γ -Fe2O3 nanoparticles with the toxicity of particulate matter originating from subway tunnels in Seoul stations, Korea

Journal of Hazardous Materials 382 (2020) 121175

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Correlation of α /γ -Fe2O3 nanoparticles with the toxicity of particulate matter originating from subway tunnels in Seoul stations, Korea

T

Le Thi Nhu Ngoca, Yongil Leeb, Hang-Suk Chunc, Ju-Young Moond, Jin Seok Choie, ⁎ ⁎ Duckshin Parkb, , Young-Chul Leea, a

Department of BioNano Technology, Gachon University, 1342 Seongnam-Daero, Sujeong-Gu, Seongnam-Si, Gyeonggi-do, 13120, Republic of Korea Korea Railroad Research Institute (KRRI), 176 Cheoldobakmulkwan-ro, Uiwang-si, 16105, Gyeonggi-do, Republic of Korea c Department of Predictive Toxicology, Korea Institute of Toxicology (KIT), Daejeon, 34114, Republic of Korea d Department of Beauty Design Management, Hansung University, 116 Samseongyoro-16gil, Seoul, 02876, Republic of Korea e Analysis Center for Research Advancement, Korea Advanced Institute of Science and Technology (KAIST), Yuseong-gu, Daejeon, 34141, Republic of Korea b

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Editor: R. Debora

According to the increasing concern about particulate matter (PM) pollution at subway systems, particularly its potentially severe effects on human health, this study investigated the constituents, characteristics, and toxicity of PM collected at underground subway stations in Seoul, Korea. It was found that α /γ -Fe2O3 NPs, which are considered as thermal products derived from the brake-wheel-rail interface, were the main components of PM (57.6% and 48% of PM10 and PM2.5, respectively). In addition, hydrothermally synthesized α /γ -Fe2O3 NPs, proposing to possess similar properties to those of Fe2O3 contained in PM, were used to investigate the correlation of these oxides with PM toxicity. In particular, the synthesized γ -Fe2O3 NPs induced a negligibly toxic, while the synthesized α -Fe2O3 NPs and PM showed remarkably toxic effects on HeLa cells and zebrafish embryos, specifically in reducing cell proliferation to 85% and 72% survival, causing high apoptosis of 29.8% and 29.3%, and inhibiting the development of embryos up to 60% and 8% after prolonged exposure, respectively. It is considered that α -Fe2O3 NPs were primarily responsible for the harmful effects of PM, resulting in significant damage to DNA due to their capacity of producing high reactive oxygen species (ROS) and, thus, deleterious effects on the human body.

Keywords: PM toxicity/-Fe2O3 nanoparticles Cytotoxicity Apoptosis Embryotoxicity

⁎

Corresponding authors. E-mail addresses: [email protected] (D. Park), [email protected] (Y.-C. Lee).

https://doi.org/10.1016/j.jhazmat.2019.121175 Received 22 July 2019; Received in revised form 3 September 2019; Accepted 5 September 2019 Available online 06 September 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.

Journal of Hazardous Materials 382 (2020) 121175

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1. Introduction

containing, carbonaceous, soil-derived, secondary sulfate, and nitrate particles were the most significant contributors to PM collected in subway tunnels in Seoul, Korea (Jung et al., 2010). Interestingly, it was reported that most Fe fractions were found in the form of oxides (α -Fe2O3, γ -Fe2O3, and Fe3O4) that accounted for 75–80% of pollutants in metro environments. Recently, some studies have revealed a correlation of these iron oxides with PM toxicity, though there are also conflicting opinions (Johansson and Johansson, 2003; Salma et al., 2009). Johansson et al. reported that PM in underground subway stations in Stockholm, Sweden, was eight times more toxic than urban street pollutants, because it contained a high magnetite mass (Fe3O4) that adversely affects human health (Johansson and Johansson, 2003). Meanwhile, hematite (α -Fe2O3) in PM collected in Budapest, Hungary, and was found to be less toxic to cells and, indeed, harmless (Salma et al., 2009). In our previous study, we investigated the chemical components of PM in subway tunnels in Seoul, Korea, through a number of specific physico-chemical observations, including transmission electron microscopy (TEM), scanning electron microscopy (SEM) with an energy-dispersive X-ray (EDX) spectrometer, and X-ray diffraction (XRD) (Lee et al., 2018a). The results showed that PM was generated as a thermal product of wheel-rail-brake and pantograph-catenary wire interfaces, and was predominantly composed of iron oxides (α -Fe2O3 and γ -Fe2O3) and small amounts of other inorganic elements (Ca, Si, Ba, Mg, Si, Al, and Zr) as well as organic compounds (e.g., polycyclic aromatic hydrocarbon). In the present study, the critical characteristics of PM were analyzed in detail, and the deleterious effects of iron oxides therein on public health were evaluated. Notably, the data obtained from the laboratory synthesis of the two phases of Fe2O3 using a hydrothermal approach, suggested that they have similar properties to those of Fecontaining PM samples originating from metro stations. These synthetic iron oxides along with PM samples were assessed for their potential toxicity to HeLa cells and zebrafish in order to clarify the correlation between them and harmful effects on railway commuters in Seoul, Korea.

Although rail subway systems are more and more popular worldwide due to their potential benefits such as reduction of traffic congestion in urban areas and environmental friendliness (Lee et al., 2018a; Moreno et al., 2015), there are a number of contentious issues, especially air quality both on station platforms and inside trains (Moreno et al., 2015; Jung et al., 2012). It has been reported that subway particles originating primarily from the movement of trains can be accumulated in the atmosphere, leading to high and higher particulate matter (PM) concentrations (Aarnio et al., 2005; Johansson and Johansson, 2003; Kim et al., 2008; Park and Ha, 2008). Therefore, there has been growing concern regarding air quality in underground subway systems and its adverse effects on human health. Undoubtedly, the potential human health risks of PM are of great concern (Kim et al., 2015; Knibbs et al., 2011; Ngoc et al., 2018; Anderson et al., 2012). Certainly, small diameter PM (PM10 and PM2.5), when inhaled, easily penetrates into the human body and deposits itself deeply into the respiratory bronchioles (lung, bronchi, and trachea) (Kim et al., 2015; Ngoc et al., 2018). Deng et al. pointed out that PM10 was mainly deposited in the tracheobronchial region by impaction, and PM2.5 was primarily settled in the pulmonary region of the lung by sedimentation and diffusion (Deng et al., 2019). Indeed, while depositing in the lung, these small particles may interfere with gas exchange and then flow into the blood, leading to serious health problems, such as diabetes, cardiovascular diseases, respiratory health effects, and premature deaths (Knibbs et al., 2011; Ngoc et al., 2018; Hansen et al., 2016). Recently, Fisher et al. revealed that there were 539 ischemic and 122 hemorrhagic out of 727 strokes over a 10-year period (1999–2010), and found that PM10 had a remarkable association with increased ischemic stroke (odds ratio = 1.26; 95% confidence interval (CI) of 1.03–1.55) for each interquartile range of 14.46 μg/m3 of PM10 (Fisher et al., 2019). Wu et al. indicated the close correlation of PM2.5 in adverse lipid level changes, as a consequence, increasing cardiovascular risks in midlife women (Wu et al., 2019). The results showed an increase of 3.8% (95% CI of 1.00–6.60) in lipoprotein and 1.4% (95% CI of 0.50–2.30) in the ratio of apolipoprotein B due to increased 3 μg/m3 exposed to PM2.5 each year (Wu et al., 2019). Particularly, the WHO (2016) estimated that there were 4.2 million premature deaths each year due to PM pollution, accounting for 26%, 25%, 17%, and 16% of cases of respiratory infection, chronic obstructive pulmonary disease, ischemic heart disease, and lung cancer deaths, respectively, as the 14th leading cause of death worldwide (Anderson et al., 2012; WHO, 2016). Burnett et al. suggested that the pollution of PM2.5 may be related to additional causes of death according to the incorporation of risk information from 41 cohorts from 16 countries. The study estimated that the mortality attributable to PM2.5 exposure of 8.9 million (95% CI of 8.8–11.9) was 120% higher than other risk factors, such as cigarette smoking (6.3 million deaths; 95% CI of 5.7–7.0) (Burnett et al., 2018). In addition, PM levels are often higher than those above ground, according to a series of recently published studies on subway systems in Los Angeles, London, Stockholm, and Shanghai (Johansson and Johansson, 2003; Seaton et al., 2005; Kam et al., 2011; Ye et al., 2010). It has been reported that the components of PM collected from subway tunnels are quite different from those in the atmosphere (Chillrud et al., 2004; Furuya et al., 2001). They are major contributions of the large amounts of Fe and Si and the small proportions of Mn, Ca, Cu, Cr, Ba, C, and K (Lee et al., 2018a; Chillrud et al., 2004; Furuya et al., 2001). Bukowiecki et al. estimated that PM samples in the Zurich metro system contained a 6.918 ng/m3 mass concentration of Fe at a distance of 10 m from outdoor railway (Bukowiecki et al., 2007). In another study, the concentration of PM10 was about 30–120 μg/m3 in underground subway stations in Tokyo, Japan during 2001 (Furuya et al., 2001). Particularly, Fe fractions were predominant compared with other chemical elements, accounting for 30–60-fold higher proportions than those in the atmosphere (Furuya et al., 2001). Jung et al. found that Fe-

2. Materials and methods 2.1. Materials Iron (III) nitrate nonahydrate (Fe(NO3)3·9H2O, 403.9972 g/mol), dimethyl sulfoxide (DMSO, 78.13 g/mol), cell proliferation reagent WST-1 solution, 2′,7′-dicholorofluorescin diacetate (DCFH-DA, 487.29 g/mol), and phosphate-buffered saline (PBS) tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bulk ethanol was purchased from Samchun Pure Chemical Co. Ltd (Gyeonggi-do, Korea). The Annexin V apoptosis detection kit 1 was obtained from BD Pharmingen® (San Diego, CA, USA). Roswell Park Memorial Institute (RPMI) 1640, trypsin-EDTA (0.05%), and fetal bovine serum (FBS) were supplied by Gibco® via Life Technologies (Rockville, MD, USA). Penicillin/Streptomycin (Pen/Strep, 100×) and L-Glutamine 200 mM (100×) were supplied from Invitrogen (Carlsbad, CA, USA). Milli-Q DI water was utilized throughout the experiments (conductivity < 18.2 MΩ cm2; Milli-Q Millipore filter system; Millipore Co., MA, USA). 2.2. Collection and investigation of PM 2.2.1. PM collection Based on the methodology employed in our previous study (Lee et al., 2018a), inorganic (metallic), ionic and carbon components of PM samples were collected in subway tunnels from 15th May to 9th June, 2017. The samples were collected on Φ 47 mm quartz filters (QMA filter, Φ 47 mm, Pall Corporation, NY, USA) utilizing a mini-volume air sampler (TAS, 5 litter per min, Airmetrics, Eugene, OR, USA) and Zeflour filters (polytetrafluoroethylene membrane filter, Φ 47 mm, Pall 2

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2.5. Zebrafish maintenance

Corp.) using a low-volume air sampler (PMS-104, 16.7 litter per min, APM, Bucheon, Korea). Lastly, for the XRD analysis, samples were collected from the platform floor in the tunnels. The quartz and Zeflour filters were changed every day at 5 pm during the measuring period (Lee et al., 2018a). In order to analyze the PM chemical composition and morphology, an aluminum foil filter (CFG-225, Φ25 mm, Dekati, Kangasala, Finland) and an electrical low-pressure impactor (ELPI, 10 litter per min, Dekati) were utilized to collect PM samples during 3 days (15th to 17th May). Floor dust in the subway tunnels also was collected and filtered under 1 mm using a sieve (Mesh 18: Φ1.00 mm) to identify crystalline material (Lee et al., 2018a).

Zebrafish embryos (strain: wild-type AB line) were maintained under 14:10 h light/dark cycles at 28.5 °C and fed newly hatched brine shrimp twice a day. The embryos were collected after spawning and cultured in egg water at 28.5 °C. The embryonic development stages were measured according to the standard procedure (Monte, 2000). This research process has been approved by the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (KIT) (KIT2016-1604-0112) and a certificate of approval is available upon request. 2.6. Bioassay for toxicity of PM and synthesized iron oxide NPs

2.2.2. Investigation of components and characteristics of PM According to our previous study (Lee et al., 2018a), PM morphology was characterized by SEM (S-4700, Hitachi, Tokyo, Japan), TEM (Talos F200X, FEI, Hillsboro, OR, USA), and XRD (SmartLab, Rigaku, Austin, TX, USA). The chemical compositions of the PM were determined using an EDX. The PM samples were sputter-coated with Pt prior to the SEM. For the TEM and EDX analyses, the filters were placed and mixed with 20 mL DI water in a tube, after sonicate for 30 min in an ultrasonic extractor, the mixture was then transferred to a TEM grid (CF200 Cu-C film, EMS, Hatfield, PA, USA). For the XRD analysis, dust collected from subway tunnels was filtered using a 1-mm sieve. Quality assurance of the ion compound (IC), metal compound (ICP-AES), and carbon (C) analyses was performed using standard solutions.

2.6.1. Cytotoxicity assay To determine cell viability, a colorimetric assay with WST-1 reagent was carried out. HeLa cells (1 × 104 cells/mL) were seeded into transparent 96-well plate (Falcon, Franklin, NJ, USA). After 24 h incubation, the medium was replaced with 80 μL of fresh medium, to which 20 μL concentrations (5, 10, 30, 50, 70, and 100 μg/mL) of synthetic iron oxide NPs and PM were added. Then, WST-1 reagent (10 μL) was added to each well after treatment periods (24 or 48 h), and the cells were kept incubated for 4 h. The absorbance was measured at wavelengths of 450 and 650 nm. Cell viability was estimated by the following:

Cell viability (%) =

2.3. Synthesis and characterization of engineered iron oxide nanoparticles (NPs)

Absorbance of treated cells × 100% Absorbance of control cells

2.6.2. Evaluation of intracellular reactive oxygen species (ROS) HeLa cells (2.5 × 105 cells/mL) seeded in a black 96-well-plate (Thermo Scientific™, USA) were treated by NPs (100 μg/mL) along with 10 μM of dipyridamole-a radical scavenger as a negative control under identical conditions (Chun et al., 2017). After 1 and 2 days of incubation, 10 μM DCFH-DA dissolved in DMSO was stained for 1 h in a dark room, and then twice washed with PBS buffer. In the literature, DCFHDA penetrates into the internal cells and then is hydrolyzed into DCFH by esters (Song et al., 2010). Immediately, this was recorded by fluorescence measurement at 485/530 nm.

2.3.1. Hydrothermal synthesis of iron oxide NPs A total of 4.03 g of Fe(NO3)3·9H2O was added to 50 mL of ethanol and stirred until the solute was completely dissolved. After complete dissolution, the mixture was reacted at 100 °C for 24 h. The brown precipitate was dried at 60 °C for 24 h. Formed NPs were heated under a mixture of argon (Ar) (96%) and hydrogen (H2) (4%) atmosphere at 300, 400, 500, 600, and 700 °C for 3 h. 2.3.2. Observation characteristics of synthetic iron oxide NPs Their size and surface characteristics were investigated under TEM (Tecnai TF30 ST, FEI Company, OR, USA), Fast Fourier Transform (FFT), and SEM (SEM-4700) equipped with an EDX. The samples were prepared for TEM observation by dropping a tiny pipetted amount on a carbon-coated Cu grid (200-mesh) and oven-drying at 50 °C. The power XRD patterns of synthetic iron oxide NPs were assessed by micro-area X-ray diffractometry (D/MAX-2500, RIGAKU, USA) at 40 kV, 300 mA, and 2 θ = 3–70 ° in 0.02 increments. The Brunauer–Emmett–Teller (BET) surface area and average pore diameters were obtained from N2 adsorption/desorption isotherms using a fully automatic physisorption analyzer (NOVA 4200 Instrument, Florida, USA). 2.4. Cell culture

2.6.3. FACS protocol for cell-death mechanism In order to investigate cell-death mechanism, Annexin V-FITC/PI staining was used to assess the externalization of phosphatidylserine during early apoptosis (Vermes et al., 1995). Briefly, after 24 h incubation in a 6-well plate, HeLa cells (1 × 106 cells/mL) were treated with 100 μg/mL of iron oxide NPs and PM for 24 and 48 h. After these treatment periods, both the supernatant and the cells adhered to the bottom of the plate were collected and washed two times with PBS buffer. The control and treated cells were suspended in a binding buffer and then stained with single Annexin V-FITC, single PI, and a mixture of Annexin V-FITC and PI in a FACS holder before detection apoptosis by CellQuest program of FACS machine (BD FACS Calibur, Becton, Dickinson and Company, USA).

Human cervical cancer cells (HeLa) were supplied from the Korean Cell Line Bank (Seoul, Korea). According to the literature (Masters, 2000), the growth culture medium was prepared by mixing RPMI with 1% pen/strep, 1% L-glutamine, and 10% FBS. These cells were subcultured every other day. When the cell density reached 80–90% confluence, after removing the supernatant of the medium and twice washing with PBS buffer, trypsin-EDTA was added to strip off cells from the bottom of the flask. After that, these cells were collected in conical tubes and mixed with a new growth RPMI medium, were then centrifuged at 1200 rpm for 3 min. The cell destiny was adjusted to between 105 and 106s cells/mL, and the cells were then incubated in T-25 culture flasks (37 °C and 5% CO2).

2.6.4. Embryotoxicity to zebrafish In order to evaluate embryotoxicity, 10 fertilized healthy zebrafish embryos were maintained in a 12-well plate containing 2 mL of E3 medium (0.17 mM KCl, 5 mM NaCl, 0.33 mM MgSO4, and 0.33 mM CaCl2). Then, the embryos were treated with different concentrations of synthetic iron oxide NPs and PM suspended in E3 medium for 3–96 h post-fertilization (hpf) at 28.5 °C. These treatment solutions were exchanged after 48 h. Tests were performed in triplicate. Zebrafish embryos exposed to E3 medium without these treatments served as a control group. The malformations and hatching rates of the embryos at 72 and 96 hpf were recorded under a stereomicroscope (Nikon, SMZ645, Japan) to evaluate the embryotoxicity of these NPs. 3

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Fig. 1. Observed characteristics of PM collected in subway stations at Seoul, Korea through SEM and TEM: (A) SEM images of PM on scales of 10 μm and 5 μm are displayed at left-/right-top, respectively, and chemical compositions of these samples are represented in left-/right-bottom EDX spectra; (B) TEM images of PM sample on scales of 200, 50, 20, and 10 nm; (C) TEM element mapping shows PM sample containing three main elements (Fe, C, and O) as derived from brake-wheelrail interface.

3. Results

emissions of the brake-wheel-rail interface showed high mass concentrations of inorganic compounds (30.41–75.1 μg/m3). Specifically, Fe (major iron oxides) represented the large quantities of 50.4 ± 13.3 and 11.9 ± 8.1 μg/m3, respectively, of PM10 and PM2.5. The morphology of the PM was characterized by SEM/EDX, TEM, and XRD recording. The SEM images showed that PM of 0.12–3.20 μm size in the form of irregular spheres was considered to be generated by mechanical abrasion at the brake-wheel-rail (Fig. 1A). According to the EDX measurement data, the PM consisted of three main elements (Fe, C, and O) together with other organic and inorganic elements (Si, S, Ca,

3.1. PM characterizations According to our previous study (Lee et al., 2018a), PM collected from the subway tunnels contained various organic (CO32−, SO42−, Na+, and NO3−) and inorganic compounds (Fe, AL, Ba, Ni, Mn, Pd, Cr, Zn, V, and Cu), in which the mean mass concentrations of anion compounds, as detected in both PM10 and PM2.5, were 11.0 ± 5.1 and 6.7 ± 3.4 μg/m3, respectively. In other words, PM obtained from the

4

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Cu, and Br) (Fig. 1A). It was found that the nano-size Fe component is generated by friction and then condensation, thereafter attaching to PM (Lee et al., 2018a; Namgung et al., 2016; Lee et al., 2018b). Indeed, it was also noted that Cu or CuO emissions arise from short circuits incurred when a catenary wire is attached to a pantograph to provide discharge (Lee et al., 2018a). It has been suggested that these NPs are generated at positions of contact with the wheel–rail, which contact points surpass a given temperature threshold when a train is braking, assuming that NPs are emitted at the wheel–rail contact rather than at the brake pad–disc interface (Lee et al., 2018a; Namgung et al., 2016; Lee et al., 2018b). Interesting, highly crystal PM was apparent under TEM observation, as evidenced by the abundance of available diffraction patterns to determine the particle structure and lattice spacing. The TEM images exhibited the presence of iron oxide nanocrystals flakes (Fig. 1B-C). According to our previous results (Lee et al., 2018a), PM showed clusters of ferruginous nanocrystals, including high-crystallinity α -Fe2O3, γ -Fe2O3, and C nanocrystals, in which γ -Fe2O3 displayed an elongated cuboctahedral morphology, while α -Fe2O3 showed a circular and rhombohedral architecture (Fig. 1B) (Lee et al., 2018a; Guo and Barnard, 2013). The chemical mapping (Fig. 1C) provided the elemental ratios of PM and the major contributions of Fe (60.2%), O (13.8%), and C (15.5%). It could be seen that these pollutants had originated from steel used in wheels, rails, brakes, and the surrounding environment (Moreno et al., 2015). Notably, Fe nanocrystals were mechanically generated by friction abrasion, especially through the slip of the two metallic surfaces, followed by oxidation (Loxham et al., 2013). Moreover, carbonaceous particles in the subway environment can be derived from the commutator brush with high resistance in electric motors and train pantographs connected to the catenary (Moreno et al., 2015); thus it was proposed that most of the carbonaceous emissions were derived from the interaction between the brake and the train wheel. In addition, other trace cations, including Mg, Al, Si, Cu, and Zr, were heterogeneously identified within the pad (Moreno et al., 2015); these are known as metallic lateral brake particles and show a typically heterogeneous PM distribution. Remarkably, the XRD analysis clearly confirmed the presence of magnetic and non-magnetic chemical compounds, including iron oxides (α -Fe2O3 and γ -Fe2O3), quartz (SiO2; JCPDS card number 01-0758322); calcium carbonate (CaCO3; 00-047-1743), and other compounds (Al2O3, Cu2O, CuO, FeS2, and MgO) (Fig. 2A–D). In fact, quartz is the second richest mineral in the continental crust, and CaCO3 is the main component of cement; thus, these components’ presence might be attributed to the surrounding environment (Zhang et al., 2011g; Choi et al., 2018). Further, Fe is a component of rails that is oxidized to iron oxides or iron hydroxides when exposed to air and then presents on the rail surfaces (Zhang et al., 2011g; Isozaki et al., 2016). Among these species, Fe2O3 was found to account for the highest proportion of total dust mass concentration, estimated at 57.6% and 48.1% in PM10 and PM2.5, respectively.

γ -Fe2O3 with an increase in the calcination temperature to 400 °C, and the peak intensities were obviously increased, implying improving crystallinity. Although at higher heating temperatures (500 and 600 °C), the γ -Fe2O3 phase remained unchanged with typical diffraction peaks at (220), (211), (400), (422), (511), and (440), and slight differences were detected in peak intensities. It was clearly apparent that the diffraction peaks of maghemite γ -Fe2O3 had a greater intensity at 500 °C, than at 400 or 600 °C, suggesting the improvement of the crystallization of the synthetic Fe2O3 at sufficient temperatures. Furthermore, by calcination of the composite at 700 °C for 3 h in the mixed Ar and H2 atmosphere, iron oxide was formed in the phase of magnetite Fe3O4 with low intensities. Notably, the patterns of the synthetic αFe2O3 and γ-Fe2O3 samples had no diffraction peaks from other crystalline forms, demonstrating the formation of high-purity and crystallinity NPs (JCPDS, No. 00-024-0072 and 00-025-1402, respectively) (Islam et al., 2012). Besides, the synthetic Fe3O4 NPs were found to have poor crystallinity due to weak peak intensities relative to the commercial powders (JCPDS, 01-086-1340) (Islam et al., 2012). TEM could provide further insight into the size and morphology of the corresponding products (Figs. 4 and S1). It has been revealed that calcination conditions play an important role in controlling the morphology, size, and crystal structure of NPs. There were remarkable changes in crystal formation with increasing temperature. At 300 °C, α -Fe2O3 NPs were well formed in the shape of regular cubic structure (∼70.6 nm) (Fig. 4), in which the common faces of α -Fe2O3, including (012), (104), and (110), were displayed through high-resolution TEM (HR-TEM) and FFT images (Fig. S1A). Particularly, there were pale areas inside the α -Fe2O3 NPs, indicating that these nanospheres consisted of single crystals, resulting in less agglomeration in aqueous solution. Furthermore, the crystalline structures of γ -Fe2O3 were found in the form of hexagonal and asymmetrical hexagonal with obviously different diameters at high heating (400–600 °C) (Fig. 4 and Table 1). The HR-TEM images also showed the presence of typical faces in γ -Fe2O3 NPs, such as (111), (220), and (311) faces (Fig. S1B–D). These Fe2O3 NPs were more agglomerated at 500 and 600 °C and were completely compressed crystals, thus reducing the purity of NPs and increasing their sizes to 127.75 and 222.05 nm respectively, as correlative to the lower heat treatment (400 °C) (∼85.86 nm) (Figure). Furthermore, at the high calcination temperature (700 °C), the Fe3O4’s morphology seemed to be heterogeneous crystal and highly accumulated in an average size of 320 nm, suggesting that its structure was not well crystallized (Figs. 4 and S1E). In order to investigate the surface areas of both synthesized iron oxide NPs, the nitrogen adsorption-desorption isotherm and BET calculations of these calcined NPs were determined. The obtained results showed completely different hysteresis patterns at relative pressure 0–1, corresponding to different pore sizes and architectures (Fig. 5A). In fact, the type IV isotherm pattern was well-defined in the synthesis of α -Fe2O3, the behavior of which corresponded to that of mesoporous hematite (α -Fe2O3) presented in other references (Zhou et al. (2009); Park et al. (2016)). Meanwhile, the hysteresis loops were poorer when the calcination temperatures were increased, due to the transformation into other oxide phases. For the sample calcined at 400 °C, the hysteresis loop was not well established, which may have been caused by the presence of inter-particle void spaces. Correspondingly, the type III isotherm pattern was clearly observed at γ -Fe2O3 obtained at 500 and 600 °C heating. Besides, the synthetic Fe3O4 NPs were completely nonabsorbable and desorption nitrogen, as was consistent with the few molecular layers in their surface area. It is undeniable that the heating temperature is a critical factor in the control of the structure of iron oxides. In addition, the pore-size distribution was estimated from the desorption isotherms of these iron oxides as demonstrated in Fig. 5B. The BET surface area was found to be the highest in the α -Fe2O3 sample (33.99 m2/g), which value was significantly reduced when the heating temperature was continuously increased to 600 °C (Table 1). Although the mesoporous structures of the γ -Fe2O3 NPs were still evident, their

3.2. Characterization of hydrothermally synthesized iron oxides In the literature, phase transformations are correlated with systemic variation in operating parameters (Liu et al., 2016; Islam et al., 2012). To confirm the formation phases of iron oxides prepared through this study, a comparable XRD analysis was conducted with commercial powder materials (Fig. 3). The results proved that under different heating conditions, particularly calcination temperature, iron oxide can be formed in various phases, including α -Fe2O3, γ -Fe2O3, and Fe3O4, consistently with those of powder materials (Wako Catalog No. 32294283, 324–94282, and 093-01035 respectively) (Islam et al., 2012). When the composite was heated at 300 °C under a mixture of Ar and H2 atmosphere, it was found that the (012), (104), (110), (113), (116), and (214) planes, which displayed great peak intensities, confirmed the formation of α -Fe2O3. Interestingly, the phase was transformed into 5

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Fig. 2. XRD patterns of PM collected in subway tunnels at Seoul stations.

detected to display an ultramicroporous architecture (BET∼0.06 m2/g) without the distribution of pore-size. After examination of the TEM images for both calcined samples (Fig. 4), it could be concluded that the mesoporous structures of α -Fe2O3 and γ -Fe2O3 were well developed, and most of the crystals were well-being prepared under the preferred temperatures (300–600 °C).

3.3. Assessment of in-vitro toxicity of engineered Fe2O3 NPs and PM According to the investigated characterization of PM, only α -Fe2O3 and γ -Fe2O3 NPs were identified as the main components of PM samples originating from the subway tunnels. Therefore, this study only performed in-vitro cytotoxicity tests on HeLa cells and in-vivo embryotoxicity assays on zebrafish of these iron oxide phases in order to assess their role in PM toxicity. In the present study, HeLa cells were exposed to synthetic α /γ -Fe2O3 NPs and PM sample collected from subway tunnels at different concentrations of 5, 10, 30, 70, and 100 μg/mL for 24 and 48 h, and the cytotoxicity was determined using the WST-1 assay. The results showed that both phases of the iron oxides led to negligible cytotoxicity to HeLa cells at low concentrations (5–50 μg/mL) (Fig. 6). Meanwhile, toxicities were obviously observed at high concentrations (≥100 μg/mL), which reduced the survival rate of the HeLa cells to 85.5% and 84.5% after 24 and 48 h exposure, respectively (P < 0.5). In the other hand, PM had a significantly adverse effect on the survival of HeLa cells, resulting in 72% and 58% dead cells after short-term and prolonged treatment, respectively.

Fig. 3. XRD patterns of synthetic iron oxides under various calcination temperatures (300, 400, 500, 600, and 700 °C).

surface areas had obviously been reduced, relative to the hematite NPs, due to the larger particles and severe agglomeration. Further, these γ -Fe2O3 NPs showed a low pore-size distribution in the range of 5–10 nm, while the α -Fe2O3 represented a broader distribution (5–40 nm) with an average pore-size of 23.18 nm and possibly a larger pore volume of 0.197 cm3/g as well. Particularly, the Fe3O4 NPs were 6

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Fig. 4. Representative TEM images of synthetic iron oxide NPs at different heating temperatures: (A) α -Fe2O3 (300 °C); (B) γ -Fe2O3 (400 °C); (C) γ -Fe2O3 (500 °C); (D) γ -Fe2O3 (600 °C); (E) magnetic-Fe3O4 (700 °C).

apoptotic and necrotic populations in the treated and untreated cells were assessed according to the flow-cytometric outcomes, and the results clearly showed different levels of DNA damage (Fig. 7B). According to the results, synthetic α -Fe2O3 induced a high apoptosis of 29.80% during 24 h exposure, as compared with the untreated cells. Conversely, all of the synthetic γ -Fe2O3 NPs not only showed low toxicity to HeLa cells but also promoted living cells, thereby reducing the rate of apoptosis (∼4.13–16.25%). A possible explanation is that the synthetic α -Fe2O3 was small in diameter and high crystallinity, which facilitated its entry of and then interaction with cells that produced a large amount of ROS radicals, thus causing the highest apoptosis level-as compared with synthetic γ -Fe2O3 NPs. Besides, PM caused significant DNA damage during the period of treatment (29.30 ± 2.10%). Although both treatments caused early apoptosis, they only negligibly made cells undergo the necrosis phase and late apoptosis (approximate rate 1.00%).

3.4. Relative ROS quantification and cell-death mechanism by FACS of engineered Fe2O3 NPs and PM The ability of synthetic Fe2O3 and PM to generate DNA-damaging ROS was evaluated using time-dependent (24 and 48 h) DCFH-DA assay (Fig. 7A). All of the synthetic γ -Fe2O3 NPs were found to be non-generative of ROS radicals on HeLa cells, which fact proved they were less toxic and able to prevent apoptosis and necrosis cell death of HeLa cells. Conversely, the synthetic α -Fe2O3 NPs significantly induced ROS radicals with prolonged exposure, reaching 133.71% in DCFH-DA fluorescence versus control samples. Besides, PM showed the same trend with the synthetic α -Fe2O3 in producing large amounts of ROS radicals over both time durations, resulting in 140.50 ± 0.93% and 150.12 ± 2.31%, respectively. Furthermore, in order to investigate the mode of cell death in HeLa cells caused by these NPs, whether viable, apoptotic or necrotic, a flowcytometric analysis using Annexin V-FITC and PI staining was conducted. Therein, apoptosis cells could easily be identified by Annexin VFITC dye, which is introduced from the inside to the outer leaflet of the plasma membrane in the early stages of apoptosis (Bohdanowicz and Grinstein, 2013; Rieger et al., 2011). Meanwhile, PI does not stain live or apoptotic cells, because it depends on membrane permeability to enter cells (Rieger et al., 2011). In the present study, the proportions of

3.5. In-vivo toxicity of Fe2O3 and PM to zebrafish embryos Hatching is the most important event in the lifecycle of zebrafish. The duration of embryonic development to hatching is a major feature of embryotoxicity (Shaw and Handy, 2011; Duan et al., 2013). In our study, zebrafish embryos were exposed to synthetic Fe2O3 NPs and PM,

Table 1 Morphologies, sizes, BET surface areas, and average pore diameters of synthetic iron oxide NPs under different calcination temperatures.

α -Fe2O3 (300 °C) γ -Fe2O3 (400 °C) γ -Fe2O3 (500 °C) γ -Fe2O3 (600 °C) Fe3O4 (700 °C)

Morphology of NPs

Average diameter (nm)

BET (m2/g)

Pore size (nm)

Pore volume (cm3/g)

Cubic Hexagonal Asymmetrical hexagonal Asymmetrical hexagonal Heterogeneous

70.60 ± 30.27 85.86 ± 31.64 127.75 ± 17.86 222.05 ± 8.34 320.18 ± 57.15

33.99 10.05 6.14 3.56 0.06

23.18 9.44 8.21 7.16

0.197 0.023 0.013 0.006 –

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Fig. 6. Cytotoxicity of engineered α /γ -Fe2O3 NPs and PM to HeLa cells during (A) 24 h and (B) 48 h exposure.

Fig. 5. (A) Nitrogen adsorption-desorption isotherms and (B) pore size distribution of iron oxides calcined at 300, 400, 500, 600, and 700 °C.

4. Discussion and it was obtained that both of the treatments induced dose-dependent inhibition of embryo hatching (Fig. 8). To begin with, the results showed that even though neither phase of the synthetic Fe2O3 NPs inhibited the growth of the zebrafish embryos (the survival rate was 100%), they did induce a slight delay in embryo development after 72 h exposure (Fig. 8A). A sharp decline in the hatching rate was found in the synthetic α -Fe2O3-treated group, which was 40% lower than in the control group (100%). Conversely, the synthetic γ -Fe2O3 NPs showed only a slight influence on the growth of embryos, allowing 90% of them to hatch. It is believed that the difference in the toxic effects of these NPs might be related to their structural characteristics. With a smaller diameter and a higher ROS production capacity, the synthetic α -Fe2O3 effected higher toxicity than did the synthetic γ -Fe2O3 NPs In other words, the zebrafish embryos were exposed to PM concentrations (30 and 300 ppm) within 3–96 hpf in order to evaluate the PM’s embryotoxicity. At 72 and 96 hpf, a difference was observed in the hatching rate between the control and PM-exposed groups (Fig. 8B). There was a negligible difference between the PM-treated (30 ppm) and control groups, with almost all embryos hatched. Meanwhile, the obvious difference was found under high concentration of PM treatment (300 ppm), the embryos did not hatch and then died within the chorion. The hatching rates dropped remarkably to 6% and 8% at 72 and 96 hpf, respectively.

In the present study, among large quantities of heavy metals and organic fractions (e.g., polyaromatic hydrocarbons and volatile compounds), α /γ -Fe2O3 NPs were identified as the primary components of PM samples obtained from subway tunnels at Seoul stations through several observations of PM characteristics (Monte, 2000; Chun et al., 2017). It is assumed that these iron oxides might be closely correlated with the harmful effects of PM on human health after prolonged exposure. These correlations could be fully elucidated via the NPs’ cellkilling mechanisms, which are known to be mainly responsible for ROS production on HeLa cells. In fact, it has been demonstrated that PMinduced health effects are mostly related to inflammation and oxidative stress, leading to DNA damage (Risom et al., 2005). Certainly, PMmediated oxidative stress arises from mixed sources, and involves the generation of ROS directly from the surfaces of NPs, dissolution of organic compounds or transition metals, alteration of mitochondrial functions or nicotinamide adenine dinucleotide phosphate (NADPHoxidase), and activation of inflammatory cells capable of producing reactive nitrogen species and ROS radicals (Fig. 9) (Prahalad et al., 2001; Ma and Ma, 2002; Voelkel et al., 2003). Recently, several studies have demonstrated that transition metals, such as Fe, Cu, Zr, Ba, and V on PM’s surface play a crucial role in creating ROS through Fenton reactions (Ghio et al., 2000). In addition, the organic fractions of urban air particles containing Quinone compounds, which may undergo redox-cycling-producing hydroxyl radicals, account for a large 8

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intrinsic pathways (cytochrome c release, caspase-9/-3 activation) but also extrinsic ones (tumor necrosis-α secretion, caspase-8/-3 activation) (Peixoto et al., 2017; Dagher et al., 2006). Induction of intrinsic apoptosis is one of the major activation pathways by micro dust exposure that disrupts the structure of DNA, dsDNA, and ssDNA, consequently leading to epithelial and mitochondrial dysfunction and inflammation of cells (Peixoto et al., 2017; Visalli et al., 2015; Danielsen et al., 2011; Khan et al., 2012; Park et al., 2014). Moreover, autophagy has been considered to contribute to cellular homeostasis and to adapt to stress by functioning as a cytoprotective response (Peixoto et al., 2017; Kubisch et al., 2013). Deng et al. revealed that PM2.5 derived from an iron-and-steel factory increased the number cytoplasmic vacuoles in lung cells, which fact suggests that autophagy induction is due to PM (Deng et al., 2013). It can be pointed out that the intracellular generation of ROS has been implicated in the activation of the autophagy process, owing to the susceptibility of oxidized proteins to uptake by autophagosomes and then lysosomes (Khan et al., 2012; Deng et al., 2013). The critical role of ROS in autophagy is to directly provide the membrane source for autophagosome biogenesis or to indirectly remove amino acid from the process of the progression/regulation of autophagy (Khan et al., 2012; Scherz-Shouval and Elazar, 2011). In addition, the autophagy process has been considered as a necrosispromoting cell-death in cells that are unable to participate in apoptotic machinery activation (Ullman et al., 2007). In the case of intense shortterm exposure to high concentrations of PM and synthetic Fe2O3, DNA damage can result from the release of cytokines, high inflammation, and the exacerbation of oxidative stress; thus, mitochondrial dysfunction and instability of cellular mechanism shift the cells’ fate to necrotic death, leading to mutagenesis in the next cell cycle (Roubicek et al., 2007). Furthermore, the present study’s results indicated that the synthetic α -Fe2O3 NPs and PM had a noticeable influence on the development of zebrafish embryos. It has been suggested that zebrafish-hatching retardation is attributable to the interference of Fe and other heavy metals presented in PM with metalloprotease hatching enzymes (Felix et al., 2017). Certainly, these NPs settle to the bottom of the vessel and subsequently interact with hatching enzymes. Moreover, the large amount of ROS induced by Fe2O3 and PM also directly destroys hatching enzymes, thus reducing this enzyme function leading to delaying of embryonic’ development (Felix et al., 2017). Overall, it can be concluded that underground subway PM has cytotoxic and inflammatory potentials as well as transient biological effects. Due to PM is found to contain not only these Fe fractions but also other heavy metals and organic compounds, PM’s toxicity is attributed to all of its components thereby PM obviously displays higher toxic to HeLa cells and zebrafish embryos, compared with the synthetic Fe2O3 NPs. According to the literature, PM is emitted as the result of a thermal process occurring at the brake-wheel-rail interface. During the operation of the train, especially when braking, the temperature at the interface between the wheels and the rail increase rapidly due to the friction at the interaction sites, thus generating PM-containing iron oxides and other compounds as thermal products. Therefore, the hydrothermally synthesized α /γ -Fe2O3 NPs can be identified as representative of the Fe2O3 presented in PM collected in underground subways. Consequently, the results of this study can be considered to be positive evidences indicative of the role of Fe2O3 in contributing to the adverse effects of PM, wherein the toxicity of subway NPs is primarily contributed by large quantities of such iron oxides, especially hematite α -Fe2O3.

Fig. 7. (A) Generation of ROS and (B) apoptotic cell death after 24 h exposure by engineered α /γ -Fe2O3 NPs and PM.

proportion of the metabolic cells arising from metabolism processes and cause the expression of cytochrome enzymes and, subsequently, enhance ROS production (Dellinger et al., 2001; Bonvallot et al., 2001). Interestingly, it has been suggested that iron oxides can enter and then interact with cells by various mechanisms, including passive diffusion, receptor-mediated endocytosis, clathrin-mediated endocytosis, and endocytosis through a caveolin intermediary (Patil et al., 2018). After entering the cell membrane, iron oxides can release to Fe2+ and Fe3+ by lysosome-present due to Fenton or Haber-Weiss reaction, therefore generating ROS radicals by altering mitochondrial and other organelle functions corresponding to the activation of inflammatory cells (Risom et al., 2005; Patil et al., 2018; Kanagesan et al., 2013; Mahmoudi et al., 2011). Additionally, these nanometer-sized NPs can easily penetrate the nuclear membrane and make a remarkable contribution to nucleic acid destruction and disruption of hydrogen bonding in the DNA structure, leading to damage of DNA (Patil et al., 2018; Mahmoudi et al., 2011). Another possible explanation for the cytotoxicological and cell-morphological changes is that the phenotypic effects of Fe2O3 internalization may strongly inhibit transcriptional regulation and protein synthesis, resulting in a loss of cell phenotype and possibly cell death (Kanagesan et al., 2013). In the present study, synthetic α -Fe2O3 presented the closest correlation with PM in that it possessed the best crystal structure and the smallest diameter (∼70 nm), and consequently, could most easily enter the cell membrane and produce huge amounts of ROS radicals, thereby severe affecting cells, as compared with the other γ -Fe2O3 NPs (85.86–222 nm). On the other hand, these NPs cause apoptosis events not only via the

5. Conclusion In the present study, α /γ -Fe2O3 NPs were successfully synthesized with high crystallinity structures after calcination at 300–600 °C, and were then confirmed by TEM, XRD, and BET. Particularly, it was determined that these oxides accounted for large amounts of the PM that 9

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Fig. 8. Morphology and hatching rate of zebrafish embryos under treatment of (A) iron oxides for 72 (hpf) and (B) PM sample for 72 and 96 hpf.

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Fig. 9. Schematic representation of possible mechanisms of iron oxide and PM interaction, induction of oxidative stress and their roles in carcinogenesis (Risom et al., 2005; Patil et al., 2018) (NADPH: Nicotinamide adenine dinucleotide phosphate; PAH: Polycyclic aromatic hydrocarbons).

had been obtained from subway tunnels in Seoul, Korea. The results of in-vitro toxicity and in-vivo embryotoxicity tests, respectively, revealed that synthetic α -Fe2O3 and PM, as compared with synthetic γ -Fe2O3, were more toxic in HeLa cells and also inhibited the hatching of embryonic zebrafish. It can be concluded that these iron oxides contributed most to the PM toxicity, specifically α -Fe2O3, by generating a huge amount of ROS radicals and causing apoptosis, autophagy, and necrosis cell death.

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Funding This research was supported by a grant from the Subway Fine Dust Reduction Technology Development Project of the Ministry of Land Infrastructure and Transport (19QPPW-B152306-01) and the Ministry of Environment as “Korea Environmental industry & Technology institute (KEITI) (NO. 2018000120004)”. This work was also supported by the Basic Science Research Program through the “National Research Foundation of Korea funded by the Ministry of Education (NRF2017R1D1A1A09000642)”. Declaration of Competing Interest None. Acknowledgment The authors thank Han Na Jeon for conducting in-vitro toxicity of synthetic Fe2O3 NPs. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2019.121175. References Aarnio, P., Yli-Tuomi, T., Kousa, A., Mäkelä, T., Hirsikko, A., Hämeri, K., et al., 2005. The concentrations and composition of and exposure to fine particles (PM2.5) in the

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