Anne Platel
email: anne.platel@pasteur-lille.fr

K Privat

S Talahari
email: smail.talahari@pasteur-lille.fr

A Delobel

G Dourdin

E Gateau

S Simar

Y Saleh
email: yara.saleh@univ-lille.fr

S Antherieu

L Canivet

L.-Y Alleman

G Garçon

J M Lo Guidice

H Amini

Trang Nhung

Schwartz, J A M Henderson

vivo genotoxic effects of air pollution fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) particles on lung models

Keywords:

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Air pollution levels remain dangerously high in many parts of the world. Both short-and long-term exposure to air pollutants have been associated with health impacts in children and adults [START_REF] Burns | Interventions to reduce ambient particulate matter air pollution and their effect on health[END_REF]Lim et al., 2012;[START_REF] Loomis | The carcinogenicity of outdoor air pollution[END_REF][START_REF] Rückerl | Health effects of particulate air pollution: A review of epidemiological evidence[END_REF][START_REF] Schraufnagel | Air Pollution and Noncommunicable Diseases[END_REF]World Health Organization, 2016). According to the World Health Organization, in 2016, around 8 million people die every year from exposure to polluted air (World Health Organization, consulted on September 2019). Of those deaths, around 4.2 million are attributable solely to ambient (outdoor) air pollution, mainly from heart disease, stroke, chronic obstructive pulmonary disease (COPD), lung cancer and acute respiratory infections in children. In 2016, 91% of the world population was living in places where the WHO air quality guidelines levels were not met (World Health Organization, consulted on September 2019). A new study published recently shows that the scale of the phenomenon could be worse. Indeed, ambient air pollution could be twice as deadly as what the scientific community admitted until then; it would thus cause 8.8 million deaths a year in the world [START_REF] Lelieveld | Cardiovascular disease burden from ambient air pollution in Europe reassessed using novel hazard ratio functions[END_REF]. Pollutants with the strongest evidence for public health concern include coarse (PM 10 , i.e. particulates of median aerodynamic diameter ≤ 10 μm) and fine particulate matter (PM 2.5 or FPs, i.e. particulates of median aerodynamic diameter ≤ 2.5 μm). PM is of major toxicological interest due to its great heterogeneity. PM consists in a complex mixture made of an inorganic core (ions, metals, inorganic carbonaceous species) coated with organic compounds such as polycyclic aromatic hydrocarbons (PAHs), semi-volatile organic compounds, polychlorobiphenyls, furans, dioxins, and micro-organisms (bacteria and fungi) [START_REF] Harrison | Particulate matter in the atmosphere: which particle properties are important for its effects on health?[END_REF]. Ultrafine PM is capable of penetrating and lodging deep inside the lungs and entering the bloodstream causing cardiovascular, cerebrovascular and respiratory effects [START_REF] Cho | In vitro and in vivo experimental studies of PM2.5 on disease progression[END_REF]. The adverse health effects of PM on the progression of diseases mainly depend on gender due to exposure differences, hormonal status and life stages [START_REF] Clougherty | A growing role for gender analysis in air pollution epidemiology[END_REF]. In 2013, PM and outdoor air pollution were classified as carcinogenic to humans (group 1) by the International Agency of Research on Cancer (International Agency for Research on Cancer, 2015). Current limit values are defined aiming to lower the concentrations of PM 10 and PM 2.5 in the air in order to improve air quality. However, ultrafine particles (PM 0.1 or UFPs, i.e. particulates of median aerodynamic diameter ≤ 0.10 μm) are still under no regulation despite their greater chemical reactivity and potentially hazardous characteristics. Indeed, they are thought to be more toxic than larger particles due to their greater number, higher surface area per unit of mass and smaller size allowing them to reach the deepest areas in lungs and enter the bloodstream resulting in their distribution to other organs [START_REF] Nemmar | Recent advances in particulate matter and nanoparticle toxicology: A review of the in vivo and in vitro studies[END_REF][START_REF] Peters | Translocation and potential neurologic al effects of fine and ultrafine particles a critical update[END_REF]. Deleterious effects of PM involve different mechanisms of toxic action connected to each other. Firstly, PM can generate reactive oxygen species (ROS) and some oxidative metabolites, causing oxidative stress. It also induces inflammatory injuries, damages DNA and causes epigenetic changes [START_REF] Cho | In vitro and in vivo experimental studies of PM2.5 on disease progression[END_REF][START_REF] Kim | Predictive and prognostic biomarkers of respiratory diseases due to particulate matter exposure[END_REF]. Although many studies have been conducted in recent years, PM toxicity mechanism is not yet clearly understood.

In this context, following our previous work on the in vitro genotoxicity of PM 2.5 from an urban surrounding [START_REF] Leclercq | Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM 2.5[END_REF], the aim of our work was to investigate the in vitro and in vivo genotoxic and mutagenic potential of air pollution FPs (2.5 µm -0.18 µm) and quasi UFPs (≤ 0.18 µm) collected in the industrial-port area of Dunkirk (Northern France), near the motorways and the conurbation. For a better human health assessment, we strove to be as close as possible to the actual in vivo exposure conditions in terms of route of exposure. For that, the collected particles were directly tested on cells or administered to animals, rather than testing the extracted organic matter (EOM) of the PM, as frequently reported in the literature for genotoxicity assessment (Abbas et al., 2019;[START_REF] Bełcik | Genotoxic and cytotoxic properties of PM2.5 collected over the year in Wrocław (Poland)[END_REF][START_REF] Billet | Chemical characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and their organic extracts on pulmonary cells[END_REF][START_REF] Bocchi | Genotoxicity of airborne PM2.5 assessed by salmonella and comet assays in five cities of the Emilia-Romagna (Italy) mutagenicity monitoring network[END_REF][START_REF] Bocchi | Characterization of urban aerosol: seasonal variation of mutagenicity and genotoxicity of PM 2.5 , PM 1 and semi-volatile organic compounds[END_REF][START_REF] Borgie | Comparison between ultrafine and fine particulate matter collected in Lebanon: Chemical characterization, in vitro cytotoxic effects and metabolizing enzymes gene expression in human bronchial epithelial cells[END_REF][START_REF] Cassoni | The Salmonella mutagenicity of urban airborne particulate matter (PM2.5) from eight sites of the Emilia-Romagna regional monitoring network (Italy)[END_REF]Gábelová et al., 2007aGábelová et al., , 2007b[START_REF] Gábelová | Genotoxicity of environmental air pollution in three European cities: Prague, Košice and Sofia[END_REF][START_REF] Lemos | Genotoxicity biomarkers for airborne particulate matter (PM2.5) in an area under petrochemical influence[END_REF][START_REF] Oh | Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells)[END_REF][START_REF] Tice | Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter[END_REF]. In vitro assessment was performed with two human lung cell models, common immortalized cell lines (BEAS-2B and NCI-H292 cells) and primary cells [normal human bronchial epithelial cells (NHBE)], by using the in vitro comet and micronucleus assays at the dose of 5 µg PM/cm 2 . These tests have been chosen for their complementarities for the detection of primary DNA damage and chromosomal aberrations, respectively. These two methodologies appear to be well adapted to highlight a broad spectrum of genetic and mutagenic events, particularly of clastogenic and aneugenic types. It is known that after nanoparticles exposure, the comet assay is the most common test for DNA damage detection, and that the micronucleus test is less time-consuming and thus more used for nanoparticles testing than the chromosomal aberration assay [START_REF] Dusinska | Immunotoxicity, genotoxicity and epigenetic toxicity of nanomaterials: New strategies for toxicity testing?[END_REF]. Moreover, they represent the most commonly used combination of tests for nanoparticles genotoxicity studies [START_REF] Magdolenova | Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles[END_REF]. From a methodological point of view, the comet assay was performed on cells exposed at the air-liquid interface (ALI) in order to obtain a differentiation state as close as possible to the cellular exposure conditions observed in vivo. ALI cell culture is a useful method for investigating the harmful effects of PM on the respiratory airway as primary cells exhibit differentiation and pseudostratified striation similar to those observed in vivo [START_REF] Cho | In vitro and in vivo experimental studies of PM2.5 on disease progression[END_REF]. On the other hand, the micronucleus test requires at least 1.5 to 2 cell divisions to reveal micronucleus formation (OECDa, 2016). However, our preliminary assays demonstrated that, cells, once at the ALI, differentiate but no longer divide, and thus, preventing the induction of the micronuclei (data not shown). The micronucleus test was therefore performed in submerged conditions, knowing that submerged cell culture is still a common method to assess adverse effects of PM in cell experiments [START_REF] Bakand | Cell culture techniques essential for toxicity testing of inhaled materials and nanomaterials in vitro[END_REF][START_REF] Fröhlich | Toxicological Assessment of Inhaled Nanoparticles: Role of in Vivo, ex Vivo, in Vitro, and in Silico Studies[END_REF]. Moreover, each assay was coupled to an assessment of cytotoxicity (trypan blue dye exclusion assay and calculation of the percentage of relative population doubling).

For in vivo assessment, after intranasal exposures of BALB/c mice to FPs and UFPs, genotoxic effects were assessed using (i) the in vivo comet assay on lung (as a primo exposed organ) and on liver (as a systemic organ, metabolizer and rich in cells capable of phagocytosis), (ii) the in vivo micronucleus test on bone marrow, and (iii) the in vivo Pig-A gene mutation assay on peripheral blood, at doses of 10 and/or 100 µg per animal. For ethical and scientific reasons, these three tests were performed on the same animals. The comet assay was performed in the frame of the acute and subacute (1 month) exposures. Indeed, as this test is rather an exposure marker, it is more relevant to perform it after a short period of treatment. It is less appropriate after a long period during which repair and cytotoxicity events may underestimate the genotoxic response. The micronucleus and Pig-A gene mutation tests, as markers of effects, were carried out in the two long treatment regimens, subacute (1 month) and subchronic (3 months) exposures.

MATERIALS AND METHODS

Chemicals and reagents

Phosphate buffered saline (PBS) was purchased from GIBCO Invitrogen (Cergy-Pontoise, France).

Giemsa reagent, mitomycin C (MMC, CAS No. 50-07-7), methyl methanesulfonate (MMS,, cyclophosphamide (CPA,, penicillin, streptomycin, amphotericin B, EDTA, propidium iodide, NaCl, Trypan Blue and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Acetic acid was from VWR (Fontenaysous-bois, France). Dimethyl sulfoxide (DMSO) was purchased from Acros Organics (Noisy le Grand, France). Hank's balanced salt solution (HBSS) without phenol red, rat tail collagen I solution and

Particles sampling

FPs and UFPs were collected at approximately 2 km south of the Dunkirk industrial harbor area (North of France) with our previously described protocol [START_REF] Saleh | Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice[END_REF]. Briefly, the sampling was conducted using a High-Volume Impactor Sampler (HVIS, flow rate of 400 L/min) in order to collect the fine fraction by impaction on Teflon stripes and the UFPs fraction by filtration on 8"x10" polycarbonate (PC) membranes. The cumulated fine fraction was transferred with a Teflon tip into an acid cleaned dry vial, weighted and stored along with all the UFPs PC membranes at -20°C until chemical analyses and toxicological assays.

Physicochemical characterization

Trace metals measurements, polycyclic aromatic hydrocarbons (PAHs) analysis, and particles size distribution were performed in both size fractions and described in our previous study [START_REF] Saleh | Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice[END_REF].

Briefly, concentrations of trace elements were measured after microwave acid digestion by ICP-MS (NeXion 300x, Perkin Elmer). For PAHs, analysis was performed after pressurized liquid extraction in acetonitrile with a Dionex ASE 200 instrument (ThermoFisher Scientific) by high-pressure liquid chromatography consisting of a Waters 2695 Alliance system (Waters SA, Saint-Quentin-en-Yvelines,

France) coupled to an on-line 996-photodiode array and a 2475-fluorimetric detector. The hydrodynamic diameter of FPs and UFPs was measured after suspending particles in HBSS (1mg/mL) using a Dynamic Laser Scatter Zetasizer nano ZS (Malvern Instruments, Orsay, France).

In vitro genotoxicity and cytotoxicity assessment

In vitro genotoxicity assays, treatment conditions and cells used in our study are summarized in Table 1.

Preparation of particles suspensions

Firstly, FP and UFP samples were suspended in HBSS. Then, these initial particles suspensions were prepared following a protocol described by Jensen et al. in the NANOGENOTOX Joint Action [START_REF] Jensen | Final Protocol for producing suitable manufactured nanomaterial exposure media[END_REF]. Briefly, UFP and FPs were suspended in 0.05% BSA-water in order to obtain a 10x initial particles solution (BSA-water was prepared by dissolving 0.05% w/v BSA in distilled water and sterilefiltered) and dispersed by probe-sonication for 16 min, at 400W and 10% amplitude using a 400 Watt [START_REF] Costa | Induction of morphological changes in BEAS-2B human bronchial epithelial cells following chronic sub-cytotoxic and mildly cytotoxic hexavalent chromium exposures[END_REF][START_REF] Haniu | Culture medium type affects endocytosis of multi-walled carbon nanotubes in BEAS-2B cells and subsequent biological response[END_REF].

In vitro comet assay

Cell treatment at the air / liquid interface (ALI)

Cells were seeded at a density of 2.5x10 5 cells/insert for NHBE cells or at a density of 8x10 4 cells/insert for BEAS-2B and NCI-H292 cells, on 24 mm Transwell ® inserts (with 0.4 μm pore polyester membrane, thickness of 10 µm) in a 6-well microplate (Corning ® Transwell ® polyester membrane cell culture inserts, Sigma Aldrich, Saint-Quentin Fallavier, France) pre-coated with 0.03 mg/mL rat tail collagen I solution.

Cells were grown submerged in medium until reaching 90-100% confluency, i.e. ∼ 5-7 days for NHBE cells in B-ALI ™ growth medium according to the manufacturer recommendations, ∼3 days in BEGM for BEAS-2B cells and ∼3 days in RPMI 10 for NCI-H292 cells.. For NBHE cells, B-ALI ™ growth medium was replaced by B-ALI ™ differentiation medium with 0.2% (v/v) B-ALI ™ inducer. For NHBE cells, differentiation at the ALI is reached after 21 days of culture. This effective differentiation was already checked by [START_REF] Leclercq | Differential responses of healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM 4[END_REF] and [START_REF] Sotty | Toxicological effects of ambient fine (PM2.5-0.18) and ultrafine (PM0.18) particles in healthy and diseased 3D organo-typic mucocilary-phenotype models[END_REF]. One experiment was performed with duplicate cultures (i.e. 2 biological replicates) for each group (i.e. 2 wells per dose). Each treatment (without metabolic activation)

was coupled to an assessment of cytotoxicity as described below. In each insert, the apical medium was removed to expose the cells. Particles suspensions (as described in § 2.4.1) were then directly deposited by pipetting on the permeable membrane inserts (300 µL/insert), for 4 h without recovery time (4h/+0h).

Then cells were washed twice with PBS and immediately harvested by trypsinization with 1 mL/well of trypsin-EDTA for NHBE cells or 1 mL/well of TrypLE ™ Express for BEAS-2B and NCI-H292 cells, for 4 min at 37°C. Then, 1 mL/well of Trypsin Neutralizing Solution for NHBE cells, 1 mL/well of RPMI 10 growth medium for NCI-H292 cells, or 1 mL/well of BEGM for BEAS-2B cells, was added in order to stop the action of trypsin. Cells were transferred to 15 mL polypropylene tubes and centrifuged at 1000 rpm for 5 min. Supernatant was discarded and the pelleted cells were immediately used for the comet assay.

Positive control was MMS (0.96 µg/cm 2 ) and negative control was culture medium.

Comet assay protocol

The comet assay was performed under alkaline conditions (pH>13) in compliance with previously described protocol [START_REF] Singh | A simple technique for quantitation of low levels of DNA damage in individual cells[END_REF][START_REF] Tice | Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter[END_REF][START_REF] Witte | Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part III: The Comet Assay as an Alternative to In Vitro Clastogenicity Tests for Early Drug Candidate Selection[END_REF]. For each group 2 duplicate slides per culture were prepared (i.e. 4 technical replicates in total per concentration). When scoring for DNA migration, slides were independently coded and analyzed after addition of 25 µL of propidium iodide (20 µg/mL in distilled water) and a coverslip. Slides were then examined at 250x magnification using a fluorescence microscope (Leica Microsystems SAS -DM 2000, Heerbrugg, Switzerland) equipped with an excitation filter of 515-560 nm and a 590 nm barrier filter, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies) to the Comet Assay IV Image Analysis System, version 4.11 (Perceptive Instruments Ltd, Suffolk, United Kingdom). Four hundred randomly selected cells per concentration (100 cells from each of the 2 duplicate slides in the 2 cultures) were scored. DNA damage was expressed as percentage of DNA in the tail (% tail intensity) [START_REF] Burlinson | Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup[END_REF][START_REF] Lovell | Statistical issues in the use of the comet assay[END_REF].

Cytotoxicity assessment

For the comet assay, cytotoxicity was assessed at harvest using the Trypan Blue dye exclusion assay.

Concentrations which led to more than 70% of viability were then submitted to a genotoxicity assessment.

In vitro micronucleus test

Cell treatment in submerged conditions

NHBE and BEAS-2B cells were seeded at a density of 1.5x10 5 cells/well in BEGM in 6-well Corning ® CellBIND ® plates (Sigma-Aldrich, Saint-Quentin Fallavier, France). NCI-H292 cells were seeded at a density of 3x10 5 cells/well in RPMI 10 in 6-well cell culture plates. In order to allow exponential cell growth and maximum cell attachment, cells were incubated for 24 h (NHBE cells) or 48 h (BEAS-2B and NCI-H292 cells) prior to treatment. One experiment was performed with duplicate cultures (i.e. 2 wells per dose). Each treatment (without metabolic activation) was coupled to an assessment of cytotoxicity as described below. Cells were treated (2 mL/well) for 24 h then washed twice with PBS and fresh medium was added. Plates were incubated at 37°C for an additional 5 days recovery period (24h/+120h) for NHBE cells or 2 days recovery period (24h/+48h) for BEAS-2B and NCI-H292 cells. At the end of the recovery time, the harvest was the same as for the comet assay (see 2.4.3.1). Positive control was MMC (0.01 µg/cm 2 ) and negative control was culture medium.

Micronucleus test protocol

At the harvest time, pelleted cells were treated with a hypotonic solution (BEGM diluted 1:3 v/v in distilled water, 2 mL/tube, 10 min for NHBE cells; BEGM diluted 1:3 v/v in distilled water, 2 mL/tube, 8 min for BEAS-2B cells; RPMI 10 diluted 1:1 v/v in distilled water, 5 mL/tube, 7.5 min for NCI-H292 cells). At the end of the hypotonic shock, a pre-fixation step was performed by adding very slowly 500 µL/tube of 5% (in medium) cold Carnoy mixture (ethanol/acetic acid, 3:1 v/v). Cells were then centrifuged at 1000 rpm for 5 min and pelleted cells were fixed for at least 15 min with 2 mL/tube of pure cold Carnoy mixture (this fixation step was performed twice for NHBE cells). After another centrifugation, cells were spread on glass slides, air-dried at room temperature, stained for 10 min with 2% (NHBE cells) or 4% (BEAS-2B and NCI-H292 cells) Giemsa mineral water solution, rinsed with distilled water and finally dried at room temperature. For each group, 2 duplicate slides per culture were prepared for analysis (i.e. 4 technical replicates in total per concentration). Slides were independently coded and analyzed. Micronuclei (MN), identified according to recommended criteria [START_REF] Fenech | The in vitro micronucleus technique[END_REF][START_REF] Fenech | HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures[END_REF], were scored in 1000 intact mononucleated cells per slide (4000 mononucleated cells/concentration) at 500x magnification.

Cytotoxicity assessment

For the micronucleus test, measurements of cell proliferation are made to assure that sufficient treated cells have undergone mitosis during the test. For this, the percentage of Relative Population Doubling Concentrations leading to a RPD greater than 45±5% were considered as being of low or no cytotoxicity and were then submitted to genotoxicity assessment.

In vivo genotoxicity assessment

The genotoxic/mutagenic potential of FPs and UFPs was analyzed by a battery of three in vivo tests composed of the comet assay, the micronucleus assay and the Pig-A gene mutation assay. Tests, endpoints, target organs and treatment schedules are summarized in Table 3.

Animal model

Experiments were conducted on male BALB/c mice (Janvier Labs, Le Genest-Saint-Isle, France), 9 weeks old. This mouse strain is described as sufficiently sensitive to the chemical induction of lung cancers [START_REF] Meuwissen | Mouse models for human lung cancer[END_REF]. Animal procedures were in agreement with European directive 2010/63/EU for the protection of animals used for scientific purposes and obtained the regional Ethical Committee on Animal Experimentation (CEEA 75) approval.

Treatments

Three exposure protocols were realized in this study: mice were exposed twice at 24-hour intervals (acute exposure) or three times/week during 1 and 3 months (subchronic exposures). Animals were anesthetized with 2% isoflurane and exposed to FPs or UFPs by intranasal instillation at the selected dose of 100 µg for acute exposure and 10 µg for subchronic exposures. Particles were suspended in 37 µL of sterile HBSS (i.e. approximately 2 mL/kg). A negative control group received intranasally 37 µL of HBSS and 3 positive

In vivo comet assay

The potential genotoxic activity of FPs and UFPs was tested using the in vivo comet assay in isolated lung and liver cells in compliance with the OECD test guideline No. 489 (OECD, 2016b), using 2 successive administrations at 24-hour intervals (see 2.5.2), followed by one sampling time 2-6 hours after the last treatment. At the sampling time, treated animals were anaesthetized with 10 µL/g of a mix of ketamine (100 mg/kg) / xylazine (20 mg/kg) administered intraperitoneally, and exsanguined. 'V' shaped incision was made from the center of the lower abdomen to the rib cage. The skin and muscles were removed to reveal the abdominal cavity. A portion of the lung and liver were removed and washed in the cold fresh mincing buffer (10% v/v EDTA 200 mM, 80% v/v HBSS without phenol red and 10% v/v DMSO; pH 7.5) until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. Cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected. Cells were enumerated on a haemocytometer and 2 x 10 4 viable cells per slide were sampled from each cell suspension for proceeding to slide preparation, electrophoresis and scoring (as described previously for the in vitro comet assay. See 2.4.3.2). Three slides were prepared per animal (5 animals per group). After coding slides, 750 randomly selected cells per group (i.e. 50 cells per slide, 3 slides per animal, 5 animals per group), were analysed.

In vivo micronucleus test

The potential genotoxic activity of FPs and UFPs was tested using the in vivo micronucleus test in the bone marrow of treated mice during 1 and 3 months, in compliance with the OECD test guideline No. 474 (OECD, 2016c). At the sampling time, animals were sacrificed (5 animals per group). The femurs were removed and the bone marrow was extracted with 1 mL of fetal calf serum. The supernatant was removed and after homogenisation, the centrifugate was spread on slides (2 slides per animal). The smears were stained using a technique derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between immature and mature erythrocytes, i.e. polychromatic (PCE) and normochromatic erythrocytes (NCE), respectively. PCE are purple whereas NCE are red. After coding slides, the number of PCE having one or more micronuclei (micronucleated polychromatic erythrocytes, MNPCE) was determined from the microscopic examination of at least 4000 PCE per animal (i.e. 2000 PCE per slide). The PCE/NCE ratio was determined by analyzing at least 1000 erythrocytes per animal (i.e. 500 erythrocytes per slide).

In vivo Pig-A gene mutation assay

The purpose of the in vivo erythrocytes-based Pig-A gene mutation assay is to quantify in vivo mutation through a flow cytometry-based approach in somatic cells of treated animals (Bryce et al., 2008;Dobrovolsky et al., 2010;Kimoto et al., 2011). Blood samples were collected at the end of each chronic exposure, i.e. on the 28 th day and the 45 th day, by retro-orbital bleeding after isoflurane anaesthesia.

Approximately 120 μL of blood were collected into heparinized capillary tubes (Fisher Scientific).

Immediately upon collection, 60 μL exactly of each blood sample were transferred into tubes containing 100 µL kit-supplied heparin solution where they remained at room temperature for less than 2 hours until sample preparation according to supplier's protocol (MutaFlowPLUS Kits Mouse Blood (Litron, Rochester, New York). A FACSCanto II flow cytometer (BD Biosciences) providing 488 nm and 633 nm excitation, running FACSDiva ™ v7.0 software, was used for data acquisition and analysis. Frequencies of mutant reticulocytes (RET) and mutant red blood cells (RBC) were determined by examining at least 1 x 10 6 of total RET or RBC, respectively, per animal. All mutant cell frequencies, %RET, averages and standard error calculations were performed with the Excel ® spreadsheet developed by the supplier (Litron).

Statistical Analysis

For each test, differences with a p<0.05 were considered statistically significant.

For the in vitro and in vivo comet assay, it has previously been shown that the tail intensity does not follow a Gaussian distribution [START_REF] Bauer | The distribution of the tail moments in single cell gel electrophoresis (comet assay) obeys a chi-square (χ2) not a gaussian distribution[END_REF]. Thus, the non-parametric Mann-Whitney U-test was used to evaluate the statistical difference between groups (i.e. between each concentration vs. the respective negative control). All statistical analyses were performed with StatView  Software (version 5.0, SAS Institute Inc., SAS Campus Drive, Cary, North Carolina 27513, USA).

For the in vitro micronucleus test, the statistical significance of difference between groups (i.e. each concentration vs. the negative control) was determined using the CHI-squared test.

For the in vivo micronucleus test, statistical analyses were performed for micronucleus number with StatView ® Software using the non-parametric Mann-Whitney U-test. The statistical comparison for the PCE/NCE ratio was performed using the Student's t test.

For the in vivo Pig-A gene mutation assay, a pair-wise comparison using the Dunnet's t-test was performed.

RESULTS

In vitro genotoxicity and cytotoxicity assessment

Results of the in vitro tests are summarized in Table 4.

In vitro comet assay

Primary DNA damage was measured with the in vitro comet assay after a 4 h treatment. Results of the means of medians of percentage of tail intensity (TI) for each group and the corresponding relative cell viability obtained in BEAS-2B, NCI-H292 and NHBE cells are presented in Figure 1. To exclude cytotoxicity as a confounding factor, the cell viability should be more than 70 % for the comet assay [START_REF] Collins | The comet assay: topical issues[END_REF][START_REF] Tice | Genotoxicity of urban air pollutants in the Czech Republic. Part II. DNA adduct formation in mammalian cells by extractable organic matter[END_REF][START_REF] Witte | Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part III: The Comet Assay as an Alternative to In Vitro Clastogenicity Tests for Early Drug Candidate Selection[END_REF]. Figure 1 shows that both types of airborne particles did not induce any significant cytotoxic effects at the tested concentration of 5 µg PM/cm 2 , as assessed by the trypan blue dye exclusion assay. Moreover, under tested conditions, no increase in DNA strand breaks was observed in NHBE cells, whatever the type of particles. On the contrary, slight but statistically significant increases (p<0.05) in TI were obtained in BEAS-2B cells (6.55 % and 4.19 % for FPS and UFPs, respectively, vs. 1.54 % for the negative control). For NCI-H292 cells, increases in TI

were observed for both particles, statistically significant only for FPs [12.24% for FPs (p<0.05) and 10.25%

for UFPs, vs. 4.97 % for the negative control].

In vitro micronucleus test

The in vitro micronucleus test was used to assess the induction of chromosomal aberrations (clastogenicity and/or aneuploidy) after a 24 h treatment.. Micronuclei frequencies and the corresponding cytotoxicity (% RPD) are shown in Figure 2. No cytotoxic effect was observed, except for UFPs on NCI-H292 for which cell viability was only 30.50 %. For both airborne particles tested, no genotoxic activity were noted since no statistically or biologically significant effect was observed, in any of the three cell lines (even with UFPs in NCI-H292 cells despite a high level of toxicity noticed).

In vivo genotoxicity assessment

Results of the in vivo tests are summarized in Table 5.

Acute exposure

The genotoxic potential of the airborne particles was investigated in the in vivo comet assay, on isolated lung and liver cells of mice after an acute exposure (2 treatments at 24-hour intervals at the highest dose of 100 µg of particles per animal). Results for the mean of medians of percentage of TI are given in Figure 3. For FPs and UFPs, very low levels of TI (< 2.5 %), close to those obtained for the negative controls, were observed in the two selected organs.

Subacute exposure

Results of genotoxicity/mutagenicity assessment after the subacute exposure of mice (1 month, 10 µg of particles per animal, 3 times a week) are presented in Figure 4.

For FPs and UFPs, no statistically significant increase in the level of DNA damage was observed (TI < 1.2 %), in either the liver or the lungs (Figure 4A and4B).

Regarding the frequency of MNPCE, no significant increase was found in animals treated with FPs or UFPs ( 1 ‰) when compared to the control group (0.6 ‰) (Figure 4C).. Airborne particles treatment did not significantly affect the ratio PCE/NCE indicating the absence of cytotoxic effects on the bone marrow at this dose level.

The frequencies of mutants RET (< 1.6 x 10 -6 cells) and mutants RBC (< 0.7 x 10 -6 cells) did not shown any statistically significant increase in the animals treated with FPs or UFPs when compared to the control group (RET = 0.6 x 10 -6 cells and RBC = 0.9 x 10 -6 cells) (Figure 4D). The percentage of RET is the ratio of newly formed RNA-positive erythrocyte relative to all erythrocytes, and is used as a measure of bone marrow cytotoxicity. FPs or UFPs treated mice did not exhibit any significant changes in % RET at day 28, thus confirming the absence of toxicity.

Subchronic exposure

Summarized results obtained after the subchronic exposure of animals (3 months, 10 µg of particles per animal, 3 times a week) are presented in Figure 5.

No induction of MN formation was observed in mice treated with FPs or UFPs (< 0.7 ‰). No decrease of the ratio PCE/NCE was observed (Figure 5A).

No statistically significant increase in mutant frequencies of RBC and RET (< 3 x 10 -6 cells) was observed for the UFPs compared to the negative control (RET = 0.5 x 10 -6 cells and RBC = 0.7 x 10 -6 cells) (Figure 5B). For FPs, a slight increase in frequencies of mutants RBC and mutants RET (< 22 x 10 -6 ) was found, but it remained non-statistically significant compared to the negative control. The absence of toxicity on the bone marrow was confirmed (only very slight decreases, non-statistically significant, in the % RET).

DISCUSSION

Respiratory diseases are one of the leading causes of death in the world and constitute a major challenge in terms of public health [START_REF] Ferkol | The Global Burden of Respiratory Disease[END_REF][START_REF] Schraufnagel | Air Pollution and Noncommunicable Diseases[END_REF]. The prevalence of these diseases is constantly increasing in the Hauts-de-France region (northern France), where the tested particles were collected (Agence Régionale de Santé des Hauts-de-France, 2017; Lapôtre- [START_REF] Lapôtre-Ledoux | Estimations régionales et départementales d'incidence et de mortalité par cancers en France[END_REF]. Numerous epidemiological studies have highlighted the role of air quality in respiratory morbidity and mortality [START_REF] Shamsipour | National and sub-national exposure to ambient fine particulate matter (PM2.5) and its attributable burden of disease in Iran from 1990 to 2016[END_REF][START_REF] Nabizadeh | Characteristics of cohort studies of longterm exposure to PM2.5: a systematic review[END_REF][START_REF] Shamsipour | National and sub-national exposure to ambient fine particulate matter (PM2.5) and its attributable burden of disease in Iran from 1990 to 2016[END_REF][START_REF] Weber | Assessing the impact of fine particulate matter (PM 2.5 ) on respiratory-cardiovascular chronic diseases in the New York City Metropolitan area using Hierarchical Bayesian Model estimates[END_REF][START_REF] Xing | The impact of PM2.5 on the human respiratory system[END_REF][START_REF] Yitshak-Sade | Estimating the causal effect of annual PM2.5 exposure on mortality rates in the Northeastern and mid-Atlantic states[END_REF]. Anthropogenic air pollution appears to be responsible for increasing the incidence and aggravation of diseases such as asthma, allergies or COPD [START_REF] Guerreiro | Air quality in Europe -2018 report[END_REF][START_REF] Xia | Pulmonary diseases induced by ambient ultrafine and engineered nanoparticles in twenty-first century[END_REF]. Although air pollution is now a well-suspected risk factor of lung cancer [START_REF] Loomis | The carcinogenicity of outdoor air pollution[END_REF], to our knowledge no work has been conducted to specifically study in vitro and in vivo genotoxic and mutagenic effects induced by FPs and UFPs. To fill this gap, assays were implemented to better determine whether genetic/mutagenic endpoints, in in vitro lung cell models and in an animal model, would be impacted after exposure to atmospheric FPs and UFPs collected in an area characterized by intense industrial activity and heavy high traffic roads.

For the in vitro approach, we used two types of lung cell models: standard cell lines (BEAS-2B and NCI-H292 cells) and human primary cells (NHBE cells). In order to be as close as possible to the "real" in vivo exposure conditions, the comet assay was performed on cells exposed at the ALI. Because of the absolute need of proliferative cells for the study of chromosomal aberrations, the in vitro micronucleus test cells were performed on cells grown in submerged conditions (under these conditions, cells could not be differentiated). The choice of the PM concentration for cell exposure was based on preliminary cell viability assays (measures of the amount of intracellular ATP and release of G6PD in the culture medium). The concentration of 5 µg PM/cm 2 corresponds to the 10% lethal concentration (LC 10 ) [START_REF] Sotty | Toxicological effects of ambient fine (PM2.5-0.18) and ultrafine (PM0.18) particles in healthy and diseased 3D organo-typic mucocilary-phenotype models[END_REF]. This was confirmed by our data, since, in our test conditions, FPs and UFPs did not induce any significant cytotoxic effects, neither in the comet assay nor in the micronucleus test, in the three cell types (except for UFPs which induced ca. 70% of cytotoxicity on NCI-H292 in the micronucleus test). We have shown that for primary NHBE cells, negative results were obtained for both endpoints, as observed in vivo. As expected, primary cells appeared to be more predictive of an in vivo effect. The lack of genotoxic activity observed with NHBE cells was also consistent with our previously published data on the in vitro genotoxicity of air pollution-derived PM 2.5 collected in Lille (northern France) during two different seasons (i.e. autumn-winter and spring-summer) [START_REF] Leclercq | Genetic and epigenetic alterations in normal and sensitive COPD-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived PM 2.5[END_REF]. Otherwise, immortalized BEAS-2B and NCI-H292 cells induced a positive response in the comet assay but no effect was noticed in the micronucleus test. In these cell lines, the observed primary DNA damage did not lead to chromosomal aberrations suggesting a potential involvement of DNA repair processes. BEAS-2B and NCI-H292 are both well characterized P53 proficient cells. In terms of metabolic capacity, NCI-H292 and BEAS-2B cell lines exhibited high homology in gene expression pattern with primary cells (xenobiotic metabolizing enzymes, transporters and nuclear receptors) [START_REF] Courcot | Xenobiotic metabolism and disposition in human lung cell models: comparison with in vivo expression profiles[END_REF][START_REF] Garcia-Canton | Metabolic characterization of cell systems used in in vitro toxicology testing: Lung cell system BEAS-2B as a working example[END_REF]. Thus, they could be used as surrogates for investigating the metabolism and bioactivation of toxicants in bronchial epithelium. However, we can hypothesize that their origin (cancerous cells for NCI-H292 and 12-SV40 hybrid virus transformed cells for BEAS-2B) and their poor detoxification capabilities (especially in antioxidant systems) make them more sensitive to primary DNA damage. Indeed, as stated by Bhowmick and Gappa-Fahlenkamp ( 2016), when NCI-H292 cells are cultured at ALI and exposed to cigarette smoke, they suffer considerable DNA damage. Moreover, it is known that BEAS-2B and NCI-H292 cells were relatively inefficient regarding the formation of tight intercellular junctions.

On the other hand, it is well known that chemical composition of the particles plays an important role in induction of toxicity. Despite the occurrence of PAHs (benzo[a]pyrene, indenopyrene, fluoranthene, benzo[b]fluoranthene…) and metals (As, Ni, Pb, V, Cd, Cu, Zn, Fe, Cr…) in our FPs and UFPs samples [START_REF] Saleh | Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice[END_REF], some of which being classified by IARC as carcinogenic, no genotoxicity was observed in NHBE cells. Our results, obtained with native particles samples, may seem at odds with numerous studies conducted mainly almost exclusively on PM extractable organic matter (PM-EOM) (Abbas et al., 2019;[START_REF] Bełcik | Genotoxic and cytotoxic properties of PM2.5 collected over the year in Wrocław (Poland)[END_REF][START_REF] Billet | Chemical characterization of fine and ultrafine PM, direct and indirect genotoxicity of PM and their organic extracts on pulmonary cells[END_REF][START_REF] Bocchi | Genotoxicity of airborne PM2.5 assessed by salmonella and comet assays in five cities of the Emilia-Romagna (Italy) mutagenicity monitoring network[END_REF][START_REF] Gábelová | Genotoxicity of environmental air pollution in three European cities: Prague, Košice and Sofia[END_REF][START_REF] Lemos | Genotoxicity biomarkers for airborne particulate matter (PM2.5) in an area under petrochemical influence[END_REF][START_REF] Marabini | Ultrafine particles (UFPs) from domestic wood stoves: genotoxicity in human lung carcinoma A549 cells[END_REF][START_REF] Marabini | Ultrafine particles (UFPs) from domestic wood stoves: genotoxicity in human lung carcinoma A549 cells[END_REF][START_REF] Pereira | Toxicogenetic monitoring in urban cities exposed to different airborne contaminants[END_REF]. We can thus hypothesize that chemicals adsorbed on particles, in particular PAHs, nitro-PAHs and other related compounds (dioxins and furans), were probably not enough bioavailable, or in too small amounts compared to the sensitivity of test systems, to exert their genotoxic effect. However, as explained by [START_REF] André | Mutagenicity and genotoxicity of PM2.5 issued from an urbano-industrialized area of Dunkerque (France)[END_REF] 2014) also observed positive responses in the comet and in the micronucleus assays, as well as for histone H2AX phosphorylation, in the BEAS-2B cell line exposed for 72 h to native PM samples from industrial, urban, and rural areas of Dunkirk, and exclusively at the higher concentration tested of 15 µg PM/cm 2 (no genotoxic effect appeared at the lower dose of 3.75 µg PM/cm 2 ). This suggests that, in our study, the concentration (5 µg PM/cm 2 ) and treatment times (4 h for the comet assay and 24 h for the micronucleus test), although more realistic, were probably not high and not long enough, respectively, to provide measurable genotoxic effects in all cellular models and in all end-points because of their sensitivity. However, we are aware that the PM concentrations we used in our in vitro study are quite far from environmental exposure expected in humans. Recently, [START_REF] Gualtieri | Is it the time to study air pollution effects under environmental conditions? A case study to support the shift of in vitro toxicology from the bench to the field[END_REF] studied the effects of air pollution on BEAS-2B cells exposed at the ALI under 'real' environmental conditions. They showed that the total theoretically deposited mass, being in the order of 10 -2 ng/cm 2 is roughly 3 orders of magnitude lower than the doses usually reported in PM in vitro toxicological studies performed under laboratory conditions [START_REF] Gualtieri | Is it the time to study air pollution effects under environmental conditions? A case study to support the shift of in vitro toxicology from the bench to the field[END_REF].

In summary, for the in vitro approach, various results have been reported in the literature proving the complexity of the study of the genotoxic response after exposure to a heterogeneous mixture represented by PM. Our work suggests the need to develop appropriate and standard methodologies for assessing the in vitro genotoxicity of PM providing more reliable and reproducible data. Few years ago, as stated by Magdalenova et al. (2014), although the number of studies on the genotoxicity of engineered NPs was increasing, many conflicting results were published. However, conditions influencing the results of NP genotoxicity studies are now well known [START_REF] Magdolenova | Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles[END_REF] p53 deficient, high inter-individual variability of the cell donors [START_REF] Sotty | Toxicological effects of ambient fine (PM2.5-0.18) and ultrafine (PM0.18) particles in healthy and diseased 3D organo-typic mucocilary-phenotype models[END_REF], variation in metabolic activities, antioxidant and DNA repair capabilities, internalization/phagocytosis properties…], methods of exposure (ALI vs. submerged conditions), treatment times (short vs. long, acute vs. repeated), doses (low and realistic vs. high), and cytotoxicity measurements. In addition, it is important to avoid interference of PM with the genotoxicity assay and to include sufficient controls to avoid false positive/negative results.

For the in vivo approach, animal models, especially rodents, have already been the subject of many studies to assess health effects of airborne PM 2.5 such as respiratory diseases, cardiovascular dysfunctions, diabetes mellitus and allergic sensitization [START_REF] Cho | In vitro and in vivo experimental studies of PM2.5 on disease progression[END_REF]. However, no data has been found in the literature regarding the in vivo genotoxicity assessment of PM 2.5 and PM 0.1 administered by the pulmonary route, which highlights the innovative nature of our work. Indeed, we performed a complete study of the genotoxic potential of FPs and UFPs both in terms of genotoxic endpoints studied (primary DNA damage, chromosomal aberrations, gene mutations) and duration of treatment performed (acute, subacute and subchronic). Animals were exposed to particles at the doses of 100 µg and 10 µg for acute and subchronic exposures, respectively. We have estimated that intratracheal instillation of 10 µg of PM 2.5

to BALB/c mice [whose minute ventilation is 57 mL/min according to de Hennezel et al. (2001)] is equivalent to human exposure to 121.83 µg/m 3 of air for 24 h. As explained in our previously published study, such a daily exposure could correspond approximately to that reached during a pollution spike in the North of France (no data are available for UFPs concentration) [START_REF] Saleh | Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice[END_REF]. This assumption is possible if no clearance of particles is considered and if the dose of exposure at the lung alveolar epithelium is not considered (i.e. the same mass deposited on the alveolar epithelial surfaces of mice vs.

humans corresponds to a very different exposure dose in these two species). Moreover, Chen et al.

(2010) have shown that in road tunnels, during rush hours (i.e. during holidays between 8 am and 11 am), PM 2.5 concentration can increase up to 132 µg/m 3 (46.5 µg/m 3 for PM 0.1 ). According to the very recent update (April 2018) of the WHO ambient (outdoor) air quality database containing average annual estimates for PM 2.5 exposure globally (covering more than 4000 cities in 108 countries), the concentration of atmospheric PM 2.5 of 121.83 µg/m 3 corresponds to a moderate to high exposure to these particles. In all regions of the world, apart from the high-income region of the Americas, populations are exposed to levels of PM 2.5 that are higher than the annual mean value of 10 μg/m 3 recommended in the WHO air quality guideline. In addition, during air pollution peak episodes, as PM 2.5 concentration frequently exceeds 10 times the annual average, we have chosen to expose mice to 100 µg for the single treatment. Therefore, the doses of 10 µg and 100 µg used in our study for chronic and acute exposures, respectively, are realistic compared to the different PM 2.5 concentration scenarios found in the atmosphere..

Our exhaustive investigation were realized in BALB/c mice after intranasal administration which provides homogeneous pulmonary delivery of particles and causes pulmonary inflammation (increase in IL-6 level in plasma) and tissue remodeling at the molecular, cellular and histological level, as observed in our previously published study [START_REF] Saleh | Exposure to Atmospheric Ultrafine Particles Induces Severe Lung Inflammatory Response and Tissue Remodeling in Mice[END_REF]. Our results revealed that UFPs and FPs induced no biologically and no statistically significant increases in DNA strand breaks, chromosomal aberrations and gene mutations, whatever the duration of exposure. Therefore, these airborne particles are considered having no genotoxic/mutagenic activity under our experimental conditions. These negative results, although they may seem surprising knowing that PM is classified as carcinogenic, may be explained by the metabolization and inactivation of mutagenic compounds before they can reach the target cells. In two old studies, similar negative results were found. Crebelli et al. (1988) performed a micronucleus test on bone marrow in Swiss mice exposed to EOM-PM 10 by intraperitoneal (i.p.) injection. No increase in micronuclei frequency was observed. Sprague-Dawley rats were also treated with EOM-PM 10 by gastric intubation or i.p. injection in order to study body-fluid (blood, urine and feces) mutagenicity (Ames test on TA98 strain). Negative results were also obtained. This study demonstrated the rapid and total disappearance of mutagens and pro-mutagens in body fluids of treated animals and thus suggesting an extensive in vivo inactivation, and/or a probably limited bioavailability of mutagens adsorbed onto airborne PM. [START_REF] Krishna | Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice[END_REF] showed no significant increase in the sister-chromatid exchange (SCE) assay performed in mouse bone marrow and spleen cells after airborne PM oral or i.p. administration. The authors suggested that it may be due to the inactivation of mutagenic compounds in the body, the inability of mutagenic compounds to reach the bone marrow and spleen, or the relative low concentration of mutagenic material in these organs to cause significant increase in SCE level. Furthermore, some studies reported a cancer promoter effect of air pollution in mice, which could also explain our negative results obtained in "healthy" cells and organs [START_REF] Cury | Urban levels of air pollution modifies the progression of urethane-induced lung tumours in mice[END_REF][START_REF] Reymão | Urban Air Pollution Enhances the Formation of Urethane-Induced Lung Tumors in Mice[END_REF]. Low levels of air pollution are able to favor the development of tumors, i.e. to accelerate the process of progression towards malignancy. This suggests that mechanisms of mutagenesis may not be directly involved in the induction of lung cancer, and therefore that FPs and UFPs would probably not be initiating agents at this dose level.

It is important to highlight the original nature of our work which provides a complete study of the in vitro and in vivo genotoxic/mutagenic potential of FPs and UFPs. However, in order to continue to investigate the mechanism of action of these particles in the appearance and/or development of lung cancer, further studies would be implemented.

For in vitro assays, we used three different lung cell models (primary and immortalized cells), cultured both in submerged conditions and at the ALI. Cells were exposed to FPs and UFPs suspensions which have advantages (control of the dose actually used, relatively easy to perform, moderate cost…) but unfortunately also limitations [START_REF] Fröhlich | Toxicological Assessment of Inhaled Nanoparticles: Role of in Vivo, ex Vivo, in Vitro, and in Silico Studies[END_REF]. Indeed, suspension exposure remains a non-physiological exposure and it is well known that NPs (and PM) suspended in medium often form aggregates. Therefore, for further refinement of our study it would be interesting to expose cells with aerosols generated by commercially available ALI-based exposure systems (such as VITROCELL ® ).

Moreover, as the lung is composed of approximately 40 cell types, a co-culture model with bronchial and immune (macrophages, monocytes...) cells could be used in order to preserve biological conditions and intercellular signaling. It is also known that PM can induce oxidative stress via ROS generation through a Fenton-type reaction involving transition metals (and more particularly Fe) but also through reactions involving organic compounds, such as PAHs, dioxins and furans [START_REF] André | Mutagenicity and genotoxicity of PM2.5 issued from an urbano-industrialized area of Dunkerque (France)[END_REF][START_REF] Baulig | Role of Paris PM2.5 components in the pro-inflammatory response induced in airway epithelial cells[END_REF][START_REF] Boublil | Development of a repeated exposure protocol of human bronchial epithelium in vitro to study the long-term effects of atmospheric particles[END_REF][START_REF] Kim | Predictive and prognostic biomarkers of respiratory diseases due to particulate matter exposure[END_REF]. We can suppose that the "standard" comet assay, as performed in our study, may not be sensitive enough to detect oxidative damage. The modified version of the comet assay using endonucleases (e.g. Fpg, hOgg1 or Endo III) could have been useful, as shown by [START_REF] Oh | Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells)[END_REF]. At last, since air pollution appears to be responsible for increasing the incidence and aggravation of pulmonary diseases, our present data could be supplemented by toxicological study of FPs and UFPs on sensitive experimental models such as human bronchial epithelial cells from patients with COPD or asthma.

Regarding our in vivo study, we performed a full assessment of the genotoxicity of FPs and UFPs by performing 3 different tests (comet assay, micronucleus test and Pig-A test), while only one is normally recommended by the French national agency of medicine and health products safety for drugs in nanoparticular form (AFSSAPS, 2011). Nevertheless, our study could be improved by the use of another mode of treatment. Indeed, mice were treated by intranasal instillation. Although not invasive and not requiring deep anesthesia, this technique does not correspond to a physiological route of exposure. It would be interesting to carry out further investigations with systems such as whole-body or nose only exposure [START_REF] Fröhlich | Toxicological Assessment of Inhaled Nanoparticles: Role of in Vivo, ex Vivo, in Vitro, and in Silico Studies[END_REF].

CONCLUSION

To conclude, this work aimed to provide new relevant insights about the possible role of the genotoxicity and mutagenicity of airborne FPs and UFPs, from an industrial-influenced urban area, in lung carcinogenesis. We have developed a relevant experimental strategy allowing us to be closer to human mode of exposure (i.e. native PM, ALI exposed cells, animals treated by pulmonary route) and levels of exposure (i.e. moderate doses). Under these experimental conditions, FPs and UFPs are devoid of in vitro genotoxic effect in NHBE primary cells, which therefore seem to be more predictive of an in vivo effect. In return, positive results were obtained in the comet assay in BEAS-2B and NCI-H292 immortalized cell lines. Our work suggests the need to develop appropriate and standard methodologies for assessing the in vitro genotoxicity of PM. Moreover, FPs and UFPs exhibit no in vivo genotoxic/mutagenic activity in our tested conditions. No differences appear between both types of particles. However, UFPs are known to be relatively more toxic than larger FPs because they can reach deeper in the airways thanks to their lower aerodynamic diameter. These data suggest the potential promotion role of FPs and UFPs in pulmonary carcinogenesis at this dose level, and thus the involvement of other mechanisms than genotoxicity, such as epigenetic alterations. the percent ratio of viable unstained cells to non-viable stained cells in exposed versus control groups).

TABLE AND FIGURE LEGENDS

The control groups were treated with culture medium. MMS was used as positive control (0.96 µg/cm 2 ). * p<0.05 (Mann-Whitney U-test). One experiment, 2 biological replicates, 4 technical replicates. Table 1. Summary of in vitro genotoxic/mutagenic tests performed.

Cells were treated with 5 µg PM/cm 2 of airborne FPs and UFPs. Comet assay was performed immediately after 4 hours exposure at the ALI whereas micronucleus test was performed after 24 hours exposure in submerged conditions followed by a 2 days (BEAS-2B and NCI-H292 cells) or a 5 days (NHBE cells) recovery period. X: test performed. For acute exposure (24 hours), mice received 2 treatments at 24-hours intervals at the dose of 100 µg of particles. For subacute (1 month) and subchronic (3 months) exposures, animals were treated with 10 µg of particles, 3 times a week. X: test performed. Cells were treated with 5 µg/cm2 of airborne FPs and UFPs. Comet assay was performed immediately after 4h exposure at the ALI whereas micronucleus test was performed after 24h exposure in submerged conditions followed by a 2 days (BEAS-2B and NCI-H292 cells) or a 5 days (NHBE cells) recovery period.

-: negative result; +: positive result. For acute exposure, mice received 2 treatments at 24-hours interval at the dose of 100 µg of particles. For subacute (1 month) and subchronic (3 months) exposures, animals were treated with 10 µg of particles, 3 times a week. n.a.: not assessed; -: negative result. For acute exposure (24 hours), mice received 2 treatments at 24-hours intervals at the dose of 100 µg of particles. For subacute (1 month) and subchronic (3 months) exposures, animals were treated with 10 µg of particles, 3 times a week. X: test performed. For acute exposure, mice received 2 treatments at 24-hours interval at the dose of 100 µg of particles.

For subacute (1 month) and subchronic (3 months) exposures, animals were treated with 10 µg of particles, 3 times a week. n.a.: not assessed; -: negative result.

(

  RPD) as recommended by OECD test guideline No. 487 (OECD, 2016a) was calculated: RPD = [(Number of Population doublings in treated cultures) ÷ (Number of Population doublings in control cultures)] x 100 where Population Doubling = [log (Post-treatment cell number ÷ Initial cell number)] ÷ log 2.

  control groups were treated (1) orally with MMS [100 mg/kg b.w/day (x2) in sterile water] for the comet assay (combined or not to the micronucleus test), (2) intraperitoneally with CPA [50 mg/ kg b.w/day (x1) in 0,9 % NaCl] for the micronucleus test, or (3) ENU [40 mg/ kg b.w/day (x3) in sterile water] for the Pig-A assay. Animal body weights were recorded before each administration while clinical signs were monitored daily (data not shown).

Figure 1 .

 1 Figure 1. Results of in vitro comet assays. Primary DNA damage was determined in BEAS-2B, NCI-H292 and NHBE cells with the alkaline comet assay immediately after 4 h exposure to 5 µg PM/cm 2 of airborne particles, at the ALI. Values are depicted as means of medians of % of tail intensity and standard deviations. Cytotoxicity was assessed at harvest using the Trypan Blue dye exclusion assay. Results are expressed as relative percent of cell survival (i.e.

Figure 2 .

 2 Figure 2. Results of in vitro micronucleus tests.Chromosomal aberrations were determined with the micronucleus test after 24 h exposure to 5 µg PM/cm 2 of airborne particles in submerged conditions, followed by a 2 days (BEAS-2B and NCI-H292 cells) or a 5 days (NHBE cells) recovery period. Values are depicted as means of number of micronuclei (MN) per 1000 cells and standard deviations. Cytotoxicity was evaluated by calculating the percentage of relative population doubling (RPD). The control groups were treated with culture medium. MMC was used as positive control (0.01 µg/cm 2 ). * p<0.05 (CHI-squared test). One experiment, 2 biological replicates, 4

Figure 3 .

 3 Figure 3. Results of in vivo genotoxicity assessment after acute exposure of airborne particles in mice. Animals (n=5) received 2 treatments at 24-hour interval at the dose of 100 µg of particles. The control group received 37 µL of HBSS. MMS was used as positive control [(100 mg/kg b.w./day)x2]. The level of primary DNA damage is expressed as the mean of medians of % of tail DNA intensity. ** p<0.01 (Mann-Whitney U-test).

Figure 4 .

 4 Figure 4. Results of in vivo genotoxicity/mutagenicity assessment after subacute exposure of airborne particles in mice.

Figure 5 .

 5 Figure 5. Results of in vivo genotoxicity/mutagenicity assessment after subchronic exposure of airborne particles in mice. Animals (n=5) received 10 µg of particles, 3 times a week, during 3 months. The control group received 37 µL of HBSS at each treatment. CPA [(50 mg/kg b.w./day)x1] and ENU [(40 mg/kg b.w./day)x3] were used as positive controls. (A) The chromosomal aberrations frequency is expressed as the number of micronucleated polychromatic erythrocytes (MNPCE) per 1000 cells. (B) The gene mutations frequency is expressed as the number of red blood cells (RBC) or reticulocytes (RET) per 10 6 cells. The polychromatic erythrocytes (PCE) / normochromatic erythrocytes (NCE) ratio and % RET are used as a measure of bone marrow cytotoxicity. * p<0.5; ** p<0.01 (Mann-Whitney U-test for the micronucleus test, Dunnet's t-test for the Pig-A test).

Figure 1 .

 1 Figure 1. Results of in vitro comet assays.Primary DNA damage was determined in BEAS-2B, NCI-H292 and NHBE cells with the alkaline comet assay immediately after 4 h exposure to 5 µg PM/cm 2 of airborne particles, at the ALI. Values are depicted as means of medians of % of tail intensity and standard deviations. Cytotoxicity was assessed at harvest using the Trypan Blue dye exclusion assay. Results are expressed as relative percent of cell survival (i.e. the percent ratio of viable unstained cells to non-viable stained cells in exposed versus control groups). The control groups were treated with culture medium. MMS was used as positive control (0.96 µg/cm 2 ). * p<0.05 (Mann-Whitney U-test). One experiment, 2 biological replicates, 4 technical replicates.

Figure 2 .

 2 FIGURE 2

Figure 3 .Figure 4 .

 34 FIGURE 3

Figure 5 .

 5 FIGURE 5

  TryPLE™ Express were obtained from ThermoFisher Scientific (Illkirch, France). RPMI 1640, horse serum, fetal calf serum, non-essential amino acids and phosphate buffered saline (PBS) were purchased from GIBCO Invitrogen (Cergy-Pontoise, France). BEGM

TM BulletKit TM , B-ALI ™ BulletKit ™ , B-ALI ™ growth basal medium, B-ALI ™ differentiation basal medium, B-ALI ™ inducer, Trypsin Neutralizing Solution were purchased from Lonza (Verviers, Belgium).

2.4.2. Cell lines and culture conditions 2.4.2.1. Primary cells: NHBE cells

	(this may vary depending on the donor). Their characteristics were described recently by Rayner et al.
	(2019).
	2.4
	UFPs and FPs suspensions
	were cooled on ice to minimize heat-development during sonication. These initial suspensions were then
	diluted 1:9 v/v in cell culture medium, supplemented with amphotericin B (20 mg/mL) at 2% (v/v), at the
	time of cell treatment to obtain final suspensions at 5 µg PM/cm 2 . Initial and final suspensions were
	carefully vortexed before each pipetting.
	NHBE cells (Lonza, Verviers, Belgium) were isolated from donated human tissue (from epithelial lining of
	airways above bifurcation of the lungs), after obtaining permission for their use in research applications by
	informed consent or legal authorization. The characteristics of the healthy donor and cells are shown in
	Table 2. Cells were cultured in 75 cm 2 Corning ® CellBIND ® surface cell culture flasks (Sigma-Aldrich, St.
	Quentin Fallavier, France) in bronchial epithelial growth medium BEGM according to the manufacturer
	recommendations. Medium was renewed every 3 days and cells were subcultured once a week and
	seeded at a density of 5x10 5 cells per flask. The doubling time of NHBE cells was approximately 30 hours

Branson Sonifier S-450D (Branson Ultrasonics Corp., Danbury, CT, USA).

.2.2. Immortalised cells: BEAS-2B and NCI-H292 cells NCI-H292 cells. The

	human muco-epidermoid bronchiolar carcinoma cell line NCI-H292 was obtained
	from the American Type Culture Collection (ATCC	® CRL-1848™). Cells were seeded at a density of 5x10	5
	cells per culture flask (75 cm	2 ; Falcon TM , Becton Dickinson Biosciences, France) and maintained at 37°C
	in a humidified 5% CO 2 atmosphere in RPMI 1640 supplemented with 10% (v/v) heat-inactivated fetal calf
	serum, 200 UI/mL penicillin, 50 mg/L streptomycin, 2.5 mg/L amphotericin B. This medium is hereafter
	referred to as RPMI 10 growth medium. Medium was renewed every 2 to 3 days and cells were
	subcultured when they reached 80-90 % confluence. The ATCC indicates that the doubling time of NCI-
	H292 cells is approximately 48 hours.
	BEAS-2B cells. The human bronchial epithelial cell line BEAS-2B was obtained from the American Type
	Culture Collection (ATCC	® CRL-9609™). Comparable to the NHBE cells, BEAS-2B cells were seeded at a
	density of 5x10 5 cells in 75 cm	2 Corning ® CellBIND ® surface cell culture flasks and maintained at 37°C in a
	humidified 5% CO 2 atmosphere in BEGM. Medium was renewed every 2 to 3 days and cells were
	subcultured before reaching confluence since confluent cultures rapidly undergo squamous terminal
	differentiation. The doubling time of BEAS-2B cells is approximately 24 hours

  the particles is not assessed. Nevertheless, increases in DNA damage and in chromosomal aberrations were not systematically reported since some negative genotoxicity results were also found in the literature. For example,[START_REF] André | Mutagenicity and genotoxicity of PM2.5 issued from an urbano-industrialized area of Dunkerque (France)[END_REF] have directly tested suspensions of PM 2.5 collected in

	Dunkirk. Exposure of A549 immortalized pulmonary cells to PM 2.5 for 72 h at the LC50 (31.6 µg PM/cm	2 )
	did not lead to quantifiable PAH bulky DNA adducts (although A549 cells are cancerous cells with highly
	active phase I metabolism enzymes). Recently, Billet et al. (2018) observed neither the formation of DNA
	bulky adducts after a 72 h-exposure of BEAS-2B cells to PM 0.3-2.5 or to PM-EOM, despite the high
	sensitivity of the 32 P post-labeling used method. Abbas noticed slight but significant decreases of
	phosphorylated-Histone 2AX (H2AX) in BEAS-2B cells 72 h after their exposure to the organic fraction of
	air pollution-derived PM 2.5 from 3 to 30 μg PM 2.5 -eq/cm 2 (Abbas et al., 2019). In return, 8-OHdG adducts
	were measured as significantly increased after 48 h of treatment but exclusively at the highest
	concentration of 15 µg PM/cm 2 (not at 3 µg PM/cm 2 ). However, 8-OHdG formation was shown in A549
	cells after incubation for 24 h, 48 h and 72 h with whole PM 2.5 from Dunkirk at concentrations
	corresponding to the LC10 (6.33 µg PM/cm
	, extrapolations
	of results obtained with organic extracts to potential effects on human health are limited for three main
	reasons: (i) there are too high chemical concentrations in the PM-EOM compared to environmental

concentrations; (ii) PM-EOM does not exactly match what is found in "real life", i.e. in the whole complex mixture-coated onto the particles (especially metals not extractable by organic solvents); (iii) toxicity of the core of 2 ) and LC50 (31.6 µg PM/cm 2 )

[START_REF] André | Mutagenicity and genotoxicity of PM2.5 issued from an urbano-industrialized area of Dunkerque (France)[END_REF]

.

Lepers et al. (

  and recommendations on method standardization and assay modifications have been made. The same observation can be done for airborne PM since a diversity of genotoxicity assays and test systems has been used leading to a multiplicity of results. Thus, it is difficult to compare results without standardized methods. Variability in the results of genotoxicity studies can be attributed to the emission sources of PM (natural vs. anthropogenic, rural vs. industrial, heavy fuel combustion vs. road traffic…) conditioning their physicochemical characteristics (size, solubility, aggregation/agglomeration, composition, presence of mutagens and transition metals...), and to the experimental conditions. This last point is of major importance when comparing studies. Indeed, several factors in in vitro experimental conditions could influence the results of genotoxicity testing, such as types of PM extraction (aqueous media, organic solvent, thermal

desorption…) and thus types of particles tested (fractions more or less polar, or native PM), preparation of PM suspensions (solvents and methods used for suspending and dispersing them), cellular models used [primary vs. immortalized cultures, normal vs. cancerous cells, human vs. rodent origin, p53 proficient vs.

Table 2 . Characteristics of NHBE cells used in this study.

 2 NHBE cells (Lonza) were isolated from the epithelial lining of the airway above bifurcation of the lungs of normal donors who have not been diagnosed asthma, COPD or cystic fibrosis.

Table 3 . Summary of in vivo genotoxic/mutagenic tests performed.

 3

Table 4 . Summary of in vitro genotoxicity/mutagenicity tests results.

 4

Table 5 . Summary of in vivo genotoxicity/mutagenicity tests results.

 5

TABLE 1 Table 1 : Summary of in vitro genotoxic/mutagenic tests performed.

 11 Cells were treated with 5 µg PM/cm 2 of airborne FPs and UFPs. Comet assay was performed immediately after 4 hours exposure at the ALI whereas micronucleus test was performed after 24 hours exposure in submerged conditions followed by a 2 days (BEAS-2B and NCI-H292 cells) or a 5 days (NHBE cells) recovery period. X: test performed.

			Treatment conditions		Cell types
	In vitro tests	End-points
			Culture	Duration	NHBE	BEAS-2B	NCI-H292
	Comet assay	Primary DNA damage	ALI	4 hours	X	X	X
	Micronucleus test	Chromosomal aberrations	Immersion	24 hours	X	X	X

TABLE 2 Table 2 : Characteristics of NHBE cells used in this study.

 22 Lonza) were isolated from the epithelial lining of the airway above bifurcation of the lungs of normal donors who have not been diagnosed asthma, COPD or cystic fibrosis.

	SPECIFICATIONS	RESULTS
	Donor characteristics
	Age (years)	49
	Sex	Female
	Race	Caucasian
	Smoking habits	Non-smoker
	Alcohol use	Non-alcohol user
	Virus Testing
	HIV Test	Not detected
	HBV Test	Not detected
	HCV Test	Not detected
	Microbial Testing
	Sterility Test	Negative
	Mycoplasma	Negative
	Cell Performance Testing
	Cell passage frozen	1
	Viability	84 %
	Cell count (cells/vial)	879 000
	Seeding efficiency	27 %
	Doubling time (hours)	28
	NHBE cells (

TABLE 3 Table 3 : Summary of in vivo genotoxic/mutagenic tests performed.

 33

				Treatment schedule
	In vivo tests	End-points	Target organs
				24 hours	1 month	3 months
	Comet assay	Primary DNA damage	Liver, lung	X	X
	Micronucleus test	Chromosomal aberrations	Bone marow		X	X
	Pig-A gene mutation assay	Gene mutations	Erythrocytes		X	X

TABLE 4 Table 4 : Summary of in vitro genotoxicity/mutagenicity tests results.

 44 Cells were treated with 5 µg/cm 2 of airborne FPs and UFPs. Comet assay was performed immediately after 4h exposure at the ALI whereas micronucleus test was performed after 24h exposure in submerged conditions followed by a 2 days (BEAS-2B and NCI-H292 cells) or a 5 days (NHBE cells) recovery period. -: negative result; +: positive result.

	In vitro tests	End-points	Treatment conditions	NHBE	Cell types BEAS-2B	NCI-H292
	Comet assay	Primary DNA damage	ALI	-	+	+
	Micronucleus test	Chromosomal aberrations	Immersion	-	-	-

TABLE 5 Table 5 : Summary of in vivo genotoxicity/mutagenicity tests results.

 55

				Treatment schedule
	In vivo tests	End-points	Target organs
				24 hours	1 month	3 months
	Comet assay	Primary DNA damage	Liver, lung	-	-	n.a.
	Micronucleus test	Chromosomal aberrations	Bone marow	n.a.	-	-
	Pig-A gene mutation assay	Gene mutations	Erythrocytes	n.a.	-	-

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work benefited grants from the Region Hauts-de-France (PRSE, Convention N°13004366), the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) (PNREST ANSES, CANCER ITMO AVIESAN, Convention N°2014/1/183) and the European Funds for Regional Economical Development (CPER CLIMIBIO). We thank E. Vernet for technical assistance. We thank Dr Helene Bauderlique from the BioImaging Center Lille for facility access to the cytometer BD Canto II (BICeLcampus Pasteur, 1 rue du Prof Calmette, 59019 Lille).