Polycyclic aromatic hydrocarbons are associated with ...

Science of the Total Environment 574 (2017) 1649–1658

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Polycyclic aromatic hydrocarbons are associated with increased risk of chronic obstructive pulmonary disease during haze events in China Lingyan Yang a,1, Wen-Cheng Wang b,1, Shih-Chun Candice Lung b, Zhelin Sun a, Chongjun Chen c, Jen-Kun Chen d, Qiang Zou e, Yu-Hsin Lin f, Chia-Hua Lin g,⁎ a

Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China Research Center for Environmental Changes, Academia Sinica, Taipei 11529, Taiwan c School of Environmental Science and Engineering, Suzhou University of Science and Technology, Suzhou 215009, China d Institute of Biomedical Engineering & Nanomedicine, National Health Research Institutes, Miaoli 35053, Taiwan e Suzhou Environmental Monitor Center, Suzhou 215004, China f Department of Food and Beverage Management, Taipei College of Maritime Technology, Taipei 11174, Taiwan g Department of Biotechnology, National Formosa University, Yunlin 63208, Taiwan b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• The deterioration of air quality is an increasing concern in China. • The normal human lung epithelial cells were used to elucidate the association between COPD and the components of PM2.5. • The emission of PAHs with high BaPequivalent concentrations should be strictly controlled during the haze in China.

a r t i c l e

i n f o

Article history: Received 4 June 2016 Received in revised form 28 July 2016 Accepted 31 August 2016 Available online 7 September 2016 Editor: D. Barcelo Keywords: PM2.5 Polycyclic aromatic hydrocarbons

a b s t r a c t Although exposure to particulate matter with a diameter of b2.5 μm (PM2.5) is associated with chronic obstructive pulmonary disease (COPD), the major components of PM2.5 in COPD pathogenesis are controversial. Here we employed the human lung epithelial cell line BEAS-2B to elucidate the association between COPD and the organic and water-soluble components of PM2.5. We found that the PM2.5 organic extract was a potential major risk factor for pulmonary epithelial barrier dysfunction through the depletion of proteins from the zonula occludens. This extract induced severe oxidative stress that increased DNA damage and the production of proinflammatory cytokines by BEAS-2B cells as well as decreased α1-antitrypsin expression, suggesting a mechanism that increases the risk of COPD. These effects were mainly mediated by polycyclic aromatic hydrocarbons (PAHs) through the aryl hydrocarbon receptor pathway. PAHs with high benzo(a)pyrene (BaP)-equivalent concentrations, but not major PAH components, have an increased risk of causing COPD, suggesting that BaP-equivalent concentrations

⁎ Corresponding author. E-mail address: [email protected] (C.-H. Lin). 1 Lingyan Yang and Wen-Cheng Wang contributed equally to this work.

http://dx.doi.org/10.1016/j.scitotenv.2016.08.211 0048-9697/© 2016 Elsevier B.V. All rights reserved.

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L. Yang et al. / Science of the Total Environment 574 (2017) 1649–1658

Oxidative stress COPD Aryl hydrocarbon receptor

represent a PM2.5-induced COPD risk metric, which may contribute to provide a rationale for the remediation of air pollution. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The deterioration of air quality is an increasing concern in China, particularly because only five of the largest 500 cities in China meet the World Health Organization's (WHO) air quality standards (Asian-Development-Bank, 2013; Li and Zhang, 2014). Haze is the most serious environmental issue, and its main component is airborne fine particulate matter b2.5 μm in diameter (PM2.5). The lungs are a primary target of PM2.5 (Schwarze et al., 2006) and exposure to PM2.5 increases the risk of acute and chronic respiratory disease and lung cancer (Dominici et al., 2006; Ni et al., 2015; Zhou et al., 2015). Chronic obstructive pulmonary disease (COPD) is a significant public health issue in China and is predicated to represent the third and fourth leading causes of death in rural and urban areas, respectively (Fang et al., 2011). Therefore, it is important to define the association between COPD and PM2.5 exposure and the mechanisms of PM2.5-induced COPD. The development of biomarkers is required to develop novel therapeutics and risk-assessment methodologies. Generations of reactive oxygen species (ROS) by PM2.5 have drawn much attention in recent years as one of the prime bases for PM2.5 toxicity. Evidence indicates that ROS are produced from the surface of PM2.5 that absorbs pollutants such as polycyclic aromatic hydrocarbons (PAHs) and transition metals (Zuo et al., 2011; Zuo et al., 2012). Interactions of PAHs with the aryl hydrocarbon receptor (AhR) and the subsequent activation of cytochrome P450 1A1 (CYP1A1) significantly contribute to the induction of ROS through the P450 catalytic cycle (Kopf and Walker, 2010). PAHs widely vary in their ROS inducing ability. Furthermore, ROS are formed via Fenton or Fenton-like reactions (Lloyd and Phillips, 1999). The damage caused by ROS to DNA, proteins and lipids is implicated in adverse effects on health (Rahman, 2007). Moreover, inflammation contributes to the pathogenesis of human diseases (MacNee, 2000), and excessive ROS levels trigger inflammatory responses through the activation of transcription factors that increase the synthesis of proinflammatory cytokines (Riva et al., 2011). These cytokines may enhance local and systemic inflammatory responses and contribute to COPD and asthma (Lodovici and Bigagli, 2011; MacNee, 2001; Oh et al., 2011). Reduced intake of antioxidants may influence COPD (Barnes et al., 2003; Schunemann et al., 2001). It seems that the accumulation of ROS has important consequences for the pathogenesis of COPD (Zuo et al., 2014). The airway epithelial cell serves as a natural barrier, preventing the entry of inhaled xenobiotics into tissues (Gao et al., 2015). However, excessive accumulation of ROS decreases the tight junction protein levels such as zona occludens (ZO) and occludin, resulting in further disruption of the barrier in the lung epithelium (Chen et al., 2015). An aberrant epithelial barrier may serve as a potential marker to identify the early signs of pulmonary disease (Lucas et al., 2009), and exposure to PM2.5 increases the permeability of the epithelial barrier of the lung (Brauner et al., 2009; Jacquemin et al., 2009). An alteration of the protease-antiprotease ratio is an index of pathogenesis, as reported by Barnes et al. (2003), and proteases degrade connective tissue in the lung parenchyma to induce COPD (Sinden et al., 2015). The elastase inhibitor α1-antitrypsin (AAT) encoded by the gene serpin peptidase inhibitor, clade A, member 1 (SERPINA1) protects alveoli and is primarily synthesized by bronchial epithelial cells, hepatocytes and macrophages (Silverman and Sandhaus, 2009; Stoller and Aboussouan, 2005). Oxidation of AAT because of excess ROS levels reduces the ability of AAT to protect barrier integrity and converts AAT into an inducer of proinflammatory cytokines (Zuo et al., 2016). These

responses may accelerate the progression of COPD (Alam et al., 2011; Zuo et al., 2016). The chemical composition of PM2.5 likely contributes to its adverse health effects (Bell et al., 2009; Lodovici and Bigagli, 2011; Oh et al., 2011). Although various studies demonstrate the adverse effects of PM2.5 extracts (Huang et al., 2014; Oh et al., 2011), limited studies propose their role in COPD. Therefore, the aim of the present study was to employ the epithelial cell line BEAS-2B, derived from normal human lung tissue, to define the pathogenic risk and mechanism of COPD after exposure to organic and water-soluble extracts of PM2.5. PM2.5 was collected during haze events in the urban area of Suzhou, China to evaluate its effects on lung barrier integrity, oxidative and inflammatory responses, and the expression of early markers of COPD. The present findings indicate that organic components of PM2.5 contribute to COPD pathogenesis and facilitate our understanding of PM2.5 exposure and to evaluate its effect on the risk of COPD. 2. Materials and methods 2.1. Chemicals Thiazolyl blue tetrazolium bromide (MTT), α-naphthoflavone (αNF), 2′,7′-dichlorofluorescin diacetate (DCFH-DA), propidium iodide (PI), L-ascorbic acid and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lactate dehydrogenase (LDH) cytotoxicity assay kit, anti-β-actin antibody, horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG and HRP-labelled goat antimouse IgG were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti-Bax, anti-Bcl2 and anti-AAT antibodies were purchased from ImmunoWay Biotechnology Company (Newark, DE, USA). LHC-9 medium, phosphate buffered saline (0.01 M, pH 7.2), and TRIzol Reagent were purchased form Gibco (Life Technologies, Thermo Fisher Scientific, MA, USA). The PAH standards Benzo[a]pyrene, Benzo[b + k]fluoranthene, Dibenz[a,h]anthracene and Pyrene were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 2.2. Collection and characterisation of PM2.5 PM2.5 samples from an urban region (31°17′10.16″ N, 120°37′47.49″ E) of Suzhou in Eastern China were collected on glass fibre filters (Whatman, EPM2000) and teflon (quartz) filters (Whatman) using high (1.13 m3/min) (Tisch Environmental, Cleves, OH, USA) and low (10.0 L/min)-volume samplers (Partisol 2300, Thermo). Sampling was performed daily from 25th to 31st December 2013. The quartz filters were preheated at 400 °C for 4 h. All filters used to collect PM2.5 were weighed and conditioned at 20 °C ± 2 °C and 40% ± 5% humidity. The organic components were analysed using gas chromatography–mass spectrometry (Finnigan TSQ700 with a Hewlett Packard Model 5890A gas chromatograph). Inorganic constituents of PM2.5 were analysed using inductively coupled plasma-mass spectrometry (ELEMENT, Finnigan MAT, Bremen, Germany) and ion chromatography (Metrohm IC, Switzerland). 2.3. Preparations of organic and water soluble PM2.5 extracts PM2.5 filters were cut into small pieces and extracted according to Yang et al. (2011) and Courtois et al. (2014) with modifications. The organic extracts of PM2.5 were prepared using dichloromethane in an ultrasonic bath and rotary evaporation to 1–2 mL, evaporated to dry


using nitrogen, and the residues were re-dissolved in DMSO. Water-soluble PM2.5 extracts were extracted using ddH2O in an ultrasonic bath, freeze dried, passed through a 0.22 μm filter and stored at −20 °C. 2.4. Cell culture The human lung epithelial cell line BEAS-2B was maintained in LHC9 medium in a 37 °C incubator in a humidified atmosphere containing 5% CO2. The medium was changed twice weekly, and trypsinised cells were passaged daily. 2.5. MTT and LDH assays Cells were treated with PM2.5 extracts (10 and 100 μg/mL) for 24 h, and viability was determined using MTT and LDH assays according to the manufacturer's protocol. The samples were analysed using a spectrophotometer (Victor ×4, 2030 Multilabel Reader, Perkin Elmer), and absorbance at 490 nm was recorded using a 96-well plate reader. Cell viability is expressed as the percentage of controls. 2.6. Measurement of transepithelial electrical resistance (TEER) After 24 h of exposure to PM2.5 extracts (10 and 100 μg/mL), TEER was measured with a Millicell® ERS-2 Volt-Ohm meter (Millipore) to evaluate the epithelial cell barrier integrity. The values of barrier resistance (ohm/cm2) were obtained after subtracting the resistance of the blank membrane. Three independent biological replicates were performed. 2.7. DCFH-DA assay ROS levels were determined according to Lin and Lin (2006) with modifications. BEAS-2B cells were seeded in 96-well plates (8 × 103 cells per well) for 24 h and then treated with PM2.5 extracts (10 or 100 μg/mL) for 24 h. Fresh media containing 5 μM DCFH-DA were added for 0.5 h at 37 °C. ROS generation relative to controls was determined using a fluorescence plate reader (λex = 485 nm, λem = 535 nm). H2O2 (100 mM) was used as a positive control.

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denaturation for 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Melting curve analysis was performed to characterise the amplicons. Values were obtained from the threshold cycle (Ct) number. The relative target gene mRNA levels were derived using the equation 2−ΔΔCt, where ΔΔCt = ΔCt target gene − ΔCt GAPDH. Data represent the ratio between test and control values. 2.10. Western blotting BEAS-2B cells were treated with PM2.5 extracts (10 or 100 μg/mL) for 24 h, washed three times with cold PBS and lysed in 200 μL of protein extraction buffer (1 M Tris-HCl, pH 7.9, 3 M NaCl, 1% aprotinin, 2 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol) for 30 min on ice. The lysate was centrifuged at 12,000g for 20 min at 4 °C and the supernatant was subjected to Western blotting. Lysate protein concentrations were measured using the BCA protein assay kit (Beyotime, Shanghai, China). Proteins (40 μg/lane) were electrophoretically separated using 6–12% (w/v) SDS-PAGE gradient gels at 100 V and electroblotted onto PVDF membranes. Membranes were blocked for 2 h in 5% non-fat milk in TBS. The Bcl2, Bax, Heme oxygenase 1 (HO-1), ZO-1, ZO-2 and AAT levels were detected using the cognate antibodies (1:1000), respectively. Secondary anti-mouse or anti-rabbit antibodies (1:1000) were coupled to HRP. An LAS4000EPUVmini (FUJIFILM, JAPAN) was used to quantitate chemiluminescence. The membranes were probed with an anti-β-actin antibody as a loading control. 2.11. ELISA assay After 24 h of exposure to PM2.5 extracts (10 or 100 μg/mL), the cell culture medium was collected for analysis of oxidised α1-antitrypsin (Ox-AT) according to the manufacturer's protocol (Sinobestbio, Shanghai, China). Absorbance was recorded at 450 nm using an ELISA plate reader (Victor ×4, 2030 Multilabel Reader, Perkin Elmer). 2.12. Statistical analysis The statistical significance of differences between experimental and control data were evaluated using the Student's t-test. Statistical significance was defined as *p b 0.05.

2.8. Comet assay 3. Results DNA single-strand breaks were quantified using the Comet assay (Tice et al., 2000). After 24 h of exposure to PM2.5 extracts (10 or 100 μg/mL), cells were mixed with 1.2% low-melting agarose and added to slides coated with 1% normal-melting agarose. Cells were lysed in 4 °C lysis buffer (pH 10) for 1 h and subjected to electrophoresis at 25 V/300 mA for 20 min. The slides were soaked twice in 0.4 M Trizma base, pH 7.5, 4 °C for 15 min. DNA was stained with propidium iodide (5 μg/mL) and analysed using a fluorescence microscope (Zeiss A1; Zeiss, Germany). Randomly captured cells from each sample were analysed using CASP software (University of Wroclaw, Poland). DNA migration was measured by calculating the percentage of DNA in the tail (%Tail DNA). H2O2 (100 mM) was used as a positive control. 2.9. Quantitative real-time PCR assay Cells were exposed to PM2.5 extracts (10 or 100 μg/mL) for 24 h, total RNA was prepared using RNAzol and cDNA was synthesized from 1 μg RNA using the HiFiScript 1st Strand cDNA Synthesis kit (ComWin Biotech, Beijing, China). Primers for tumour necrosis factor-alpha (TNFα), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), CYP1A1, serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and glyceraldehyde-3phosphate dehydrogenase (GAPDH) are shown in Table S1. Quantitative real-time PCR was performed using the UltraSYBR Mixture (With Rox; ComWin Biotech) and data were analysed using an ABI 7500 RealTime PCR System (Applied Biosystems, Foster City, CA, USA) as follows:

3.1. Level and composition of PM2.5 The daily mean PM2.5 concentration was 129.4 ± 73.5 μg/m3 (50– 261 μg/m3) during the sampling period (Table S2). The daily PM2.5 alveolar deposition dose for humans was 401.1 ± 227.9 μg/day (154.9– 808.8 μg/day) (Table S1 and S2). The daily mean PM2.5-bound benzo[a]pyrene (BaP) concentration was 7.47 ng/m3 (Table S3). The chemical composition of PM2.5 is shown in Table S3. Among 18 PAHs, fluoranthene (5.71 ng/m3), pyrene (6.14 ng/m3), chrysene (5.41 ng/m3), benzo[b + k]fluoranthene (9.96 ng/m3), benzo[e]pyrene (4.73 ng/m3), indeno[123-cd]pyrene (4.10 ng/m3) and benzo[ghi]perylene (5.08 ng/m3) were the major species. The data show that Fe (342.57 μg/m3), Zn (171.16 μg/m3), Al (259.70 μg/m3) and Pb (102.96 μg/m3) were the most abundant elemental metals 3 2− (Table S4). NO− (19.45 μg/m3), and NH+ 3 (28.78 μg/m ), SO4 4 (15.37 μg/m3) were the most abundant ions (Table S5). 3.2. Epithelial barrier dysfunction induced by PM2.5 extracts To determine whether PM2.5 extracts can disrupt the epithelial barrier, BEAS-2B cells were exposed to 10 or 100 μg/mL of PM2.5 organic and water-soluble extracts for 24 h. After exposure, TEER, ZO-1 and ZO-2 were analysed by Millicell® ERS-2 Volt-Ohm meter and western blotting. TEER, ZO-1 and ZO-2 levels were significantly reduced in the

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epithelial barrier and BEAS-2B cells treated with the organic solvent extracts, but not water-soluble extracts (Fig. 1). The cytotoxicity of PM2.5 extracts to BEAS-2B cells was also assessed using MTT assay. A significant reduction in cell viability was only observed in cells treated with the PM2.5 organic extract (100 μg/mL; Fig. 2A). The effect of PM2.5 extracts on cell membrane integrity was assessed using an LDH assay. A significant increase in LDH activity was detected only when cells were treated with the organic extract (100 μg/mL; Fig. 2B). Only the PM2.5 organic extracts induced apoptosis, indicated by the significant shift in the Bax:Bcl2 ratio (Fig. 2C and D).

treated with the PM2.5 organic extract, but not by the water-soluble extract (Fig. 5A), and SERPINA1 expression was correlated with AAT levels. Furthermore, exposure to PM2.5 organic extracts significantly increased the level of Ox-AT (Fig. 5B). The AAT levels were significantly decreased after exposure to PM2.5 organic extracts (Fig. 5C and D). In order to further confirmation that oxidative stress leads to protein damage nonspecifically, 100 mM H2O2 was used as a positive control and 50 ng/ mL L-ascorbic acids treated with PM2.5 organic extracts as a negative control. The results reveal that the depletion of AAT and ZO is mainly as a result of oxidative stress (Fig. S1).

3.3. Oxidative and inflammatory responses induced by PM2.5 extracts 3.5. Effects of PAHs on the expression of COPD markers Oxidative and inflammatory responses in BEAS-2B cells after exposure to PM2.5 extracts were measured using DCFH-DA, Western blotting, real-time RT-PCR and comet assays. Although significant ROS formation mainly occurred in BEAS-2B cells treated with PM2.5 organic extracts (100 μg/mL), other treatments were weakly positive (Fig. 3A, and B). Significant increases in HO-1 activity were detected under all conditions (Fig. 3D and E). We observed significant DNA damage in response to PM2.5 organic extract (Figs. 3C and S3). When we studied the effects of PM2.5 extracts on proinflammatory cytokine levels, we detected increased TNF-α, IL-1β and IL-8 levels (Fig. 4) that were higher in organic extract-treated cells. 3.4. Association of PM2.5 components with COPD pathogenesis Oxidation of AAT, a neutrophil elastase inhibitor within the lung, may contribute to the pathogenesis of COPD (Li et al., 2009). Therefore, we determined the levels of biochemical markers of early COPD, such as SERPINA1, AAT and Ox-AT, produced by BEAS-2B cells exposed to PM2.5 extracts. SERPINA1 mRNA levels were significantly decreased in cells

Our result showed that BEAS-2B cells treated with the PM2.5 organic extract expressed CYP1A1 mRNA (Fig. 6A). To assess the role of PAHs in mediating COPD pathogenesis, the cells were incubated with 100 μg/mL PM2.5 organic extract in the presence of the AhR inhibitor α-NF. The level of biochemical markers of COPD, CYP1A1 mRNA expression and ROS was markedly inhibited by the PM2.5 organic extract (Figs. 6 and S2). We tested the ability of individual PAHs present in high BaPequivalent concentrations (Table S3) on the induction of expression of early markers of COPD at concentrations equivalent to those present in PM2.5 organic extracts (100 μg/mL). Pyrene was used as the negative control. Most individual PAHs except for pyrene induced significant increases in the surrogate marker levels for the risk of COPD (elevation of Ox-AT expression, reduction of SERPINA1 mRNA and AAT expression). BaP, dibenz[a,h]anthracene and benzo[b]fluoranthene had the greatest effects (Fig. 7). The decrease and increase in AAT and CYP1A1 mRNA expression, respectively, were significantly correlated (r2 = 0.97) (S3).

Fig. 1. Effect of 24 h exposure to PM2.5 extracts on markers of epithelial barrier dysfunction in BEAS-2B cells. (A) TEER values. (B) ZO-1 and ZO-2 expression. (C) Quantitative analysis of ZO1 expression. (D) Quantitative analysis of ZO-2 expression. Data represents the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicates statistically significant differences from the control.


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Fig. 2. Effect of 24 h exposure to PM2.5 extracts on cytotoxicity and apoptotic response of BEAS-2B cells. (A) Cell viability. (B) Cell membrane integrity. (C) Bcl2 and Bax expression. (D) The Bax:Bcl2 ratio (index of apoptosis). Data represents the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control.

4. Discussion Our PM2.5 samples were collected during intensive haze events. The daily mean PM2.5 concentration (129.4 ± 73.5 μg/m3, 50–261 μg/m3) was significantly higher than the Chinese national and WHO Interim target-1 standards (75 μg/m3) for PM2.5 (Zhang et al., 2015). A 10 μg/ m3 increase in the concentrations PM2.5 increases by 6%, 15%–21% and 12%–14% non-accidental mortality, lung cancer mortality and cardiovascular mortality, respectively (Chen et al., 2008). The daily PM2.5 alveolar deposition dose for humans was 401.1 ± 227.9 μg/day, which is equivalent to 196.6 ng/day for mice (S1). Gorr et al. (2014) have shown that exposure to PM2.5 (49.1 ng/day) early in life induces persistent cardiac dysfunction in mice (Gorr et al., 2014), suggesting that residents of our sampling area are at a risk for disease. However, the toxicity of PM2.5 is mainly related to its composition (Harrison and Yin, 2000). Therefore, we evaluated the carcinogenic potential of PM2.5 using the benzo[a]pyrene (BaP)-equivalent concentration of BaP (Collins et al., 1998), which is calculated by multiplying the concentration of each PAH by their toxic equivalency factors (TEFs) relative to BaP (Table S3). The daily mean PM2.5-bound BaP concentration was significantly higher than the Chinese national standard value (2.5 ng/m3) (Bandowe et al., 2014), suggesting that residents of our sampling area were at a risk of cancers (Table S3). Fluoranthene, pyrene, chrysene, benzo[b + k]fluoranthene, benzo[e]pyrene, indeno[123-cd]pyrene and benzo[ghi]perylene were the major species in our PM2.5 samples (Table S3). The concentrations of all PAHs were lower than the Chinese national standard (10 ng/m3) (Li et al., 2014). High fluoranthene and pyrene levels are associated with coal combustion emissions (Liu et al., 2003); benzo[b + k]fluoranthene, benzo[e]pyrene, indeno[123-cd]pyrene and benzo[ghi]perylene are associated with vehicular emissions (Slezakova et al., 2013); and chrysene is associated with combustion of natural

gas (Simcik et al., 1999). Natural gas is commonly used as fuel in taxis in China and for domestic cooking. Fe, Zn, Al and Pb were the most abundant elemental metals in our PM2.5 samples (Table S4). Most of these metals are associated with motor vehicle emissions (Liu et al., 2− + 2014). NO− 3 , SO4 , and NH4 were the most abundant ions (Table S5), which are converted from NOx, SO2 and NH3, respectively, on the surface of PM2.5 components (Li et al., 2007) and are associated with vehicle emissions (Ameur-Bouddabbous et al., 2012; Chen et al., 2003; Durbin et al., 2002). These data indicate that motor vehicle emissions are a major contributor to PM2.5. Evidence indicates that the chemical composition of PM2.5 contributes to its adverse effects (Bell et al., 2009; Oh et al., 2011). Therefore, we investigated the ability of PM2.5 water-soluble and organic extracts to induce markers of pulmonary adverse effects in BEAS-2B cells. The pulmonary epithelial barrier is the first line of defence against invasion by PM2.5, and barrier dysfunction alters pulmonary permeability and increases susceptibility to lung injury (You et al., 2012; Zhang et al., 2007). The tight junction is a primary barrier that prevents molecules from diffusing across the epithelial sheet between adjacent cells. The tight junction protein ZO seals the paracellular pathway to form the basic structure of the tight junction (Denker and Nigam, 1998) and changes in ZO expression in response to various stimuli may disrupt the pulmonary epithelial barrier (You et al., 2012). In our experiment, the TEER and tight junction proteins (ZO-1 and ZO-2) were only significantly reduced in cells treated with the organic solvent extracts (Fig. 1). These data indicate that PM2.5 organic extracts may disrupt the pulmonary barrier by depleting tight junction proteins. Although controversial, epithelial cell apoptosis was proposed as a major contributor to epithelial barrier dysfunction (Pastorelli et al., 2013). In our present study, a break in the integrity of cell membrane was associated with apoptosis in BEAS-2B cells treated with the high dose of PM2.5 organic extract (Fig. 2). Although the low dose of PM2.5

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Fig. 3. Effect of 24 h exposure of PM2.5 extracts exposure on the oxidative response of BEAS-2B cells. (A) and (B) ROS formation induced by PM2.5 extracts. (C) Oxidative DNA strand breaks. (D) HO-1 expression. (E) Quantitative analysis of HO-1 expression. Data represent the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control.

organic extract induced only a slightly, but not significantly, cytotoxic effects (Fig. 2A and B), epithelial barrier dysfunction was detected, implying that apoptosis was a potential major contributor to epithelial barrier dysfunction (Figs. 1 and 2). Therefore, the organic components on the PM2.5 surface were the major contributors to PM2.5 induced-pulmonary epithelial barrier dysfunction. Oxidative stress and inflammation are the hallmarks of pulmonary disease (Valavanidis et al., 2013). In our present study, PM2.5 organic extracts induced higher ROS levels (Fig. 3A and B). HO-1 is a ubiquitous inducible enzyme in response to oxidative stress (Lin et al., 2011). HO-1 activity was higher in cells exposed to PM2.5 organic extracts because

of homeostatic mechanisms that maintain physiological redox potential (Fig. 3D and E). However, exposure of cultures to the 100 μg/mL of PM2.5 organic extracts caused massive ROS formation resulting in oxidative stress and potential oxidative damage to DNA. Indeed, extensive DNA strand breaks were observed after exposure to high dose of PM2.5 organic extract (Figs. 3C and S3). We also observed significant DNA damage in response to 10 μg/mL of the PM2.5 organic extract (Figs. 3C and S3). This effect might be explained by the conversion of PAHs to active metabolites that reacted with DNA (Ran et al., 2008). Unrepaired DNA damage leads to mutations that may cause lung cancer, a common complication of COPD (Sin et al., 2007). We also detected increased TNF-α, IL-1β and


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Fig. 4. Effect of 24 h exposure to PM2.5 on markers of inflammation expressed by BEAS-2B cells. (A) TNF-α mRNA expression. (B) IL-1β mRNA expression. (B) IL-8 mRNA expression. Data represents the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control.

Fig. 5. Effect of 24 h exposure of PM2.5 extracts on the levels of predictive biomarkers for COPD expressed by BEAS-2B cells. (A) SERPINA1 mRNA expression. (B) Ox-AT expression (C) AAT expression. (D) Quantitative analysis of AAT expression. Data represent the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control.

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Fig. 6. Effect of 24 h exposure to PM2.5 organic extracts on (A) CYP1A1 mRNA expression and expression of predictive biomarkers for COPD in the presence of α-NF in BEAS-2B cells. (B) SERPINA1 mRNA expression. (C) AAT expression. (D) Quantitative analysis of AAT expression. (E) Ox-AT expression. Data represent the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control. #p b 0.05 indicates statistically significant differences from cells treated with PM2.5.

IL-8 levels (Fig. 4) that were higher in organic extract-treated cells. Overall, these results indicate that PM2.5 organic extracts play a major role in adverse oxidative response induction in pulmonary epithelial cells. Moreover, these results are consistent with findings that oxidative responses are associated with increased risk of COPD (MacNee, 2000; Ni et al., 2015). Some epidemiological studies show that chronic exposure to air pollution increases the prevalence and incidence of COPD (Andersen et al., 2011; Atkinson et al., 2015). However, most of these epidemiological studies do not define the risk factors and molecular mechanisms of PM2.5-induced COPD. Oxidation of AAT may contribute to the pathogenesis of COPD (Li et al., 2009). In present study, the total protein levels of AAT were significantly decreased due to changes in the level of SERPINA1 gene and Ox-AT protein after epithelial exposure to PM2.5 organic extracts (Fig. 5). These data indicate that PM2.5 organic extracts impaired the normal function of AAT. If AAT does not effectively inhibit neutrophil elastase, excess neutrophil elastase accelerates the elastin degradation in the lung parenchyma, causing an increased risk of COPD (Alam et al., 2011; Li et al., 2009; Taggart et al., 2000). Therefore, organic compounds present in PM 2.5 represent the major contributor to the induction of COPD.

Although individual PAHs are not major constituents of PM2.5 organic extracts, evidence indicates that they adversely affect human health (Chiba et al., 2012). PAHs induce uncoupling of the catalytic cycle of CYP1A1 through AhR to induce oxidative responses in human (Costa et al., 2010). The expression of biochemical markers of COPD and CYP1A1 mRNA was markedly inhibited by the PM2.5 organic extract in the presence of α-NF (Figs. 6), suggesting that PAHs play a major role in the pathogenesis COPD through an AhR-dependent mechanism. Further, we investigated the potential relationships between individual PAHs and COPD. In present study, most high BaP-equivalent concentration of individual PAHs induced significant increases in the surrogate marker levels for the risk of COPD (Fig. 7). The decrease and increase in AAT and CYP1A1 mRNA expression, respectively, were significantly correlated (r2 = 0.97) (S3), indicating that a high BaP-equivalent concentration of an individual PAH increases the risk of COPD. BaP, dibenz[a,h]anthracene and benzo[b]fluoranthene had the greatest effects. Dibenz[a,h]anthracene and benzo[b]fluoranthene are present in vehicular emissions, and BaP is generated by wood combustion (Kulkarni and Venkataraman, 2000). Therefore, we recommend the imposition of stringent regulations to reduce the risk of COPD caused by PM2.5 exposure in the sampling area.


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Fig. 7. Effect of 24 h exposure to individual PAHs on the expression of predictive biomarkers for COPD in BEAS-2B cells. (A) SERPINA1 mRNA expression. (B) Ox-AT expression (C) AAT expression. (D) Quantitative analysis of AAT expression. 1. Control; 2. PM2.5 organic extracts (100 μg/mL); 3. Benzo[a]pyrene (19.2 ng/mL); 4. Benzo[b]fluoranthene (58.6 ng/mL); 5. Dibenz[a,h]anthracene (3.0 ng/mL); 6. Pyrene (45.2 ng/mL). Data represent the mean ± standard deviation of three determinations. *p b 0.05 and **p b 0.01 indicate statistically significant differences from the control.

5. Conclusions In conclusion, we show here that PM2.5 induced markers of oxidative stress through the AhR pathway that deplete ZO and AAT, indicating that PM2.5 increases the risk of COPD. Further, determining the BaPTEF of PAHs may provide more accurate assessment risk of COPD caused by PM2.5 exposure and to screen for major PAHs that mainly contribute to the pathogenesis of COPD. For example, although the PAH concentration of BaP is lower compared with pyrene, it should be remediated before other pollutants. We believe that the metric of the BaP-TEF will provide valuable information to justify the remediation of regional air pollution/emissions. Notes The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements This work was supported by the Academia Sinica thematic project [grant number AS-104-SS-A02] and Suzhou Science & Technology Bureau of China [grant number SS201445]. We also thank Nai-Chun Huang of the Institute of Biomedical Engineering and Nanomedicine (NHRI-BN-104-PP-27) for managing ICP-MS analysis. Appendix A. Supplementary data Supplementary material describes the calculation of the daily PM2.5 alveolar deposition dose, the effective concentrations (EC50) of PM2.5 extracts and the correlation between AAT depletion and induced CYP1A1 expression. Tables S1–S5 present the RT-PCR primer sequences;

the concentration of PAHs, metals and ions in PM2.5; and TEF and BaPequivalent concentration of PAHs in PM2.5. Figs. S1–S3 show the oxidative stress leads to AAT and ZO protein depleted, oxidative response with AhR inhibitor (α-NF), oxidative DNA strand breaks. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.scitotenv.2016.08.211.

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