Prooxidant and Proinflammatory Potency of Air Pollution Particulate

Article pubs.acs.org/crt

Prooxidant and Proinflammatory Potency of Air Pollution Particulate Matter (PM2.5−0.3) Produced in Rural, Urban, or Industrial Surroundings in Human Bronchial Epithelial Cells (BEAS-2B) Mona Dergham,†,‡ Capucine Lepers,†,‡ Anthony Verdin,†,‡ Sylvain Billet,†,‡ Fabrice Cazier,†,§ Dominique Courcot,†,‡ Pirouz Shirali,†,‡ and Guillaume Garçon*,†,‡,∥ †

Université Lille Nord de France, Lille, France Université du Littoral-Côte d’Opale, EA 4492, Dunkerque, France § Centre Commun de Mesures, Université du Littoral-Côte d’Opale, Dunkerque, France ∥ Université de Lille 2, EA 4483, Lille, France ‡

ABSTRACT: Compelling evidence indicates that exposure to air pollution particulate matter (PM) affects human health. However, how PM composition interacts with PM-size to cause adverse health effects needs elucidation. In this study, we were also interested in the physicochemical characteristics and toxicological end points of PM2.5−0.3 samples produced in rural, urban, or industrial surroundings, thereby expecting to differentiate their respective in vitro adverse health effects in human bronchial epithelial cells (BEAS-2B). Physicochemical characteristics of the three PM2.5−0.3 samples, notably their inorganic and organic components, were closely related to their respective emission sources. Referring also to the dose/response relationships of the three PM2.5−0.3 samples, the most toxicologically relevant exposure times (i.e., 24, 48, and 72 h) and doses (i.e., 3.75 μg PM/cm2 and 15 μg PM/cm2) to use to study the underlying mechanisms of action involved in PM-induced lung toxicity were chosen. Organic chemicals adsorbed on the three PM2.5−0.3 samples (i.e., polycyclic aromatic hydrocarbons) were able to induce the gene expression of xenobiotic-metabolizing enzymes (i.e., Cytochrome P4501A1 and 1B1, and, to a lesser extent, NADPH-quinone oxidoreductase-1). Moreover, intracellular reactive oxygen species within BEAS-2B cells exposed to the three PM2.5−0.3 samples induced oxidative damage (i.e., 8-hydroxy-2′deoxyguanosine formation, malondialdehyde production and/or glutathione status alteration). There were also statistically significant increases of the gene expression and/or protein secretion of inflammatory mediators (i.e., notably IL-6 and IL-8) in BEAS-2B cells after their exposure to the three PM2.5−0.3 samples. Taken together, the present findings indicated that oxidative damage and inflammatory response preceeded cytotoxicity in air pollution PM2.5−0.3-exposed BEAS-2B cells and supported the idea that PM-size, composition, and origin could interact in a complex manner to determine the in vitro responsiveness to PM.

■

INTRODUCTION Air pollution is now a well-recognized human health risk factor.1−5 Many time-series studies have shown that air pollution particulate matter (PM), generally measured as particles with aerodynamic diameter ≤10 μm (PM10) and as those with aerodynamic diameter ≤2.5 μm (PM2.5), is associated with an increased risk of death from cardiovascular and/ or respiratory causes in both Europe and the United States of America.1,4,6 PM-size has also been the most available metric used in epidemiology to assess PM-related health effects. The adverse health effects of air pollution PM may relate to its physicochemical characteristics, including mass, size, number, surface area, composition, concentration, and source.7−10 PM is also a very complex and heterogeneous mixture of metals, salts, organic chemicals, and biological materials generally adsorbed on carbonaceous cores.11−13 However, the majority of published studies dealing with the adverse health effects of air pollution PM have generally been realized using complex mixtures, yet their © 2012 American Chemical Society

physicochemical characteristics are not well established, and the possible influence of the chemicals adsorbed on inhalable particles is most often neglected.7 Although these studies provided some evidence regarding the role of PM components and their associated adverse health effects, there is still no consensus within the scientific community as to which specific components are the most significant determinants of the toxicological response.8,14,15 How PM components interact with PM-size to cause health effects, therefore, still needs elucidation. Less work has been reported using air pollution PM from specific emission sources, which is a critical need for regulators. A few years ago, we undertook an extensive short-term exposure in vitro study by sampling air pollution PM2.5−0.3 in Dunkerque City (France) and studied its toxicologically relevant physicochemical characteristics.16,17 In vitro short-term exposure to air pollution Received: December 6, 2011 Published: March 9, 2012 904

dx.doi.org/10.1021/tx200529v | Chem. Res. Toxicol. 2012, 25, 904−919

Chemical Research in Toxicology

Article

direction), and the second, also called I PM, under industrialrelated emission sources (i.e., SW/W prevailing wind direction). With regards to the prevailing wind direction, Dunkerque City, a French sea-side city located on the southern coast of the North Sea is subjected to air pollution PM arising not only from the nearby industrial activities (e.g., iron and steel industry, aluminum industry, oil refinery, basic chemistry, pharmaceutical industry, food industry), located at a distance range between about 3 and 12 km, but also from motor vehicle traffic from the urban road network. A third air pollution PM2.5−0.3 sample, also called R PM, was collected in Rubrouck Village (France; 50°50′21″ N/2°21′19″E) under rural-related emission sources. The three PM2.5−0.3 samples were collected by using high volume cascade impaction air samplers (Staplex, New-York, USA), as published elsewhere.23 Briefly, plates were mounted without any backup filter to maintain a constant aspiration flow rate (i.e., 80 m3/h). The lowest stage was doubled to increase the efficiency of smallest particles sampling (i.e., PM0.3). Collection was done continuously during spring/ summer 2008, as follows: R PM2.5−0.3, from February 07, 2008 until July 24, 2008; U PM2.5−0.3, from February 13, 2008 until August 13, 2008; and I PM2.5−0.3, from April 05, 2008 until September 13, 2008. The difference between the collection periods mainly relies on the need to collect sufficient masses of the three PM2.5−0.3 samples to study both physicochemical characteristics and toxicological end points. The longer collection periods needed to collect sufficient masses of the U PM2.5−0.3 and I PM2.5−0.3, produced in urban and industrial surroundings respectively, were related to their alternating collection, depending on the prevailing wind direction. Impacting systems were changed every week. Meteorological data (i.e., wind speed, wind direction, temperature, and hygrometry) were recorded. After sampling, impacting plates were dried under a laminar flow bench for 48 h; thereafter, PM were carefully removed from plates and immediately stored at −20 °C. PM Physicochemical Characterization. The physicochemical characterization of the three PM2.5−0.3 samples was carried out by studying their size distributions (i.e., scanning electron microscopy, SEM), specific surface areas (i.e., Brunauer−Emmett−Teller method; PM2.5−0.3 samples were outgassed under vacuum at room temperature, and, thereafter, volumes of pure nitrogen gas adsorbed to their surface at −196 °C were correlated to their specific surface area, including pores), inorganic chemicals (i.e., inductively coupled plasma-mass spectrometry, ICP-MS), ionic species (i.e., ionic chromatography, IC), and organic chemicals (i.e., VOCs: thermal desorption preconcentration method and gas chromatography−mass spectrometry, GC-MS; PAHs, Soxhlet extraction with dichloromethane and GC-MS; polychlorinated dibenzo-p-dioxins, PCDDs; polychlorinated dibenzofurans, PCDFs; polychlorinated biphenyls, PCBs, ASE extraction with toluene and high resolution (HR) GC/HRMS.34 PM Outgassing. To better elucidate the role of chemicals adsorbed on PM, PM having undergone a thermal desorption at 400 °C under a secondary vacuum (i.e., also called desorbed PM, dPM) and having thereby kept inorganic structures and lost their organic chemicals, were included as the inorganic fraction control in the experimental design of this study.16 Cell Line and Culture Conditions. Cell Line. The BEAS-2B cell line was obtained from European Collection of Cell Cultures (ECACC, Wiltshire, UK; reference, 95102433) and was originally derived from normal bronchial epithelial human cells obtained from the autopsy of noncancerous individuals. Culture Conditions. BEAS-2B cells were cultured in CellBIND surface plastic flasks (Corning; ThermoFisher Scientific, Illkirch, France), in bronchial epithelial cell basal medium (BEBM, LONZA Verviers SPRL), supplemented with 0.1% (v/v) retinoic acid, 0.1% (v/v) epinephrine, 0.1% (v/v) triiodothyronine, 0.1% (v/v) human recombinant epidermal growth factor, 0.1% (v/v) insulin, 0.1% (v/v) gentamicin sulfate amphotericin-B, 0.1% (v/v) transferrin, 0.1% (v/v) hydrocortisone, and 0.4% bovine pituitary extract (BEGM Bronchial Epithelial BulletKit, LONZA Verviers SPRL), as published elsewhere.35 The cells were kept in

PM2.5−0.3 induced concentration- and/or time-dependent toxicological end points in human lung cell target models.14,18−26 Thereafter, to contribute to a better knowledge of the relative role of PM components in producing lung injury, physicochemical and toxicological characteristics of three PM2.5−0.3 samples collected under different types of emission sources near Dunkerque City (France) were determine to discriminate their respective potential in triggering a variety of oxidative and inflammatory end points in human bronchial epithelial cells (i.e., BEAS-2B cell line). The three PM2.5−0.3 samples were, respectively, produced in rural, urban, or industrial surroundings (i.e., also called R, U, and I, respectively). After the determination of their most toxicologically relevant physicochemical characteristics, the overall cytotoxicity of the three PM2.5−0.3 samples was evaluated in BEAS-2B cells. Hence, to better elucidate the role of the chemical components within air pollution PM in causing lung injuries, PM2.5−0.3-induced gene expression of polycyclic aromatic hydrocarbons (PAHs) and/or volatile organic compound (VOC)-metabolizing enzymes (i.e. cytochrome P450 (CYP) 1A1, CYP1B1, CYP2E1, CYP2S1, and NADPH quinone oxido-reductase-1, NQO1) was studied in BEAS-2B cells. Nowadays, the underlying mechanisms for PM-induced lung injury are still not clear, but oxidative stress and inflammatory reaction are considered as key events.27 Lung disorders are often associated with prooxidant/antioxidant imbalance and the proinflammatory reaction, and there is increasing evidence that air pollution PM induces acute responses as well as exacerbates existing inflammatory diseases within the lung.3,27,28 Excessive production of reactive oxygen species (ROS) exceeds the detoxification capacity of cell antioxidant defenses, thereby triggering a cascade of events closely associated with inflammation.8,13,28−33 Attention was also focused on the ability of the chemical components of the three PM2.5−0.3 samples to induce prooxidative and/or proinflammatory reaction in BEAS-2B cells, through the determination of PM2.5−0.3 sample-induced intracellular ROS formation (i.e., cleavage of 5 (and 6-)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, H2DCFDA), DNA damage (i.e., production of 8-hydroxy-2′-deoxyguanosine, 8-OHdG), lipid peroxidation (i.e., production of malondialdehyde, MDA), and glutathione status alteration (i.e., glutathione disulfide/ reduced glutathione), on the one hand, and both the gene expression and the protein secretion of some inflammatory mediators (i.e., tumor necrosis factor-alpha, TNF-α; interleukine-1 beta, IL-1β; interleukine-6, IL-6; interleukine-8, IL8), on the other hand.

■

EXPERIMENTAL PROCEDURES

Reagents. Cell culture reagents were provided by LONZA Verviers SPRL (Verviers, Belgium). Titanium(IV) oxide powder (anatase; purity, 99%; primary particle size, 0.2 μm; surface area, 14 m2/g; surface not coated) was from Acros Organics (Noisy le Grand, France). H2DCFDA was from Invitrogen (Paisley, UK). All other chemicals were from SigmaAldrich (Saint-Quentin Fallavier, France). Cell Proliferation Reagent ELISA BrdU kit, cell proliferation reagent (WST-1), and Cytotoxicity LDH Detection Kit were from Roche Diagnostics (Neuilly surSeine, France). TaqMan Gene Expression Cells-to-CT kits and TaqMan Gene Expression Assays were from Applied Biosystems (Life Technologies, Courtaboeuf, France). DNeasy Tissue Kits were from Qiagen (Courtaboeuf, France). Highly sensitive 8-OHdG Check was from Gentaur France SARL (Paris, France). Quantikine colorimetric sandwich ELISAs were from R&D Systems Europe (Abingdon, UK). Methods. Site Description, PM Sampling, and Physicochemical Characterization. Site Description and PM Sampling. Two PM2.5−0.3 samples were from Dunkerque City (France; 51°2′16″N/2°22′35″E), the first, also called U PM, under urban-related emission sources (i.e., SE/E prevailing wind 905



Article

a humidified atmosphere at 37 °C and 5% CO2. Before reaching 80% confluence, cells were subcultured by using 0.25% trypsin/0.53 mM versene solution containing 0.5% (w/v) polyvinylpyrrolidone (SigmaAldrich). The medium was refreshed every 2−3 days, and cells were subcultured weekly (1,500−3,000 cells/cm2). All of the BEAS-2B cells we used derived from the same cell culture. Culture Supernatant and Cell Sampling for the Study of the Cytotoxicity. Cell Exposure. BEAS-2B cells were seeded in 96well CellBIND surface cell culture microplates (Corning) at a density of 1 × 104 cells/200 μL BEGM and incubated at 37 °C for 24 h in a humidified atmosphere containing 5% CO2. Culture supernatants were removed to eliminate nonadherent cells. Nonexposed cells were used as negative controls (i.e., 100% viability) and Triton X-100 (2%, v/v)-exposed cells as positive controls (i.e., 100% mortality). Accordingly, 16 replicates were chosen at random as negative control cells (i.e., only with BEGM), 8 replicates/concentration/PM2.5−0.3 sample (i.e., R, U, or I) as exposed cells (i.e., with increasing concentrations of PM2.5−0.3, ranging from 3.75 to 120 μg PM/ cm2, or from 6.25 to 200 μg/mL), and 8 replicates as positive controls (i.e., Triton X-100, 2% v/v). Cytotoxicity Study. After 24, 48, or 72 h of exposure, the cytotoxicity in BEAS-2B cells was evaluated by studying extracellular lactate dehydrogenase (LDH) activity in cell-free culture supernatants (Cytotoxicity Detection Kit LDH, Roche Diagnostics), mitochondrial dehydrogenase (MDH) activity in cells (Cell Proliferation Reagent WST-1, Roche Diagnostics), and 5-bromo-deoxyuridinz (5-BrdU) incorporation in cells (Cell Proliferation ELISA BrdU, Roche Diagnostics), as published elsewhere.16 Culture Supernatant and Cell Sampling for the Study of PAHand/or VOC-Metabolizing Enzymes and Inflammatory Mediators. Cell Exposure. Depending on the exposure time (i.e., 24, 48, or 72 h), BEAS-2B cells were seeded in 6-well CellBIND surface cell culture microplates (Corning) at different densities (i.e., 25 × 103, 12.5 × 103, or 6.25 × 103 cells/well/1.5 mL of BEGM, respectively) and incubated for 24 h, at 37 °C, in a humidified atmosphere containing 5% CO2. Culture supernatants were removed to eliminate nonadherent cells. Only living BEAS-2B cells were exposed to each of the three PM2.5−0.3 samples at two concentrations (i.e., 3.75 μg PM/cm2 or 6.25 μg/mL and 15 μg PM/cm2 or 25 μg/mL) for 24, 48, or 72 h, without renewing the culture media. Nonexposed cells were used as negative controls, TiO2-exposed cells (i.e., 11.33 μg/cm2 or 18.88 μg/mL) as particle controls, dPM2.5−0.3-exposed cells (i.e., 2.83 μg/cm2 or 4.72 μg/mL and 11.33 μg/cm2 or 18.88 μg/mL) as inorganic fraction controls, and benzo[a]pyrene (B[a]P (1 μM)), benzene (7 μM), and lipopolysaccaride (LPS; 10 μg/mL)exposed cells as positive controls.36−39 For each exposure time, 8 wells were chosen at random as negative controls (i.e., only with supplemented BEBM), 4 wells as particle controls (i.e., TiO2-exposed), 4 wells/concentration/dPM2.5−0.3 sample (i.e., R, U, or I) as inorganic fraction controls, 4 wells/concentration/PM2.5−0.3 sample (i.e., R, U, or I) as exposed cells, and 4 wells/chemical (i.e. benzene, B[a]P or LPS-exposed cells) as positive controls. Culture Supernatant and Cell Sampling. After 24, 48, or 72 h of exposure, 300 μL-aliquots of cell-free culture supernatants were collected and quickly frozen at −80 °C until further study of TNF-α, IL-1β, IL-6, and IL-8 concentrations or total protein contents. Adherent cells were washed twice with 2-mL aliquots of cold phosphate-buffered saline (PBS; 0.01 M; pH 7.2), and quickly frozen at −80 °C until further study of the gene expression of PAHs and/or VOCs-metabolizing enzymes and inflammatory mediators. Xenobiotic-Metabolizing Enzyme and Inflammatory Mediator Gene Expression. Gene expression profiles of xenobiotic-metabolizing enzymes (i.e. CYP1A1, CYP1B1, CYP2S1, CYP2E1, and NQO1) and inflammatory mediators (i.e. TNF-α, IL-1β, IL-6, and IL-8) were studied by using the TaqMan Gene Expression Cells-to-CT kit and predefined TaqMan Gene Expression Assays (i.e., Hs00153120_m1, Hs0016164383_m1, Hs00559368_m1, Hs00258076_m1,

Hs00168547_m1, Hs99999043_m1, Hs00174097_m1, Hs00174131_m1, and Hs00174103_m1, respectively ; Applied Biosystems). Real-time quantitative polymerase chain reactions (qPCR) were carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems). The relative changes in gene expression were calculated by the ΔΔCt method and normalized against endogenous ribosomal 18S RNA (i.e., Hs_99999901_s1), using the Sequence Detection 7500 Software v2.0.3 (Applied Biosystems). Briefly, in a real-time qPCR assay, the Cycle threshold (Ct) is defined as the number of cycles required for the fluorescent signal to cross the limit threshold, therefore exceeding the background level. Hence, Ct levels are inversely proportional to the amount of target nucleic acid (e.g., mRNA) in the sample. The relative quantitation (RQ) of target mRNA, which corresponds to the differences in target mRNA levels in exposed cells (i.e., TiO2-exposed cells, dPM2.5−0.3-exposed cells, PM2.5−0.3-exposed cells, or benzene, B[a]P, or LPS-exposed cells) versus in nonexposed cells, corrected by the level of endogenous ribosomal 18S RNA, used as the housekeeping gene, wase calculated using the following equation: RQ = 2−Δ(ΔCt), where ΔCt = Ct(target gene) − Ct(18S RNA), and Δ(ΔCt) = ΔCt(exposed cells) − ΔCt(nonexposed cells).18,20,25 Cytokine Concentration. TNF-α, IL-1β, IL-6, and IL-8 concentrations in cell-free culture supernatants were determined using commercially available enzyme immunoassays (Quantikine Colorimetric Sandwich ELISA, R&D Systems Europe), following the manufacturer’s instructions. Total protein contents in cell-free culture supernatants were studied using the bicinchoninic acid (BCA) kit (Sigma-Aldrich). Cells Sampling for the Study of Intracellular ROS. Cell Exposure. BEAS-2B cells were seeded in 96-well CellBIND surface cell culture microplates (black with clear flat bottom; Corning) at a density of 1 × 104 cells/200 μL BEGM and incubated at 37 °C in a humidified atmosphere containing 5% CO2, for 24 h. Culture supernatants were removed to eliminate nonadherent cells. Only living BEAS-2B cells were exposed to each of the three PM2.5−0.3 samples at two concentrations (i.e., 3.75 μg PM/cm2 or 6.25 μg/mL, and 15 μg PM/cm2 or 25 μg/mL) for 24, 48, or 72 h, without renewing the culture media. Nonexposed cells were used as negative controls, TiO2exposed cells (i.e., 11.33 μg/cm2 or 18.88 μg/mL) as particle controls, dPM2.5−0.3-exposed cells (i.e., 2.83 μg/cm2 or 4.72 μg/mL, and 11.33 μg/cm2 or 18.88 μg/mL) as inorganic fraction control, and B[a]P (1 μM)-, benzene (7 μM)-, LPS (10 μg/mL)-, and H2O2 (100 μM)-exposed cells as positive controls.36−40 For each exposure time, 16 wells were chosen at random as negative controls (i.e., only with BEGM), 8 wells as particle controls (i.e., TiO2-exposed), 8 wells/concentration/dPM2.5−0.3 sample (i.e., R, U, or I) as inorganic fraction controls, 8 wells/ concentration/PM2.5−0.3 sample (i.e., R, U, or I) as exposed cells, and 8 wells/chemical (i.e. benzene, B[a]P-, LPS-, or H2O2exposed cells) as positive controls. Intracellular ROS. Production of intracellular ROS within cells were carried out by a fluorimetric assay using carboxy-H2DCFDA as the probe, as publisher elsewhere.40 Carboxy-H2DCFDA was chosen because it carries an additional negative charge that improves its retention compared to noncarboxylated forms. Briefly, after the specified exposure time (i.e., 24, 48, or 72 h), the plates were washed with 100 μL/well of cold-PBS, and thereafter, 100 μL/well of 10 μM carboxy H2DCFDA were added. The plates were incubated at 37 °C for a period of 40 min. Intracellular oxidation of carboxy-H2DCFDA to DCF was monitored fluorimetrically (excitation wavelength, 490 nm; emission wavelength, 520 nm) by using a computerized microplate reader (software, Ascent v2.6; hardware, Fluoroskan Ascent; Thermo Fisher Scientific, Courtaboeuf, France). Cells Sampling for the Study of Oxidative Damage. Cell Exposure. Depending on the exposure time (i.e., 24, 48, or 72 h), BEAS-2B cells were seeded in CellBIND surface cell culture flasks (75 cm2; Corning) at different densitiesy (i.e., 4 × 106, 2 × 106, or 1 × 106 cells/flask/20 mL of supplemented BEBM, respectively), and incubated during 24 h, at 37 °C, in a humidified atmosphere containing 5% CO2. Culture supernatants were removed to eliminate nonadherent cells. Only living BEAS-2B cells were 906



Article

Figure 1. Size distribution of the three air pollution particulate matter samples produced in rural, urban, or industrial surroundings (i.e., also called R, U, and I, respectively), as depicted as relative frequency (%; A) and cumulative frequency (%; B). washed twice with 5-mL aliquots of cold PBS (0.01 M; pH 7.2) and quickly frozen at −80 °C until further study of oxidative damage and total protein contents. Oxidative Damage. DNA adduct 8-OHdG concentrations were studied in cell lysates using commercially available enzyme immunoassays (Highly Sensitive 8-OHdG Check, Gentaur France SARL), according to the method originally described by Toyokuni et al.41 and modified by Garçon et al.14 MDA concentrations and glutathione status (i.e., glutathione disulfide, GSSG/reduced glutathione, GSH) were studied in cell lysates by using high-performance liquid chromatography with fluorescence detection, as published elsewhere.42−44 Statistical Analysis. Results are expressed as mean values and standard deviations. For each exposure time (i.e., 24, 48, 72 h), data from particle control cells (i.e., TiO2-exposed cells), inorganic fraction control cells (i.e., R, U, or I dPM2.5−0.3-exposed cells), PM2.5−0.3exposed cells (i.e., R, U, or I), and positive control cells (i.e., B[a]P-, benzene-, LPS-, and/or H2O2-exposed cells) were compared to those

exposed to each of the three PM2.5−0.3 samples at two concentrations (i.e., 3.75 μg PM/cm2 or 6.25 μg/mL and 15 μg PM/cm2 or 25 μg/mL) for 24, 48, or 72 h, without renewing the culture media. Nonexposed cells were used as negative controls, TiO2-exposed cells (i.e., 11.33 μg/cm2 or 18.88 μg/mL) as particle controls, dPM2.5−0.3-exposed cells (i.e., 2.83 μg/cm2 or 4.72 μg/mL and 11.33 μg/cm2 or 18.88 μg/mL) as inorganic fraction control, and B[a]P (1 μM)-, benzene (7 μM)-, and LPS (10 μg/mL)exposed cells as positive controls.36−39 For each exposure time, 8 culture flasks were chosen at random as negative controls (i.e., only with BEGM), 4 culture flasks as particle controls (i.e., TiO2exposed), 4 culture flasks/concentration/dPM2.5−0.3 sample (i.e., R, U, or I) as inorganic fraction controls, 4 culture flasks/ concentration/PM2.5−0.3 sample (i.e., R, U, or I) as exposed cells, and 4 culture flasks/chemical (i.e. benzene-, B[a]P-, or LPSexposed cells) as positive controls. Cell Sampling. After 24, 48, or 72 h of exposure, adherent cells were removed and centrifuged (500g, 10 min, 4 °C). Cell pellets were 907



Article

to a lesser extent in U PM2.5−0.3 (i.e., 1,577 ng/m3), as compared to R PM2.5−0.3 (i.e., 263 ng/m3) (Table1). As shown in

from nonexposed cells. Thereafter, we looked for correlation between the biological markers under study. Statistical analyses were performed by the Mann−Whitney U test, with p value correction for multiple comparisons, and the nonparametric Spearman’s rho test (Software: SPSS for Windows, v10.05, 2000; Paris, France). Statistically significant differences were reported with p < 0.05.

Table 1. Inorganic Chemical Concentrations in the Three Air Pollution Particulate Matter (PM2.5‑0.3) Samples

■

concentration: ng/m3 (% m/m)a

RESULTS PM Physicochemical Characteristics. Total masses of the three PM2.5−0.3 samples produced in R, U, or I were 357.1 mg, 623.4 mg, and 310.3 mg, respectively. Average concentrations of air pollution PM2.5−0.3 during the collection period were as follows: 2.8 μg/m3 for R PM2.5−0.3, 9.9 μg/m3 for U PM2.5−0.3, and 11.5 μg/m3 for I PM2.5−0.3. Figure 1A and B shows their size distribution, as depicted as cumulative frequency (%) and relative frequency (%), respectively. Accordingly, 96.45%, 78.97%, and 93.82% of the total PM number were ≤2.5 μm in R PM2.5−0.3, U PM2.5−0.3, and I PM2.5−0.3, respectively. The highest particle numbers in the three PM2.5−0.3 samples were also detected in the lowest size classes. Figure 2A, B, and C

inorganic chemicals

rural

Ag

0.004 (0.0001)

urban 0.017 (0.0001)

industrial 0.027 (0.0002)

Alb

57.2 (2.068)

140(1.413)

452 (3.936)

As

0.057 (0.002)

0.205 (0.002)

0.251 (0.002)

Bac

0.65 (0.023)

3.04 (0.030)

5.77 (0.050)

Bi

0.021 (0.0007)

0.055 (0.0005)

0.065 (0.0005)

Ca

55.2 (1.992)

439 (4.434)

698 (6.076)

Cd

0.028 (0.001)

0.082 (0.0008)

0.226 (0.001)

Ce

0.086 (0.003)

0.292(0.002)

0.469 (0.004)

Co

0.026 (0.0009)

0.183 (0.001)

0.264 (0.002)

Cr

0.60 (0.021)

3.85 (0.038)

4.76 (0.041)

Cu

1.10 (0.039)

8.65 (0.087)

19.2 (0.167)

Fe

73.4 (2.650)

415 (4.194)

1,234 (10.734)

In

Mn >Zn) and medium elements (e.g., Ti > Pb > Ba > Cr > Ni > Sr > V > Sn) were the most abundantly present in I PM2.5−0.3 (i.e., 3,059 ng/m3) and

rural

urban

industrial

F−

1.25 (0.045)

1.49 (0.015)

0.58 (0.005)

Cl−

26.0 (0.938)

306 (3.092)

254 (2.212)

NO3−

139 (5.054)

855 (8.644)

783 (6.814)

SO42‑

91.9 (3.321)

480 (4.848)

827 (7.193)

Na+

28.5 (1.029)

325 (3.287)

192 (1.673)

NH4+

28.5 (1.029)

193 (1.953)

59.2 (0.515)

K+

13.1 (0.472)

33.9 (0.342)

20.9 (0.182)

Mg2+

5.7 (0.205)

46.5 (0.470)

42.8 (0.372)

Ca2+

48.1 (1.739)

390 (3.940)

608 (5.293)

total ionic species

382 (13.832)

2,632 (26.591)

2,790 (24.259)

a

Ionic species concentrations are expressed as mass composition per volume unit (i.e., ng/m3) and, within parentheses, percentage composition per mass (i.e., % m/m).

PM2.5−0.3 (i.e., 2,790 ng/m3 and 2,632 ng/m3, respectively) versus R PM2.5−0.3 (i.e., 382 ng/m3). In the three PM2.5−0.3 908



Article

samples under study, lots of organic chemicals (i.e., VOCs and/ or PAHs) were found (Table 3). The highest concentrations of

Table 4. Polychlorinated Dibenzo-p-dioxin and Furan Concentrations in the Three Air Pollution Particulate Matter (PM2.5‑0.3) Samples

Table 3. Organic Chemical Concentrations in the Three Air Pollution Particulate Matter (PM2.5‑0.3) Samples

concentration (fg/m3) congeners

concentration: ng/m3 (% m/m)a organic chemicals

rural

urban

industrial

VOCsb toluene

3.2 ( Ba > Cr > Ni > Sr > V > Sn) were the most abundantly present in I PM2.5−0.3 (i.e., 3,059 ng/m3 or 26.6% m/m), and to a lesser degree in U PM 2.5−0.3 (i.e., 1,577 ng/m 3 or 15.9% m/m), versus R PM2.5−0.3 (i.e., 263 ng/m3 or 9.5% m/m). There were also not only inorganic chemicals usually associated with natural environment (e.g., Na, Mg, Ca, Ti) but also so-called anthropogenic elements (e.g., Fe, Al, Mn, Ba, Cr, Zn, Pb, Cu). Anthropogenic elements found in I PM2.5−0.3 and to a lesser extent in U PM2.5−0.3 are related to the collection area characterized by industrial activities (i.e., iron and steel industry, aluminum industry, oil refinery, basic chemistry, pharmaceutical industry, food industry, etc.) and/or by heavy motor vehicle traffic.12,16 The concentrations of the

ionic species were higher in I PM2.5−0.3 and U PM2.5−0.3 (i.e., 2,790 ng/m3 or 24.2% m/m, and 2,632 ng/m3 or 26.5% m/m, respectively) versus R PM2.5−0.3 (i.e., 382 ng/m3 or 13.8% m/m). These ionic species revealed the influence of North-Sea emissions (i.e., Na+, Cl−), natural erosion (i.e., Ca2+, Mg2+, K+), and anthropogenic activities (i.e., SO42‑, NO3−, F−).16,34 The highest concentrations of organic chemicals (i.e., VOCs, PAHs, PCDDs/PCDFs, and PCBs) were reported in I PM2.5−0.3 (i.e., ∼ 0.020% m/m), and to a lesser degree in U PM2.5−0.3 915



Article

(i.e., ∼ 0.016% m/m), versus in R PM2.5−0.3 (i.e., ∼ 0.007% m/m). These organic chemicals could also be linked to the industrial activities and heavy vehicle traffic.12,13,34 Overall, the respective physicochemical characteristics of the three PM2.5−0.3 samples under study were closely related to their respective natural and/or anthropogenic emission sources. Thereafter, we focused our attention on the cytotoxic end points of the three PM2.5−0.3 samples under study. BEAS-2B cell exposure to R PM2.5−0.3, U PM2.5−0.3, and I PM2.5−0.3 led to statistically significant changes in the extracellular LDH activity, MDH activity, and BrdU incorporation into DNA. These changes referred not only to the relatively late alteration of membrane integrity and/or permeability but also to earlier alteration of mitochondrial metabolism, and cell cycle, respectively.24 However, with regard to their respective physicochemical characteristics, the relatively high cytotoxicity of U PM2.5−0.3 and I PM2.5−0.3, and even R PM2.5−0.3 is somewhat surprising, thereby indicating the possible involvement of other agents than chemicals, including notably biological materials (e.g., bacteria and fungi). By referring to the above-mentioned results, it is also reasonable to consider the role of condensation nuclei, which could serve as the physical vector, notably favor the penetration and the retention of organic chemicals within lung cells, and also modulate their metabolic activation.38,39,52,53 Hence, it appeared difficult to predict the efficiency of the metabolic activation of the VOCs and/or PAHs adsorbed on air pollution PM2.5−0.3 by BEAS-2B cells. Indeed, organic chemicals within the three PM2.5−0.3 samples under study, mainly PAHs, induced statistically significant gene expression of CYP1A1 and CYP1B1, and to a lesser extent, NQO1. As mentioned above, it is important to consider the longer and/or higher gene transcription of these parameters in BEAS-2B cells exposed to I PM2.5−0.3, and to a lesser degree U PM2.5−0.3, versus R PM2.5−0.3. These data also supported the hypothesis that the three PM2.5−0.3 samples, even with different specific area (i.e., 2.8, 3.5, and 5.2 m2/g, respectively), could act as a physical vector, thereby increasing both the penetration and the retention of PAHs into the cells, and enabling them to exert a more durable gene induction.38,39 These data were consistent with the relatively high concentrations of PAHs within I PM2.5−0.3 and U PM2.5−0.3 (i.e., 1,021 ng/m3 or 1.9% m/m, and 689 ng/m3 or 1.5% m/m, respectively), versus R PM2.5−0.3 (i.e., 146 ng/m3 or 0.6% m/m).53 While the underlying mechanisms responsible for air pollution PM-related injury in the lung are still incompletely understood, a hypothesis currently under investigation is that many of them may derive from oxidative stress, initiated by the excessive production of ROS within affected cells.15,21,22,55,56 As shown by the statistically significant oxidative damage we reported, there were intracellular ROS within BEAS-2B cells after their exposure to PM2.5−0.3 produced in rural, urban, or industrial surroundings, and even their dPM2.5−0.3, thereby supporting the possible role played by the inorganic chemicals (i.e., Al, Fe, Mn, and Zn) as such as the organic chemicals (i.e., PAHs) as exogenous sources of ROS. Accordingly, in the literature, it is believed that air pollution induces oxidative stress either directly via transition metals or indirectly via inflammation caused by inhaled particles, as well as by the reactive quinones arising from PAH metabolism.14,15,21 However, with regard to the results from the Figure 6, no obvious difference in the intracellular ROS was observed within BEAS-2B cells after their exposure to dPM2.5−0.3 versus

PM2.5−0.3, whatever the production surroundings (i.e., rural, urban, or industrial surroundings). Moreover, as shown in Tables 1 and 3, I PM2.5−0.3, for which the highest concentrations of both inorganic and organic chemicals (e.g., metals, 1,021 ng/m3 or 1.9% m/m, and PAHs, 3,059 ng/m3 or 26.6% m/m) were found, was precisely the one that induced the lowest response with regard to intracellular ROS. Moreover, as shown in Figures 7, 8, and 9, no clear difference was reported between I PM2.5−0.3- and U PM2.5−0.3- nor R PM2.5−0.3-induced oxidative damage within BEAS-2B cells. Taken together, these results did also support not only the role of inorganic chemicals rather than organic chemicals as the exogenous source of ROS but also the possible role of other factors that remain to be determined (e.g., interaction of PM2.5−0.3 physical characteristics and cell uptake efficiency and/or role of other chemicals).57−59 Another important result of this in vitro study was the statistically significant increases of both the gene expression and/or the protein secretion of inflammatory mediators (i.e., IL-6 and/or IL-8) in BEAS-2B cells 24, 48, and 72 h after their exposure to the three PM2.5−0.3 samples under study. In view of the above-described results about their physicochemical characterization, there was a clear relationship between their physicochemical characteristics and their ability to activate the dose-dependent secretion of IL-6 and IL-8 by stimulated BEAS2B cells.8,15,59 By referring to the results from Figures 12 and 13, an obvious difference in the dose-dependent secretion of both IL-6 and IL-8 by BEAS-2B cells was reported after their exposure to dPM 2.5−0.3 versus PM2.5−0.3 , whatever the production surroundings (i.e., rural, urban, or industrial surroundings). Recent data in the literature reported that some of the main covalent metals (i.e., Al, Fe, Mn, Zn) mainly detected in I PM2.5−0.3 and U PM2.5−0.3 samples could be firmly involved in redox systems, which can lead to the initiation of radical reactions.12−15,19,55 Moreover, the metabolic activation by enzyme-catalyzed reactions of the PAHs-adsorbed on PM2.5−0.3 could result in the excessive production of ROS capable of interfering with cell homeostasis.21,54,57 However, because of the previously mentioned PM2.5−0.3-induced oxidative damage in BEAS-2B cells, the lowest secretion of both IL-6 and IL-8 was observed in I PM2.5−0.3-exposed BEAS2B cells, versus U PM2.5−0.3 and even R PM2.5−0.3, despite the highest concentrations of both inorganic and organic chemicals (e.g., metals, 1,021 ng/m3 or 1.9% m/m; and PAHs, 3,059 ng/m3 or 26.6% m/m) found within I PM2.5−0.3 (i.e., Tables 1 and 3). All together, these results did again highlight not only the role of inorganic chemicals rather than organic chemicals as the exogenous inducer of inflammatory secretion but also the possible role of other factors that remain to be determined (e.g., interaction of PM2.5−0.3 physical characteristics and cell uptake efficiency and/or role of other chemicals).57−59 However, although several studies provide some evidence regarding the role of PM components and their associated cell effects, there is still no consensus within the scientific community as to which specific components are the most significant determinants of the cell response.7 Gualtieri et al. remind us that the in vitro responsiveness to air pollution PM may be cell line dependent and suggest that the PM different properties may trigger different end points such as inflammation, perturbation of cell cycle, and cell death.15 Nevertheless, one of the strengths of the present pluridisciplinary in vitro approach is to study the adverse lung effects of air pollution PM2.5−0.3 samples by referring to their physicochemical characterization together with some of the underlying mechanisms of action 916



Article

Notes

possibly involved in their lung toxicity in BEAS-2B cells. However, before concluding, caution is necessary when tackling the question of the helpfulness of in vitro models to improve the extrapolation to the experiments in animals and/or the situations in humans.9,10,15 Despite these limitations, in vitro experiments continue to be a prerequisite for a preliminary understanding of the underlying mechanism of action that, in general, is currently obtainable only through animal and/or human studies. In conclusion, the physicochemical characteristics of the PM2.5−0.3 samples produced in rural, urban, or industrial surroundings were consistent with their respective natural and/or anthropogenic emission sources. Taken together, the present findings indicated that oxidative damage and inflammatory response preceeded cytotoxicity in air pollution PM2.5−0.3-exposed BEAS-2B cells and supported the growing evidence that PM-size, composition, and origin interact in a complex manner to generate the toxicity of inhalable PM on human health. One of the most striking observations is that the variable concentrations of transition metals (i.e., Fe, Al, Mn, and Zn) and organic compounds (i.e., PAHs, after their effective metabolic activation) found in the three PM2.5−0.3 samples under study, respectively, produced in rural, urban, or industrial surroundings, might be firmly involved in both the oxidative damage and the inflammatory response reported in PM2.5−0.3-exposed BEAS-2B cells. However, the almost same or even lowest biological responses observed in I PM2.5−0.3exposed BEAS-2B cells versus U PM2.5−0.3 and indeed R PM2.5−0.3, despite the highest concentrations of both inorganic and organic chemicals found within I PM2.5−0.3, highlight not only the role of inorganic chemicals rather than organic chemicals as the exogenous inducer of oxidative damage and/or inflammatory response but also the possible role of other factors that remain to be determined (e.g., interaction of PM2.5−0.3 physical characteristics and cell uptake efficiency and/or role of other chemicals).57,58 Accordingly, much effort has been exerted by several authors to identify some characteristic patterns of in vitro toxicological response related to air pollution PM by focusing attention only on the inorganic composition of air pollution PM.8,15,60−64 Further toxicological and epidemiological research on the impacts of PMsize and composition is essential to better understand these phenomena and direct and prioritize pollution control efforts. Such information may in the long term lead to improved strategies to reduce the most critical elements of air pollution PM.

■

The authors declare no competing financial interest.

■

ABBREVIATIONS 8-OHdG, 8-hydroxy-2′-deoxyguanosine; BrdU, 5-bromodeoxyuridine; CYP, cytochrome P450; H2DCFDA, 5- (and 6-) carboxy-2′,7′-dichloro-dihydroflourescein diacetate; IL-1β, interleukine-1 beta; IL-6, interleukine-6; IL-8, interleukine-8; LDH, lactate dehydrogenase; MDA, malondialdehyde; MDH, mitochondrial dehydrogenase; NQO1, NADPH quinone oxido-reductase-1; PAHs, polycyclic aromatic hydrocarbons; PCBs, polychlorinated biphenyls; PCDDs, polychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated dibenzofurans; PM, particulate matter; ROS, reactive oxygen species; TNF-α, tumor necrosis factor-alpha; VOCs, volatile organic compounds

■

REFERENCES

(1) Pope, C. A. III, Burnett, R. T., Krewski, D., Jerrett, M., Shi, Y., Calle, E. E., and Thun, M. J. (2009) Cardiovascular mortality and exposure to airborne fine particulate matter and cigarette smoke: shape of the exposure-response relationship. Circulation 120, 941−948. (2) Brook, R. D., Rajagopalan, S., Pope, C. A. III, Brook, J. R., Bhatnagar, A., and Diez-Roux, A. V. (2010) Particulate matter air pollution and cardiovascular disease: an update to the scientific statement from the American Heart Association. Circulation 121, 2331−2378. (3) Peacock, J. L., Anderson, H. R., Bremner, S. A., Marston, L., Seemungal, T. A., Strachan, D. P., and Wedzicha, J. A. (2011) Outdoor air pollution and respiratory health in patients with COPD. Thorax 66, 591−596. (4) Raaschou-Nielsen, O., Andersen, Z., Hvidberg, M., Jensen, S. S., Ketzel, M., Sørensen, M., Loft, S., Overvad, K., and Tjønneland, A. (2011) Lung cancer incidence and long-term exposure to air pollution from traffic. Environ. Health Perspect. 19, 860−865. (5) Rusconi, F., Catelan, D., Accetta, G., Peluso, M., Pistelli, R., Barbone, F., Di Felice, E., Munnia, A., Murgia, P., Paladini, L., Serci, A., and Biggeri, A. (2011) Asthma symptoms, lung function, and markers of oxidative stress and inflammation in children exposed to oil refinery pollution. J. Asthma 48, 84−90. (6) Maynard, D., Coull, B. A., Gryparis, A., and Schwartz, J. (2007) Mortality risk associated with short-term exposure to traffic particles and sulfates. Environ. Health Perspect. 115, 751−755. (7) Osornio-Vargas, A. R., Serrano, J., Rojas-Bracho, L., Miranda, J., García-Cuellar, C., Reyna, M. A., Flores, G., Zuk, M., Quintero, M., Vázquez, I., Sánchez-Pérez, Y., López, T., and Rosas, I. (2011) In vitro biological effects of airborne PM2.5 and PM10 from a semi-desert city on the Mexico−US border. Chemosphere 83, 618−626. (8) Perrone, M. G., Gualtieri, M., Ferrero, L., Lo Porto, C., Udisti, R., Bolzacchini, E., and Camatini, M. (2010) Seasonal variations in chemical composition and in vitro biological effects of fine PM from Milan. Chemosphere 78, 1368−1377. (9) Sawyer, K., Mundandhara, S., Ghio, A. J., and Madden, M. C. (2010) The effects of ambient particulate matter on human alveolar macrophage oxidative and inflammatory responses. J. Toxicol. Environ. Health, Part A 73, 41−47. (10) Yoshida, T., Yoshioka, Y., Fujimura, F., Kayamuro, H., Yamashita, K., Higashisaka, K., Nakanishi, R., Morishita, Y., Nabeshi, H., Yamashita, T., Muroi, M., Tanamoto, K., Nagano, K., Abe, Y., Kamada, H., Kawal, Y., and Mayumi, T. (2010) Urban aerosols induce pro-inflammatory cytokine production in macrophages and cause airway inflammation in vivo. Biol. Pharm. Bull. 33, 780−783. (11) Alfaro-Moreno, E., Ponce-de-León, S., Osornio-Vargas, A. R., García-Cuellar, C., Martínez, L., and Rosas, I. (2007) Potential toxic effects associated to metals and endotoxin present in PM10: an ancillary study using multivariate analysis. Inhal. Toxicol. 19, 49−53. (12) Baulig, A., Singh, S., Marchand, A., Schins, R., Barouki, R., Garlatti, M., Marano, F., and Baeza-Squiban, A. (2009) Role of Paris

AUTHOR INFORMATION

Corresponding Author

*EA 4483, Département de Toxicologie, Santé publique et Environnement, Faculté des Sciences Pharmaceutiques et Biologiques de Lille, 3, rue du Professeur Laguesse, BP83 59006 Lille Cedex, France. Tel: +33-362283030. E-mail: [email protected]. Funding

The “Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV)”, EA 4492, participates in the Institut de Recherche en ENvironnement Industriel (IRENI), which is financed by the Communauté Urbaine de Dunkerque, the Région Nord Pas-de-Calais, the Ministère de l′Enseignement Supérieur et de la Recherche, the CNRS and European Regional Development Fund (ERDF). The research described in this article benefited from grants from the Agence Française de Sécurité Sanitaire de l′Environnement et du Travail (AFSSET; Convention no. EST-2007-48) and the Lebanon National Council for Scientific Research. 917



Article

in multiple critical regions of 3p chromosome in human epithelial lung cells (L132). Toxicol. Lett. 187, 172−179. (27) Lonkar, P., and Dedon, P. C. (2011) Reactive species and DNA damage in chronic inflammation: reconciling chemical mechanisms and biological fates. Int. J. Cancer. 128, 1999−2009. (28) Maestrelli, P., Canova, C., Scapellato, M. L., Visentin, A., Tessari, R., Bartolucci, G. B., Simonato, L., and Lotti, M. (2011) Personal exposure to particulate matter is associated with worse health perception in adult asthma. J. Invest. Allergol. Clin. Immunol. 21, 120− 128. (29) Terzano, C., Di Stefano, F., Conti, V., Graziani, E., and Petroianni, A. (2010) Air pollution ultrafine particles: toxicity beyond the lung. Eur. Rev. Med. Pharmacol. Sci. 14, 809−821. (30) Jomova, K., and Valko, M. (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 283, 65−87. (31) Ziech, D., Franco, R., Pappa, A., and Panayiotidis, M. I. (2011) Reactive Oxygen Species (ROS)-induced genetic and epigenetic alterations in human carcinogenesis. Mutat. Res. 711, 167−177. (32) Gualtieri, M., Mantecca, P., Cetta, F., and Camatini, M. (2008) Organic compounds in tire particle induce reactive oxygen species and heat-shock proteins in the human alveolar cell line A549. Environ. Int. 34, 437−442. (33) Oh, M., Kim, A. R., Park, Y. J., Lee, S. Y., and 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. 723, 142−151. (34) Cazier, F., Dewaele, D., Delbende, A., Nouali, A., Garçon, G., Verdin, A., Courcot, D., Bouhsina, S., and Shirali, P. (2011) Sampling analysis and characterization of particles in the atmosphere of rural, urban and industrial areas. Proc. Environ. Sci. 4, 218−227. (35) Diémé, D., Garçon, G., Cabral-N’Dior, M., Billet, S., Verdin, A., Cazier, F., Courcot, D., Diouf, A., and Shirali, P. (2011) Relationship between physicochemical characterization and toxicity of fine Particulate Matter (PM2.5) collected in Dakar City (Senegal). Environ. Res.,in press. (36) Powley, M. W., and Carlson, G. P. (1999) Species comparison of hepatic and pulmonary metabolism of benzene. Toxicology 139, 207−217. (37) Garçon, G., Zerimech, F., Hannothiaux, M. H., Gosset, P., Martin, A., Marez, T., and Shirali, P. (2001) Antioxidant defense disruption by polycyclic aromatic hydrocarbons-coated onto Fe2O3 particles in human lung cells (A549). Toxicology 166, 129−137. (38) Garçon, G., Gosset, P., Zerimech, F., Grave-Descampiaux, B., and Shirali, P. (2004) Effect of Fe2O3 on the capacity of benzo(a)pyrene to induce polycyclic aromatic hydrocarbon-metabolizing enzymes in the respiratory tract of Sprague-Dawley rats. Toxicol. Lett. 150, 179−189. (39) Garçon, G., Gosset, P., Maunit, B., Zerimech, F., Creusy, C., Müller, J. F., and Shirali, P. (2004) Influence of iron (56Fe2O3 or 54 Fe2O3) in up-regulation of polycyclic aromatic hydrocarbonmetabolizing cytochrome P4501A1 hemoproteins. J. Appl. Toxicol. 24, 249−256. (40) Naha, P. C., Davoren, M., Lyng, F. M., and Byrne, H. J. (2011) Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells. Toxicol. Appl. Pharmacol. 246, 91−99. (41) Toyokuni, S., Tanaka, T., Hattori, Y., Nishiyama, Y., Yoshida, A., Uchida, K., Hiai, H., Ochi, H., and Osawa, T. (1997) Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N451: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab. Invest. 76, 365−374. (42) Garçon, G., Shirali, P., Garry, S., Fontaine, M., Zerimech, F., Martin, A., Haguenoer, J. M., and Hannothiaux, M. H. (2000) Polycyclic aromatic hydrocarbons-coated onto Fe2O3 particles: assessment of cellular membrane damage and antioxidant system disruption in human epithelial lung cells (L132) in culture. Toxicol. Lett. 117, 25−35.

PM2.5 components in the pro-inflammatory response induced in airway epithelial cells. Toxicology 26, 126−135. (13) Danielsen, P. H., Møller, P., Jensen, K. A., Sharma, A. K., Wallin, H., Bossi, R., Autrup, H., Mølhave, L., Ravanat, J. L., Bried, J. J., de Kok, T. M., and Loft, S. (2011) Oxidative stress, DNA damage, and inflammation induced by ambient air and wood smoke particulate matter in human A549 and THP-1 cell lines. Chem. Res. Toxicol. 24, 168−184. (14) Garçon, G., Dagher, Z., Zerimech, F., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., and Shirali, P. (2006) Dunkerque city air pollution particulate matter-induced cytotoxicity, oxidative stress and inflammation in human epithelial lung cells (L132) in culture. Toxicol. in Vitro 20, 519−528. (15) Gualtieri, M., Øvrevik, J., Holme, J. A., Perrone, M. G., Bolzacchini, E., Schwarze, P. E., and Camatini, M. (2010) Differences in cytotoxicity versus pro-inflammatory potency of different PM fractions in human epithelial lung cells. Toxicol. in Vitro 24, 29−39. (16) Billet, S., Abbas, I., Le Goff, J., Verdin, A., Andre, V., Lafargue, P. E., Hachimi, A., Cazier, F., Sichel, F., Shirali, P., and Garçon, G. (2008) Genotoxic potential of polycyclic aromatic hydrocarbons-coated onto airborne particulate matter (PM2.5) in human lung epithelial A549 cells. Cancer Lett. 270, 144−155. (17) Billet, S., Garçon, G., Dagher, Z., Verdin, A., Ledoux, F., Courcot, D., Aboukais, A., and Shirali, P. (2007) Ambient particulate matter (PM2.5): physicochemical characterization and metabolic activation of the organic fraction in human lung epithelial cells (A549). Environ. Res. 105, 212−223. (18) Abbas, I., Saint-Georges, F., Billet, S., Verdin, A., Mulliez, P., Shirali, P., and Garçon, G. (2009) Air pollution Particulate Matter (PM2.5)-induced gene expression of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in an in vitro coculture lung model. Toxicol. in Vitro 2, 37−46. (19) Abbas, I., Garçon, G., Saint-Georges, F., Billet, S., Verdin, A., Gosset, P., Mulliez, P., and Shirali, P. (2010) Occurrence of molecular abnormalities of cell cycle in L132 cells after in vitro short-term exposure to air pollution PM2.5. Chem.-Biol. Interact. 188, 558−565. (20) Abbas, I., Garçon, G., Saint-Georges, F., André, V., Gosset, P., Billet, S., Le Goff, J., Verdin, A., Mulliez, P., Sichel, F., and Shirali, P. (2011) Polycyclic aromatic hydrocarbons within airborne particulate matter (PM2.5) produced DNA bulky stable adducts in a human lung cell coculture model. J Appl Toxicol., DOI: 10.1002/jat.1722. (21) André, V., Billet, S., Pottier, D., Le Goff, J., Pottier, I., Garçon, G., Shirali, P., and Sichel, F. (2010) Mutagenicity and genotoxicity of PM2.5 issued from an urbano-industrialized area of Dunkerque (France). J. Appl. Toxicol. 31, 131−138. (22) Dagher, Z., Garçon, G., Gosset, P., Ledoux, F., Surpateanu, G., Courcot, D., Aboukais, A., Puskaric, E., and Shirali, P. (2005) Proinflammatory effects of Dunkerque city air pollution particulate matter 2.5 in human epithelial lung cells (L132) in culture. J. Appl. Toxicol. 25, 166−175. (23) Dagher, Z., Garçon, G., Billet, S., Gosset, P., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., and Shirali, P. (2006) Activation of different pathways of apoptosis by Dunkerque city air pollution particulate matter (PM2.5) in human epithelial lung cells (L132) in culture. Toxicology 225, 12−24. (24) Dagher, Z., Garçon, G., Billet, S., Verdin, A., Ledoux, F., Courcot, D., Aboukais, A., Puskaric, E., and Shirali, P. (2007) Role of Nuclear Factor-kappa B Activation in the adverse effects induced by air pollution particulate matter (PM2.5) in human epithelial lung cells (L132) in culture. J. Appl. Toxicol. 27, 284−290. (25) Saint-Georges, F., Abbas, I., Billet, S., Verdin, A., Gosset, P., Mulliez, P., Shirali, P., and Garçon, G. (2008) Gene expression induction of volatile organic compound and/or polycyclic aromatic hydrocarbon-metabolizing enzymes in isolated human alveolar macrophages in response to airborne particulate matter (PM2.5). Toxicology 244, 220−230. (26) Saint-Georges, F., Garçon, G., Escande, F., Abbas, I., Verdin, A., Gosset, P., Mulliez, P., and Shirali, P. (2009) Role of air pollution particulate matter (PM2.5) in the occurrence of loss of heterozygosity 918



Article

́ R., Ź akov́a, P., Lotkova, H., Kučera, O., and Č ervinkova, (43) Kandar, Z. (2007) Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection. J. Pharmaceut. Biomed. Anal. 43, 1382−1387. (44) McMenamin, M. E., Himmelfarb, J., and Nolin, T. D. (2009) Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection. J. Chromatogr., B 877, 3274−3281. (45) Schwarze, P. E., Ovrevik, J., Hetland, R. B., Becher, R., Cassee, F. R., Lag, M., Lovik, M., Dybing, E., and Refsnes, M. (2007) Importance of size and composition of particles for effects on cells in vitro. Inhal. Toxicol. 19, 17−22. (46) Cho, E. C., Zhang, Q., and Xia, Y. (2011) The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles. Nat. Nanotechnol. 6, 385−391. (47) European Parliament and the Council of the European Union (2008) Air Quality-Existing Legislation. Directive 2008/50/EC of the European Parliament and of the Council of 21 May 2008 on Ambient Air Quality and Cleaner Air for Europe, http://eur-lex.europa.eu/ LexUriServ/LexUriServ.do?uri=CELEX:32008L0050:EN:NOT (retrieved 01.12.2011). (48) US Code of Federal Regulations (2008) Title 40: Protection of Environment Chapter I, Environmental Protection Agency, continued Part 50, national primary and secondary ambient air quality standards, http://www.access.gpo.gov/nara/cfr/waisidx_08/40cfr50_08.html (retrieved 01.12.2011). (49) World Health Organization (2006) World Health Organization Country Health System Fact Sheet 2006, http://www.afro.who.int/ home/countries/fact_sheets/ guinea.pdf (retrieved 01.12.2011). (50) World Health Organization (2006) World Health Organization, Public Health and Environment, PHE, Air Quality Guidelines: Global Update 2005, WHO Air Quality Guidelines for Particulate Matter, Ozone, Nitrogen Dioxide and Sulfur Dioxide, WHO/SDE/PHE/ OEH/06.02, http://www.who.int/phe/health_topics/outdoorair_ aqg/en/ (retrieved 01.12.2011). (51) Sillanpäa,̈ M., Hillamo, R., Saarikoski, S., Frey, A., Pennanen, A., Makkonen, U., Spolnik, Z., Van Grieken, R., Braniš, M., Brunekreef, B., Chalbot, M. C., Kuhlbusch, T., Sunyer, J., Kerminen, V. M., Kulmala, M., and Salonen, R. O. (2006) Chemical composition and mass closure of particulate matter at six urban sites in Europe. Atmos. Environ. 40, 212−223. (52) Vakharia, D. D., Liu, N., Pause, R., Fasco, M., Bessette, E., Zhang, Q. Y., and Kaminsky, L. S. (2001) Effect of metals on polycyclic aromatic hydrocarbon induction of CYP1A1 and CYP1A2 in human hepatocyte cultures. Toxicol. Appl. Pharmacol. 170, 93−103. (53) Vakharia, D. D., Liu, N., Pause, R., Fasco, M., Bessette, E., Zhang, Q. Y., and Kaminsky, L. S. (2001) Polycyclic aromatic hydrocarbon/metal mixtures: effect on PAH induction of CYP1A1 in human HEPG2 cells. Drug Metab. Dispos. 29, 999−1006. (54) Totlandsdal, A. I., Cassee, F. R., Schwarze, P. E., Refsnes, M., and Làg, M. (2010) Diesel exhaust particles induce CYP1A1 and proinflammatory responses via differential pathways in human bronchial epithelial cells. Part. Fibre Toxicol. 7, 41. (55) Araujo, J. A., and Nel, A. E. (2009) Particulate matter and atherosclerosis: role of particle size, composition and oxidative stress. Part. Fibre Toxicol. 6, 1−19. (56) Rossner, P. Jr., Rossnerova, A., and Sram, R. J. (2011) Oxidative stress and chromosomal aberrations in an environmentally exposed population. Mutat. Res. 707, 34−41. (57) Caldorera-Moore, M., Guimard, N., Shi, L, and Roy, K. (2010) Designer nanoparticles: Incorporating size, shape, and triggered release into nanoscale drug carriers. Expert Opin. Drug Delivery 7 (4), 479− 495. (58) Øvrevik, J., Arlt, V. M., Øya, E., Nagy, E., Mollerup, S., Phillips, D. H., Låg, M., and Holme, J. A. (2010) Differential effects of nitroPAHs and amino-PAHs on cytokine and chemokine responses in human bronchial epithelial BEAS-2B cells. Toxicol. Appl. Pharmacol. 242, 270−280.

(59) Baulig, A., Poirault, J. J., Ausset, P., Schins, R., Shi, T., Baralle, D., Dorlhene, P., Meyer, M., Lefevre, R., Baeza-Squiban, A., and 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. (60) Alfaro-Moreno, E., Ponce-de-León, S., Osornio-Vargas, A. R., García-Cuellar, C., Martínez, L., and Rosas, I. (2007) Potential toxic effects associated to metals and endotoxin present in PM10: an ancillary study using multivariate analysis. Inhal. Toxicol. 19, 49−53. (61) Osornio-Vargas, A. R., Serrano, J., Rojas-Bracho, L., Miranda, J., García-Cuellar, C., Reyna, M. A., Flores, G., Zuk, M., Quintero, M., Vázquez, I., Sánchez-Pérez, Y., López, T., and Rosas, I. (2011) In vitro biological effects of airborne PM2.5 and PM10 from a semi-desert city on the Mexico−US border. Chemosphere 83, 618−626. (62) Quintana, R., Serrano, J., Gómez, V., de Foy, B., Miranda, J., Garcia-Cuellar, C., Vega, E., Vázquez-López, I., Molina, L. T., Manzano-León, N., Rosas, I., and Osornio-Vargas, A. R. (2011) The oxidative potential and biological effects induced by PM10 obtained in Mexico City and at a receptor site during the MILAGRO Campaign. Environ. Pollut. 159, 3446−3454. (63) Rosas Perez, I., Serrano, J., Alfaro-Moreno, E., Baumgardner, D., Garcia-Cuellar, C., Martin, Miranda., del Campo, J. M., Raga, G. B., Castillejos, M., Colin, R. D., and Osornio-Vargas, A. R. (2007) between PM10 composition and cell toxicity: a multivariate and graphical approach. Chemosphere 67, 1218−1228. (64) Veranth, J. M., Moss, T. A., Chow, J. C., Labban, R., Nichols, W. K., Walton, J. C., Watson, J. G., and Yost, G. S. (2006) Correlation of in vitro cytokine responses with the chemical composition of soil-derived particulate matter. Environ. Health Perspect. 114, 341−349.

919


Prooxidant and Proinflammatory Potency of Air Pollution Particulate

Recommend Documents