Role of Paris PM2.5 components in the pro-inflammatory response induced in airway epithelial cells

Toxicology 261 (2009) 126–135

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Role of Paris PM2.5 components in the pro-inflammatory response induced in airway epithelial cells Augustin Baulig a , Seema Singh b,1 , Alexandre Marchand c,d , Roel Schins b , Robert Barouki c,d , Michèle Garlatti c,d , Francelyne Marano a , Armelle Baeza-Squiban a,∗ a

Univ Paris Diderot-Paris 7, Unit of Functional and Adaptive Biology (BFA) EAC CNRS 7059, Laboratory of Molecular and Cellular Responses to Xenobiotics, 5 rue Thomas Mann, case 7073, 75205 Paris Cedex 13, France Particle Research, Institut für Umweltmedizinische Forschung GmbH (IUF), Aufˇım Hennekamp 50, 40225 Düsseldorf, Germany c INSERM UMR-S 747, 45 rue des Saints-Pères, 75 270 Paris Cedex 06, France d Université Paris Descartes, Centre Universitaire des Saints-Pères, 45 rue des Saints pères, 75270 Paris Cedex 06, France b

a r t i c l e

i n f o

Article history: Received 31 March 2009 Received in revised form 7 May 2009 Accepted 8 May 2009 Available online 19 May 2009 Keywords: PM2.5 Polyaromatic hydrocarbons Metals Diesel exhaust particles Pro-inflammatory cytokines Reactive oxygen species

a b s t r a c t Particulate matter (PM) is suspected to play a role in environmentally-induced pathologies. Due to its complex composition, the contribution of each PM components to PM-induced biological effects remains unclear. Four samples of Paris PM2.5 having different polyaromatic hydrocarbons and metals contents were compared with each other and with their respective aqueous and organic extracts used alone or in combination. The four PM2.5 samples similarly induced granulocyte macrophage-colony stimulating factor (GM-CSF) release, a pro-inflammatory cytokine, by human bronchial epithelial cells. It results from the activation of upstream signalling pathways and the modulation of the cellular redox state that is different according to PM2.5 samples. The PM-aqueous extracts contained soluble metals involved in hydroxyl radical production in abiotic conditions. However they slightly contributed to the intracellular reactive oxygen species production and GM-CSF release by comparison with organic extracts. Organic compounds transactivated the xenobiotic responsive element (XRE) and antioxidant responsive element (ARE), leading to increased cytochrome P450 1A1 expression and NADPH-quinone oxydoreductase-1 expression respectively but to different extend according to PM samples underlying differences in their bioavailability. Our study underlines that chemical composition of particles per se is insufficient to predict cellular effects and that the interaction and the bioavailability of the various components were critical. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Air quality has become a major public health issue, particularly in large cities. Particulate matters have been suspected to play a role in environmentally-induced pathologies. Several epidemiological studies have highlighted the association between PM2.5 (particulate matter sampled with an impactor having 50% efficiency for 2.5 ␮m aerodynamical diameter particles) pollution and an increase of cardiopulmonary morbidity, mortality and of lung cancer (Dominici et al., 2006; Pope et al., 2002).

Abbreviations: PM2.5 , particulate matter with an aerodynamic diameter <2.5 ␮m; DEP, diesel exhaust particles; CB, carbon black particles; 16-HBE, 16-HBE14o- cell line; GM-CSF, granulocyte macrophage-colony stimulating factor; PAH, polycyclic aromatic hydrocarbons; EPR, electron paramagnetic resonance. ∗ Corresponding author. Tel.: +33 1 57 27 83 35; fax: +33 1 57 27 83 29. E-mail address: [email protected] (A. Baeza-Squiban). 1 Current address: TERI, Delhi, India. 0300-483X/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2009.05.007

One of the most critical short-term effects of PM is to trigger airway and systemic inflammation which could account for the cardiovascular disorders induced by these pollutants. Exposure of human volunteers to concentrated ambient particles (CAPs) (Ghio et al., 2000) or PM2.5 (Schaumann et al., 2004) induces an increase in inflammatory cells in both the bronchial and alveolar compartments. In vitro, epithelial cells and macrophages (particles target cells) exposed to PM, developed a pro-inflammatory response characterized by the release of inflammatory cytokines (Baulig et al., 2003a; Hetland et al., 2004; Jalava et al., 2008). However, particles are composed of different constituents and the contribution of each of these components to PM-induced biological effects remains unclear. Addressing this question is a real challenge because (i) particulate matter is a very complex mixture containing various components such as carbonaceous core, polyaromatic hydrocarbons (PAH), quinones, metals, endotoxins and many other components (Harrison and Yin, 2000) and (ii) because it should improve the regulation of emissions and potential sources of the most toxic components and it could possibly

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lead to a more adapted therapeutic approach in compromised persons. The complex composition of PM results initially from its origin which varies according to the location and is further modified by physico-chemical transformations in the atmosphere. Such variations in PM composition according to the sampling site and season have been confirmed in one of our previous works studying PM2.5 seasonally sampled in two Paris sites (Baulig et al., 2004). Other studies using specific particles either rich in PAH (diesel exhaust particles (DEP)) or metals (residual oil fly ash) have highlighted the role of these components in the pro-inflammatory response (Bonvallot et al., 2001; Nel et al., 2001; Schaumann et al., 2004). Endotoxins can also be present on particles and induce inflammation (Huang et al., 2002; Schins et al., 2004). In this context, it appears necessary to perform thorough experiments with realistic particles to which we are exposed in our cities. It is now well known that oxidative stress plays an important role in the pro-inflammatory response induced by particles (Nel et al., 2001). Oxidative stress could arise as a result of Fenton-type reactions catalyzed by transition metal present in particles which typically include Fe, V, Cr, Co, Ni, Cu, Zn and Ti (Shi et al., 2003). It could also result from a direct generation on particle surface, especially for ultrafine particles. Finally, reactive oxygen species (ROS) could be produced by reactions involving organic compounds. Some evidence has incriminated quinones through radical generation due to redox cycling (Squadrito et al., 2001) or PAH through its metabolization because the activity of the monooxygenase cytochrome P450 1A1 (CYP1A1) is known to produce ROS (Perret and Pompon, 1998) and this enzyme was shown to be induced in DEP-treated epithelial cells (Baulig et al., 2003b). The aim of the present work was to compare four samples of PM2.5 with each other and with their respective aqueous and organic extracts used alone or in combination in order to: (1) determine the contribution of each fraction in the different steps implicated in the pro-inflammatory response and (2) evaluate whether the biological effects induced by the different particle samples could be explained by both their chemical characteristics and the bioavailability of these chemicals. For this purpose we used PM2.5 seasonally sampled in two Paris sites (urban background and kerbside) that have been previously characterized for their metals and PAH contents and their ability to induce a concentration and time dependent pro-inflammatory response in human bronchial epithelial cells (Baulig et al., 2004). Abiotic and biotic ROS production, activation of signalling pathways and transcription factors, as well as cytokine release by human bronchial epithelial cells were investigated following treatment with PM2.5 or its organic and aqueous components. The expression of CYP1A1 and NADPH-quinone oxydoreductase (NQO-1), two xenobiotic metabolizing enzymes, was studied to appreciate chemical bioavailability. 2. Materials and methods 2.1. Particles collection and preparation Urban atmospheric particulate matter sampled with an impactor having 50% efficiency for 2.5 ␮m aerodynamical diameter particles (PM2.5 ), was collected at ground-level with a high volume sampler machine (DA-80, Megatec, Paris, France), equipped with a PM2.5 selective-inlet head, in two locations of Paris: (1) an urban background station at Vitry-sur-Seine, a suburbs of Paris and (2) a kerbside station at Porte d’Auteuil in border to a great highway which is a ring road of Paris. In the two stations, particles were sampled in summer and winter (2002–2003). The machine operated at the flow rate of 30 m3 /h and particles were recovered on 150 mm diameter nitrocellulose filters (HAWP, Millipore, Molsheim, France). Particles were detached from filters as already described (Baulig et al., 2004). The distribution of PM diameter in the collected aerosols has already been shown elsewhere (Baulig et al., 2004). PM2.5 were compared with diesel exhaust particles (DEP): standard reference materiel 1650a purchased from National Institute for Standard Technology (NIST, Interchim, Montluc¸on, France) and carbon black particles purchased from

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Degussa (Paris, France). All these particles were prepared at 2 mg/mL in dipalmitoyl phosphatidyl lecithin (DPL, Sigma–Aldrich, Saint Quentin Fallavier, France), a surfactant component which allows to keep particles in suspension, and were used at 10 ␮g/cm2 (respectively 50 ␮g/mL). PM2.5 samples were extracted by dichloromethane with a ASE 200® accelerated solvent extraction system (Dionex, Aix en Provence, France). The obtained extract was dried and then redissolved in dimethyl sulfoxide (DMSO). Organic extracts were used at a concentration equivalent to that found on native particles. This value was calculated according to the proportion of the soluble organic fraction (SOF) in the total dry weight of PM2.5 determined for each particle samples. Winter and summer background PM2.5 contained 12% of SOF whereas winter and summer kerbside PM2.5 contained 9% and 11% of SOF respectively. PM2.5 samples were also washed with ultrapure water in order to collect the water-soluble components. After two centrifugations at 12,000 × g, the supernatants were filtered on 0.2 ␮m filter to eliminate all the particles. Cells were exposed to a volume of aqueous extract equivalent to the volume of particle suspension used. Soluble metals present in PM-aqueous extracts were depleted by filtering on chelating ion exchange resin columns according with the manufacturer procedure (Chelex 100, Bio-Rad, Marnes la Coquette, France). PAH and metal contents are known and previously published elsewhere (Baulig et al., 2004). Negative controls were made by using DPL for particles and aqueous extracts, and DMSO for organic extracts. 2.2. Chemicals and reagents All chemicals were purchased from Sigma–Aldrich (Saint Quentin Fallavier, France) except when otherwise specified. For culture experiments, several antioxidants were used. N-acetylcysteine (NAC) and mannitol were dissolved in water and used at 10 mM. The antioxidant enzyme catalase (from bovine liver, activity: 3400 U/mg prot), a scavenger of H2 O2 was tested at 1400 U/mL medium. NAC, mannitol and catalase were added to cell cultures 20 min before toxic treatment. Four signalling pathways inhibitors were also used: an inhibitor of the epidermal growth factor (EGFR) tyrosine kinase (AG1478, Tebu Bio, Le Perray en Yvelines, France) at 1 ␮M, a selective mitogen-activated protein (MAP) kinase/extracellular regulated kinase (Erk) inhibitor (PD98059, Tebu Bio) at 10 ␮M, a p38 MAP kinase inhibitor (SB203580, Calbiochem, VWR, Fontenay sous bois, France) at 1 ␮M and a Janus kinase inhibitor (SP600125, Calbiochem) at 10 ␮M. Recombinant endotoxins neutralizing protein (rENP) is an 12.2 kD protein purified from the amebocytes of the horseshoe crab, Limulus polyphemus (Cape Cod Associates, Cape Cod, MA, USA). This protein neutralizes the bioactivity of LPS when used in a 1:1 ratio (weight) of ENP/LPS. RENP was diluted in water and was used at 2 ␮g/mL. 2.3. Measurement of OH• generation using EPR Generation of hydroxyl radicals by particles suspensions was studied in the presence of hydrogen peroxide (for initiating the Fenton reaction) and the spin trap 5,5-dimethyl-1-pyroline-N-oxide (DMPO) according to Shi et al. (2003). 100 ␮l of each particles suspension at 1000 ␮g/mL was sonicated (Sonorex water bath) and then mixed with 100 ␮l H2 O2 (0.5 M in phosphate buffer saline (PBS, Invitrogen, Cergy-Pontoise, France)) and 200 ␮l DMPO (0.05 M in PBS). The mixture was incubated in the dark and shaken continuously at 37 ◦ C for 15 min in presence or absence of the metal chelator desferioxamin at a final concentration of 100 ␮M or the hydroxyl-radical scavenger DMSO at 5%, v/v. Following incubation, the suspensions were filtered through a 0.1 ␮m pore filter (Acrodisc 25 mm syringe filter, Pall Gelman Laboratory, Ann Arbor, USA), and transferred immediately to a 100 ␮l glass capillary and measured with a miniscope MS100 EPR spectrometer (Magnettech, Berlin, Germany). The EPR-spectra were recorded at room temperature using the following instrumental conditions: microwave frequency: 9.39 GHz, magnetic field: 3360 G, sweep width: 100 G, scan time: 30 s, number of scans: 3, modulation amplitude: 1.8 G, receiver gain: 1000. Quantitation was carried out on first derivation of EPR signal of DMPO-OH quartet as the sum of total amplitudes, and outcomes are expressed as the total amplitude in arbitrary units. 2.4. Cell culture conditions Dr. Gruenert (Cozens et al., 1994) (San Francisco, CA, USA) kindly provided the human bronchial epithelial cell subclone 16HBE14o-. The cell line was cultured in DMEM/F12 culture medium supplemented with penicillin (100 U/mL), streptomycin (100 ␮g/mL), glutamine (1%), fungizone (0.125 ␮g/mL, Invitrogen, Cergy-Pontoise, France) and UltroserG (UG) (2%, Invitrogen). Cells were cultured on collagen (type I, 4 ␮g/cm2 ) coated 25 or 75 cm2 flasks, 6- or 96-well plates (Costar, Cambridge, MA, USA) at 20,000 cells/cm2 . At the time of treatment, UG was not added to DMEM/F12. Human nasal turbinates were obtained from patients undergoing turbinectomy and were cultured as previously described (Million et al., 2001). Cells were cultured on 6well plates at 500,000 cells/well for 6 days. All cultures were incubated in humidified 95% air with 5% CO2 at 37 ◦ C.

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2.5. Analysis of intracellular peroxide levels Intracellular peroxide levels were assessed using H2 DCF-DA (Molecular Probes, Eugene, OR), an oxidation-sensitive fluorescent probe as previously described (Baulig et al., 2003b). Briefly, cells were loaded for 20 min with 20 ␮M H2 DCF-DA in Hank’s balanced salt solution (HBSS) and further incubated with PM2.5 for 4 h. The fluorescence analysis was performed with an EPICS-Elite-ESP flow cytometer (Coultronics, Margency, France) equipped with a 15 mW air cooled argon-ion laser tuned at 488 nm. 2.6. Cytokine assay Subconfluent cultures were exposed for 24 h to particles. The concentration of granulocyte macrophage-colony stimulating factor (GM-CSF) released into the culture supernatants was measured with human GM-CSF Duoset ELISA development system respectively (R&D Systems Europe, Abingdom, United Kingdom). 2.7. Electrophoretic mobility shift assay (EMSA) After treatment, nuclear extracts were isolated from subconfluent 16-HBE cells (3 × 106 ) cultured in 25 cm2 flasks according to Baulig et al. (2003b). They were complexed with different radiolabeled double-stranded oligonucleotides: (1) simple-strand human consensus antioxidant responsive element (ARE), synthetized by Invitrogen and annealed as already described (Baulig et al., 2003b) and (2) double-strand human consensus NF-␬B (Invitrogen) and human consensus AP-1 (Invitrogen) oligonucleotides. Shifted complexes were electrophoresed on 5% polyacrylamide gel. 2.8. Real time PCR Triplicate PCR reactions were performed in a 384-well microplate. Total reaction (10 ␮l) each containing 0.02 ␮g cDNA, 300 nM “sens” and “antisens” primers and 5 ␮l PCR Master Mix (containing the taq polymerase enzyme and the SYBR green, Applied Biosystem, Foster City, CA, USA). NQO-1 sens 5 -GAA GGC AGT GCT TTC CAT CA-3 ; antisens 5 -GAA TAC GGT CGA TTC CCT CT-3 . CYP1A1 sens 5 -ATC AGG CTG TCT GTG ATG TC-3 ; antisens 5 -AAG GAT GAG CCA GCA GTA TG-3 . PCR and fluorescence analysis were performed using the thermocycle AB prism (Applied Biosystem). Amplification conditions included at 50 ◦ C for 2 min, at 95 ◦ C for 10 min then 35 cycles at 95 ◦ C 15 s and at 60 ◦ C for 1 min (for probe/primer hybridization and DNA synthesis). The CT values of samples were reported to the CT values of the RPL 13 control gene. 2.9. Evaluation of CYP1A1 activity by EROD assay This assay was performed on subconfluent primary nasal cells as 16-HBE cells lacked functional CYP1A1 activity. After washing with phosphate buffer saline (PBS) (Invitrogen), cells were incubated with DMEM/F12 containing 5 ␮M ethoxyresorufine and 2 mM salicylamide. Ethoxyresorufine is metabolized by CYP1A1 in resorufin, a fluorescent compound. Kinetic fluorescence measurements were made with a microspectrofluorimeter (Fluostar galaxy, BMG, Champigny-sur-Marne, France) with an excitation wavelength of 530 nm and an emission wavelenght of 590 nm for 40 min. 2.10. Transient transfection and luciferase activity measurement The plasmid pCt-131-3-XRE, pCt-131-2-ARE and pCt-131-3-AP1 were a generous gift from Professor Barouki. For transfection experiments, we used the cationic lipid reagent lipofectamine 2000 (Invitrogen). 16-HBE cells were cultured on 12well plate and have been transfected when the growth attempts 90–95% confluent. For each transfection sample, DNA–lipofectamine 2000 complexes were prepared according to the manufacturer procedure (1.6 ␮g DNA and 4 ␮l lipofectamine 2000 in OPTI-MEM). Cells were incubated for 24 h with the complex at 37 ◦ C in a CO2 incubator. After washing, the toxic treatment was added for 4 h. Cells were washed another time and incubated for 24 h just with DMEM/F12 medium. 2.11. Statistical analysis For in vitro experiments, all data were expressed as the mean ± standard error of the mean (SEM) of 3 cultures from a representative experiment. Means were compared by analysis of variance. The equal variance test is significant with alpha = 0.05 (p < 0.001). All pairwise multiple comparisons were made with the Student–Newman–Keuls method (t-test, p < 0.05).

3. Results 3.1. Production of reactive oxygen species by PM The ability of PM2.5 to generate hydroxyl radicals in abiotic conditions was measured by EPR in presence of DMPO, a spin trap, and

Fig. 1. EPR signal induced by PM2.5 . DMPO-OH generation by winter urban kerbside PM2.5 . Hydroxyl radical formation was measured indirectly by the DMPO-OH adduct generation in presence of H2 O2 . (A) Dose dependent (␮g/mL) formation of DMPOOH• adduct in presence of PM2.5 . (B) Comparison of the EPR signal induced by PM2.5 (1000 ␮g/mL), the corresponding PM-aqueous extract prepared from a 1000 ␮g/mL PM2.5 suspension, PM2.5 incubated with metal chelator desferioxamin (100 ␮M) and PM2.5 incubated with the hydroxyl-radical scavenger DMSO (5%, v/v).

H2 O2 in order to initiate the Fenton reaction. PM2.5 induce a dose dependent significant OH• production as illustrated in Fig. 1A with particles sampled in the kerbside site during the winter season. The same dose-dependent effect was obtained with winter urban background particles (data not shown). In addition, the aqueous extract induces OH• production similarly to PM2.5 (Fig. 1B). When PM2.5 were preincubated with the metal chelator desferioxamin or the radical scavenger DMSO, no EPR signal was detected (Fig. 1B). The ability of PM2.5 to induce ROS production in human bronchial epithelial cells was measured using a specific fluorescent probe H2 DCF-DA. All PM2.5 at 10 ␮g/cm2 induced a significant increase of DCF fluorescence after 4 h of exposure that is significantly higher with PM2.5 sampled in winter as compared to PM2.5 sampled in summer. The effect was also significantly higher with particles from the urban background than with the kerbside particles at the same season (Fig. 2A). Organic extracts exhibit different responses when compared to their respective PM2.5 . For the winter samples the effects of PM2.5 was similar to their corresponding organic extracts. In contrast, for the summer urban background particles the organic fraction caused significantly lower ROS generation than particles, whereas for the summer kerbside particles the organic extract was significantly stronger than particles (Fig. 2A). The aqueous extracts of all PM2.5 samples showed a significantly similar increase in DCF fluorescence relative to control, but this effect was only modest as compared to that induced by their corresponding particles (Fig. 2A). In addition, the effect was abolished after aqueous extract was filtered through an ion exchange resin in order to remove metals (see Fig. 2B for winter urban background particles (WUB)). The combination of PM-aqueous and organic extracts tended to increase the DCF fluorescence level induced by the organic extract alone (Fig. 2C). In order to characterize the mechanisms of ROS production by PM2.5 , different antioxidants, i.e. the ROS scavenger and GSH pro-

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Fig. 2. DCF fluorescence induced by PM2.5 or their extracts in 16-HBE cells. Cells were loaded with 20 ␮M H2 DCFH-DA for 20 min and washed with HBSS. Then the cells were incubated with 10 ␮g/cm2 of PM2.5 or their respective organic or aqueous extract for 4 h. After treatment, cells were detached and the DCF fluorescence was measured on 10,000 viable cells by flow cytometry. (A) Comparison of PM2.5 with their respective organic and aqueous extracts. (B) Effect of metal depletion on the DCF fluorescence induced by the aqueous extract of winter urban background (WUB) PM2.5 . (C) Effect of the combination of metal and organic extracts from WUB PM2.5 on the DCF fluorescence. (D) Effects of antioxidants on WUB PM2.5 -induced DCF fluorescence. NAC (10 mM), CAT (1400 U/mL) and MAN (10 mM) were added to cell culture 20 min before PM2.5 . Values are means ± S.D. (n = 3): (a) different from respective control (DPL for particles and aqueous extract, DMSO for organic extract); (b) different from other PM2.5 ; (c) different from respective PM2.5 ; (d) different from aqueous extract (p < 0.05).

duction activator NAC, the extracellular H2 O2 scavenger catalase and the extracellular • OH scavenger mannitol were used. NAC and catalase were both found to reduce the DCF fluorescence induced by winter urban background particles, whereas mannitol had no effect (Fig. 2D). 3.2. PM2.5 components involved in GM-CSF release GM-CSF was chosen as a biomarker of the pro-inflammatory response as this cytokine is found to be a major regulator of both macrophages and neutrophils activation and survival in the lungs (Vlahos et al., 2006) and is involved in the maturation of dendritic cells (Ritz et al., 2002). Its increased release has been observed in the bronchoalveolar lavage of rodents exposed to DEP (Takano et al., 1997) as well as in the culture medium of bronchial epithelial cells exposed in vitro to PM (Reibman et al., 2002; Ramgolam et al., 2008). All PM2.5 at 10 ␮g/cm2 induced a similar increase in GM-CSF after 24 h treatment that is significantly lower than the release induced by DEP. In contrast, CB particles were ineffective (Fig. 3A). Aqueous extracts of particles did not cause a significant GM-CSF production except for the winter urban background sample, which induced a slight GM-CSF release (Fig. 3A). By comparison, organic extracts from all samples induced a clear release of GM-CSF, but this always tended to be lower than the GM-CSF release induced by their corresponding PM2.5 (Fig. 3A). The combination of PM-aqueous and organic extracts did not significantly modify the GM-CSF release induced by the organic extract alone (Fig. 3B). In contrast, metal removal from aqueous extracts using chelex column reduced the GM-CSF release (Fig. 3B). The role of endotoxins in this pro-inflammatory response was investigated using a recombinant endotoxin neutralizing protein

(rENP). In presence of 2 ␮g/mL rENP, the PM2.5 induced GM-CSF release was only reduced (approx. 30%) for summer kerbside particles (Table 1). 3.3. Signalling pathways involved in GM-CSF release The implication of different transduction pathways in the effects of PM2.5 was assessed using specific inhibitors of various protein kinases. In the presence of the JNK signalling pathway inhibitor SP600125 (10 ␮M) and the EGFR tyrosine kinase inhibitor AG1478 (1 ␮M), the GM-CSF release induced by the four different PM2.5 samples was found to be decreased (Table 2). In the presence of the ERK1/2 pathway inhibitor PD98059 (10 ␮M), the GM-CSF release was reduced whereas the p38 inhibitor SB203358 (1 ␮M) failed to modify the GM-CSF release (Table 2). The involvement of ROS in PM2.5 induced GM-CSF release was determined by treating 16-HBE cells with PM2.5 in the presence or absence of antioxidants. NAC and catalase were found to reduce the GM-CSF release as induced by 10 ␮g/cm2 winter urban background particles whereas mannitol was without effect (Fig. 3C). 3.4. Activation of transcription factors by particles As TPA response element (TRE) and NF-␬B response element (NRE) are redox sensitive gene regulatory sequences localized in the promoter of genes encoding inflammatory mediators, the effects of particle treatment of 16-HBE cells on transcription factor binding to these sequences were studied by electrophoretic mobility shift assay (EMSA) (Fig. I supplementary material). Whereas after DEP treatment, an increase of the protein/DNA complex was only detected for NRE, in CB treated cells, only increased binding to TRE

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Fig. 3. Release of GM-CSF. (A) 16-HBE cells were treated with 10 ␮g/cm2 of PM2.5 or their respective aqueous or organic extracts for 24 h. (B) Effect on GM-CSF release of (i) WUB PM2.5 organic and aqueous extracts combination and (ii) of metal removal from the aqueous extract of WUB PM2.5 . (C) Effects of antioxidants on WUB PM2.5 -induced GM-CSF release. NAC (10 mM), CAT (1400 U/mL) and MAN (10 mM) were added to cell culture 20 min before PM2.5 . Values are means ± S.D. (n = 3): (a) different from control (DPL for particles and aqueous extract, DMSO for organic extract); (b) different from native PM2.5 (p < 0.05).

Table 1 Effect of recombinant endotoxins neutralizing protein on GM-CSF releasea . Control

Without rENP With rENP

Urban background PM2.5

5.83 ± 0.31 5.30 ± 0.30

Kerbside PM2.5

Winter

Summer

Winter

Summer

8.52 ± 0.93 7.69 ± 0.23

9.92 ± 1.24 9.70 ± 0.43

9.77 ± 0.38 9.39 ± 0.63

8.94 ± 0.62 6.17 ± 0.93b

a GM-CSF release (ng/mL) was measured after 24 h of treatment by 10 ␮g/cm2 of each PM2.5 samples in presence or not of 2 ␮g/mL recombinant endotoxins neutralizing protein (rENP). b Statistically significant in relation to treatment without rENP.

was detected. In cells that were treated with winter urban background particles, both complexes were increased.

regulatory sequence which controls the expression of the CYP1A1 gene, (ii) the expression of CYP1A1 mRNA and (iii) the CYP1A1 enzymatic activity. The XRE transactivation activity was studied by transfection of a plasmid containing 3 XRE and the luciferase reporter gene. All PM2.5 samples induced an increased luciferase activity (around 5 fold increase). This increase was more important than that obtained with DEP and also tended to be stronger with winter particles

3.5. Effects of particles on the phase I metabolization enzyme gene CYP1A1 The study of CYP1A1 expression was performed by measurement of (i) the activity of xenobiotic response element (XRE), a gene Table 2 Signalling pathways involved in PM2.5 -induced GM-CSF releasea . Control

Urban background PM2.5 Winter

Without inhibitors With i JNK With i p38 With i ERK1/2 With i EGFR

SP600125 SB203358 PD98059 AG1478

5.76 4.17 6.43 5.20 4.51

± ± ± ± ±

0.43 0.31b 0.94 1.51 1.21

12.61 3.67 12.76 9.24 7.57

± ± ± ± ±

Kerbside PM2.5 Summer

0.52 0.92b 3.34 1.31b 0.21b

14.69 5.00 14.20 9.09 7.08

± ± ± ± ±

Winter 2.32 1.01b 0.58 2.24b 1.43b

14.47 6.40 13.52 9.51 6.32

± ± ± ± ±

Summer 0.18 0.54b 2.20 0.91b 0.89b

13.23 5.87 13.11 7.65 7.35

± ± ± ± ±

0.13 0.94b 0.73 1.68b 0.81b

GM-CSF release (ng/mL) was measured after 24 h of treatment by 10 ␮g/cm2 of each PM2.5 samples in presence or not of different signalling protein inhibitors. Statistically significant reduction of PM-induced GM-CSF release in presence of signalling pathways inhibitors by comparison to PM-induced GM-CSF release in absence of inhibitors. a

b

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Fig. 4. CYP1A1 expression. (A) XRE activity. 16-HBE cells were transfected with a pct131-3XRE-Luc construction and treated with PM2.5 or DEP at 10 ␮g/cm2 and with the aqueous and organic extracts of WUB PM2.5 . After 24 h, the luciferase activity was specifically detected. Values are means ± S.D. (n = 3): (a) different from control (p < 0.05). (B) CYP1A1 mRNA expression. 16-HBE cells were treated with PM2.5 or DEP at 10 ␮g/cm2 for 4 h. Total RNA were extracted and mRNA CYP1A1 levels were quantified by real time RT-PCR. Results are expressed as ratio relatively to control. (C) CYP1A1 enzymatic activity. Primary nasal cells were treated with the different PM2.5 samples at 10 ␮g/cm2 , their respective aqueous and organic extracts for 24 h. Resorufin fluorescence over time (0–40 min) was quantified with a microplate reader.

than with their corresponding summer samples (Fig. 4A). Considering selective extracts of winter urban background particles, it was found that the aqueous extract was without effect whereas the organic extract induced an increased luciferase activity which was similar to particles (Fig. 4A). The expression of CYP1A1 mRNA measured by real time PCR appeared to be well correlated to XRE transactivation. Each PM2.5 sample induced an increased CYP 1A1 mRNA expression which

again appeared to be higher with the winter samples particularly from urban background (Fig. 4B). The same pattern was also seen with regard to CYP1A1 enzymatic activity (Fig. 4C). The aqueous extracts were ineffective, whereas all organic extracts induced a notable effect. However according to PM2.5 sample, the effect of the organic extract was either similar to the corresponding particle sample or higher (Fig. 4C). Coexposure of cells to aqueous and organic extracts

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Fig. 5. NQO-1 expression. (A) EMSA. ARE oligonucleotides and nuclear proteins binding in 16-HBE cells exposed to DEP, CB, WUB PM2.5 at 10 ␮g/cm2 and its aqueous extract prepared with an equivalent dose of PM2.5 . (B) ARE activity. 16-HBE cells were transfected with a pct131-2ARE-Luc construction and treated with PM2.5 , DEP or CB at 10 ␮g/cm2 and with the aqueous and organic extracts of WUB PM2.5 . After 24 h, the luciferase activity was specifically detected. Values are means ± S.D. (n = 3): (a) different from control (p < 0.05). (C) NQO-1 mRNA expression. 16-HBE cells were treated with PM2.5 or DEP at 10 ␮g/cm2 for 4 h. Total RNA were extracted and mRNA NQO-1 levels were quantified by real time RT-PCR. Results are expressed as ratio relatively to control.

induced CYP1A1 enzymatic activity similarly to the organic extract alone (data not shown). 3.6. Effects of particles on the phase II metabolization enzyme gene NQO-1 The NQO-1 expression study was performed by analysis of (1) the activity of the antioxidant response element (ARE), a redox sensitive gene regulatory sequence which controls the expression of the NQO-1 gene; (2) the mRNA expression of NQO-1. Increased binding of proteins to ARE oligonucleotides was observed with nuclear extracts from DEP as well as from winter urban background PM2.5 -treated cells (Fig. 5A). This binding was

similar for the PM2.5 samples in their native form or their aqueous extract. By contrast, CB has no effect (Fig. 5A). In order to verify whether this complex induces gene expression, a plasmid containing the ARE and the reporter gene luciferase was transfected into 16-HBE cells before particle treatment. All PM2.5 samples induced an increase of luciferase activity but this increase was lower than the one induced by DEP. CB induced a slight effect, which was lower than that seen with the PM2.5 (Fig. 5B). Aqueous and organic extracts of winter urban background particles both induce a similar and significant increase of luciferase activity but lower than with native particles (Fig. 5B). The mRNA expression of NQO-1 was measured by real time PCR. All PM2.5 samples induced an increased expression compared

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to control, which tended to be stronger with the winter samples (Fig. 5C). 4. Discussion The aim of our study was to determine the mechanisms of the cellular effects of different PM2.5 fractions from the Parisian area. Our objectives were to compare the effects of the different samples with each other and to evaluate the contribution of the different components of these samples. Such studies were meant to assess whether chemical compositions of particles per se were sufficient to predict cellular effects or whether the interaction and the bioavailability of the various components were critical. We have shown that the pro-inflammatory response induced by urban PM2.5 in bronchial epithelial cells is mainly due to its organic component. In contrast, the aqueous fraction of PM2.5 , which was shown to induce free radical production from available transition metals, contributed only slightly to intracellular ROS production and cytokine release. This organic component has also been shown to play the main role in the induction of other cytokines (Gro␣ and IL1␣, Baulig et al., 2007) and growth factors of the EGF receptor ligand family (Rumelhard et al., 2007a,b). PAH from the different urban PM2.5 samples were found to exhibit differences in their bioavailability, and this might partially explain why these samples, which differ in their PAH concentrations (Baulig et al., 2004), induce similar cytokine release. More probably, the pro-inflammatory response results from complex interactions between PM components linked to their bioavailability. Several studies have provided evidence for the involvement of ROS in the signal transduction activation and cytokine release induced by different particles (Gonzalez-Flecha, 2004). These ROS were thought to originate from either their organic compounds (Baulig et al., 2003b), metals (Donaldson et al., 1997) or the carbonaceous core of the particles (Dick et al., 2003). In our samples, metals were found to be responsible for the • OH production as measured by EPR in abiotic conditions since the EPR signal disappears when PM2.5 are co-treated with the metal chelator desferioxamin and the antioxidant DMSO. The EPR signal obtained with particles appeared to be similar to the one obtained with their corresponding aqueous extracts suggesting that the water soluble metal fraction is predominantly involved in Fenton mediated • OH production. The pattern of ROS production induced by the four PM2.5 samples at the intracellular level is different from the pattern previously observed by EPR (Baulig et al., 2004). Several reasons can be given for these differences. Firstly, the EPR experimental conditions measure • OH in the presence of H2 O2 , in order to promote the Fenton reaction, whereas cellular conditions are far more complex e.g. due to the presence of intracellular antioxidants and metal binding proteins. Secondly, the H2 DCF-DA probe measures other ROS than • OH; these ROS could emanate directly from PM but also indirectly from the alteration in cellular functions triggered by PM exposure (Knaapen et al., 2004). Finally, while metals were found to be responsible for the EPR signal as well as for part of the intracellular DCF fluorescence elicited by treatment with the aqueous fraction, this fraction was found to be only marginally involved in the intracellular ROS production by comparison with the organic fraction. In line with previous observations (Baulig et al., 2004), there was no difference in the GM-CSF release induced by the four samples. In the current study, we showed that this PM2.5 cytokine release involves the activation of the same signalling pathways such as MAP kinases (Erk and JNK) and EGF receptor as already described with DEP on these cells (Blanchet et al., 2004; Bonvallot et al., 2001). The role of oxidative stress in the GM-CSF release was underlined by the effect of antioxidants. Mannitol, an extracellular • OH scavenger, failed to reduce the PM2.5 induced GM-CSF release and ROS pro-

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duction, suggesting that extracellular • OH are not involved in these biological effects. By contrast, catalase and NAC both reduced the GM-CSF release as well as ROS production induced by PM2.5 indicative of the involvement of both intracellular ROS and extracellular H2 O2 . Endotoxins have been shown to be associated to the insoluble fraction of PM (Soukup and Becker, 2001) and are considered to explain some of the pro-inflammatory properties of PM (Huang et al., 2002; Schins et al., 2004). rENP only reduces the GM-CSF release induced by PM2.5 sampled in the kerbside site in winter suggesting that it is the sole sample contaminated with endotoxins at concentrations that could contribute to the pro-inflammatory effects of PM in epithelial cells. The organic extracts induced a significant GM-CSF release which was similar for each sample. In contrast, the aqueous extracts only showed a slight contribution to GM-CSF release which could not be attributed to metals since their removal from the extracts did not change the GM-CSF release. In addition, the combination of the organic extract and aqueous fraction did not modify the GM-CSF release induced by the organic extract alone, suggesting that neither synergistic nor antagonistic effects occur. The involvement of the organic extracts in GM-CSF release by 16-HBE cells corroborates previous observations made with DEP (Bonvallot et al., 2001). An important question raised is why the different PM2.5 samples induced a similar GM-CSF release even though (1) they induce different intracellular ROS production and (2) they differ by their chemical composition, especially PAH (Baulig et al., 2004). To further address this issue, we investigated the expression of CYP1A1 and NQO-1. CYP1A1 is a phase I metabolizing enzyme specifically inducible by PAH but also capable to metabolize PAH. NQO-1 is a phase II metabolizing enzyme involved in the quinones detoxification and its expression is induced by ROS and electrophiles that activate the antioxidant responsive element ARE (Itoh et al., 1999). PAH can be desorbed from particles after their phagocytosis (Baulig et al., 2003b; Boland et al., 2000) and become bioavailable, since XRE was found to be activated and associated with an increased CYP1A1 mRNA expression and CYP1A1 enzymatic activity in cells treated with the four PM2.5 samples. In addition, expression of the phase II metabolizing enzyme NQO-1 was also found to be increased by particles in association with activation of ARE as demonstrated by the increased binding of nuclear proteins on the regulatory sequence and the increased promoter activity. However, the activation of NQO-1 cannot be attributed only to PAH as aqueous extracts both increase the complex detected by EMSA and the luciferase activity. An interesting observation in our study was that whatever the methodological approach used to study CYP1A1 and NQO-1 expression, the response profile was the same: (1) winter particles induced a higher effect than summer particles and (2) urban background particles induced a higher effect than kerbside particles. Notably however, these results did not correlate with the PAH analysis (Baulig et al., 2004), which would suggest that the bioavailability of organic compounds from particles could differ according to PM2.5 samples. Indeed, comparative analysis of the CYP1A1 activity induction by the organic fraction and their corresponding particles indicated that the kerbside particles did not show a strong bioavailability of CYP1A1-inducible PAH, whereas in contrast, those present on urban background particles appeared to be more bioavailable. Although winter kerbside particles have twice as much PAH as winter urban background particles, the organic extract of winter kerbside induced a similar induction of EROD activity. In addition, interaction between CYP1A1 and metals has been investigated as copper is known to inhibit the enzyme EROD of the CYP1A family (Ghosh et al., 2001). Moreover, copper is present in the aqueous fraction as we have previously shown a positive correlation between copper concentrations in the different PM2.5 samples and

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the EPR signal (Baulig et al., 2004). The coincubation of aqueous and organic extracts did not change the EROD activity, suggesting that the metals present in the aqueous extract did not inhibit CYP1A1 activity. Although our results show that the organic fraction mainly contribute to the biological response, we have to keep in mind that the organic extracts used to show this contribution do not reflect the cellular situation. With these organic extracts, cells are exposed to all organic compounds extractable by dichloromethane, whereas exposure of cells to PM2.5 leads to the release of the organic compounds that can be readily desorbed within the cellular milieu. In the latter case, it is more than likely that all organic compounds are not totally desorbed from PM2.5 . It underlines cautions towards experiments only performed with organic extracts. Similarly, for the aqueous extract, cellular conditions can be different since only metals that can dissolve in the aqueous epithelial lining fluid overlaying the cells will be effective. In conclusion, we have shown that in the airway epithelial cells, Paris PM2.5 modify the cellular redox state and induce a pro-inflammatory response characterized by GM-CSF release. This effect is similar to that of DEP. As suspected, both PM2.5 and DEP activate similar signalling pathways including ERK 1/2, JNK and EGFR. Using selective fractions of multiple samples of PM2.5 , we provide evidence that the organic fraction contains the most important contributors (by contrast to water-soluble metals and endotoxins) to the biological effects according to their bioavailability. Indeed, they transactivate the XRE and ARE, leading to increased CYP1A1 expression and NQO-1 expression respectively. Our study underlines that, with complex mixtures, it is very difficult to correlate their effect with the presence of a single component (Mauderly, 2004) and that chemical monitoring of PM is not sufficient to predict its biological reactivity. Acknowledgments We acknowledge Dr. Gruenert for human bronchial cell line, Professor P. Herman (Centre hospitalier universitaire Lariboisière, Paris, France), Professor J. Soudant (Service ORL, hospital PitiéSalpétrière, Paris, France) for providing turbinates and Tingming Shi for advice on EPR analysis. This work was supported by Renault, Ademe and Primequal grants. This work was also supported by Caisse d’Assurance Maladie des Professions Libérales de Provinces, Paris, France. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tox.2009.05.007. References Baulig, A., Sourdeval, M., Meyer, M., Marano, F., Baeza-Squiban, A., 2003b. Biological effects of atmospheric particles on human bronchial epithelial cells. Comparison with diesel exhaust particles. Toxicol. In Vitro 17, 567–573. Baulig, A., Garlatti, M., Bonvallot, V., Marchand, A., Barouki, R., Marano, F., BaezaSquiban, A., 2003a. Involvement of reactive oxygen species in the metabolic pathways triggered by diesel exhaust particles in human airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 285, L671–679. Baulig, A., Poirault, J.J., Ausset, P., Schins, R., Shi, T., Baralle, D., Dorlhene, P., Meyer, M., Lefevre, R., Baeza-Squiban, A., Marano, F., 2004. Physicochemical characteristics and biological activities of seasonal atmospheric particulate matter sampling in two locations of Paris. Environ. Sci. Technol. 38, 5985–5992. Baulig, A., Blanchet, S., Rumelhard, M., Lacroix, G., Marano, F., Baeza-Squiban, A., 2007. Fine urban atmospheric particulate matter modulates inflammatory gene and protein expression in human bronchial epithelial cells. Front. Biosci. 12, 771–782. Blanchet, S., Ramgolam, K., Baulig, A., Marano, F., Baeza-Squiban, A., 2004. Fine particulate matter induces amphiregulin secretion by bronchial epithelial cells. Am. J. Respir. Cell. Mol. Biol. 30, 421–427. Boland, S., Bonvallot, V., Fournier, T., Baeza-Squiban, A., Aubier, M., Marano, F., 2000. Mechanisms of GM-CSF increase by diesel exhaust particles in human airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278, L25–32.

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