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Environmental Processes 3+
Interactions of Gaseous 2-Chlorophenol with Fe -Saturated Montmorillonite and their Toxicity to Human Lung Cells Anping Peng, Juan Gao, Zeyou Chen, Yi Wang, Hui Li, Lena Q. Ma, and Cheng Gu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06664 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018
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Interactions of Gaseous 2-Chlorophenol with Fe3+-Saturated
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Montmorillonite and their Toxicity to Human Lung Cells
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Anping Peng,†,§ Juan Gao,‡ Zeyou Chen,† Yi Wang,† Hui Li,§ Lena, Q. Ma,† and Cheng
4
Gu,†,*
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†
8
Nanjing University, Nanjing 210023, P.R. China
9
‡
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,
Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science,
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Chinese Academy of Sciences, Nanjing, Jiangsu 210008, P. R. China
11
§
12
Michigan 48824, United States
Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing,
13 14 15 16
*To whom correspondence should be addressed:
[email protected] 17
School of the Environment, Nanjing University, Nanjing, Jiangsu, 210023, P. R. China,
18
Phone/Fax: +86-25-89680636
19
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Abstract
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In the present study, the interactions of gaseous 2-chlorophenol with Fe3+-saturated
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montmorillonite particles in a gas-solid system was investigated to simulate the reactions of
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mineral dusts with volatile organic pollutants in atmosphere. Results suggested that
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Fe3+-saturated montmorillonite mediated the dimerization of gaseous 2-chlorophenol to form
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hydroxylated polychlorinated biphenyls, hydroxylated polychlorinated diphenyl ethers, and
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hydroxylated polychlorinated dibenzofurans. The toxicity of Fe3+-montmorillonite particles to
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A549 human lung epithelial cells before and after interaction with 2-chlorophenol was
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examined to explore their adverse impact on human health. Based on cell morphological
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analysis, cytotoxicity tests and Fourier-transform infrared imaging spectra, surface-catalyzed
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reactions of
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montmorillonite particle on A549 cells. This was supported by increased cellular membrane
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permeability, release of extracellular lactate dehydrogenase, and cell damages on cellular
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DNA, proteins and lipids. Since mineral dusts are important components of particulate matter,
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our results help to understand the interactions of volatile organic pollutants with particulate
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matter in atmosphere, and their adverse impacts on human health.
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Key words: Fe3+-saturated montmorillonite; 2-chlorophenol; heterogeneous surface reaction
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A549 cell line; cytotoxicity; mineral dust; particulate matter.
Fe3+-montmorillonite with 2-chlorophenol increased the toxicity of
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Introduction
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Mineral dust, an important component of atmospheric aerosols, is made of soil particles
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suspended in the atmosphere by wind or human activities.1 Depending on sources, mineral
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composition of dust particles is different.2 Montmorillonite, illite, kaolinite and quartz are the
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most common components in mineral dusts.2 Especially for montmorillonite, it is a 2:1
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layered aluminosilicate clay mineral often used to represent mineral dust particles,3 and
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considered as an important condensation nucleus for cloud and rain formation.4,5 Due to
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isomorphic substitution in the tetrahedral Si and/or octahedral Al layers, montmorillonite
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generally possesses structural negative charges that are compensated by exchangeable cations
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in the interlayer regions, resulting in some transition metals incorporated in mineral dusts.3 It
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was reported that ~95% of global atmospheric Fe budgets are in mineral aerosols as iron
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oxide grains, clay-associated iron, and soluble iron.6 Studies showed that Fe species in
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mineral dust aerosols can catalyze the oxidative reactions of NO2 and SO2 to form nitrate and
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sulfate species.7,8 However, limited efforts have focused on gaseous reactions of volatile
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organic pollutants with Fe-enriched mineral dusts under atmospheric conditions.
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Chlorophenols are a class of chlorinated organic pollutants that are widely used in
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industrial and agricultural practice as wood preservatives, herbicides and fungicides.9
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Biodegradation of highly chlorinated aromatic compounds by microorganisms or disinfection
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of drinking water via chlorine can produce chlorophenols.10 As a result, chlorophenols have
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been frequently detected in various environmental compartments.10,11 In ambient air,
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chlorophenols are present as vapors coming from combustion of wastes, coal or wood.10 In
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general, the concentration of chlorophenol in the air is an effect of local emission sources and 3
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high concentration can be found in air samples from certain areas. For instance,
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pentachlorophenol has been detected in urban air at levels of 5.7–7.8 ng m-3.12 The gaseous
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chlorophenol may expose to human especially for the residents living near the pesticide
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manufacturing industries by inhalation, ingestion, eye and dermal contact, causing potential
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adverse effects on health, which has attracted much attention.10 However, little information is
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available for the interactions between gaseous chlorophenols with suspended mineral dusts in
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the atmosphere and their human health effects.
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Prior studies have manifested that transition metal oxides or fly ash surface can catalyze
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chlorophenols to form dioxins at high temperature.13-15 Whereas, our previous studies found
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that Fe3+-montmorillonite clay could oxidize chlorophenol to form dioxin and dioxin-like
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compounds through a single electron transfer process under environmentally relevant
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conditions.16,17 Dioxin and dioxin-like compounds are often highly toxic contaminants due to
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their potency to activate aryl hydrocarbon receptor.18 Here, we hypothesized that similar
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reactions could occur between gaseous chlorophenols and mineral dust surfaces, potentially
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posing risks to human health.
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In this study, Fe3+-montmorillonite was used to simulate mineral dusts since it is one of
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the most abundant soil minerals. Because of its high vapor pressure (0.308 kPa at 25 ℃),
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2-chlorophenol was utilized as a model volatile organic pollutant. The gas-phase reactions
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were conducted in a self-constructed reactor where 2-chlorophenol vapor could interact
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directly with the suspended Fe3+-montmorillonite particles under environmentally relevant
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conditions. The reaction process was monitored by an in situ diffuse reflectance infrared
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Fourier transform spectroscopy (DRIFTS) system. The toxicity of montmorillonite particles 4
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to human lung cells before and after reacting with 2-chlorophenol were evaluated based on
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cell morphology, cytotoxicity tests and Fourier transform infrared imaging spectroscopy
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(FT-IRIS).
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Materials and Methods
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Chemicals and clay minerals. Chemicals and cell line used in this study are described in the
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Supporting Information (SI). Fe3+- and Na+-montmorillonite (particle size of 20–80 nm) were
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prepared from commercial montmorillonite clay mineral, and the detailed information for
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preparation process along with selected physicochemical properties is summarized in the text
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and Table S1 in SI.
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Heterogeneous reaction system. The schematic diagram of the heterogeneous reaction
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system is shown in Sections I and II of Figure S1. To simulate the evaporation of
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chlorophenol and water, compressed air streams 1 and 2 were introduced into two stainless
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steel cylinder jars with 40 mm in diameter and 120 mm in height (parts A and B in Figure S1),
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which contained 1 mL of 2-chlorophenol and 10 mL of ultrapure water, respectively. The air
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flow rate of stream 1 was 0.2 L min−1. The relative humidity of the reaction system was
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controlled via changing the flow rate of air stream 2. Air stream 3 was used to balance the
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total air flow rate of 1.0 L min−1 for three air streams, which was monitored and controlled by
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a S49 32/MT digital mass flow controller (Horiba Metron Instruments, China). The three air
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streams were homogeneously mixed in a 150 mL diffusion jar (part C in Figure S1) before
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reaching the reaction tube. 0.1 g Fe3+-montmorillonite powder was placed in the sample
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holder that was mounted in the center of the quartz reaction tube (10 mm in diameter and 150
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mm in height, part D in Figure S1). The reaction was initiated by passing the 2-chlorophenol 5
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air mixture through the prepared Fe3+-montmorillonite for 2 h. Preliminary experiment
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showed that montmorillonite particles were suspended at air flow rate of 1.0 L min−1 in the
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reactor. Two tandem bottles with 50 mL of 1.0 M NaOH were connected to the outlet to
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collect residual 2-chlorophenol (parts E and F in Figure S1). The temperature for all gas lines
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and the reactor (Sections I and II) was maintained at 25 ℃ in a constant temperature chamber.
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Na+-saturated montmorillonite was used as experimental control.
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In situ FTIR analysis. To trace the whole reaction process on clay surface, real time infrared
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(IR) spectra of the Fe3+-montmorillonite particles at different reaction time were collected by
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a Bruker tensor 27 FTIR spectrometer (Bruker Optik, Germany) equipped with a DRIFTS
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chamber (ZnSe windows, Harrick Scientific) and a mercury cadmium telluride detector. For
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in situ FTIR measurement, Fe3+-montmorillonite powder (0.05 g) was loaded into a DRIFTS
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cell that was connected to a similar air feeding system as described above (Section III in
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Figure S1). The air flow rate and the reaction time were set as 0.5 L min−1 and 320 min,
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respectively. The measured IR frequency region was 400–4000 cm−1, and a total of 64 scans
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were collected for each spectrum with a resolution of 4 cm−1. The interferences from the IR
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absorption of water (~1600 cm−1) and montmorillonite (< 1250 cm−1) were eliminated via
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subtraction of their spectra from the spectrum of the spent montmorillonite using OPUS
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software 7.0 (Bruker Optics, Germany).19
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Identification of reaction products. Spent Fe3+-montmorillonite particles collected from the
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heterogeneous systems were extracted with 2 mL of acetone/hexane mixture (1:1 v/v) for 10
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h. The extract was further acetylated with 5 mL of sodium carbonate (0.5 M) and 120 µL of
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acetic anhydride for 2 h, followed by centrifugation at 1250 g for 20 min. Preliminary 6
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experiment indicated that the recoveries of the extraction method for 2-chlorophenol
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adsorbed on montmorillonite particles under different humidity conditions are 88.3 ± 2.78% –
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107 ± 7.71%. The reaction products were identified using a Thermo Fisher 1310 gas
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chromatograph coupled with an ISQ mass spectrometer (GC-MS) on a full scan mode with
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molecular weight ranging from 40 to 1000 amu. A TR-5 MS capillary column (length = 30 m;
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internal diameter = 0.25 mm; film thickness = 0.25 µm) was used. The carrier gas is helium
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at a flow rate of 1.0 mL min−1 with splitless injection at 290 ℃. The oven temperature was
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programmed initially at 60 ℃ (2 min hold), then increasing to 200 ℃ (15 ℃ min−1, 5 min
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hold), and finally to 320 ℃ (15 ℃ min−1, 15 min hold). The concentrations of Fe2+ and Cl−
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generated during the reaction process were also measured. Further detailed information is
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described in SI.
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Density functional theory (DFT) calculations were also carried out to evaluate the
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possible reaction site for 2-chlorophenol. The frontier electron densities (FED) of the highest
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occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO)
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of 2-chlorophenol were determined by DFT method (B3LYP)20 in Gaussian 09 program.21
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The 6-311G (d,p) basis set was applied to C, H, O and Cl atoms. Molecular structure of
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2-chlorophenol was constructed with Gview program based on the optimized result.
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Cell culture. To evaluate the toxicity of Fe3+-montmorillonite particles collected from
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gas-phase reaction system, human pulmonary epithelial cell line (A549), a widely used
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human lung cancer cell model for investigating cytotoxicity and genotoxicity of nanoparticles
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or organic pollutants was used.22,23 A549 cells were cultured in Dulbecco's modified eagle
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medium
(DMEM)
containing
10%
(v/v)
fetal
bovine
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and
1%
(v/v)
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antibiotic-antimycotic solution (GIBCO/BRL) in an incubator with 5% CO2 at 37 ℃ for 2 d
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for further use.
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Exposure experiment. Before the exposure experiment, 500 µL of A549 cell culture
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suspension was transferred to each well of 24-well or 96-well microtiter plate. For selected
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wells of 24-well plate, CaF2 window (11 mm in diameter and 1 mm in thickness) was
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preloaded at the bottom of 24-well plate for the subsequent microscopic examination. The
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concentration of A549 cells in each well was 1 × 102 cells µL−1, which was confirmed by a
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hemacytometer. After 48 h cultivation, all A549 cells grew at the bottom of wells or on the
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CaF2 window. The culture media were then carefully decanted, and replaced with the same
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volume of fresh DMEM media containing 0.1 to 500 µg mL−1 of the reacted
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Fe3+-montmorillonite particles. Before exposed to the cells, an intermittent ultrasonic
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decomposition process (2 min with the interval of 30 s) was applied to the DMEM medium
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containing montmorillonite particles to ensure the clay particles evenly distributed in media,
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which was confirmed by an atomic force microscopy (AFM) measurement (Figure S2). For
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comparison, the same amount of Fe3+-montmorillonite and 0.8–4000 ng mL−1 of
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2-chlorophenol were also exposed to A549 cells, respectively. The amount of 2-chlorophenol
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exposed to the cell was the same as the total residual 2-chlorophenol on Fe3+-montmorillonite
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surface after the reaction. The concentration ranges of montmorillonite and chlorophenol
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used in this study covered most of the exposure levels employed in previous studies,24-26 and
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would be appropriate to evaluate the potential health effects. The exposure experiments for
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Na+-montmorillonite and the corresponding spent Na+-montmorillonite were also conducted
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using the same methods. All of the experiments were conducted in four replicates. 8
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Examination of cell morphology. After 24 h exposure, the cell morphology of A549 cell
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grown in 24-well plate was examined using a Nikon TS-100 inverted microscope (Tokyo,
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Japan). The cells growing on the CaF2 window were recorded as FT-IRIS image using a
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Bruker HYPERION 2000 infrared microscope (Ettlingen, Germany) with IR spectral range of
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600–4000 cm−1 at 4 cm−1 resolution and 32 scans per pixel. The spectrum of CaF2 window
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was used to correct the signal from the instrumental and atmospheric background. The
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hyperspectral images were analyzed by OPUS 7.0 software.
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Cytotoxicity analysis. To determine the cytotoxicity of the reacted montmorillonite, the
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viability of A549 cell grown in 96-well plate and the corresponding extracellular cytoplasmic
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enzyme lactate dehydrogenase (LDH) concentration in media after 24 h exposure were
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measured using Cell Counting Kit-8 (CCK-8) kit and LDH kit following the manufacturers’
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instruction, respectively. For all the analyses, the cell grown in the media without
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montmorillonite or 2-chlorophenol exposure was considered as control. To eliminate the
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potential interference of montmorillonite particle on the toxicity analysis, the treatment with
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the corresponding amount of Fe3+-montmorillonite, Na+-montmorillonite, DEME medium,
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and CCK-8 or LDH detection solution without A549 cell was set as blank control and the
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measurement was conducted under the same conditions.
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Statistical analysis. The experimental data were presented as mean ± standard deviation of
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four independent measurements and were evaluated by one-way ANOVA followed by
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Fisher’s least significant difference (LSD) test. Significant differences were established at p
