Winter Polycyclic Aromatic Hydrocarbon-Bound Particulate Matter from

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Winter polycyclic aromatic hydrocarbon (PAH)-bound particulate matter (PM) from peri-urban North China promotes lung cancer cell metastasis Huifeng Yue, Yang Yun, Rui Gao, Guangke Li, and Nan Sang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506280c • Publication Date (Web): 26 May 2015 Downloaded from http://pubs.acs.org on June 2, 2015

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Environmental Science & Technology

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Title page

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Winter polycyclic aromatic hydrocarbon (PAH)-bound particulate matter (PM)

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from peri-urban North China promotes lung cancer cell metastasis

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Huifeng Yue , Yang Yun , Rui Gao, Guangke Li, Nan Sang*

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College of Environment and Resource, Research Center of Environment and Health, Shanxi University,

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Taiyuan, Shanxi 030006, PR China

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* Corresponding author. Tel.: +86-351-7011932

These authors contributed equally to this work

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Fax: +86-351-7011932

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E-mail: [email protected]

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Mailing address: Nan Sang

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College of Environment and Resource,

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Shanxi University, Taiyuan, Shanxi 030006

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People’s Republic of China

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ACS Paragon Plus Environment


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Table of Contents (TOC) Art

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Exposure to winter polycyclic aromatic hydrocarbon (PAH)-bound particulate matter (PM) in peri-urban

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areas, mainly from coal combustion, induces concentration- and time-dependent migration and

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invasion of A549 cells through reactive oxygen species (ROS)-dependent epithelial-to-mesenchymal

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transition (EMT) activation and extracellular matrix (ECM) degradation.

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ABSTRACT: Based on the close relationship between human exposure to high concentrations of small

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particulate matter (PM) and increased lung cancer mortality, PM was recently designated as a Group I

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carcinogen. Considering that PM is highly heterogeneous, the potential health risks of PM promoting

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tumor metastasis in lung cancer, as well as its chemical characteristics, remain elusive. In the present

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study, we collected PM2.5 and PM10 in a peri-urban residential site of Taiyuan and determined the

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concentration and source of polycyclic aromatic hydrocarbons (PAHs). The results indicated that 18

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PAHs, ranging from 38.21 to 269.69 ng/m (for PM2.5) and from 44.34 to 340.78 ng/m (for PM10),

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exhibited seasonal variations, and the PAHs in winter PM mainly originated from coal combustion. We

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calculated the benzo(a)pyrene-equivalent (BaPeq) and found that the PAH-bound PM in winter

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exhibited higher carcinogenic risks for humans. Following this result, in vitro bioassays demonstrated

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that PM2.5 and PM10 induced A549 cell migration and invasion, and the mechanism involved reactive

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oxygen

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extracellular matrix (ECM) degradation. Our data indicate the potential risk for winter PAH-bound PM

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from peri-urban North China promoting lung cancer cell metastasis and reveal a mechanistic basis for

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treating, ameliorating, or preventing outcomes in polluted environments.

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species

(ROS)-mediated

epithelial-to-mesenchymal

3

transition

(EMT)

activation

and

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INTRODUCTION

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Atmospheric particulate matter (PM) has been a global environmental problem due to the

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numerous harmful effects on individuals in developed and developing countries. Epidemiological

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studies have demonstrated a close, quantitative relationship between long-term residential exposure to

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small particulates and increased mortality or morbidity.1-3 In 2013, the International Agency for

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Research on Cancer (IARC) of the World Health Organization (WHO) concluded that outdoor air

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pollution is carcinogenic to humans, and the airborne particulate mixture is most closely associated

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with increased cancer incidence, especially lung cancer.4 The global fraction of adult mortality

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attributable to the anthropogenic component of PM2.5 (95% CI) was 12.8% (5.9-18.5%) for lung

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cancer.5 The Global Burden of Disease study estimated that in 2010, ambient air pollution alone cost

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more than 1.2 million lives and 24 million healthy years of life in China,6 and PM in particular was found

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to be related to a remarkable increase in the prevalence of lung cancer in past decades.

7

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Based on data from 2009, the rising energy demands of China have predominantly led to

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increased ambient PM pollution from coal-fired power plants by approximately 69-76% in

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northern China.8

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National Ambient Air Quality Standards (NAAQS) in one-third of the routinely monitored cities, most of

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which were located in northern China. In northern Chinese cities, although coal combustion has been

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limited and well controlled in past decades, there remains an extensive and dispersive use of coal

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combustion in rural areas that do not have effective control technologies in place, especially for the

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extensive consumption of coal in residential stoves for cooking and heating, particularly in winter.10 The

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situation is worsening with the combination of an exponential population growth and the urbanization

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and industrialization of Taiyuan,

Consequently,

the

mean

PM10

concentrations

exceeded

the

9

11

one of the major cities in China for energy production and chemical 4


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and metallurgical industries.

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PM is a complex mixture of extremely small particles and liquid droplets that consists of a number

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of components, including acids (such as nitrates and sulfates), organic chemicals, metals, and soil or

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dust particles. Several studies have confirmed the pivotal role of the organic compounds adsorbed in

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PM;

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aromatic hydrocarbons (PAHs) and nitro- and oxygenated-PAHs.15-17 Although contributing minimally to

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PM mass (less than 0.1%), PAHs are the major toxic constituents of PM, and they contribute primarily

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to the toxicity of PM10 in urban areas.18 Additionally, the overproduction of reactive oxygen species

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(ROS) by PAHs bound to PM following oxidative stress have been identified as important signals

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modulating the harmful effects of particulate mixture.19-21 Considering that inhalation exposure to

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ambient PAHs is related to increased lung cancer risk,22, 23 it is inevitable that PAH-bound PM plays an

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important role in the increased cancer incidence from PM exposure.

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12-14

other studies have shown that the biological effects of PM are directly related to polycyclic

Tumor migration and invasion at an early stage is the major reason for the high mortality of lung 24

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cancer, especially for non-small-cell lung carcinoma (NSCLC).

However, the biological process

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requires several steps, including vessel formation, cell attachment, invasion, and cell proliferation, and

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is modulated by intricate signals.25 Recently, the activation of the epithelial-to-mesenchymal transition

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(EMT) program and the degradation of basement membranes and stromal extracellular matrix (ECM)

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have been implicated as crucial steps in lung tumor metastasis.26 The EMT process involves the loss of

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E-cadherin (E-cad) expression and the up-regulation of mesenchymal molecular markers, such as

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fibronectin (Fib).27 ECM degradation is characterized by changes in molecular pathways and is

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accompanied by the elevation of matrix metalloproteinase (MMP) expression and the down-regulation

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of metalloproteinase inhibitors (TIMPs).

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Starting in November 2006, the Taiyuan government made various efforts to reduce air pollution, 28

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and energy structure-adjustment strategies were undertaken to accomplish this goal.

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time, 1,235 coal-fired boilers were demolished, 6-km2 coal-free areas were built, and nearly 400 boilers

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were renovated and modified to burn clean fuel.

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12 industrial sources of pollution were shut down in 2005, and a cumulative 121 closures were

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completed by 2012.28 The annual average PM10 concentrations decreased from 196 µg/m3 in 2001 to

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89 µg/m in 2010.

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situation, coupled with the increased morbidity of lung cancer, indicates that the current pollution

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situation is not ideal.

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sources in peri-urban Taiyuan and to clarify the potential capacity and mechanism by which

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PAH-bound PM promotes lung cancer cell metastasis.

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MATERIALS AND METHODS

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Following this

According to the government reports of Taiyuan city,

However, boilers are still used for heating in winter in peri-urban Taiyuan, and this

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Therefore, we sought to analyze the PAH-bound PM concentrations and

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PM Sample Collection. Samplings were performed between 2012 and 2013: in spring, March 1,

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2012 to April 30, 2012; in summer, May 1, 2012 to July 31, 2012; in autumn, August 1, 2012 to October

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31, 2012; and in winter, November 1, 2012 to February 28, 2013 (considering the annual heating period

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in Northern China). The samples were collected onto glass filters (GF/A-1820-090, Whatman, UK)

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using two PM middle volume air samplers (TH-150CIII and TH-150C, Wuhan, China). Then, we pooled

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the PM samples from volume filters collected every Monday, Tuesday, Wednesday, Thursday and

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Friday during a particular season as one group. Following this action, we cut 1/8-volume filters for PAH

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measurement and another 1/8-volume filters for in vitro experiments. Additional details of PM collection

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and filter treatment are shown in the Text and Figure S1 of the Supporting Information (SI).

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Determination of PAHs in PM. The samples were extracted, concentrated and resuspended 6


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according to previously reported methods.32, 33 Subsequently, the concentrations of the following 18

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PAHs in PM2.5 and PM10 were determined using GC-MSD (6890 N, HP; 5973, HP): naphthalene (NA);

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acenaphthylene (ACL); acenaphthene (AC); fluoranthene (FA); phenanthrene (PHE); anthracene (AN);

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fluorene (FL); pyrene (PY); benzo(a)anthracene (BaA); chrysene (CHR); benzo(b)fluoranthene (BbFA);

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benzo(k)fluoranthene (BkFA); benzo(a)pyrene (BaP); benzp(e)pyrene (BeP); indeno(1,2,3-cd)pyrene

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(IP), dibenzo(a,h)anthracene (DBahA); benzo(g,hi)perylene (BghiP); and coronene (COR). The

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detailed procedures are presented in the SI Text.

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Cell Culture and Exposure. The A549 NSCLC cell line, a widely used human lung cancer cell

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model for investigating cancer cell migration and invasion in the malignant progression of human lung

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carcinoma,26, 34-36 was purchased from the Cell Bank of Type Culture Collection (CAS, China) and kept

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in a monolayer culture in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum

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(FBS, Gibco, USA), 100 U/mL streptomycin (Gibco, USA) and 100 U/mL penicillin (Gibco, USA). Cells

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were cultured at 5% CO2 and 95% humidity at 37 °C.37 Cells were divided randomly into control and

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different treatment groups: one control group was incubated only in RPMI 1640, and other groups were

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treated with PM (10 µg/mL) from different seasons or winter PM at different concentrations (0.1, 0.3, 1,

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3 and 10 µg/mL). For the interference experiments, the groups were treated with winter PM (10 µg/mL)

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for 24 h in the absence or presence of a ROS inhibitor, N-acetyl-L-cysteine (NAC, Sigma, USA), which

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was added to the medium 1 h before PM treatment.38 5

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Wound-Healing Assay. To assess cell motility, A549 cells (5 × 10 cells per mL) were seeded in

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24-well plates (Corning, USA) and cultured as confluent monolayers in RPMI 1640 medium without

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FBS. The monolayers were scraped with a sterile white micropipette tip (0 h) to create a denuded zone

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with a constant width and were washed twice with PBS to remove cellular debris.

39

The cells were 7



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exposed to winter PM2.5 and PM10 at various concentrations (0.1, 0.3, 1, 3 and 10 µg/mL) for 6, 12 and

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24 h, and the scratched monolayers were imaged using an inverted microscope (Olympus, Japan) at

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200× magnification in a blinded fashion. The distance between cells in the scraped zone was

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determined, and three independent experiments were performed.

40

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Cell Invasion Assay. Cell invasion assay was conducted to investigate A549 cell invasion using

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24-well Matrigel-coated transwell inserts. Briefly, the upper surface of the filter (8.0-µm pore size) was

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coated with Matrigel before being air-dried overnight at room temperature. A549 cells (5 × 10 cells per

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mL) were seeded into 35-mm dishes, and winter PM2.5 and PM10 were added at a final concentration of

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0.1, 0.3, 1, 3 and 10 µg/mL; or spring, summer and autumn PM2.5 and PM10 were added at a final

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concentration of 10 µg/mL for 24 h prior to invasion. Subsequently, the PM-treated cells were

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harvested, and approximately 8 × 104 cells were added to the upper chamber and cultured in RPMI

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1640 without FBS. RPMI 1640 with 10% FBS was added to the lower chamber. After a 24-h incubation,

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the cells that had migrated through the Matrigel were fixed with 4% paraformaldehyde and stained with

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crystal violet. Migrated cells in randomly selected fields were counted by light microscopy (Olympus,

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Japan) at a magnification of 400x.37

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ROS Assay. After seeding of 5 × 105 cells per mL to 35-mm petri dishes in RPMI 1640 with 10%

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FBS, the cells were treated with winter PM2.5 and PM10 at various concentrations (0.1, 0.3, 1, 3 and 10

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µg/mL) for 6 h. Then, the cells were harvested, washed with PBS twice, resuspended in RPMI 1640

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and incubated with 2′,7′-dichlorofluoresceindiacetate (DCFH-DA, 10 µM) at 37 °C for 30 min in the dark.

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Finally, the cells were washed with PBS, and the formation of the fluorescent-oxidized derivative of

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2′,7′-dichlorofluorescein (DCF) was monitored using a flow cytometer (C6, BD, USA) at an emission

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wavelength of 530 nm and an excitation wavelength of 488 nm. ROS generation was quantified by the 8


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median fluorescence intensity of formed fluorescent compound DCF for 10,000 cells.41, 42 5

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Real-time Quantitative Reverse Transcription-PCR. Approximately 5 × 10 A549 cells per mL

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were seeded in 35-mm petri dishes for 24 h in RPMI 1640 with 10% FBS. Winter PM2.5 and PM10 at

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various concentrations (0.1, 0.3, 1, 3 and 10 µg/mL) in RPMI 1640 without FBS were added to

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PBS-washed cells (2 mL in each well), and cell samples were harvested after 24 h of incubation. To

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determine the mRNA expression of E-cad, Fib, MMP-2 and TIMP-2, total RNA was extracted by TRIzol

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Reagent (Invitrogen, USA), and first-strand complementary DNA (cDNA) was synthesized using the

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reverse transcription kit (TaKaRa, China).43 The primer sequences are shown in Table S1, SI. The

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relative quantification of the gene expression was determined on a qTOWER 2.2 Real-Time PCR

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(Analytik Jena AG, Jena, Germany), with β-actin as an internal control.44 Detailed procedures are

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presented in the SI Text.

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Statistical Analyses. The data are presented as the mean ± standard deviation of three

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independent experiments and were evaluated by one-way ANOVA followed by Fisher's least significant

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difference (LSD) test. Significant differences were established at p < 0.05.

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RESULTS AND DISCUSSION

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Analysis of PAH-bound PM Concentration and Source. To characterize the PAHs in the PM

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samples, we first determined the concentrations of PAH bound to PM. The levels of 18 PAHs are listed

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in Table S2, SI. The total concentration of PAHs in PM2.5 and PM10 ranged 38.21-269.69 ng/m3 and

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44.34-340.78 ng/m , respectively. The mean concentration of PAHs in PM2.5 was at least 10-fold higher

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than that in southern China cities, including Hong Kong (3.35 ng/m3), Xiamen (4.35 ng/m3), and

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Guangzhou (6.77 ng/m3),45 but was not higher than that of other cities (Shenyang, 612.16 ng/m3;

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Anshan, 615.43 ng/m ; Fushun, 1081.71 ng/m ; Jinzhou, 149.68 ng/m ; Dalian, 75.32 ng/m ) in

3

3

3

3

3

9



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Liaoning province – another heavily industrialized site of northern China.46 Meanwhile, the mean

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concentration of PAHs in PM10 observed in this study was higher than that of many cities, including

194

Nanjing (80.60 ng/m3),47 Chiang Mai (14.50 ng/m3),48 Shanghai (64.85 ng/m3),49 Tianjin (116.00

195

ng/m ),

196

ng/m3), and Dalian (23.12 ng/m3).46 The highest concentrations of PAHs in PM2.5 and PM10 were

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observed in winter, and the lowest concentrations were observed in summer. Similar seasonal trends

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have also been observed in some Asian cities such as Guangzhou,

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of Liaoning,46 Taichung,54 Shizuoka,55 Zonguldak,56 Delhi57 and Kaohsiung58 and cities on other

200

continents, such as Atlanta

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concentration of PAHs in PM2.5 collected from the winter of 2012 was 4-fold higher than that of Beijing

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(80.45 ng/m3) and Xian (68.05 ng/m3),45 and the value in PM10 was 9-fold higher than that of Shenzhen

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(35.32 ng/m ),

3 50

3

3

3

Shenyang (63.00 ng/m ), Anshan (66.87 ng/m ), Fushun (124.16 ng/m ), Jinzhou (19.04

3 61

59

51

52

Hongkong,

53

and Beijing,

cities

60

and Florence . Compared with the data from the references, the total

3 62

Chengdu (58.56 ng/m ),

3 63

and Shanghai (34.49 ng/m ).

204

Subsequently, we analyzed the similarities between winter and other seasons for the PAH

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compositions of PM2.5 and PM10 and between PM2.5 and PM10 in winter by applying the coefficient of

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divergence (CD) analysis.46 Wongphatarakul64 noted that a CD value of 0.269 reflects similarities of

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particle compositions between two cities. In the present study, for both PM2.5 and PM10, the CDjk values

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between winter and other seasons were all higher than 0.437 (Figure 1), indicating that there are

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significant differences in PAH composition, which may be ascribed to the fossil fuel application for

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domestic heating

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winter was 0.217, which is lower than 0.269; therefore, the PAH compositions between the fine and

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coarse fraction in winter were similar.

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53, 65, 66

during winter in Shanxi. In contrast, the CDjk value between PM2.5 and PM10 in

The diagnostic ratio has been reported to be a convenient method for identifying possible 10


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emission sources.46 To address the above issue, five diagnostic ratios were calculated, and the results

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are presented in Table S3 (see SI). For winter, the ratios IP/(IP + BghiP) (PM2.5/0.59, PM10/0.59),

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BaP/BghiP (PM2.5/0.98, PM10/0.97) and BaA/(BaA + CHR) (PM2.5/0.49, PM10/0.46) indicated that coal

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burning and non-burned fossil fuels contributed to the major sources of emission. Coupled with the

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data of the ring distribution of PAHs (Figure S2, SI), we confirmed that PAHs in PM2.5 and PM10 in

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winter were generated by coal combustion. Other studies46, 67 have also reported that coal combustion

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is the major source of PAHs in industrial agglomeration regions of China, such as E'erduosi and the

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cities of Liaoning.

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Lung Cancer Risk of PAH-bound PM. Epidemiological findings coupled with an animal inhalation

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study have revealed that PAHs, especially those emitted from coal combustion, are related to lung

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cancer risk, and PAH-bound PM from air pollution may also result in a high risk of lung cancer.45, 68, 69

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Therefore, we estimated the lung cancer risk of PAH-bound PM from peri-urban areas, using the BaP

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equivalent concentration (BaPeq). Here, the calculated total BaPeq for PM2.5 were 12.74, 5.54, 6.97 and

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43.45 ng/m for spring, summer, autumn and winter, respectively, whereas the values for PM10 were

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14.07, 6.12, 8.26 and 52.59 ng/m3, respectively (Table S4, SI). The BaPeq of PAHs in both PM2.5 and

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PM10 exhibited seasonal variation, and the values in winter were 4-8 times higher than in other seasons.

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We compared our values with those from other cities in China and abroad (Table S5, SI).

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The total BaPeq values in winter were similar to that of Liaoning, a coal-based industrial region, and

232

were significantly higher than that of other cities. Considering that PAH inhalation is associated with

233

lung cancer risk, the PAH-bound PM exposure in the area might play an important role in increased

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lung cancer mortality or morbidity.

235

3

45-47, 60, 67, 70-72

It has been reported that indoor PAH exposure is a major contributor to the overall exposure 11



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compared with outdoor exposure.73, 74 For the urban residents in Taiyuan,73 the contributions of indoor

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PAH inhalation exposure were 73% during the heating season and 65% during the non-heating season,

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whereas for the rural residents, the indoor contribution was as high as 92% in the heating season and

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only 40% in the non-heating season. These results suggest that the present health risk assessment

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has an exposure measurement limitation, and indoor PAH sources, such as cooking and smoking, were

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not taken into account.

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PAH-bound PM-Induced Motility and the Invasion of A549 Cells. Because the high mortality of

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lung cancer is attributed to its early metastasis, we investigated whether PM samples could cause

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human lung cancer cell metastasis. As shown in Figure 2, the total concentrations of 18 PAHs

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adsorbed on PM2.5 and PM10 in winter were 3.4-7.0 times (for PM2.5) and 3.8-7.7 times (for PM10) higher

246

than that in the other seasons. Interestingly, the cell numbers in the lower chamber treated by PM2.5

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and PM10 exhibited similar seasonal variations; the greatest migration was observed for cells treated

248

with winter PM2.5 and PM10. These results suggest a positive association between the level of PAHs

249

adsorbed on PM and the potential capacity for cell metastasis induced by PM, and winter samples

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caused the most obvious effects.

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Cell proliferation and migration are important biological processes in tumor metastasis and

252

progression. Therefore, we examined the concentration- and time-dependent effects for winter

253

PM-induced motility of A549 cells using a wound-healing assay. After exposure to winter PM2.5 and

254

PM10 for 6, 12 and 24 h, the cells in the denuded zone obviously and gradually increased, and nearly

255

filled the wounded area at higher concentrations (Figure 3A). The migration rate appeared to increase

256

rapidly after exposure to PM at 0.1, 0.3, 1, 3 and 10 µg/mL for 24 h and reached 1.57, 15.03, 22.51,

257

35.31 and 48.73%, respectively, for PM2.5 and 8.34, 8.18, 9.95, 25.62 and 38.18%, respectively, for 12


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PM10 (Figure S3, SI). The quantitative data revealed that winter PM2.5 and PM10 from the peri-urban

259

area of Taiyuan stimulated A549 cell migration in a concentration- and time-dependent manner.

260

Following the above results, we further examined the effect of winter PM2.5 and PM10 on the

261

invasive ability of A549 cells. In Figure 3B, the invaded cell numbers were elevated in a concentration-

262

dependent manner after a 24-h treatment with PM (3-, 4-, 6-, 15- and 20-fold of control for PM2.5, and

263

1.5-, 2-, 2.5-, 10.5- and 11-fold of control for PM10 at 0.1, 0.3, 1, 3 and 10 µg/mL, respectively), and

264

significant differences occurred after 3 and 10 µg/mL PM exposure.

265

Previous studies have documented the advantages of utilizing bioassays for indicating specific 75

266

effects and providing results that clearly separated the regions with different types of pollution.

267

we coupled chemical analysis of PAH-bound PM with specific biological effects for promoting lung

268

cancer metastasis. First, the total concentrations of PAHs in winter PM were higher than that in other

269

cities of China and abroad. Accordingly, the present PM samples showed a similar capability for

270

stimulating lung cancer cell motility and invasion, and this finding was consistent with other studies on

271

exposure to PM2.5 extracts from Xiamen and Beijing.

272

the fine and coarse fraction in winter were similar, and the concentration of PAH in PM2.5 was lower

273

than that of PM10, the enhanced migration and invasion caused by PM2.5 were more significant relative

274

to that caused by PM10. The difference in sources between PM2.5 and PM10 may be the secondary

275

organic carbon (SOC) absorbed in PM2.5 and mineral dust in PM10. The SOC level of PM2.5 from

276

Taiyuan in 2012 (4.3 ± 3.0 µg/m ) was lower than that of other cities,

277

concentrations of atmospheric PAHs in Taiyuan.77 These results indicate that primary organic carbon

278

(POC) was the most important contributor to PM2.5 pollution in Taiyuan. The difference in toxicity

279

between PM2.5 and PM10 was not attributed to the SOC levels of PM2.5 from Taiyuan in 2012. In detail,

45

Here,

Second, even though the PAH source between

3

76

and there were very high

13



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the SOC concentration of summer PM2.5 samples from Taiyuan in 2012 (5.0 ± 3.0 µg/m3) was higher

281

than that of winter samples (4.4 ± 2.6 µg/m ),

282

-4.62-9.98 °C, relative high wind speed at an average daily value of over 14.2 km/h with dense haze

283

and a short duration of sunshine in winter (Table S6). However, the opposite trend was found in the cell

284

invasion assay. This result indicates the presence of a specific effect (lung cancer metastasis) for winter

285

PAH-bound PM and the difference of molecular modulation for PM2.5 and PM10.

3 76, 78

because of the temperature at Taiyuan ranged

286

Mechanism for A549 Cell Migration and Invasion Induced by PAH-bound PM. The activation

287

of the EMT program has been implicated as an important step in the metastasis of lung and other

288

tumors,

289

program. To clarify the possible mechanism for PAH-bound PM-induced A549 cell metastasis, we

290

evaluated E-cad and Fib expression at the transcriptional level. The results indicate that PM2.5 and

291

PM10 caused a concentration-dependent change in the levels of both markers, with E-cad expression

292

decreasing to 0.48- and 0.39-fold of control levels (Figure 4A) and Fib expression increasing to 1.89-

293

and 1.37-fold of control levels (Figure 4B) at the highest concentration after a 24-h treatment. Because

294

the lack of E-cad and the increased Fib induces the loss of the epithelial phenotype and acquisition of

295

the mesenchymal phenotype,80 PM-induced alterations suggest that the cancer cells lost desmosomes

296

and adherens junctions and increased motility.

79

and the loss of E-cad expression emerges as a critical step that drives this developmental

297

ECM degradation is crucial to cellular invasion and requires matrix-degrading proteinases.81

298

Therefore, we examined MMP-2 and TIMP-2 levels. MMP-2 expression was significantly elevated

299

(Figure 4C), and TIMP-2 expression was reduced at the transcriptional level (Figure 4D) after the

300

winter-PM treatment. Interestingly, the MMP-2 mRNA levels were significantly amplified at different

301

concentrations of PM2.5 treatment relative to PM10 treatment. Our findings suggest that the PM sample 14


Page 15 of 37


302

might cause proteolysis of ECM components, thereby releasing stored angiogenic signaling molecules

303

from the ECM and facilitating the migration process.

304

These observations were consistent with the results of wound-healing and cell invasion assays

305

and confirmed that the winter PM from the peri-urban area of Taiyuan significantly induced lung cancer

306

cell migration and invasion and that EMT activation and ECM were involved in the process. It has been

307

well documented that an increase in intracellular ROS leads to changes in cytoskeletal organization,82

308

cell junctions,

309

ROS levels have been associated with tumor clinical staging.85 Importantly, EMT progression and ECM

310

degradation may be activated in cancer cells by ROS.

311

correlation between ROS generation and PAH concentration, with the exception of three low molecular

312

weight compounds (ACL, AC, NA) for PM2.5 (Table S7, SI). This finding indicates that ROS generation

313

might be an important modulator for winter PAH-bound PM to promote lung cancer metastasis. To

314

examine this hypothesis, we first analyzed intracellular ROS levels after exposure to winter PM. As

315

shown in Figure 5, the ROS level was significantly enhanced after a 6-h treatment in a

316

concentration-dependent manner, and the elevation caused by PM2.5 was higher than that of PM10 after

317

treatment at the same concentration. The current result was consistent with a previous report that PM2.5

318

induced greater oxidative stress than PM10 due to a greater surface at the same weight

319

explain the differences between PM2.5 and PM10 with respect to A549 cell metastasis and the

320

transcriptional changes of EMT and ECM markers (E-cad, Fib, MMP-2 and TIMP-2).

83

84

and migration

during cancer progression. In clinical specimens of NSCLC, higher

86, 87

Interestingly, we observed a positive

88

and might

321

Subsequently, to provide further evidence for the involvement of ROS in winter PM-induced cell

322

metastasis, we pretreated A549 cells with an ROS inhibitor (NAC) and measured A549 cell motility and

323

invasion after PM treatment in the absence or presence of NAC. Following a 24-h treatment with the 15



Page 16 of 37

324

winter PM (10 µg/mL), A549 cells pretreated with NAC exhibited significant attenuations in the denuded

325

zone (68.4%) and lower chamber (63.5%; Figure 5). These results provided further evidence that

326

winter PAH-bound PM2.5 and PM10 from the peri-urban area of Taiyuan induced the migration and

327

invasion of A549 cells and that the action was involved in ROS-dependent EMT activation and ECM

328

degradation. This finding indicates that winter PM2.5 and PM10 exposure in peri-urban areas,

329

characterized by coal burning-produced PAHs, poses a serious risk for lung cancer development and

330

reveals a mechanistic basis for treating, ameliorating, or preventing these outcomes in a polluted

331

environment.

332

AUTHOR INFORMATION

333

Corresponding Author

334

* Fax: +86-351-7011932. E-mail: [email protected] (N.S.)

335

Notes

336

The authors declare no competing financial interest.

337

ACKNOWLEDGMENTS

338

This study was supported by National Science Foundation of PR China (Nos. 21477070, 21377076,

339

21307079, 21222701), Specialized Research Fund for the Doctoral Program of Higher Education

340

(SRFDP, No. 20121401110003, 20131401110005), Program for the Top Young and Middle-aged

341

Innovative Talents of Higher Learning Institutions of Shanxi (TYMIT, No. 20120201).

342

SUPPORTING INFORMATION AVAILABLE

343

Additional information on the experimental methods, some supporting figures and tables can be found

344

in the Supporting Information document. This information is available free of charge via the Internet at

345

http://pubs.acs.org/. 16


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junction of epithelial cells by oxidative stress. Genes. Cells. 2009, 14(6), 703-716.

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in lung cancer tissue and pulmonary parenchyma. Respir. Med. 2000, 94(8), 800-805.

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extracellular matrix remodeling during tumor progression. Food. Chem. Toxicol. 2013, 61, 178-186.

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PM2.5- and PM10-induced oxidative stress in rat lung epithelial cells. J. Vet. Sci. 2004, 5(1):11-18.

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FIGURE LEGENDS

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Figure 1. Analysis of the similarities between different PM samples. (A-C) The similarities between

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winter and other seasons for the PAH compositions of PM2.5 based on CD analysis. (D-F) The

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similarities between winter and other seasons for the PAH compositions of PM10 based on CD analysis.

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(G) The similarities of the PAH compositions between PM2.5 and PM10 in winter based on CD analysis.

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Figure 2. The motility and invasion of A549 cells induced by PAH-bound PM from different seasons. (A)

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The histogram indicates the PAH content of PM2.5 in different seasons, and the scattergram indicates

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the number of cells that invaded the lower chamber. (B) The histogram indicates the PAH content of

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PM10 in different seasons, and the scattergram indicates the number of cells that invaded the lower

597

chamber. After sample collection, the filters were extracted, and the concentrations of 18 PAHs in PM

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extracts were determined by using GC-MSD (6890 N, HP; 5973, HP). A549 cells were treated with PM

599

extracts (10 µg/mL) from different seasons for 24 h, and the invasion ability was evaluated by the cell

600

invasion assay.

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Figure 3. The effects of winter PM on A549 cell migration and invasion. (A) The effect of winter PM on

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A549 cell migration determined by a wound-healing assay (200×). A549 cells were treated with winter

603

PM at various concentrations (0, 0.1, 0.3, 1, 3 and 10 µg/mL) for different times (0, 6, 12 and 24 h), and

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the micrographs show representative images of cell migration. (B) The effect of winter PM on A549 cell

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invasion as detected by the cell invasion assay (400×). A549 cells were incubated with winter PM at

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various concentrations (0, 0.1, 0.3, 1, 3 and 10 µg/mL) for 24 h; subsequently, the invasive ability was

607

evaluated using the cell invasion assay. The top is the micrograph of the representative images of the

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cell migration under different treatment conditions, and the bottom is the calculation of the mean

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number of invasive cells in each group. For PM2.5: * p < 0.05, ** p < 0.01 vs. the control group; For PM10: 28


Page 29 of 37


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# p < 0.05 vs. the control group.

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Figure 4. Effects of winter PM on E-cad (A), Fib (B), MMP-2 (C) and TIMP-2 (D) mRNA expression in

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A549 cells. A549 cells were treated by winter PM at various concentrations (0, 0.1, 0.3, 1, 3 and 10

613

µg/mL) for 24 h, and the mRNA levels of E-cad, Fib, MMP-2 and TIMP-2 were determined. The black

614

lines represent the effects of PM2.5 on the gene expression, and the red lines represent those of PM10.

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For PM2.5: * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the control group; For PM10: # p < 0.05, ## p

Winter Polycyclic Aromatic Hydrocarbon-Bound Particulate Matter from

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