Fine and ultrafine atmospheric particulate matter at a

Environmental Pollution 221 (2017) 130e140

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Fine and ultrafine atmospheric particulate matter at a multi-influenced urban site: Physicochemical characterization, mutagenicity and cytotoxicity* de ric Ledoux a, *, Ve ronique Andre b, Fabrice Cazier c, Paul Genevray c, Yann Landkocz a, Fre c a e Dewaele , Perrine J. Martin , Capucine Lepers a, Anthony Verdin a, Dorothe ^d Boushina a, François Sichel b, Maurizio Gualtieri a, Pirouz Shirali a, Lucie Courcot d, Saa Dominique Courcot a, Sylvain Billet a ^te d'Opale, EA 4492 - UCEIV - Unit Univ. Littoral Co e de Chimie Environnementale et Interactions sur le Vivant, F-59140, Dunkerque, France Univ. Caen-Normandie, Aliments, Bioproc ed es, Toxicologie, Environnements, EA 4651, Centre François Baclesse, F-14032, Caen, France c ^te d'Opale, CCM - Centre Commun de Mesures, F-59140, Dunkerque, France Univ. Littoral Co d ^te d'Opale, CNRS UMR8187 - LOG - Laboratoire d'Oc Univ. Littoral Co eanologie et de G eosciences, F-62930, Wimereux, France a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 July 2016 Received in revised form 18 November 2016 Accepted 19 November 2016 Available online 30 November 2016

Particulate Matter (PM) air pollution is one of the major concerns for environment and health. Understanding the heterogeneity and complexity of fine and ultrafine PM is a fundamental issue notably for the assessment of PM toxicological effects. The aim of this study was to evaluate mutagenicity and cytotoxicity of a multi-influenced urban site PM, with or without the ultrafine fraction. For this purpose, PM2.5-0.3 (PM with aerodynamic diameter ranging from 0.3 to 2.5 mm) and PM2.5 were collected in Dunkerque, a French coastal industrial city and were extensively characterized for their physico-chemical properties, including inorganic and organic species. In order to identify the possible sources of atmospheric pollution, specific criteria like Carbon Preference Index (CPI) and PAH characteristic ratios were investigated. Mutagenicity assays using Ames test with TA98, TA102 and YG1041 Salmonella strains with or without S9 activation were performed on native PM sample and PM organic extracts and watersoluble fractions. BEAS-2B cell viability and cell proliferation were evaluated measuring lactate dehydrogenase release and mitochondrial dehydrogenase activity after exposure to PM and their extracts. Several contributing sources were identified in PM: soil resuspension, marine emissions including seasalt or shipping, road traffic and industrial activities, mainly related to steelmaking or petro-chemistry. Mutagenicity of PM was evidenced, especially for PM2.5, including ultrafine fraction, in relation to PAHs content and possibly nitro-aromatics compounds. PM induced cytotoxic effects at relatively high doses, while alteration of proliferation with low PM doses could be related to underlying mechanisms such as genotoxicity. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Air pollution PM2.5 Physicochemical characterization Ames test Cytotoxicity Mutagenicity

1. Introduction Particulate Matter (PM) air pollution is now a well-recognized carcinogen for humans and is one of the most important environment and health concerns (Loomis et al., 2013). World Health Organization (WHO) estimates that exposure to PM may cause 3.7

*

This paper has been recommended for acceptance by Eddy Y. Zeng. * Corresponding author. E-mail address: [email protected] (F. Ledoux).

http://dx.doi.org/10.1016/j.envpol.2016.11.054 0269-7491/© 2016 Elsevier Ltd. All rights reserved.

million premature deaths worldwide (WHO, 2014). In the past decade, European and US authorities established new directives to reduce PM exposure limits (Krzyzanowski, 2008). Chronic and acute exposure to PM is related to lung cancer morbidity and mortality in industrialized countries (Carey et al., 2013; Katanoda et al., 2011; Li et al., 2015). However, both the underlying mechanisms of PM toxicity and the influence of PM composition and size distribution on such health effects still remain unclear. Understanding the heterogeneity and complexity of fine and ultrafine PM is a fundamental issue for the assessment of PM toxicological effects. PM sampled at a multi-influenced site

Y. Landkocz et al. / Environmental Pollution 221 (2017) 130e140

contains organic compounds, such as Polycyclic Aromatic Hydrocarbons (PAHs), furans and dioxins, inorganic components, such as ions and metals, black carbon, crustal and biogenic elements (Harrison and Yin, 2000). PM size and composition are linked to their origins, to the atmospheric photochemistry and meteorological conditions. Primary pollutants can indeed react together to form new xenobiotics, including compounds with low relative amount but suspected high toxic potential. These interactions also occur after inhalation. Additive, antagonist, and synergistic effects can modify PM toxicity (Claxton and Woodall, 2007; DeMarini, 2013). Furthermore, due to the diversity of emission sources and the chemical transformation of pollutants in the atmosphere, multiple pathways are often involved in the toxicity of PM and in the onset of adverse health effects (Ding et al., 2014; Longhin et al., 2016; Vaccari et al., 2015). Lung carcinogenicity of PM is often related to their mutagenic and/or genotoxic properties (Sørensen et al., 2003). Several studies have demonstrated that PM2.5 extractable organic matter could be mutagenic, using several Salmonella typhimurium strains in the Ames test (de Kok et al., 2006; Traversi et al., 2015). Short-term mutagenicity bioassays of particulate samples from different PM size fractions and/or different PM components (i.e. organic fraction, water-soluble fraction or total PM) may help understanding the contribution of different PM size fractions or chemicals on health et al., 2011; Skarek et al., 2007; Topinka et al., 2013). (Andre In this context, the present work aimed to study the sizedependent toxicity of PM sampled at a multi-influenced site. PM was extensively characterized for physical and chemical properties, in order to identify the main contributing sources. Secondly, native PM2.5-0.3 (PM with aerodynamic diameter ranging from 0.3 to 2.5 mm), as well as Organic Extract (OE) and Water-soluble Fraction (WF) of PM2.5-0.3 and PM2.5, were investigated for mutagenicity, cytotoxicity and cell proliferation. Mutagenicity was evaluated using the Ames test on three different Salmonella typhimirium strains able to evidence frameshift mutation, oxidative stress or PAHs effects. Cytotoxicity and effects on proliferation were measured on BEAS-2B bronchial epithelial cell line for which metabolic ability has been previously demonstrated (Uppstad et al., 2010). 2. Materials and methods 2.1. PM sampling PM were sampled from March to July 2011 in the city center of Dunkerque, northern France (latitude: 51 201000 N; longitude: 2 220 4600 E), a coastal, industrial and urban site with 200,000 inhabitants (Kfoury et al., 2016). A high volume (68 m3/h) five-stages plus back up cascade impactor (model 235 TFIA-2, Staplex®, USA) was used to collect PM2.5-0.3, as described in Cazier et al. (2016). Briefly, a quartz fiber filter (TFAQS810, Staplex®, USA) was used as impaction substrate on the first stage while the following stages were kept nude to collect particles in their native form. During the whole sampling campaign, two impaction systems were run in parallel to maximize the mass of PM2.5-0.3 available to perform physicochemical characterization and toxicological studies. Particles were continuously collected for seven days; impaction plates were then removed and placed in a laminar flow clean bench for two days to allow complete drying. Finally, particles impacted on stage 2e5, were brushed from the plates and pooled together. All particle batches were gathered in Teflon-PFA vessels and carefully homogenized using two Teflon coated magnetic balls and a magnetic stirrer for 2 h. The resulting sample was finally stored at 20 C until use. PM2.5 were collected in parallel using the high volume (30 m3/h) aerosol sampler DHA-80 (DHA-80, DIGITEL®, Switzerland)

131

equipped with the PM2.5 inlet (DPM2,5/30/00, DIGITEL®, Switzerland). The sampler was loaded with quartz fiber filters (QMA, Whatman®, GE Healthcare Life Sciences, United Kingdom), preheated to 450 C for 8 h before sampling in order to reduce blank carbon values. After sampling, filters were kept in a horizontal laminar flow clean bench for 24 h before storage at 20 C until further handling. 2.2. Characterization of PM Both PM2.5-0.3 and PM2.5 samples were extensively characterized as published elsewhere (Billet et al., 2007; Cazier et al., 2016; Ledoux et al., 2006). PM2.5-0.3 size distribution, morphology and single particle analysis were performed using the scanning electron microscopy (438 VP; LEO Electron Microscopy Ltd, UK) coupled with energy dispersive X-ray analysis (IXRF, Oxford Instruments, UK) (SEM-EDX). Metals and ionic species were quantified in PM and WF by Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES, iCAP 6000 Series, Thermo Scientific, UK), ICP-Mass Spectrometry (ICP-MS, Varian® 820-MS, Varian, USA) and Ion Chromatography (IC, Dionex® DX 100, Thermo Scientific, UK), respectively. Polycyclic aromatic hydrocarbons (PAHs) and linear alkanes were quantified after PM Soxhlet extraction using dichloromethane by gas chromatography-mass spectrometry (GCMS, model 1200 TQ, Varian, USA). The carbon content was measured using the CHNS/O analyzer (FLASH, 2000; Fisher Scientific®, UK). 2.3. Sample preparation for toxicological studies To better elucidate the specific role of the organic and/or water soluble component of PM, two fractions were considered. PM2.5-0.3 and PM2.5 organic extracts (OEs) were obtained by Soxhlet extraction using dichloromethane at 40 C for 16 h. Dichloromethane was then evaporated under nitrogen flow and the organic extract reconstituted in dimethyl sulfoxide (DMSO). Water-soluble fractions (WFs) were obtained by lixiviation of PM2.5-0.3 and portions of PM2.5 filters in ultra-pure water, followed by 30 min of sonication. Mutagenicity tests were performed on OEs and WFs of PM2.5-0.3 and PM2.5. Native PM2.5-0.3 particulate samples were also tested. In this latter case, PM2.5-0.3 was suspended in DMSO (final concentration 0.05), results for cytotoxicity and proliferation assays were expressed as mean and standard deviation. For each exposure time and concentration, data were compared with the corresponding negative controls. Statistical analyses were performed using the MannWhitney U test (p < 0.05) and correlations between cytotoxicity and proliferation parameters were performed using the Spearman test (p < 0.05) via SPSS software (SPSS 20.0, IBM, France). Effective Concentration (EC) values were calculated by concentrationresponse curve fitting using the Hill equation model (Khinkis et al., 2003). Results were expressed as the concentrations inducing 50% of cytotoxicity (EC50) and their 95% confidence interval. 3. Results and discussion 3.1. PM elemental and water-soluble species composition The average PM2.5 concentration during the sampling period was estimated at 14 mg/m3, in agreement with the data recorded by the regional air quality monitoring network, ATMO Nord-Pas-deCalais. This value was higher than the WHO annual PM2.5 guidelines (10 mg m3) and below the PM2.5 annual limit value (LV) of

Table 1 Elemental and ionic species concentrations (in mg/g and ng/m3) in PM2.5-0.3 and in the water-soluble fraction of PM2.5-0.3 and PM2.5 collected at Dunkerque in MarcheJuly 2011. PM2.5-0.3 Total

Al Ba Cr Cu Fe Mn Ni Pb Sr Ti V Zn Ca2+ K+ Mg2+ Na+ Cl SO2 4 NO 3 NH+4 C total

PM2.5

Water-soluble

Water-soluble

(mg/g)

(ng/m3)

(mg/g)

(ng/m3)

(mg/g)

(ng/m3)

14,818 213 89 322 30,513 3,503 73 144 94 412 64 1,602

121 1.74 0.73 2.63 249 28.6 0.60 1.18 0.77 3.36 0.52 13.1

82 39 5 26 612 473 11 14 82 20 3 98 14,515 4,523 3,515 19,824 32,121 32,254 180,071 44,220

0.67 0.32 0.04 0.21 4.99 3.86 0.09 0.11 0.67 0.16 0.02 0.80 118 36.9 28.7 162 262 263 1,469 361

497 45 19 103 1,086 475 165 398 24 22 383 537 9,920 4,716 2,330 12,315 21,143 72,769 329,460 98,993

6.99 0.63 0.27 1.45 15.3 6.68 2.32 5.60 0.34 0.31 5.39 7.56 140 66 32.8 173 297 1,024 4,635 1,393

112,700

920


3.2. Individual particle composition and morphology Scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) analysis evidenced particles with different size and morphology (Fig. 1). Angular shape particles related to mechanical formation processes were predominant. Ca rich particles or Ca-S rich particles corresponding to calcite or gypsum particles (elements with atomic number below 9, such as C, N, O were not quantified in the SEM-EDX analysis) were frequently detected. Gypsum has both natural and anthropogenic origins, due to its formation from sea spray (De Hoog et al., 2005) and the erosion of building materials, respectively. These Ca based compounds are also well known raw materials for the sintering process in steelmaking activities (Machemer, 2004). Aluminosilicates, i.e. particles containing Al, Ca, Fe, Si, Mg, Ti and Fe, were also detected and could be mainly related to crustal dust resuspension. Fe-rich particles, especially spherical ones that are formed during high temperature processes, and particles containing Fe associated to Mn or Zn could be linked to the metallurgic industry near the sampling site (Hleis et al., 2013; Machemer, 2004). Submicron agglomerated carbon particles with smooth shapes were also identified and possibly derived from aged soot (Courcot et al., 2009).

133

3.3. Polycyclic aromatic hydrocarbons and paraffins PM composition PAHs were 12-fold more concentrated in PM2.5 containing the ultrafine fraction than in PM2.5-0.3, but the distribution between the PAHs was relatively similar in both sample types (Table 2). Higher PAHs concentrations in PM2.5 may be explained by combustion processes known to generate PAHs rich ultrafine particles (Kawanaka et al., 2009; Schnelle-Kreis et al., 2001). The total atmospheric PAHs concentration in PM2.5 was estimated as 7.7 ng m3. This value is in agreement with the values observed at Douai, northern France in winter (17.2 ng m3) or summer (0.58 ng m3) (Crenn et al., 2016). PAHs concentration in Munich air samples ranged between 1.24 and 5.74 ng m3, with higher values observed close to busy roads (Schnelle-Kreis et al., 2001). The values reported in the present study are higher than the average one (1.05 ng m3) observed in a coastal region in northern Spain, Gipuzkoa, (Villar-Vidal et al., 2014). Benzo[b]fluoranthene (BbF) was the most abundant PAH, followed by chrysene (Chr) and benzo [k]fluoranthene (BkF) (Table 2). Similar trends were reported in Munich, whereas a different concentration profile was reported in Gipuzkoa. These results were also in agreement with those

Fig. 1. Scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX) analysis of collected PM2.5-0.3. Numbers in brackets indicate the relative mass percentage of elements (elements with atomic number below 9 are not quantified in the SEM-EDX analysis).

134


Table 2 Polycyclic aromatic hydrocarbons (PAHs) and n-alkanes concentrations (in mg/g and pg/m3) in PM2.5-0.3 and PM2.5 collected at Dunkerque in MarcheJuly 2011. PM2.5

PM2.5-0.3 (mg/g)

(pg/m3)

(mg/g)

(pg/m3)

Naphthalene (Nap) Acenaphthylene (Acy) Acenaphthene (Ace) Fluorene (Flu) Phenanthrene (Phe) Anthracene (Ant) Fluoranthene (Fla) Pyrene (Pyr) Benz[a]anthracene (BaA) Chrysene (Chr) Benzo[b]fluoranthene (BbF) Benzo[k]fluoranthene (BkF) Benzo[a]pyrene (BaP) Indeno[1,2,3-c,d]pyrene (InPy) Dibenz[a,h]anthracene (DahA) Benzo[ghi]perylene (BghiP)

0.5 e e 0.1 2.4 1.5 2.5 2.2 3.2 5.0 9.5 5.3 3.2 5.3 2.0 5.2

7 e e 2 33 21 35 31 46 71 134 74 44 74 28 73

e e e e 13.2 e 26.9 23.9 21.3 62.7 189 64.2 24.4 52.8 13.4 55.2

e e e e 186 e 378 336 330 882 2,670 904 343 743 189 777

Total PAHs

48.5

669

548

7,708

S n-alkanes

2,166

30,500

1,635

23,000

observed in Grande-Synthe, a town very close to our sampling site (Crenn et al., 2016). In this latter study, PAHs relative abundance and absolute concentrations were dependent on the season with values 5 to 10 fold higher in winter than in summer. The major source of BbF is diesel exhaust emission (Duval and Friedlander, 1981) supporting the impact of vehicular traffic emission on the PAHs composition in Dunkerque. It should also be mentioned that heavier PAHs were also detected, in particular the highly carcinogenic dibenzopyrene isomers (DbP). Their concentration could be estimated at 72 pg/m3 in PM2.5-0.3 and 2.3 ng/m3 in PM2.5, showing that such compounds are mainly associated with ultrafine particles. Very few data regarding DbP atmospheric concentrations have been reported in the literature. Compared to those measured at Stockholm, ranging from 14 to 80 pg/m3 (in total suspended particles and PM10) (Bergvall and Westerholm, 2007; Masala et al., 2016), values observed in Dunkerque are two orders of magnitude higher. These latter are nevertheless in the range of that observed in the megacity of Beijing, 0.5 to 4 ng/m3 (Layshock et al., 2010). Such unexpected results could be explained by the presence of several potential sources in Dunkerque, in addition to the wellknown diesel particles, such as coking plant, petrochemical, and heavy fuel refining. The identification of PAHs sources is made possible by using specific PAHs ratios, especially when the selected PAHs have similar properties regarding oxidation or photochemistry. Isomers possessing such identical properties and concentration ratios such as indeno[1,2,3-c,d]pyrene/benzo[ghi]perylene (InPy/BghiP) or fluoranthene/pyrene (Fla/Pyr) maintain the source characteristic value over time (Borgie et al., 2016; Guillon, 2011). Diagnostic PAHs ratios such as InPy/BghiP, BbF/BkF and benzo[a]pyrene/benzo[ghi]perylene (BaP/BghiP) were respectively close to 1, superior to 0.5 and between 0.5 and 0.6 (Table 3) confirming the contribution of traffic sources (Ravindra et al., 2008). Moreover, a coal combustion contribution was suspected from the Fla/(Fla þ Pyr) and Fla/Pyr ratio values, equal to 0.53 and 1.14, respectively. Such conclusions were consistent with the location of the sampling site in Dunkerque city center, the proximity of two highways and also the influence of emissions from a coke plant associated with an integrated steelworks plant. Low molecular weight PAHs (i.e. naphthalene, acenaphthylene, acenaphthene and fluorene) showed very low

concentrations or were below the detection limit. CombustionPAHs versus Total-PAHs (CPAH/TPAH) ratio was 0.87 and 0.96 for PM2.5-0.3 and PM2.5 respectively (Table 3). According to the literature, a typical CPAH/TPAH ratio reference value is 0.7 for an urban area and 0.96 for a highly industrialized area (Gogou et al., 1996; Yue and Fraser, 2004). Thus, the values obtained for the collected particles confirmed the influence of industrial emissions on PM composition. Linear alkanes, known as paraffins, were more prominent in PM2.5-0.3 (Fig. 2) but showed almost similar distributions and the highest concentrations of C25, C27, C29, C31 and C33 in both PM2.50.3 and PM2.5 samples. The Carbon Preference Index (CPI) is defined as the ratio between the total concentration of odd carbon number n-alkanes and the total concentration of even ones. The CPI gives information about the nature of the paraffin sources (petrogenic, natural or mixed) and the CPI value is typically 1 in urban environments and above 2 in rural areas due to the contribution of biogenic sources (Rissanen et al., 2006). Three Carbon Preference Index were calculated to discriminate between the biogenic and petrogenic influences (Chen et al., 2014): CPI19-35 (representative of all n-alkanes) ¼ S(C19C35) /S(C18C34); CPI19-25 (representative of petrogenic n-alkanes) ¼ S(C19C25) /S(C18C24); CPI27-35 (representative of biogenic n-alkanes) ¼ S(C27C35) /S(C26C34). The CPI19-35, CPI19-25, CPI27-35 values (Table 2) obtained for PM2.5-0.3 and PM2.5 evidenced that detected alkanes were mainly related to biogenic sources (CPI19-35>2 and CPI27-35>2). Such values could appear high when considering the urban characteristics of the sampling site. Nevertheless, PM samples were collected during the Spring/Summer period and high CPI values were normally observed during the warm season compared to the colder one. The season dependence has been clearly reported in a study conducted in Vienna, comparing a traffic-influenced and a suburban site: as a function of the site characteristics, CPI varies from 1.05 to 1.33 in March to 2.63 to 3.56 in July (Kotianov a et al., 2008). CPI values of 1.9 and 3.4 were also observed in the city of Milan in summer, for PM2.5 and PM10 respectively (Pietrogrande et al., 2010). The petrogenic contribution was evidenced by the CPI19-25 indicator slightly larger than 1, with a similar value in PM2.5-0.3 and PM2.5. The highest contribution of the odd terms C27, C29, C31 could be linked related to vegetal emissions which are maximal in the spring and summer season (Simoneit, 1984). The CPI27-35 value appeared higher in PM2.5 than in PM2.5-0.3 sample (3.23 versus 2.95) and suggested a more important contribution of biogenic sources in the PM2.5 including the ultrafine particles. In addition, biomass burning (e.g., wood combustion) could also be involved as such a source has been reported to lead to high CPI values (Wang et al., 2009). 3.4. Mutagenicity of PM Mutagenicity of native PM2.5-0.3 and of WF and OE of PM2.5-0.3 and PM2.5 was evaluated using the Ames test on TA102, TA98 and YG1041 Salmonella typhimurium tester strains, with or without metabolic activation. Native PM2.5-0.3 did not cause any significant variation of the Induction Factor (IF), in all tested conditions (Table 4). The same results were obtained for WF of PM2.5 and PM2.5-0.3 (data not shown). For OE, significant increases of IF were observed for TA98 and YG1041 tester strains, while no variation was detected for TA102 strain, at the tested doses. In particular, for the PM2.5-0.3 OE, significant increases of IF were only observed for the YG1041 tester strain without metabolic activation (-S9) at highest concentrations of exposure (20 and 50 mg PM-equivalent/plate). Addition of S9 annihilated this induction. Exposure to PM2.5 OE led to a significant increase (p < 0.05) of IF for YG1041 strain for all concentrations tested (dose-dependent) without S9 activation (Table 4). The


135

Table 3 Polycyclic aromatic hydrocarbons (PAHs) characteristics ratios and Carbon Preference Index (CPI) for PM2.5-0.3 and PM2.5 collected at Dunkerque in MarcheJuly 2011 e Comparison with source characteristic values found in the literature. Ratio

PM2.5-0.3

PM2.5

Potential source according to the literature

InPy/(InPy þ BghiP)

0,50

0,49

Fla/(Fla þ Pyr)

0,53

0,53

0.5 Coale >0.5 grass, wood, coal combustiond 0.73 Gasolinea >0.5 Diesela 0.6e1.7 Refineryc ~1 Diesela ~1 gasoline enginea ~1 coke ovenb 0.6 coking plantb

>>1 biomass burningk

(CPAH ¼ Combustion PAHs ¼ Fla, Pyr, BaA, Chr, BbF, BkF, BaP, BeP, InPy and BghiP; TPAH ¼ Total PAHs). a Ravindra et al., 2008; bCazier et al., 2016; cMasclet et al., 1986; dDe La Torre-Roche et al., 2009; eChen et al., 2014; fYang et al., 1998; gManoli et al., 2004; hGogou et al, 1996; i Yue and Fraser, 2004; jRissanen et al., 2006; kWang et al., 2009.

PM2.5-0.3 PM2.5

300

200 100

0

C12 C13 C14 C15 C16 C17 C18 C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C32 C33 C34 C35 C36 C37 C38 C39 C40

Concentration (μg/g)

400

n-alkanes Fig. 2. n-alkanes concentrations (mg/g) for the PM2.5-0.3 and PM2.5 collected in Dunkerque in MarcheJuly 2011.

addition of S9 modulated the IF values while keeping a significant difference for 20 and 50 mg PM-equivalent/plate. A significant increase (p < 0.05) of IF in TA98 tester strain was also observed, but only for the PM2.5 OE, for 20 and 50 mg PM-equivalent/plate in absence of S9 fraction and only for 50 mg PM-equivalent/plate with S9 metabolic activation. The obtained results showed higher values of induction factor without S9 metabolic activation compared to those with S9 metabolic activation, suggesting the importance of PAHs, especially nitro-, amino- and hydroxylamino-PAHs in the mutagenicity of PM2.5. Differences observed between mutagenicity of native PM and OE could probably be related to the higher bioavailability of PAHs after extraction. In fact, even if equivalent concentrations were used

in this study, it is not taken into account that PAHs are more bioavailable in an organic extract than in associated PM as is the case for realistic human exposure. As far as we know, only a few studies using the Ames test have been carried out with native PM. As a comparison, Lepers et al. also observed an increase of IF for TA98 and YG1041, after exposure to the same concentrations of PM sampled in Dunkerque (Lepers et al., 2014). The YG1041 strain possesses O-acetyltransferase and nitroreductase activity (Hagiwara et al., 1993), and our results may be explained by the role of nitro-, amino- and hydroxylamino et al., 2004; Traversi aromatics, as already reported (Pastorkova et al., 2015). An increase of nitro-PAHs concentration may result from chemical reactions between aromatic compounds and a high

136


Table 4 Induction factor (IF, number of revertants /number of spontaneous revertants) of native and Organic Extract (OE) of PM2.5-0.3 and OE of PM2.5 on TA102, TA98 and YG1041 tester strains, in absence or presence of metabolic activation by S9 fraction of Aroclor1245-induced rat hepatocytes. Concentrations are given in mg/plate for native PM and in mg PM-equivalent/plate for Organic Extract. Statistical analysis were performed using the one-tailed Dunnett test and ANOVA (*p < 0.05). Number of spontaneous revertants were determined exposing tester strains to 0.5% DMSO (control). PM2.5-0.3 Native

PM2.5

Organic Extract

Organic Extract

5 mg 20 mg 50 mg 5 mg 20 mg 50 mg 5 mg 20 mg 50 mg TA102

-S9 þS9 TA98 -S9 þS9 YG1041 -S9 þS9

1.1 1.0 1.0 0.9 0.9 1.0

1.0 1.1 0.8 0.9 1.2 0.9

1.1 1.1 1.2 0.7 1.3 1.0

1.2 1.0 1.3 0.8 1.2 1.0

1.1 1.0 1.2 1.0 2.1* 1.2

1.1 1.0 1.3 1.0 2.5* 1.3

1.0 1.0 1.2 1.2 4.5* 1.3

0.9 1.0 2.1* 1.9 12.9* 2.4*

1.0 1.0 4.8* 3.8* 23.8* 8.3*

level of NOx in the atmosphere (Alves et al., 2016). The bioavailability of nitro-PAHs in Ames strains has been previously reported to be higher than the parent PAHs (Goldring et al., 1987). PAHs

require metabolic activation to exhibit their mutagenicity (Mortelmans and Zeiger, 2000). For TA98 strain, the slight decrease of IF after S9 metabolic activation suggested that PAHs were not the only PM components responsible for the IF increase and were not the most mutagenic compounds in PM samples as also reported by Claxton et al. (2004). A decrease of IF after S9 metabolic activation was also observed for particles collected in Italy (Gilli et al., 2007), in Spain (Bayona et al., 1994), in India (Dubey et al., 2015) and in Brazil (Umbuzeiro et al., 2008) supporting the importance of other compounds than PAHs. Sensitivity of TA98 to frameshift mutagens that do not require metabolic activation is known and the observed responses suggest the presence of mutagens and promutagens in the studied PM. TA102 strain was used to investigate oxidative stress. This latter could be related to Reactive Oxygen Species (ROS) generation at the particle surface or in the presence of transition metals in PM. The absence of significant variation of IF for this strain suggests that PM components were not able to promote oxidative stress damages at et al., 2011; Woodruff et al., the studied concentrations (Andre 2012). Finally when comparing the effects of PM2.5-0.3 and PM2.5 OEs, significant differences were observed and highest IF values were

120%

A. Viability (%)

100% 80%

* *

60%

* *

* *

40%

20%

* *

24 h

0% 0

B.

10

20

30

40

50

60

70

80

120%

Viability (%)

100% 80% 60% 40%

* * *

20%

* * *

48 h

0% 0

C.

* * *

* *

10

20

30

40

50

60

70

80

120%

Viability (%)

100% 80%

*

*

60%

* * *

* *

* * *

*

40%

* *

20%

0

10

20

30

40

* * *

**

72 h

0%

**

50

60

70

80

PM equivalent concentra on (μg PM-eq/cm²) WF PM2.5

OE PM2.5

WF PM2.5-0.3

OE PM2.5-0.3

PM2.5-0.3

Fig. 3. Viability deduced from extracellular LDH release for BEAS-2B cells exposed to increasing concentrations of PM2.5-0.3 (native, organic extract (OE) and water soluble fraction (WF)) and PM2.5 OE and WF during 24 h (A), 48 h (B) and 72 h (C). Values are reported as mean ± Standard Deviation. Statistical analysis was performed using Mann-Whitney U test versus control (*p < 0.05). Negative controls: for native PM and WF tests: unexposed cells; for OE test: cells in 0.1% DMSO (no significant difference compared to unexposed cells).


obtained for the PM2.5 OEs showing that mutagenicity was more induced by PM including the ultrafine fraction. This related to higher concentrations of PAHs in PM2.5 compared to PM2.5-0.3 (Table 1). Moreover, several studies have also pointed out that PAHs, oxy- and nitro-PAHs were mainly associated with ultrafine particles (Di Filippo et al., 2010, 2015). Since TA98 and YG1041 are known to have a specific response to such organic compounds, it could be suggested that the obtained results were related to differences in PAHs concentrations between the two PM fractions. 3.5. Effects of PM on cells viability and proliferation Cell viability was evaluated by measurement of LDH release in cell-free supernatants after 24, 48 and 72 h in BEAS-2B cells exposed to native PM2.5-0.3, OE of PM2.5-0.3 and PM2.5, WF of PM2.50.3 and PM2.5 (Fig. 3). Mitochondrial succinate dehydrogenase (MDH) activity was measured under the same conditions in order to evaluate cell proliferation and metabolic activity (Fig. 4). Cell exposure to native PM2.5-0.3 led to an increase of LDH release in a time- and dose-dependent manner. In parallel, a dosedependent inhibition of cell growth was also observed. Previous studies highlighted a similar response of BEAS-2B cells after exposure to PM2.5-0.3 collected in the Dunkerque area in 2008

(Dergham et al., 2015). Winter PM2.5 collected in Italy were also able to reduce cell viability in BEAS-2B cultures (Gualtieri et al., 2010) or in A549 cells (Perrone et al., 2013), and to inhibit BEAS2B cell proliferation (Gualtieri et al., 2011). Jalava et al. reported a dose dependent cytotoxicity of fine and ultrafine PM collected in Finland, Spain, Greece and in the Netherlands (Jalava et al., 2007), showing that PM with similar size, but collected in different locations can have the same effects on RAW 264.7 cells. Finally, PM2.5 collected in Lebanon (Borgie et al., 2015), in Senegal (Dieme et al., 2012), and in Benin (Cachon et al., 2014) showed similar cytotoxic responses in BEAS-2B cells. Concerning OE cytotoxicity, compared to native PM2.5-0.3, a greater decrease of cell viability was observed after 24 h of exposure and the response was time and dose dependent. Cytotoxicity was 90% after 72 h of exposure at the highest concentration tested. A more pronounced effect was observed with OE of PM2.5 which is in line with the higher PAHs content evidenced by PM chemical characterization. Indeed, organic compounds linked to PM, such as PAHs, were often associated with cytotoxic and genotoxic responses (Høgsberg et al., 2013; Ramos de Rainho et al., 2013). Furthermore, the cytotoxicity of PM with high PAHs content has been reported for samples collected in Italy (Perrone et al., 2010), in Asia (Oh et al., 2011) and in Lebanon (Borgie et al., 2015).

1.50

MDH Activity vs Control

A.

137

1.00

** ** *

0.50

** ** *

24 h

0.00 0

10

20

30

40

50

60

70

80

1.50


B.

** ** *

1.00

** * *

0.50

** **

48 h

0.00 0

10

20

**

** *

10

20

30

40

50

60

70

80

1.50


C.

** **

1.00

**

0.50

** ** *

** **

* * ***

72 h

0.00 0

30

40

50

60

70

80

PM equivalent concentra on (μg PM-eq/cm²) WF PM2.5

OE PM2.5

WF PM2.5-0.3

OE PM2.5-0.3

PM2.5-0.3

Fig. 4. MDH activity deduced from WST-1 assay in BEAS-2B cells exposed to increasing concentrations of PM2.5-0.3 (native, organic extract (OE) and water soluble fraction (WF)) and PM2.5 OE and WF during 24 h (A), 48 h (B) and 72 h (C). Values are reported as mean ± Standard Deviation. Statistical analysis was performed using Mann-Whitney U test versus control (*p < 0.05). Negative controls: for native PM and WF tests: unexposed cells; for OE test: cells in 0.1% DMSO (no significant difference compared to unexposed cells).

138


Table 5 Effective Concentration (in mg/cm2) generating 50% (EC50) of cytotoxicity or inhibition of proliferation, calculated by concentration-response curve fitting using the Hill equation model, and their 95% confidence interval (CI95) in BEAS-2B cells exposed to an increasing concentration of PM2.5-0.3 (native, organic extract (OE) and water soluble fraction (WF)) and PM2.5 OE and WF during 24, 48 and 72 h. EC50 (mg/cm2) [CI95%] 24 h

48 h Proliferation

Cytotoxicity

Proliferation

Cytotoxicity

Proliferation

>80

>80

>80

>80 >80

68.4 [66.8; 27.9 [25.2; 21.3 [19.8; 67.4 [65.2; 62.8 [60.1;

18.3 [16.8; 36.5 [35.2; 12.4 [10.8; 34.9 [32.1; 35.4 [33.7;

Water-soluble fraction PM2.5-0.3

77.4 [74.8; 79.4] 43.6 [41.5; 45.7] >80

>80

70.4 [67.2; 73.6] 61.1 [58.4; 63.8] 30.2 [27.1; 33.3] >80

Water-soluble fraction PM2.5

>80

>80

>80

PM2.5-0.3 Organic extract PM2.5-0.3 Organic extract PM2.5

72 h

Cytotoxicity

>80 >80

For cells exposed to the water-soluble fraction of PM2.5-0.3 and PM2.5 (Fig. 3), no significant LDH release was observed, except for 72 h of exposure at the highest concentration. Nevertheless, effects of PM2.5 WF on cell viability have been previously reported (Zou et al., 2016). Aqueous fractions of PM2.5 collected in Baton-Rouge and Port Allen (USA) decreased cell viability and proliferation of A549 in a dose-dependent manner (Bourgeois and Owens, 2014). WF of PM2.5 collected in Asia also exhibited cytotoxicity and inhibition of proliferation in A549 pulmonary cells (Deng et al., 2014; Huang et al., 2015). Using a concentration-response curve fitting, cytotoxic EC50 [95%CI] were calculated (Table. 5). Native PM2.5-0.3 and WFs showed cytotoxic effects at high concentrations of exposure with values from 62.8 mg PM-equivalent/cm2. OEs of PM2.5-0.3 and PM2.5 exhibit maximum EC50 values after 72 h of exposure equal to 27.9 and 21.3 mg PM-equivalent/cm2, respectively. A time-dependent response was also observed with the two OEs tested in this study. These results confirm the leading effect of organic compounds in the cytotoxicity of PM as the most pronounced effects were obtained with PM2.5 OE in which PAHs concentrations were 12 times higher than in PM2.5-0.3. WF enriched in metals and ions showed a minor cytotoxic effect at the doses and exposure times tested in this work. Differences in EC50 between native PM and OE could be attributed to the higher bioavailability of organic compounds in the extract. Furthermore, several studies have pointed out the highest mutagenic, cytotoxic and /or genotoxic properties of organic extract of PM, which contain most of the PAHs derivatives (Oh et al., 2011; Topinka et al., 2013). Cell proliferation assay showed significant decreases in MDH activity in BEAS-2B cells (Fig. 4), indicating that PM2.5-0.3 were able to cause an alteration of mitochondrial metabolism in addition to the loss of membrane integrity. As evaluated for cytotoxicity, EC50 [95%CI] were processed for proliferation assay (Table 5). OEs of PM2.5-0.3 and PM2.5 exhibit maximum EC50 values after 72 h of exposure equal to 36.5 and 12.4 mg PM-equivalent/cm2, respectively. Interestingly, WFs showed an inhibitory effect on cell proliferation at a lower dose than for cytotoxicity. This observation provides evidence that disruption of cellular homeostasis may occur at doses below the cytotoxic ones. These results are also consistent with the work of Bourgeois and Owen which observed a decrease of MDH activity of pulmonary cells exposed to the aqueous fraction of PM (Bourgeois and Owens, 2014). Native PM2.50.3 exposure also caused decreases of MDH activity. The EC50 value, 18.3 mg/cm2, which appeared lower than those of OE and WF taken separately, could highlight the synergic effects of the whole atmospheric PM content (organic compounds, metals, ions, …). It

>80 >80

70.0] 30.6] 22.8] 69.6] 65.5]

19.8] 37.8] 14.0] 37.7] 37.1]

also evidenced the importance of considering PM in their entirety for evaluation of toxicity. Moreover, a strong correlation between cytotoxicity and cell proliferation parameters was found, especially for native PM and OEs, with significant Spearman's rho factors ranging from 0.6 to 0.8, revealing a global effect of PM2.5-0.3 exposure on cellular homeostasis. Cytotoxicity and cell proliferation results were consistent with the conclusion reached for mutagenicity and physicochemical characterization. Indeed, PM linked organic compounds, such as PAHs, were often associated with cytotoxic and genotoxic responses (Høgsberg et al., 2013; Ramos de Rainho et al., 2013) and the water-soluble fraction of PM2.5 was often responsible for effects on cell proliferation (Alessandria et al., 2014). 4. Conclusion Physicochemical characterization revealed that PM2.5 is a complex mixture with inorganic and organic compounds deriving from natural as well as anthropogenic related sources. Contributions of several sources such as soil resuspension, marine emissions including sea-salt and shipping, road traffic, industrial activities mainly related to steelmaking or petrochemistry have been evidenced in the PM2.5 composition. Our results pointed out the mutagenic and cytotoxic properties of PM, with differences between PM2.5-0.3 and PM2.5, including the ultrafine fraction, and between native PM, organic extracts and water soluble fractions, that could be related to variation in composition, especially in organic compounds. Indeed, PAH levels were found to be 12-times higher in PM including the ultrafine fraction. This study showed how important it is to study ultrafine fraction whose mutagenic and cytotoxic effects appears more pronounced. Moreover, specific response of the YG1041 strains suggest a possible contribution of other mutagenic compounds including PAH derivatives such as nitro-, amino-, hydroxyamino- and oxy-PAHs which were not monitored in the present study. The preliminary toxicity results evaluated in this study also requires further investigation focusing on possible genotoxicity and epigenetic effects in BEAS-2B exposed to fine PM. Acknowledgements This work was supported by the Institut National Du Cancer (INCa; Convention no. 2010-368) the Hauts-de-France Region (Convention no. 14003399) and the French Agency of the Environment and Energy (ADEME; Convention no. 1494c0082-83-84).


References , T., Degan, R., Traversi, D., Gilli, G., 2014. Cytotoxic response Alessandria, L., Schiliro in human lung epithelial cells and ion characteristics of urban-air particles from Torino, a Northern Italian City. Environ. Sci. Pollut. Res. Int. 21, 5554e5564. Alves, D.K.M., Kummrow, F., Cardoso, A.A., Morales, D.A., Umbuzeiro, G.A., 2016. Mutagenicity profile of atmospheric particulate matter in a small urban center subjected to airborne emission from vehicle traffic and sugar cane burning. Environ. Mol. Mutagen 57, 41e50. , V., Billet, S., Pottier, D., Le Goff, J., Pottier, I., Garçon, G., Shirali, P., Sichel, F., Andre 2011. Mutagenicity and genotoxicity of PM2.5 issued from an urbanoindustrialized area of Dunkerque (France). J. Appl. Toxicol. JAT 31, 131e138. ndez, P., Solanas, A.M., Albaige s, J., 1994. Sources and Bayona, J.M., Casellas, M., Ferna seasonal variability of mutagenic agents in the Barcelona City aerosol. Chemosphere 29, 441e450. Bergvall, C., Westerholm, R., 2007. Identification and determination of highly carcinogenic dibenzopyrene isomers in air particulate samples from a street canyon, a rooftop, and a subway station in Stockholm. Environ. Sci. Technol. 41, 731e737. Billet, S., Garçon, G., Dagher, Z., Verdin, A., Ledoux, F., Cazier, 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, 212e223. Borgie, M., Dagher, Z., Ledoux, F., Verdin, A., Cazier, F., Martin, P., Hachimi, A., Shirali, P., Greige-Gerges, H., Courcot, D., 2015. 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. Environ. Pollut. 205, 250e260. Borgie, M., Ledoux, F., Dagher, Z., Verdin, A., Cazier, F., Courcot, L., Shirali, P., GreigeGerges, H., Courcot, D., 2016. Chemical characteristics of PM2.5-0.3 and PM0.3 and consequence of a dust storm episode at an urban site in Lebanon. Atmos. Res. 180, 274e286. Bourgeois, B., Owens, J.W., 2014. The influence of Hurricanes Katrina and Rita on the inflammatory cytokine response and protein expression in A549 cells exposed to PM2.5 collected in the Baton Rouge-Port Allen industrial corridor of Southeastern Louisiana in 2005. Toxicol. Mech. Methods 24, 220e242. Cachon, B.F., Firmin, S., Verdin, A., Ayi-Fanou, L., Billet, S., Cazier, F., Martin, P.J., Aissi, F., Courcot, D., Sanni, A., et al., 2014. Proinflammatory effects and oxidative stress within human bronchial epithelial cells exposed to atmospheric particulate matter (PM(2.5) and PM(>2.5)) collected from Cotonou. Benin. Environ. Pollut. Barking Essex 1987 (185), 340e351. Carey, I.M., Atkinson, R.W., Kent, A.J., van Staa, T., Cook, D.G., Anderson, H.R., 2013. Mortality associations with long-term exposure to outdoor air pollution in a national English cohort. Am. J. Respir. Crit. Care Med. 187, 1226e1233. Cazier, F., Genevray, P., Dewaele, D., Nouali, H., Verdin, A., Ledoux, F., Hachimi, A., Billet, S., Bouhsina, S., Shirali, P., et al., 2016. Characterization and seasonal variations of particles in the atmosphere of rural, urban and industrial areas: organic compounds. J. Environ. Sci. 44, 45e56. Chen, Y., Cao, J., Zhao, J., Xu, H., Arimoto, R., Wang, G., Han, Y., Shen, Z., Li, G., 2014. nAlkanes and polycyclic aromatic hydrocarbons in total suspended particulates from the southeastern Tibetan Plateau: concentrations, seasonal variations, and sources. Sci. Total Environ. 470e471, 9e18. Claxton, L.D., Woodall, G.M., 2007. A review of the mutagenicity and rodent carcinogenicity of ambient air. Mutat. Res. 636, 36e94. Claxton, L.D., Matthews, P.P., Warren, S.H., 2004. The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity. Mutat. Res. 567, 347e399. Courcot, D., Laversin, H., Ledoux, F., Cazier, F., Matta, J., Cousin, R., Aboukaïs, A., 2009. Composition and textural properties of soot and study of their oxidative elimination by catalytic process. Int. J. Environ. Pollut. 39, 253e263. Crenn, V., Fronval, I., Petitprez, D., Riffault, V., 2016. Fine particles sampled at an urban background site and an industrialized coastal site in Northern France d Part 1: seasonal variations and chemical characterization. Sci. Total Environ. http://dx.doi.org/10.1016/j.scitotenv.2015.11.165. n, J., Szalo ki, I., Eyckmans, K., Worobiec, A., Ro, C.U., Van Grieken, R., De Hoog, J., Osa 2005. Thin-window electron probe X-ray microanalysis of individual atmospheric particles above the North Sea. Atmos. Environ. 39, 3231e3242. , J.J., 2006. Toxicological de Kok, T.M.C.M., Driece, H.A.L., Hogervorst, J.G.F., Briede assessment of ambient and traffic-related particulate matter: a review of recent studies. Mutat. Res. 613, 103e122. De La Torre-Roche, R.J., Lee, W.-Y., Campos-Díaz, S.I., 2009. Soil-borne polycyclic aromatic hydrocarbons in El Paso, Texas: analysis of a potential problem in the United States/Mexico border region. J. Hazard. Mater. 163, 946e958. DeMarini, D.M., 2013. Genotoxicity biomarkers associated with exposure to traffic and near-road atmospheres: a review. Mutagenesis 28, 485e505. Deng, X., Zhang, F., Wang, L., Rui, W., Long, F., Zhao, Y., Chen, D., Ding, W., 2014. Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells. Apoptosis Int. J. Program. Cell Death 19, 1099e1112. Dergham, M., Lepers, C., Verdin, A., Cazier, F., Billet, S., Courcot, D., Shirali, P., Garçon, G., 2015. Temporalespatial variations of the physicochemical characteristics of air pollution Particulate Matter (PM2.5e0.3) and toxicological effects in human bronchial epithelial cells (BEAS-2B). Environ. Res. 137, 256e267. Di Filippo, P., Riccardi, C., Pomata, D., Buiarelli, F., 2010. Concentrations of PAHs, and nitro- and methyl- derivatives associated with a size-segregated urban aerosol. Atmos. Environ. 44, 2742e2749.

139

Di Filippo, P., Pomata, D., Riccardi, C., Buiarelli, F., Gallo, V., 2015. Oxygenated polycyclic aromatic hydrocarbons in size-segregated urban aerosol. J. Aerosol Sci. 87, 126e134. Dieme, D., Cabral-Ndior, M., Garçon, G., Verdin, A., Billet, S., Cazier, F., Courcot, D., Diouf, A., Shirali, P., 2012. Relationship between physicochemical characterization and toxicity of fine particulate matter (PM2.5) collected in Dakar city (Senegal). Environ. Res. 113, 1e13. Ding, X., Wang, M., Chu, H., Chu, M., Na, T., Wen, Y., Wu, D., Han, B., Bai, Z., Chen, W., et al., 2014. Global gene expression profiling of human bronchial epithelial cells exposed to airborne fine particulate matter collected from Wuhan, China. Toxicol. Lett. 228, 25e33. Dubey, J., Kumari, K.M., Lakhani, A., 2015. Chemical characteristics and mutagenic activity of PM₂.₅ at a site in the Indo-Gangetic plain, India. Ecotoxicol. Environ. Saf. 114, 75e83. Duval, M.M., Friedlander, S.K., 1981. Source Resolution of Polycyclic Aromatic Hydrocarbons in the Los Angeles Atmosphere Application of a CMB with Firstorder Decay (Washington, DC, USA). , T., Bono, R., La Rosa, A., Traversi, D., 2007. The mutaGilli, G., Pignata, C., Schiliro genic hazards of environmental PM2.5 in Turin. Environ. Res. 103, 168e175. Gogou, A., Stratigakis, N., Kanakidou, M., Stephanou, E.G., 1996. Organic aerosols in Eastern Mediterranean: components source reconciliation by using molecular markers and atmospheric back trajectories. Org. Geochem 25, 79e96. Goldring, J.M., Ball, L.M., Sangaiah, R., Gold, A., 1987. Mutagenic activity of nitrosubstituted cyclopenta-fused polycyclic aromatic hydrocarbons towards Salmonella typhimurium. Mutat. Res. 187, 67e77. Gualtieri, M., Øvrevik, J., Holme, J.A., Perrone, M.G., Bolzacchini, E., Schwarze, P.E., Camatini, M., 2010. Differences in cytotoxicity versus pro-inflammatory potency of different PM fractions in human epithelial lung cells. Toxicol. Vitro 24, 29e39. Gualtieri, M., Ovrevik, J., Mollerup, S., Asare, N., Longhin, E., Dahlman, H.J., Camatini, M., Holme, J.A., 2011. Airborne urban particles (Milan winter-PM2.5) cause mitotic arrest and cell death: effects on DNA, mitochondria, AhR binding and spindle organization. Mutat. Res. 713, 18e31. culaire (d13C) comme Guillon, A., 2011. Etude de la composition isotopique mole traceur de source qualitatif et quantitatif des hydrocarbures aromatiques polre (PhD thesis). University of ycycliques (HAP) particulaires dans l’atmosphe Bordeaux, p. 374. Hagiwara, Y., Watanabe, M., Oda, Y., Sofuni, T., Nohmi, T., 1993. Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity. Mutat. Res. 291, 171e180. Harrison, R.M., Yin, J., 2000. Particulate matter in the atmosphere: which particle properties are important for its effects on health? Sci. Total Environ. 249, 85e101. Hleis, D., Fern andez-Olmo, I., Ledoux, F., Kfoury, A., Courcot, L., Desmonts, T., Courcot, D., 2013. Chemical profile identification of fugitive and confined particle emissions from an integrated iron and steelmaking plant. J. Hazard Mater 250e251, 246e255. Høgsberg, T., Jacobsen, N.R., Clausen, P.A., Serup, J., 2013. Black tattoo inks induce reactive oxygen species production correlating with aggregation of pigment nanoparticles and product brand but not with the polycyclic aromatic hydrocarbon content. Exp. Dermatol 22, 464e469. Huang, M., Kang, Y., Wang, W., Chan, C.Y., Wang, X., Wong, M.H., 2015. Potential cytotoxicity of water-soluble fraction of dust and particulate matters and relation to metal(loid)s based on three human cell lines. Chemosphere 135, 61e66. Jalava, P.I., Salonen, R.O., Pennanen, A.S., Sillanp€ a€ a, M., H€ alinen, A.I., Happo, M.S., Hillamo, R., Brunekreef, B., Katsouyanni, K., Sunyer, J., et al., 2007. Heterogeneities in inflammatory and cytotoxic responses of RAW 264.7 macrophage cell line to urban air coarse, fine, and ultrafine particles from six European sampling campaigns. Inhal. Toxicol. 19, 213e225. Katanoda, K., Sobue, T., Satoh, H., Tajima, K., Suzuki, T., Nakatsuka, H., Takezaki, T., Nakayama, T., Nitta, H., Tanabe, K., et al., 2011. An association between longterm exposure to ambient air pollution and mortality from lung cancer and respiratory diseases in Japan. J. Epidemiol. Jpn. Epidemiol. Assoc. 21, 132e143. Kawanaka, Y., Tsuchiya, Y., Yun, S.-J., Sakamoto, K., 2009. Size distributions of polycyclic aromatic hydrocarbons in the atmosphere and estimation of the contribution of ultrafine particles to their lung deposition. Environ. Sci. Technol. 43, 6851e6856. Kfoury, A., Ledoux, F., Roche, C., Delmaire, G., Roussel, G., Courcot, D., 2016. PM2.5 source apportionment in a French urban coastal site under steelworks emission influences using Constrained Non-Negative Matrix Factorization receptor model. J. Environ. Sci. 40, 114e128. Khinkis, L.A., Levasseur, L., Faessel, H., Greco, W.R., 2003. Optimal design for estimating parameters of the 4-parameter hill model. Nonlinearity Biol. Toxicol. Med. 1, 363e377. , P., Puxbaum, H., Bauer, H., Caseiro, A., Marr, I.L., Cík, Kotianova G., 2008. Temporal patterns of n-alkanes at traffic exposed and suburban sites in Vienna. Atmos. Environ. 42, 2993e3005. Krzyzanowski, M., 2008. WHO air quality guidelines for europe. J. Toxicol. Environ. Health A 71, 47e50. Layshock, J., Simonich, S.M., Anderson, K.A., 2010. Effect of dibenzopyrene measurement on assessing air quality in Beijing air and possible implications for human health. J. Environ. Monit. 12, 2290e2298. Ledoux, F., Laversin, H., Courcot, D., Courcot, L., Zhilinskaya, E.A., Puskaric, E.,

140


Aboukaïs, A., 2006. Characterization of iron and manganese species in atmospheric aerosols from anthropogenic sources. Atmos. Res. 82, 622e632. Lepers, C., Dergham, M., Armand, L., Billet, S., Verdin, A., Andre, V., Pottier, D., Courcot, D., Shirali, P., Sichel, F., 2014. Mutagenicity and clastogenicity of native airborne particulate matter samples collected under industrial, urban or rural influence. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 28, 866e874. Li, J., Li, W.X., Bai, C., Song, Y., 2015. Particulate matter-induced epigenetic changes and lung cancer. Clin. Respir. J. Longhin, E., Capasso, L., Battaglia, C., Proverbio, M.C., Cosentino, C., Cifola, I., Mangano, E., Camatini, M., Gualtieri, M., 2016. Integrative transcriptomic and protein analysis of human bronchial BEAS-2B exposed to seasonal urban particulate matter. Environ. Pollut. Barking Essex 1987 (209), 87e98. Loomis, D., Grosse, Y., Lauby-Secretan, B., El Ghissassi, F., Bouvard, V., BenbrahimTallaa, L., Guha, N., Baan, R., Mattock, H., Straif, K., et al., 2013. The carcinogenicity of outdoor air pollution. Lancet Oncol. 14, 1262e1263. Machemer, S., 2004. Characterization of airborne and bulk particulate from iron and steel manufacturing facilities. Environ. Sci. Technol. 38, 381e389. Manoli, E., Kouras, A., Samara, C., 2004. Profile analysis of ambient and source emitted particle-bound polycyclic aromatic hydrocarbons from three sites in northern Greece. Chemosphere 56, 867e878. Masala, S., Lim, H., Bergvall, C., Johansson, C., Westerholm, R., 2016. Determination of semi-volatile and particle-associated polycyclic aromatic hydrocarbons in Stockholm air with emphasis on the highly carcinogenic dibenzopyrene isomers. Atmos. Environ. 140, 370e380. Masclet, P., Mouvier, G., Nikolaou, K., 1986. Relative decay index and sources of polycyclic aromatic hydrocarbons. Atmos. Environ. (1967) 20, 439e446. Mazzei, F., D'Alessandro, A., Lucarelli, F., Nava, S., Prati, P., Valli, G., Vecchi, R., 2008. Characterization of particulate matter sources in an urban environment. Sci. Total Env. 401, 81e89. Mortelmans, K., Zeiger, E., 2000. The Ames Salmonella/microsome mutagenicity assay. Mutat. Res. 455, 29e60. Oh, S.M., Kim, H.R., Park, Y.J., Lee, S.Y., Chung, K.H., 2011. Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells). Mutat. Res. Toxicol. Environ. Mutagen 723, 142e151. , A., Cerna , M., Smíd, J., Vrbíkov Pastorkova a, V., 2004. Mutagenicity of airborne particulate matter PM10. Cent. Eur. J. Public Health 12 (Suppl), S72eS75. Perrone, M.G., Gualtieri, M., Ferrero, L., Lo Porto, C., Udisti, R., Bolzacchini, E., Camatini, M., 2010. Seasonal variations in chemical composition and in vitro biological effects of fine PM from Milan. Chemosphere 78, 1368e1377. Perrone, M.G., Gualtieri, M., Consonni, V., Ferrero, L., Sangiorgi, G., Longhin, E., Ballabio, D., Bolzacchini, E., Camatini, M., 2013. Particle size, chemical composition, seasons of the year and urban, rural or remote site origins as determinants of biological effects of particulate matter on pulmonary cells. Environ. Pollut. 176, 215e227. Pietrogrande, M.C., Mercuriali, M., Perrone, M.G., Ferrero, L., Sangiorgi, G., Bolzacchini, E., 2010. Distribution of n-alkanes in the northern Italy aerosols: data handling of GC-MS signals for Homologous Series characterization. Environ. Sci. Technol. 44, 4232e4240. Putaud, J.P., van Dingenen, R., Alastuey, A., Bauer, H., Birmili, W., Cyrys, J., Flentje, H., Fuzzi, S., Gehrig, R., Hansson, H., et al., 2010. A European aerosol phenomenology - 3: physical and chemical characteristics of particulate matter from 60 rural, urban and kerbside sites across Europe. Atmos. Environ. 44, 1308e1320. â, S., Luiz Mazzei, J., Alessandra Fortes Aiub, C., Ramos de Rainho, C., Machado Corre Felzenszwalb, I., 2013. Genotoxicity of polycyclic aromatic hydrocarbons and nitro-derived in respirable airborne particulate matter collected from urban areas of Rio de Janeiro (Brazil). Biomed. Res. Int. 2013, 765352. Ravindra, K., Sokhi, R., Van Grieken, R., 2008. Atmospheric polycyclic aromatic hydrocarbons: source attribution, emission factors and regulation. Atmos. Environ. 42, 2895e2921.

€tyla €inen, T., Kallio, M., Kronholm, J., Kulmala, M., Riekkola, M.-L., Rissanen, T., Hyo 2006. Characterization of organic compounds in aerosol particles from a coniferous forest by GCeMS. Chemosphere 64, 1185e1195. Schnelle-Kreis, J., Gebefügi, I., Welzl, G., Jaensch, T., Kettrup, A., 2001. Occurrence of particle-associated polycyclic aromatic compounds in ambient air of the city of Munich. Atmos. Environ. 35 (Suppl. 1), S71eS81. Simoneit, B.R.T., 1984. Organic matter of the tropospheredIII. Characterization and sources of petroleum and pyrogenic residues in aerosols over the western united states. Atmos. Environ. 1967 (18), 51e67. , A., Holoubek, I., Skarek, M., Janosek, J., Cupr, P., Kohoutek, J., Novotn a-Rychetska 2007. Evaluation of genotoxic and non-genotoxic effects of organic air pollution using in vitro bioassays. Environ. Int. 33, 859e866. Sørensen, M., Autrup, H., Møller, P., Hertel, O., Jensen, S.S., Vinzents, P., Knudsen, L.E., Loft, S., 2003. Linking exposure to environmental pollutants with biological effects. Mutat. Research/Reviews Mutat. Res. 544, 255e271. Thorpe, A., Harrison, R.M., 2008. Sources and properties of non-exhaust particulate matter from road traffic: a review. Sci. Total Environ. 400, 270e282. Topinka, J., Milcova, A., Schmuczerova, J., Krouzek, J., Hovorka, J., 2013. Ultrafine particles are not major carriers of carcinogenic PAHs and their genotoxicity in size-segregated aerosols. Mutat. Res. Toxicol. Environ. Mutagen 754, 1e6. Traversi, D., Festa, E., Pignata, C., Gilli, G., 2015. Aero-dispersed mutagenicity attributed to particulate and semi volatile phase in an urban environment. Chemosphere 124, 163e169. Umbuzeiro, G.A., Franco, A., Martins, M.H., Kummrow, F., Carvalho, L., Schmeiser, H.H., Leykauf, J., Stiborova, M., Claxton, L.D., 2008. Mutagenicity and DNA adduct formation of PAH, nitro-PAH, and oxy-PAH fractions of atmospheric ~o Paulo, Brazil. Mutat. Res. 652, 72e80. particulate matter from Sa Uppstad, H., Øvrebø, S., Haugen, A., Mollerup, S., 2010. Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines. Toxicol. Lett. 192, 221e228. Vaccari, M., Mascolo, M.G., Rotondo, F., Morandi, E., Quercioli, D., Perdichizzi, S., Zanzi, C., Serra, S., Poluzzi, V., Angelini, P., et al., 2015. Identification of pathwaybased toxicity in the BALB/c 3T3 cell model. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 29, 1240e1253. Vallius, M., Janssen, N.A.H., Heinrich, J., Hoek, G., Ruuskanen, J., Cyrys, J., Van Grieken, R., de Hartog, J.J., Kreyling, W.G., Pekkanen, J., 2005. Sources and elemental composition of ambient PM2.5 in three European cities. Sci. Total Environ. 337, 147e162. pez de Dicastillo, M.D., Alvarez, J.I., Santa Villar-Vidal, M., Lertxundi, A., Martinez Lo Marina, L., Ayerdi, M., Basterrechea, M., Ibarluzea, J., 2014. Air polycyclic aromatic hydrocarbons (PAHs) associated with PM2.5 in a North cantabric coast urban environment. Chemosphere 99, 233e238. Wang, G., Kawamura, K., Xie, M., Hu, S., Cao, J., An, Z., Waston, J.G., Chow, J.C., 2009. Organic molecular compositions and size distributions of chinese summer and Autumn aerosols from nanjing: characteristic Haze event caused by Wheat straw burning. Environ. Sci. Technol. 43, 6493e6499. WHO, 2014. Ambient (Outdoor) Air Quality and Health. Woodruff, R.S., Li, Y., Yan, J., Bishop, M., Jones, M.Y., Watanabe, F., Biris, A.S., Rice, P., Zhou, T., Chen, T., 2012. Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay. J. Appl. Toxicol. JAT 32, 934e943. Wu, C., Larson, T.V., Wu, S., Williamson, J., Westberg, H.H., Liu, S.L.-J., 2007. Source apportionment of PM2.5 and selected hazardous air pollutants in Seattle. Sci. Total Environ. 386, 42e52. Yang, H.-H., Lee, W.-J., Chen, S.-J., Lai, S.-O., 1998. PAH emission from various industrial stacks. J. Hazard. Mater. 60, 159e174. Yue, Z., Fraser, M., 2004. Characterization of nonpolar organic fine particulate matter in Houston. Tex. Aerosol Sci. Technol. 38, 60e67. Zou, Y., Jin, C., Su, Y., Li, J., Zhu, B., 2016. Water soluble and insoluble components of urban PM2.5 and their cytotoxic effects on epithelial cells (A549) in vitro. Environ. Pollut. 212, 627e635.