Drug Metab. Rev. 33, 1–35.

179

Musti, M., Kettunenb, E., Dragonieric, S., Lindholmd, P., Cavonee, D., Seriof, G., Knuutilad, S., 2006. Cytogenetic and molecular genetic changes in malignant mesothelioma. Cancer Genet. Cytogenet. 170, 9–15. Okutan, O., Kartaloglu, Z., Ilvan, A., Kunter, E., Cerrahoglu, K., Capraz, F., 2004. Does the primary lung cancer rate increase among females? Bull. Cancer 9, E201–E210. Omiecinski, C.J., Remmel, R.P., Hosagrahara, V.P., 1999. Concise review of the cytochrome P450s and their roles in toxicology. Toxicol. Sci. 48, 151–156. Pan, H., Califano, J., Ponte, J.F., Russo, A.L., Cheng, K., Thiagalingam, A., Nemani, P., Sidranski, D., Thiagalingam, S., 2005. Loss of heterozygosity patterns provide fingerprints for genetic heterogeneity in multistep cancer progression of tobacco smoke–induced non-small cell lung cancer. Cancer Res. 65, 1664–1669. Park, J.S., Dong, S.M., Kim, H.S., Lee, J.Y., Um, S.J., Park, I.S., Kim, S.J., Namkoong, S.E., 1999. Detection of p16 gene alteration in cervical cancer using tissue microdissection and LOH study. Cancer Lett. 136, 101–108. Peluso, M., Srivatanakul, P., Munnia, A., Jedpiyawongse, A., Meunier, A., Sangrajrang, S., 2008. DNA adduct formation among workers in a Thai industrial estate and nearby residents. Sci. Total Environ. 389, 283–288. Pope 3rd, C.A., 2004. Air pollution and health—good news and bad. N. Engl. J. Med. 351, 1057–1067. Powell, C.A., Bueno, R., Borczuk, A.C., Caracta, C.F., Richards, W.G., Sugarbaker, D.J., Brody, J.S., 2003. Patterns of allelic loss differ in lung adenocarcinomas of smokers and non-smokers. Lung Cancer 3, 23–29. Samara, K., Zervou, M., Siafakas, N.M., Tzortzaki, E.G., 2006. Microsatellite DNA instability in benign lung diseases. Respir. Med. 100, 202–211. Samet, J.M., Dominici, F., Curriero, F.C., Coursac, I., Zeger, S.L., 2000. Fine particulate air pollution and mortality in 20 U.S. cities, 1987-1994. N. Engl. J. Med. 343, 1742–1749. Sanz-Ortega, J., Saez, M.C., Sierra, E., Torres, A., Balibrea, J.L., Hernando, F., SanzEsponera, J., Merino, M.J., 2001. 3p21, 5q21, and 9p21 allelic deletions are frequently found in normal bronchial cells adjacent to non-small-cell lung cancer, while they are unusual in patients with no evidence of malignancy. J. Pathol. 195, 429–434. Schayek, H., Krupsky, M., Yaron, P., Yellin, A., Simansky, D.A., Friedman, E., 2006. Genetic analyses of non-small cell lung cancer in Jewish Israeli patients. Isr. Med. Assoc. J. 8, 159–163. Schlotterer, C., Wiehe, T., 1999. Microsatellites, a neutral marker to infer selective sweeps. In: Golstein, D.B., Schlotterer, C. (Eds.), Microsatellites: Evolutions and Applications. Oxford University Press, pp. 238–247. Schottenfeld, D., 2000. Etiology and epidemiology of lung cancer. In: Pass, H.I., Mitchell, J.B., Johnson, D.H., Turrisi, A.T., Minna, J.D. (Eds.), Lung Cancer: Principles and Practice. Lippincott Williams and Wilkins, Philadelphia, USA, pp. 367–388. Schwartz, A.G., Prysak, G.M., Bock, C.H., Cote, M.L., 2007. The molecular epidemiology of lung cancer. Carcinogenesis 28, 507–518. Sorensen, M., Autrup, H., Moller, P., 2003. Linking exposure to environmental pollutants with biological effects. Mutat. Res. 544, 255–271. Soukup, J.M., Becker, S., 2001. Human alveolar macrophage responses to air pollution particulates are associated with insoluble components of coarse material, including particulate endotoxin. Toxicol. Appl. Pharmacol. 171, 20–26. Thomas, R.K., Weir, B., Meyerson, M., 2006. Genomic approaches to lung cancer. Clin. Cancer Res. 2 (14 Suppl.), 4384s–4391s. Trimeche, M., Braham, H., Ziadi, S., Amara, K., Hachana, M., Korbi, S., 2008. Investigation of allelic imbalances on chromosome 3p in nasopharyngeal carcinoma in Tunisia: high frequency of microsatellite instability in patients with early-onset of the disease. Oral Oncol. 44, 775–783. Vineis, P., Husgafvel-Pursiainen, K., 2005. Air pollution and cancer: biomarker studies in human populations. Carcinogenesis 26, 1846–1855. Whitlock, J.P., 1999. Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol. 39, 103–125. Wistuba, I.I., Berry II, J., Behrens, C., Maitra, A., Shivapurkar, N., Milchgrub, S., Mackay, B., Minna, J.D., Gazdar, A.F., 2000. Molecular changes in the bronchial epithelium of patients with small cell lung cancer. Clin. Cancer Res. 6, 2604–2610. Yohena, T., Yoshino, I., Takenaka, T., Ohba, T., Kouso, H., Osoegawa, A., Hamatake, M., Oda, S., Kuniyoshi, Y., Maehara, Y., 2007. Relationship between the loss of heterozygosity and tobacco smoking in pulmonary adenocarcinoma. Oncol. Res. 16, 333–339. Zhivotovsky, Y., Kroemer, G., 2004. Apoptosis and genomic instability. Nat. Rev. Mol. Cell Biol. 5, 752–762. Zhou, X., Kemp, B.L., Khuri, F.R., Liu, D., Lee, J.J., Wu, W., Hong, W.K., Mao, L., 2000. Prognostic implication of microsatellite alteration profiles in early-stage nonsmall cell lung cancer. Clin. Cancer Res. 6, 559–565. Zienolddiny, S., Ryberg, D., Haugen, A., 2000. Induction of microsatellite mutations by oxidative agents in human lung cancer cell lines. Carcinogenesis 21, 1521–1526.