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Polystyrene microplastic particles: In vitro pulmonary toxicity assessment

Cheng-Di Dong, Chiu-Wen Chen, Yi-Chun Chen, Hung-Hsiang

Chen, Jin-Sun Lee, Chia-Hua Lin

PII: S0304-3894(19)31529-8
DOI: https://doi.org/10.1016/j.jhazmat.2019.121575
Reference: HAZMAT 121575

To appear in: Journal of Hazardous Materials

Received Date: 29 May 2019

Revised Date: 16 September 2019
Accepted Date: 30 October 2019

Please cite this article as: Dong C-Di, Chen C-Wen, Chen Y-Chun, Chen H-Hsiang, Lee
J-Sun, Lin C-Hua, Polystyrene microplastic particles: In vitro pulmonary toxicity assessment,
Journal of Hazardous Materials (2019), doi: https://doi.org/10.1016/j.jhazmat.2019.121575

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© 2019 Published by Elsevier.

Polystyrene microplastic particles: In
vitro pulmonary toxicity assessment

Cheng-Di Dong,a Chiu-Wen Chen,a Yi-Chun Chen,b Hung-Hsiang Chen,b Jin-Sun

Leea,* and Chia-Hua Linb,*

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a
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Department of Marine Environmental Engineering, National Kaohsiung University of Science

and Technology, Kaohsiung, 81157, Taiwan;

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b
Department of Biotechnology, National Formosa University, Yunlin, 63208, Taiwan.
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*
To whom correspondence should be addressed: Dr. Chia-Hua Lin; Tel: 886-5-6315558;

Fax: 886-5-6315502; E-mail: [email protected]; Dr. Jin-Sun Lee; Tel: 886-928-

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914905; Fax: 886-5-6315502; E-mail:[email protected].

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Graphical Abstract
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Highlights
 Polystyrene microplastics (PS-MPs) cause pulmonary cytotoxicity by inducing


ROS.
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PS-MPs is associated with impaired pulmonary barrier by depleting ZO proteins.
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 PS-MPs inhalation increases the risk for chronic obstructive pulmonary disease.
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Abstract
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Microplastics (MPs) have become a global environmental concern. Recent studies have

shown that MPs, of which the predominant type is often polystyrene (PS; known as PS-
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MPs), can extend to and affect remote, sparsely inhabited areas via atmospheric
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transport. Although exposure to inhaled MPs may induce lung dysfunction, further

experimental verification of the pulmonary toxic potential of MPs and the mechanism

underlying the toxicity is needed. Here we used normal human lung epithelial BEAS-
2B cells to clarify the association between pulmonary toxicity and PS-MPs. Results

revealed that PS-MPs can cause cytotoxic and inflammatory effects in BEAS-2B cells

by inducing reactive oxygen species formation. PS-MPs can decrease transepithelial

electrical resistance by depleting zonula occludens proteins. Indeed, decreased α1-

antitrypsin levels in BEAS-2B cells suggest that exposure to PS-MPs increases the risk

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for chronic obstructive pulmonary disease, and high concentrations of PS-MPs can

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induce these adverse responses. While low PS-MP levels can only disrupt the protective

pulmonary barrier, they may also increase the risk for lung disease. Collectively, our
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findings indicate that PS-MP inhalation may influence human respiratory health.
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Keywords: Polystyrene, Microplastic particle, COPD, Epithelial barrier integrity,
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Inflammation, Oxidative stress

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1. Introduction
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Microplastics (MPs) contain small plastic debris fragments (approximate diameter,

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<5 mm) [1]. In recent years, the accumulation of contaminated plastic particles in the

environment and in various organisms has received increased attention. Indeed, this

extensive and everlasting environmental issue has created a great concern owing to the

increasing plastics production and use despite the low biodegradation rate [2, 3].
Environmental MPs may originate from indirect degradation of plastic substances

(secondary MPs) via light, ultraviolet radiation, embrittlement, biological factors [2],

and sea-salt aerosol formation [4]. In addition, MPs may directly originate from

manufactured products (primary MPs), such as microbeads that are often added to

personal care products [2]. MPs have also been found in various foodstuffs and in the

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atmosphere, which can cause long-term physical and chemical effects, giving rise to

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concerns over their potential risks to public health [4-6].

While the most studies have focused on the impact of MPs on human health with
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regard to the digestive system [7], airborne MPs as a potent pollution source may be
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found in both terrestrial and aquatic ecosystems [8]. These airborne MPs exist in the
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atmospheric compartment and result in ecosystem contamination [9]. Many of these

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MPs have a gravity lower than that of seawater and may thus be transported as sea-salt

aerosols through sea spray and wind action to urban environments located near the coast
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[4]. Indeed, 7% of the total MP contamination is considered to occur due to the

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transmission of air from the ocean [10]. Furthermore, airborne MPs can arise from

various sources, with primary MPs originating from synthetic textiles, synthetic rubber

tyre erosion, and city dust [4]. Recently, MPs have been detected in the atmospheric
fallout from cities [9, 11]. Moreover, sources of MPs in our daily lives include plastic

scraps from clothes, furniture, buildings, traffic pollution, waste incineration [11, 12],

and dried sludge-based fertilizer and polystyrene (PS) peat in horticultural soils [13].

Some studies have also demonstrated that MPs exist in the working environment [14,

15]. With regard to transport, MPs can travel through the atmosphere for over 95 km.

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The predominant MP type found in these samples is PS. Indeed, PS-MPs can extend to

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and affect remote, sparsely inhabited areas through atmospheric transport [16].

However, information on the airborne MP levels is lacking. In addition, data

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available from relevant literature present considerable variations probably owing to the
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used sampling methodology and monitoring conditions, including sampling site,
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sampling season, sampling period, and climatic condition [9, 11, 12]. Surveys
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performed in Greater Paris have shown that MPs in atmospheric fallout are recorded at

average levels of 118 MPs m−2 day−1 [9] and that there are 53 or 110 atmospheric fallout
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particles m−2 day−1 (29% fibers) [11]. Although environmental exposure is not well
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understood, airborne MPs reportedly cause occupational diseases among factory

workers owing to the higher exposure levels [8]. For example, in the flocking area of a

microfiber manufacturing plant, the highest concentration of airborne MPs reached 7

mg/m3 [4]. These airborne MPs are small enough to be inhaled and enter the airway,

where they may interact with various organisms, leading to accumulation, inflammation,

and obstruction after translocation [17, 18]. MPs may get cleared with mucus or may

advance along the respiratory tract into the deep lung, further interacting with lung fluid

and lung cells. In addition, MPs may be deposited on tissue surfaces or may enter cells,

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thereby triggering subsequent inflammatory responses, which may lead to acute and

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chronic respiratory diseases, such as asthma, dyspnea, and chronic obstructive

pulmonary disease (COPD). -p

Numerous studies have demonstrated the adverse effects of MPs in marine
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vertebrates and invertebrates [17]. These studies have shown that zooplankton,
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invertebrates, and echinoderms are all vulnerable to MP intake [19, 20]. Indeed, MP
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leachates have been shown to cause acute toxicity in Nitocra spinipes [21]. The toxicity

of MP monomers and additives in both rodents and humans has also been studied [17,
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22-25]. Indeed, MPs have been shown to release toxic monomers and/or additives
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associated with cancer and reproductive abnormalities [24, 25]. Compared with

regarding the monomer or additive effects of MPs, less is known regarding the MP

particulate toxicity effects in humans. Therefore, here, we used normal human lung
epithelial BEAS-2B cells to investigate the potential pulmonary toxicity after exposure

to PS-MPs. As biological endpoints, cytotoxicity, oxidative stress, inflammatory

response, epithelial barrier dysfunction, and predictive biomarker levels for COPD

were measured. Collectively, our findings may provide greater insights into the

pulmonary risks of PS-MP exposure in humans.

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2. Materials and methods

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2.1 Preparation and physicochemical characterization of PS-MPs

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As described previously [26], PS-MPs were synthesized from styrene (St) while
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employing poly(N-vinylpyrrolidone) (PVP) as a stabilizer and 2,2-azobis(2-
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methylpropionitrile) (AIBN) as the initiator. A mixture of absolute alcohol, deionized

water, St monomer, and PVP was placed in a four-necked flask equipped with a reflux
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condenser and stirrer. The mixture was homogeneously mixed and heated to 70°C.
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AIBN was then added to the four-necked flask, and dispersion–polymerization was

initiated under a nitrogen atmosphere. Monodispersed PS-MPs were then obtained by

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centrifugal sedimentation (Legend Mach 1.6r, Sorvall) at 1,200 rpm. This process

produced uniformly sized PS-MPs, which were collected and dispersed in ddH2O until

use. Before each experiment, suspensions were sonicated for 6 min using a probe
sonicator (SK1200H, Shanghai KUDOS Inc., Shanghai, China). Then, the PS-MPs

were suspended in LHC-9 medium (Gibco, USA) at the required concentration.

We characterized PS-MPs in terms of size distribution, zeta potential, size, and

shape. The zeta potentials of the PS-MPs were measured using the Zetasizer Nano

system (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The shape and

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size of each PS-MP were then assessed using scanning electron microscopy (SEM) (S-

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4700, Hitachi, Japan) and atomic force microscopy (AFM) (Cambridge, UK). The

Fourier transform infrared (FTIR) spectra of the PS-MPs were then collected using a
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Thermo Nicolet AVATAR FT-IR 360 instrument (LabX, ON, Canada).
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2.2 Cell culture
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The human lung epithelial cell line BEAS-2B was maintained in LHC-9 medium
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and incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium

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was changed twice weekly, and trypsinized cells were passaged daily.

2.3 Cytotoxicity assay

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The BEAS-2B cells were treated with PS-MPs (1–1,000 μg/cm2) for 24 and 48 h.

Cell viability was determined using the trypan blue exclusion assay. Briefly, after

treatment, the cell suspensions were mixed with trypan blue and visually examined to
determine whether cells took up or excluded the dye. Viable cells are expected to have

a clear cytoplasm, whereas nonviable cells are expected to have taken up the dye,

resulting in a blue cytoplasm. Cell viability was calculated as the number of viable cells

relative to that of control cells.

2.4 Analysis of the changes in redox status by the 2’,7’-dichlorofluorescein

diacetate (DCFH-DA) assay

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We used DCFH-DA assays to determine the formation of reactive oxygen species

(ROS) in cells following exposure to PS-MPs (1–1,000 μg/cm2) as described

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previously, with slight modifications [27]. Briefly, BEAS-2B cells were seeded in 96-
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well plates (8 × 103 cells/well) for 12–16 h. The medium was then discarded from the
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plates and replaced with LHC-9 containing DCFH-DA and PS-MPs. ROS generation
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relative to the controls was periodically determined (every 30 min) using a fluorescent

plate reader (λex = 485 nm, λem = 535 nm). H2O2 (20 mM) was used as the positive
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control in this assay. H2O2 induced 15- and 18-fold increases in the amounts of ROS
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formation in the BEAS-2B and control cells, respectively, after 2 h of exposure.

2.5 Enzyme-linked immunosorbent assay (ELISA)

 BEAS-2B cells were seeded in 96-well plates (8 × 103 cells/well) for 12–16 h. PS-

MPs were then dispersed in the cell culture medium. After 24 h of exposure to PS-MPs

(10 and 1,000 μg/cm2), the cell culture medium and/or cell lysate was collected for the

analysis of interleukin (IL)-6, IL-8, ZO-1, and α1-antitrypsin (AAT), according to the

manufacturer’s protocol (Sinobestbio, Shanghai, China). Before measuring the

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absorbance, PS-MPs were eliminated from the resultant solution by centrifugation.

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2.6 Protein extraction and Western blot analysis

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Heme oxygenase-1 (HO-1) protein levels were determined by Western blot

analysis. BEAS-2B cells were treated with PS-MPs (10 and 1,000 μg/cm2). After
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treatment, the cells were washed thrice with phosphate-buffered saline, centrifuged at
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8,000 g for 5 min, and soaked in liquid N2. The resultant cell pellets were thawed and
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lysed in 80 μL of protein extraction buffer (1 M Tris-HCl, pH 7.9, 3 M NaCl, 1%

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aprotinin, 2 mM phenylmethylsulfonyl fluoride, and 5 mM dithiothreitol) for 30 min

on ice, and the extracts were centrifuged for 30 min at a relative centrifugal force of
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10,000 g. Protein concentrations were measured using the Bio-Rad protein assay kit

(Hercules, CA, USA). Proteins were loaded at 50 μg/lane, separated using 12% (w:v)

sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and blotted. Anti-HO-1,

anti-ZO-1, and anti-β-actin monoclonal antibodies (Santa Cruz, Inc., Santa Cruz, CA,

USA) were used at a concentration of 1:500. Secondary antibodies, consisting of

alkaline phosphatase-coupled anti-mouse or anti-rabbit antibody, were incubated at

room temperature for 2 h at a concentration of 1:5,000. Immunoreactive bands were

visualized by incubating the blot with nitroblue tetrazolium and 5-bromo-4-chloro-3-

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indolyl-phosphate (Sigma-Aldrich). The membranes were also probed with anti-β-actin

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antibody to correct for differences in protein loading.

2.7 Measurement of transepithelial electrical resistance (TEER)

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The barrier integrity of the epithelial cells was measured using TEER. BEAS-2B

cells were seeded in 6-well Millicell® cell culture inserts until they reached 100%
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confluence, followed by stimulation with PS-MPs (10 and 1,000 μg/cm2) for 24 h. After
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24 h of exposure to PS-MPs, TEER was measured with a Millicell ERS 2-volt

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ohmmeter (Millipore, MA, USA) to evaluate the barrier integrity of the BEAS-2B cells.

Barrier resistance values (Ω*cm2) were obtained after subtracting the blank membrane
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resistance.

2.8 Statistical analysis

 All experiments were performed thrice independently. The significance of

differences in the results was evaluated using one-way analysis of variance, followed

by Dunnett’s multiple comparison tests. All comparisons were considered significantly

different when p was <0.05.

3. Results and Discussion

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3.1 Physicochemical Properties of PS-MPs

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The synthesis routes for PS-MPs are shown in Scheme 1. In this study, the

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properties of the PS-MPs are shown in Figure 1 and Table 1. PS-MPs were
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characterized as spherical particles with a rough surface (Figure 1). The average particle

size and zeta potential (in ddH2O) of the PS-MPs were 1.72 ± 0.26 𝜇m (1.67–2.17 𝜇m)
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and −26.8 ± 4.42 mV (Table 1 and Figure S1 in the SI), respectively. In suspension,
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PS-MPs have a negative charge, indicating that they tend to repel each other and that
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aggregation should be prevented [28]. Indeed, most PS-MPs only aggregated slightly

into larger complexes in ddH2O, and the average hydrodynamic diameter of PS-MPs
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was 4.06 ± 0.44 𝜇m (3.51–4.38 𝜇m) (Table 1). The PS-MPs remained dispersed in

solution for at least 2 h (Figure S2 in the SI). The majority of the PS-MPs settled to the

bottom of the culture well and established direct contact with the BEAS-2B cells within
24 h (Figure S2 in the SI). Chemical adsorption primarily occurs due to the surface

hydrophobicity of plastics [17]; thus, the boundary value of the zeta potential implied

that our PS-MPs may flocculate inorganic ions or contaminants. The FTIR spectra for

PS-MPs are shown in Figure 2. FTIR data showed that the main bands of PS-MPs,

which are attributed to the C–H stretching vibrations in the main chain and aromatic

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rings, had peaks over 2,800–3,100 cm−1. The peaks at 1,601, 1,492, 1,451, 1,027, 757,

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and 698 cm−1 are probably due to the deformation and skeletal vibrations of C–H in the

PS-MPs (Figure 2) [29]. -p

3.2 Cytotoxic effects and cell morphology after exposure to PS-MPs in BEAS-2B
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Cells
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It is possible for MPs to be inhaled and get deposited in the respiratory system,
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which may lead to diseases in susceptible individuals and cause unknown impacts on

human health [30] . Considering the potential toxic impacts of PS-MPs on the
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respiratory system, we employed the human lung epithelial cell line BEAS-2B to
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evaluate the potential risk of pulmonary toxicity after exposure to PS-MPs (1–1,000

μg/cm2) for 24 and 48 h. The viability of the BEAS-2B cells affected by PS-MPs was

then evaluated using the trypan blue exclusion assay. After 24 h of exposure, a
significant reduction in cell viability was observed only in the PS-MP-treated BEAS-

2B cells at a concentration of 1,000 μg/cm2 (Figure 3A). Our results also showed

significant cytotoxicity when human BEAS-2B epithelial cells were exposed to PS-

MPs at concentrations ≥10 μg/cm2 for 48 h (60%–70% of control; Figure 3A). At this

time point, morphological examination using light microscopy revealed that untreated

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BEAS-2B cells were homogeneously distributed on the culture plate. Similar

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observations were obtained with low-level PS-MP (1 μg/cm2) treatment (Figure 3B).

However, many of the high-level PS-MP (≥ 10 μg/cm2)-exposed BEAS-2B cells had

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transformed to a round shape and had shrunk (Figure 3B).
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Ordinary people are generally exposed to low concentrations of MPs; in contrast,
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industry workers in specific positions may be exposed to high concentrations of MPs,

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which may lead to occupational diseases. Accordingly, the following pulmonary

toxicities were evaluated at both a low dose (10 μg/cm2) and high dose (1,000 μg/cm2)
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of PS-MPs in human BEAS-2B epithelial cells. In this study, the concentration of PS-
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MPs has been expressed as μg/cm2. A given concentration can then be converted to

particle numbers/cm2 by multiplying it by 5.12 × 103.

3.3 Oxidative and inflammatory adverse response after exposure to PS-MPs in

BEAS-2B Cells
 Owing to their small size, MPs may enter various organisms and accumulate in

other organs after translocation, which may cause obstruction and inflammation [17] .

When cells are exposed to MPs, cellular oxidative stress can occur, leading to

diminished energy metabolism and even genotoxicity in marine animals [31, 32]. In

addition, HO-1, a ubiquitous stress-response protein, may be induced by exposure to

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various types of oxidative stress [33]. Therefore, to study if inhaling PS-MPs induces

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oxidative responses, the DCFH-DA and Western blot assays were applied to measure

ROS generation and HO-1 expression in BEAS-2B cells after exposure to PS-MPs (10
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and 1,000 μg/cm2) for 24 h. ROS accumulation significantly increased only in the
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BEAS-2B cells exposed to PS-MPs at 1,000 μg/cm2 (Figure 4A). Consistent with this
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ROS trend, the protein levels of HO-1 were considered more intense in BEAS-2B cells
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exposed to high-dose PS-MPs than in those exposed to low-dose PS-MPs (Figures 4B

and 4C). The observed compensatory increase of HO-1 may represent an attempt to
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maintain the oxidative status balance in low-dose PS-MP-treated cells. In contrast,

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when BEAS-2B cells were exposed to high-dose PS-MPs, massive ROS formation

overcame the cellular antioxidant enzyme capacity, leading to considerable oxidative

stress. Imbalances in oxidants and antioxidants resulting in oxidative stress could have
a potential role in the pathogenesis of lung diseases [34, 35]. ROS is capable of inducing

oxidative damage to macromolecules, including nucleic, lipids and proteins, which

result in lung cell death, loss of alveolar units and development of COPD [35, 36].

Conditions that lead to tissue damage may be facilitated by ROS, with

inflammation being the initial reaction to tissue damage. When inhaled matter enters

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the respiratory system, the airway epithelium secretes numerous biochemical

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mediators, including ROS and cytokines, to recruit inflammatory cells [37].

Proinflammatory cytokines, such as IL-6 and IL-8, then serve to activate the immune
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system, which takes part in the acute inflammatory response [37]. Proinflammatory
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factors in the BEAS-2B cells after exposure to PS-MPs at concentrations of 10 or 1,000
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μg/cm2 were then analyzed using ELISA. The expression of IL-6 in the cell culture
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medium was significantly increased after exposure to PS-MPs (10 and 1,000 μg/cm2)

for 24 h (Figure 5A). The expression of IL-8 was increased only in the BEAS-2B cells
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exposed to PS-MPs at 1,000 μg/cm2 (Figure 5B). Considerable evidence exists that both
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IL-6 and IL-8 are related to the exacerbation of COPD [38, 39]. According to our

results, high-level PS-MPs may trigger oxidative stress and activate the immune system

by augmenting IL-8 and IL-6 expression levels. This PS-MP-induced local

inflammatory response may be amplified, which can lead to further systemic

inflammation and contribute to COPD and asthma [40, 41].

3.4 Pulmonary dysfunction after exposure to PS-MPs in BEAS-2B Cells

The inhalation of foreign matter and toxic substances initiates a pulmonary

inflammatory cascade, which comprises the activation of several cell types and the

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secretion of inflammatory mediators that can cause tissue injury without effective repair

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and alterations in airway structures [42]. The airway epithelium is the first barrier of

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the host defense system in the respiratory tract. Indeed, inflammation-induced changes
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in lung function, including increases in airway epithelial permeability and the

expression of tight junction (TJ) proteins, may be the result of epithelial barrier
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dysfunction. Therefore, foreign substances and toxins are more likely to enter the
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interstitium and bloodstream, which may give rise to COPD. To examine whether PS-
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MPs can disrupt the epithelial barrier, BEAS-2B cells were exposed to 10 or 1,000

μg/cm2 of PS-MPs. Changes in TEER and the ZO-1 protein expression, which were
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assessed using the Millicell ERS-2 volt-ohm meter and the ELISA, were used to

investigate the effects of PS-MPs on epithelial permeability. After 24 h of exposure to

both high and low levels of PS-MPs, the ZO-1 expression level was decreased (Figure
6A). The value of TEER also decreased in the epithelial barrier after exposure to PS-

MPs (Figure 6B). Our data demonstrate that not only high levels but also low levels of

PS-MPs may disrupt the protective pulmonary barrier by reducing TJ protein

expression. This disruption of the lung epithelial barrier could possibly be caused by

the PS-MPs-induced oxidative responses [43, 44]. Oxidative stress has been

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demonstrated to induce the phosphorylation of ZO-1 and thus disrupts the tight junction

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[44]. The loss of epithelial integrity is a key pathological feature of COPD development

[44]. -p
Reportedly, COPD is characterized by an irreversible and progressive airway
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obstruction associated with aberrant oxidative stress and inflammation when stimulated
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by noxious particles [45]. AAT, which protects the alveoli from elastolysis, is the best
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characterized elastase inhibitor and is synthesized primarily by liver cells,

macrophages, and bronchial epithelial cells [46-48]. AAT deficiency is a well-

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established risk factor for COPD and liver dysfunction [49]. Indeed, protease–
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antiprotease imbalance is the main cause of COPD in patients with severe AAT

deficiency, and impaired AAT function may also contribute to COPD pathogenesis

[50]. To validate the effects of BEAS-2B exposure to PS-MPs on the risk for COPD,
AAT in the cell culture medium was examined using ELISA. A significant reduction

in AAT expression was observed in the BEAS-2B cells treated with high-level PS-MPs

(Figure 6C). Inadequate amounts of AAT cause neutrophil elastase to be excessively

free such that it can degrade elastin, thereby destroying lung elasticity and eventually

resulting in COPD [51, 52]. Although low-level PS-MPs can only disrupt the protective

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pulmonary barrier, they may also serve to increase the risk for lung disease after

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prolonged exposure.

4. Conclusions -p
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While the number of applications of plastic materials continues to increase,

concerns regarding the potential accumulation of contaminated plastic particles in the

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environment and in various organisms still remain. Of special concern are ingested
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MPs, which are passed up the food chain more readily than larger plastic particles.
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However, no relevant research on the toxicity of MPs in normal human cells has been

conducted. Therefore, in the present study, we employed normal human lung epithelial
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cells to determine the potential risk of PS-MPs in developing adverse pulmonary

effects. To the best of our knowledge, this study is the first to demonstrate that exposure

to inhaled PS-MPs can potentially cause inflammatory and oxidative injuries along with
the disruption of intercellular junction proteins in the lung, which may lead to

pulmonary barrier dysfunction and subsequent PS-MP-induced COPD. Collectively,

our findings indicate that more attention should be paid to health issues associated with

the manufacture and disposal of plastic products.

Conflict of interest

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All the authors declare no conflict of interest.

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Acknowledgments

This work was supported by the Ministry of Science and Technology (MOST,
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Taiwan) [grant number 106-2314-B-150-001 and 107-2221-E-150-004-MY3]. This
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manuscript was edited by Enago.
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Tables with captions

Table 1. Physicochemical properties of PS-MPs

Parameter PS-MPs

Form Powder

Particle shape Sphere bead

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Particle surface Rough

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Particle size/distribution 1.72 ± 0.26 𝜇m (1.67–2.17 𝜇m)

Hydrodynamic diameter in ddH2O -p

4.06 ± 0.44 𝜇m (3.51–4.38 𝜇m)

−26.8 ± 4.42 mV
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Zeta potential
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 Scheme 1.

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 Figure 1

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 Figure 2

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 Figure 3

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 Figure 4

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 Figure 5

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 Figure 6

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Figure captions

Scheme 1. Schematic of the route for preparing PS-MPs.

Figure 1. Physicochemical properties of PS-MPs. (A) Low-magnitude SEM image

of PS-MPs. (B) High-magnitude SEM image of PS-MPs. (C) AFM image of PS-MPs.

(D) 3D image of the rough PS-MP surface.

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Figure 2. FTIR patterns of PS-MPs. The peaks over 2,800–3,100 cm−1 are attributed

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to the C–H stretching vibrations in the main chain and in aromatic rings and the peaks

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at 1,601, 1,492, 1,451, 1,027, 757, and 698 cm−1 are due to deformation and skeletal

vibrations of C–H of PS-MPs.

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Figure 3. Effect of 24- and/or 48-h exposure to PS-MPs on cytotoxicity of BEAS-
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2B cells. (A) Cell viability. (B) Cell morphology. Data represent the mean ± standard
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deviation of three determinations. *p < 0.05 indicates statistically significant

differences from the control.

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Figure 4. Effect of 24-h exposure to PS-MPs on oxidative adverse response of

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BEAS-2B cells. (A) ROS formation. (B) HO-1 protein expression. (C) Quantitative

analysis of HO-1 protein expression. Data represent the mean ± standard deviation of
three determinations. *p < 0.05 indicates statistically significant differences from the

control.

Figure 5. Effect of 24-h exposure to PS-MPs on pro-inflammatory factors of

BEAS-2B cells. (A) IL-6 protein expression. (B) IL-8 protein expression. Data

represent the mean ± standard deviation of three determinations. *p < 0.05 indicates a

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statistically significant difference from the control.

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Figure 6. Effect of 24-h exposure to PS-MPs on epithelial cell barrier integrity and

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predictive biomarkers for COPD of BEAS-2B cells. (A) ZO-1 protein expression.

(B) TEER values. (C) AAT protein expression. Data represent the mean ± standard
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deviation of three determinations. *p < 0.05 indicates a statistically significant
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difference from the control.

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