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Cytotoxicity and efflux pump inhibition induced by molybdenum disulfide and boron nitride nanomaterials with sheet-like structure Su Liu, Zhuoyan Shen, Bing Wu, Yue Yu, Hui Hou, Xu-Xiang Zhang, and Hong-qiang Ren Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b02463 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 28, 2017
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Environmental Science & Technology
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Cytotoxicity and efflux pump inhibition induced by molybdenum
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disulfide and boron nitride nanomaterials with sheet-like structure
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Su Liu, Zhuoyan Shen, Bing Wu, Yue Yu, Hui Hou, Xu-Xiang Zhang, Hong-qiang Ren
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State Key Laboratory of Pollution Control and Resource Reuse, School of the
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Environment, Nanjing University, Nanjing, 210023, P.R. China
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Bing Wu
Corresponding author:
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E-mail:
[email protected] 11
Tel.: 0086-25-89680720
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Address: 163 Xianlin Avenue, Nanjing, P.R. China
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ABSTRACT
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Sheet-like molybdenum disulfide (MoS2) and boron nitride (BN) nanomaterials have
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attracted attentions in the past few years due to their unique material properties.
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However, information on adverse effects and underlying mechanisms of sheet-like
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MoS2 and BN nanomaterials is rare. In this study, cytotoxicities of sheet-like MoS2 and
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BN nanomaterials on human hepatoma HepG2 cell were systematically investigated at
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different toxic endpoints. Results showed that MoS2 and BN nanomaterials decreased
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cell viability at 30 µg/mL and induced adverse effects on intracellular ROS generation
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(≥ 2 µg/mL), mitochondrial depolarization (≥ 4 µg/mL) and membrane integrity (≥ 8
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µg/mL for MoS2, and ≥ 2 µg/mL for BN). Furthermore, this study firstly found that the
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low exposure concentrations (0.2-2 µg/mL) of MoS2 and BN nanomaterials could
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increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette
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(ABC) efflux transporter activity, which make both nanomaterials act as
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chemosensitizer (increasing As toxicity). Damage of plasma membrane and release of
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soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux
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pump activities. This study provides systematical understanding of cytotoxicity of
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sheet-like MoS2 and BN nanomaterials at different exposure levels, which is
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important for their safety use.
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INTRODUCTION
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With wide studies and applications of graphene and graphene oxide, other
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nanomaterials with sheet-like structure and two-dimensional (2D) morphology
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including transition metal dichalcogenides (TMDs) and graphene analogues have
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been attracting more and more interest in the past few years.1-3 For example, the 2D
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molybdenum disulfide (MoS2) nanomaterial, one of the most important TMD
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nanomaterials, could be widely applied in electronics and optoelectronics due to its
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electrical conductivity and fast heterogeneous electron transfer.4-6 Graphene analogues
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boron nitride (BN) could exhibit higher chemical and thermal stability than carbon
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materials, which make it a promising catalyst support, as it could avoid the sintering
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of supported catalyst on hot spots.7
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Rapid development of above sheet-like nanomaterials increases the possibility of
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human exposure to them. Current literatures show that graphene-based nanomaterials
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could induce intracellular reactive oxygen species (ROS) and damage plasma
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damage.8-10 However, little knowledge is available on the effects of MoS2 and BN
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nanomaterials with sheet-like structure. Limited literatures showed MoS2 nanosheet
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exposure could not decrease cell viability of mammalian cells even when the
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concentration was up to 100 µg/mL,11-13 indicating low cytotoxicity. However, several
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studies found that MoS2 nanomaterial exposure could increase ROS, proinflammatory
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and profibrogenic responses.14-16 As for BN nanomaterial, most researches focused on
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BN nanotube, and found that BN nanotube was nontoxic (100 µg/mL) in proliferation
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and cell viability towards mammalian cells.17-19 Little information on the toxicity of
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sheet-like BN nanomaterial is available.
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Shape of nanomaterials plays important roles in their toxicity.20 The MoS2 and
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BN nanomaterials with sheet-like structure have similar shape with graphene and 3
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graphene oxide. The 2D shape of graphene-based nanomaterials makes them show a
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strong tendency to interact with cell surface and damage plasma membrane by their
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sharp edge.21, 22 Thus, MoS2 and BN nanomaterials with sheet-like structure might
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have the similar effect on plasma membrane. Although previous literatures showed
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BN nanotube could agglomerate over and under the cell membrane,23,
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information on influence of MoS2 or BN nanomaterials on plasma membrane is
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available. On the other side, unlike graphene-based nanomaterials, MoS2 and BN
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nanomaterials are not persistence in living system. For example, Wang et al.13
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reported that MoS2 nanosheet is unstable to O2-oxidation under ambient conditions in
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a variety of aqueous media, and could release soluble molybdenum (Mo) and sulfur
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species. Li et al.25 found hollow BN nanosphere could act as boron (B) reservoir for
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prostate cancer treatment. Thus, soluble Mo and B release of both nanomaterials
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could also influence their toxicity, which might be similar with metal oxide
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nanomaterials.
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no
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Properties of MoS2 or BN nanomaterials mentioned above might influence their
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toxic mechanisms. For example, plasma membrane ATP binding cassette (ABC)
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efflux transporters play important roles in pumping out of xenobiotics in the cells.26, 27
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Graphene-based nanomaterials could directly damage plasma membrane structure and
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function to inhibit ABC transporter activities.28 However, metal oxide nanomaterials
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(zinc oxide and copper oxide nanoparticles) could release ions in intracellular
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conditions and form metal conjugates to compete the ABC transporter with other
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substrates, leading to inhibition of ABC transporter activities.29 Considering the
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sheet-like structure and potential of soluble species release of MoS2 or BN
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nanomaterials, it is necessary to analyze the roles of nanomaterials and their soluble
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species release on adverse effects to understand underlying toxic mechanisms. 4
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In this study, we systemically studied cytotoxicities of sheet-like MoS2 and BN
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nanomaterials at different toxic endpoints. The human hepatoma HepG2 cell line was
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chosen as a cell model. Impacts of both nanomaterials on ABC transporter activity
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were further analyzed for the first time, and the roles of plasma membrane damage
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and soluble species release were determined. This study provides systematical
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understanding of cytotoxicity of sheet-like MoS2 and BN nanomaterials at different
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exposure levels.
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MATERIALS AND METHODS
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Nanomaterials
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MoS2 nanomaterial was obtained from Dr. Wei Jiang’s Lab at Nanjing University.
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BN nanomaterial was purchased from Nanjing XFNANO Materials Tech Co. Ltd.
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(Nanjing, China). Scanning electron microscopy (SEM) images were conducted with
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a Hitachi S-3400N II instrument (Hitachi, Japan). Transmission electron microscopy
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(TEM) images of nanomaterials in alcohol were carried out by a JEM-200CX electron
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microscope (JEOL Ltd., Japan). Dynamic light scattering (DLS) analysis was
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conducted using Malvern Zetasizer Nano-ZS (Malvern Instruments Ltd., UK). Typical
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functional groups and surface modification of MoS2 and BN were characterized by
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Fourier transform infrared (FTIR) spectra, which were conducted in a Thermo Nicolet
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NEXUS870 (Thermo, USA). Raman spectroscopic analyses were performed with a
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Renishaw InVia system (Renishaw, UK).
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Cell culture and treatment
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HepG2 cell line obtained from KeyGEN Biotech (Nanjing, China) was cultured
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in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
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under standard cell culture conditions (37 °C, and 5% CO2). Before treatment with
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MoS2 and BN nanomaterials, cells were seeded in 96-well plates for 24 h. After the 5
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cells were exposed to MoS2 or BN nanomaterials for 24 h, toxicity analyses were
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performed. Stock solutions of MoS2 and BN were prepared in ddH2O at a
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concentration of 1000 µg/mL. Before usage, the nanomaterial solutions were
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ultrasonically disintegrated for 15 minutes at power of 100 W.
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Cell viability test
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Cell viability after MoS2 and BN nanomaterial exposures was determined using a
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Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc. Japan). 10 µL of
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CCK-8 solution was added into each well of the 96-well plate after MoS2 and BN
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exposure. After incubated for 2 h at 37 oC, spectrophotometric measurement of the
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cells was performed in a microplate reader (Synergy H1, BioTek, USA) at wavelengths
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of 450 nm and 690 nm. The absorbance at 690 nm was applied to reduce the influence
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of nanomaterials’ light scattering. Cell viability in treated group was expressed as
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percentage of viable cells compared with that of control group.
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Oxidative stress analysis
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Intracellular ROS in HepG2 cells was detected using cell probe DCFH-DA
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(Molecular Probes, USA).30 Hoechst 33342 (MCE, China) was applied to count the
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number of live HepG2 cells in wells of 96-well plate to avoid influence of cell loss
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after nanomaterial exposure.31 After nanomaterial exposure, 10 µM DCFH-DA was
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first added into each well. The cells were incubated for 25 min, then, 2.5 µg/mL
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Hoechst 33342 was added into each well, and incubated for 15 min. Fluorescence
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values of DCF and Hoechst 33342 were measured using a microplate reader (Synergy
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H1, BioTek, USA). The excitation/emission wavelengths for DCF and Hoechst 33342
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were 485 nm / 530 nm and 350 nm / 460 nm, respectively. The fluorescence value of
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DCF was normalized by Hoechst 33342 fluorescence value in the same well.
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Mitochondrial membrane potential assay 6
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Mitochondrial membrane potential was analyzed by JC1 detection kit (KeyGEN,
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China). After nanomaterial exposure, 50 µL JC1 solution was added into each well of
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96-well plate. The cells were incubated for 25 min. Then cells were analyzed on a
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microplate reader (Synergy H1, BioTek, USA) at two groups of fluorescence: red
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(excitation/emission wavelengths: 530 nm / 590 nm) and green (excitation/emission
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wavelengths: 485 nm / 530 nm). Mitochondrial membrane potential was determined by
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the ratio of red to green fluorescence intensity. Decrease of the ratio indicates
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mitochondrial depolarization.
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Membrane damage test
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Cell membrane integrity was analyzed by measuring release of lactate
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dehydrogenase (LDH). After nanomaterial exposure, the LDH in culture medium was
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measured by a LDH assay kit (KeyGEN, China). Absorbance at 440 nm was recorded
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in a microplate reader (Synergy H1, BioTek, USA). The LDH release in treated group
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was presented as the percentage of control group.
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Membrane fluidity was measured by 4′-(trimethylammonio)-diphenylhexatriene
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(TMA-DPH) (AAT Bioquest Inc., USA). After 24 h nanomaterial exposure, cells in the
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culture flask were washed by PBS buffer, and then 1 mL of 1.5 µM TMA-DPH was
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added. The cells were incubated for 20 min. After washing the cells by HEPES buffer,
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polarization of TMA-DPH was measured in fluorescence spectrophotometer. The
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relationship between fluorescence polarization and membrane fluidity is an inverse one.
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A background control without the TMA-DPH probe was also measured under the same
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condition as the treated samples.
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Membrane transporter activity test
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Activity of ABC transporters was chosen as a target to analyze the influence of
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MoS2 and BN nanomaterials on transmembrane proteins by measuring accumulation 7
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of cell probe Calcein-AM (CAM, Dojindo Molecular Technologies, Japan).32, 33 After
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nanomaterial exposure, 0.25 µM CAM was added into each well of 96-well plate.
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After incubating and washing, 2.5 µg/mL Hoechst 33342 was added into each well to
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count the number of live HepG2 cells in wells. The excitation/emission wavelengths of
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CAM were 485 nm / 530 nm. Fluorescence value of CAM was normalized by
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Hoechst 33342 fluorescence value in the same well. Additionally, the CAM
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fluorescence images of HepG2 after MoS2 and BN exposure were taken by an
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inverted fluorescence microscope (Nikon Eclipse Ti-U, Japan).
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Chemosensitive effect analysis
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Arsenic (As), a special substrate of multidrug resistance proteins (MRPs,
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subfamily of ABC transporters) was chosen as target xenobiotics to analyze whether
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the inhibition of ABC transporter activity induced by MoS2 and BN nanomaterials
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could lead to chemosensitive effect.34 The ROS generation, one of main effects of As
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toxicity,35, 36 was chosen as the toxic endpoint. During combined exposures, the As
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was first exposed, after 12 h, the nanomaterials were added and exposed another 12
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h.28 The exposure strategy was applied to reduce the interactive effects between As and
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nanomaterials in culture medium. The MK571 was chosen as the positive control.
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Intracellular ROS was measured as described above.
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Gene expression test
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Total RNA isolated by the Takara RNA Kit (Takara Bio. Japan) was
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reversed-transcribed into single-stranded cDNA by a Superscript III Reverse
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Transcriptase (Invitrogen, USA). The real-time PCR was carried out on Corbett
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Real-Time PCR Machine (Australia). Amount of template was quantified with SYBR
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Green (Invitrogen, USA). Following thermal profile was used: 95 oC for 2 min
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followed by 40 cycles of 95 oC for 15 s, 54-55 oC for 30 s and 72 oC for 30 s. Relative 8
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levels of target mRNA were normalized to β-actin. Primer sequences used are shown in
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Table S1.
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Confocal microscopy analysis
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The 10 mg MoS2 or BN nanomaterial was dispersed in 1 mL DMSO with 1 mg
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lipophilic dye, DiL (Invitrogen, USA). The mixture solution was sonicated and
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vortexed to coat nanomaterials with DiL. Then labelled nanomaterials were separated
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by centrifugation at 14000 × g, and re-suspended in 1 ml DMSO. HepG2 cells were
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cultured on coverslips for 24 h and treated with DiL-labelled MoS2 or BN at 5 mg/L.
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After 24 h exposure, CAM (0.25 µM) and Hoechst 33342 (5 µg/mL) were added and
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incubated to indicate cytoplasma and nucleus, respectively. Confocal images were
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acquired with Leica TCS SP8 confocal microscope (Leica, Germany).
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Measurement of soluble Mo and B species
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The HepG2 cells were seeded in a 6-well microplate. After 24 h nanomaterial
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exposure, cells were collected, washed and homogenized by ultrasonication in ddH2O.
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The samples were centrifuged at 14000 × g for 20 min. Then the supernatants were
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filtrated with 0.22 µm membrane and applied to measure the soluble Mo and B species.
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Protein contents of cell samples were measured by BCA Protein Assay Kit (Beyotime,
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China) to normalize Mo and B concentrations. Levels of Mo and B in cells were
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determined by ICP-MS (PerkinElmer, USA).
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Statistical analysis
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For all assays, 3 independent trials were performed. Results are expressed as
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means ± standard deviation. Statistical differences were evaluated using one-way
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analysis of variance (ANOVA) test followed by Tukey’s post hoc test. A p-value
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