Article pubs.acs.org/est
Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure Su Liu, Zhuoyan Shen, Bing Wu,* Yue Yu, Hui Hou, Xu-Xiang Zhang, and Hong-qiang Ren State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210023, P. R. China S Supporting Information *
ABSTRACT: Sheetlike molybdenum disulfide (MoS2) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS2 and BN nanomaterials decreased cell viability at 30 μg/mL and induced adverse effects on intracellular ROS generation (≥2 μg/mL), mitochondrial depolarization (≥4 μg/mL), and membrane integrity (≥8 μg/mL for MoS2 and ≥2 μg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2−2 μg/mL) of MoS2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS2 and BN nanomaterials at different exposure levels, which is important for their safe use.
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nanotube was nontoxic (100 μg/mL) with regard to proliferation and viability of mammalian cells.17−19 Little information on the toxicity of sheetlike BN nanomaterial is available. The shape of nanomaterials plays important roles in their toxicity.20 The MoS2 and BN nanomaterials with sheetlike structure have a shape similar to that of graphene and graphene oxide. The 2D shape of graphene-based nanomaterials makes them show a strong tendency to interact with the cell surface and damage the plasma membrane with their sharp edge.21,22 Thus, MoS2 and BN nanomaterials with sheetlike structure might have a similar effect on plasma membrane. Although previous literature showed that BN nanotubes could agglomerate over and under the cell membrane,23,24 no information on the influence of MoS2 or BN nanomaterials on plasma membrane is available. On the other side, unlike graphenebased nanomaterials, MoS2 and BN nanomaterials are not persistent in living systems. For example, Wang et al.13 reported that MoS2 nanosheet is unstable to O2-oxidation under ambient conditions in a variety of aqueous media and could release soluble molybdenum (Mo) and sulfur species. Li et al.25 found hollow BN nanospheres could act as a boron (B) reservoir for
INTRODUCTION With wide studies and applications of graphene and graphene oxide, other nanomaterials with sheetlike structure and twodimensional (2D) morphology, including transition-metal dichalcogenides (TMDs) and graphene analogues, have been attracting more and more interest in the past few years.1−3 For example, the 2D molybdenum disulfide (MoS2) nanomaterial, one of the most important TMD nanomaterials, could be widely applied in electronics and optoelectronics due to its electrical conductivity and fast heterogeneous electron transfer.4−6 The graphene analogue boron nitride (BN) could exhibit higher chemical and thermal stability than carbon materials, which make it a promising catalyst support, as it could avoid the sintering of supported catalyst at hot spots.7 Rapid development of the above sheetlike nanomaterials increases the possibility of human exposure to them. Current literature show that graphene-based nanomaterials could induce intracellular reactive oxygen species (ROS) and damage plasma.8−10 However, little knowledge is available on the effects of MoS2 and BN nanomaterials with sheetlike structure. Limited literature showed that MoS2 nanosheet exposure could not decrease the cell viability of mammalian cells, even when the concentration was up to 100 μg/mL,11−13 indicating its low cytotoxicity. However, several studies found that MoS2 nanomaterial exposure could increase ROS and proinflammatory and profibrogenic responses.14−16 As for BN nanomaterial, most research focused on BN nanotube and found that BN © 2017 American Chemical Society
Received: Revised: Accepted: Published: 10834
May 12, 2017 August 15, 2017 August 25, 2017 August 25, 2017 DOI: 10.1021/acs.est.7b02463 Environ. Sci. Technol. 2017, 51, 10834−10842
Article
Environmental Science & Technology
scattering. Cell viability in the treated group was expressed as percentage of viable cells compared with that of the control group. Oxidative Stress Analysis. Intracellular ROS in HepG2 cells was detected using the cell probe DCFH-DA (Molecular Probes).30 Hoechst 33342 (MCE) was applied to count the number of live HepG2 cells in wells of the 96-well plate to avoid the influence of cell loss after nanomaterial exposure.31 After nanomaterial exposure, 10 μM DCFH-DA was first added into each well. The cells were incubated for 25 min and then 2.5 μg/mL Hoechst 33342 was added into each well, and the cells were incubated for 15 min. Fluorescence values of DCF and Hoechst 33342 were measured using a microplate reader (Synergy H1, BioTek). The excitation/emission wavelengths for DCF and Hoechst 33342 were 485/530 and 350/460 nm, respectively. The fluorescence value of DCF was normalized by the Hoechst 33342 fluorescence value in the same well. Mitochondrial Membrane Potential Assay. Mitochondrial membrane potential was analyzed by a JC1 detection kit (KeyGEN). After nanomaterial exposure, 50 μL of JC1 solution was added into each well of the 96-well plate. The cells were incubated for 25 min. Then cells were analyzed on a microplate reader (Synergy H1, BioTek) at two groups of fluorescence: red (excitation/emission wavelengths 530/590 nm) and green (excitation/emission wavelengths 485/530 nm). Mitochondrial membrane potential was determined by the ratio of the red to green fluorescence intensity. A decrease of the ratio indicates mitochondrial depolarization. Membrane Damage Test. Cell membrane integrity was analyzed by measuring the release of lactate dehydrogenase (LDH). After nanomaterial exposure, the LDH in the culture medium was measured by a LDH assay kit (KeyGEN). The absorbance at 440 nm was recorded in a microplate reader (Synergy H1, BioTek). The LDH release in the treated group was presented as the percentage of the control group. Membrane fluidity was measured with 4′(trimethylammonio)diphenylhexatriene (TMA-DPH) (AAT Bioquest Inc.). After 24 h of nanomaterial exposure, cells in the culture flask were washed with PBS buffer, and then 1 mL of 1.5 μM TMA-DPH was added. The cells were incubated for 20 min. After washing the cells with HEPES buffer, polarization of TMA-DPH was measured in a fluorescence spectrophotometer. The relationship between fluorescence polarization and membrane fluidity is an inverse one. A background control without the TMA-DPH probe was also measured under the same conditions as the treated samples. Membrane Transporter Activity Test. The activity of ABC transporters was chosen as a target to analyze the influence of MoS2 and BN nanomaterials on transmembrane proteins by measuring the accumulation of the cell probe calcein-AM (CAM, Dojindo Molecular Technologies).32,33 After nanomaterial exposure, 0.25 μM CAM was added into each well of a 96-well plate. After incubating and washing, 2.5 μg/mL Hoechst 33342 was added into each well to count the number of live HepG2 cells in the wells. The excitation/ emission wavelengths of CAM were 485/530 nm. The fluorescence value of CAM was normalized by the fluorescence value of Hoechst 33342 in the same well. Additionally, CAM fluorescence images of HepG2 after MoS2 and BN exposure were taken by an inverted fluorescence microscope (Nikon Eclipse Ti-U). Chemosensitive Effect Analysis. Arsenic (As), a special substrate of multidrug resistance proteins (MRPs, a subfamily
prostate cancer treatment. Thus, soluble Mo and B release from both nanomaterials could also influence their toxicity, which might be similar to that of metal oxide nanomaterials. The properties of MoS2 or BN nanomaterials mentioned above might influence their toxic mechanisms. For example, plasma membrane ATP binding cassette (ABC) efflux transporters play important roles in pumping xenobiotics out of cells.26,27 Graphene-based nanomaterials could directly damage the plasma membrane structure and function to inhibit ABC transporter activities.28 However, metal oxide nanomaterials (zinc oxide and copper oxide nanoparticles) could release ions under intracellular conditions and form metal conjugates to compete with other substrates of the ABC transporter, leading to inhibition of ABC transporter activities.29 Considering the sheetlike structure and the potential for soluble species release from MoS2 or BN nanomaterials, it is necessary to analyze the roles of nanomaterials and their soluble species release on adverse effects to understand underlying toxic mechanisms. In this study, we systemically studied the cytotoxicities of sheetlike MoS2 and BN nanomaterials at different toxic end points. The human hepatoma HepG2 cell line was chosen as a cell model. Impacts of both nanomaterials on ABC transporter activity were further analyzed for the first time, and the roles of plasma membrane damage and soluble species release were determined. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS2 and BN nanomaterials at different exposure levels.
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MATERIALS AND METHODS Nanomaterials. MoS2 nanomaterial was obtained from Dr. Wei Jiang’s Lab at Nanjing University. BN nanomaterial was purchased from Nanjing XFNANO Materials Tech Co. Ltd. (Nanjing, China). Scanning electron microscopy (SEM) images were conducted with a Hitachi S-3400N II instrument. Transmission electron microscopy (TEM) images of nanomaterials in alcohol were captured by a JEM-200CX electron microscope (JEOL Ltd.). Dynamic light scattering (DLS) analysis was conducted using a Malvern Zetasizer Nano-ZS. Typical functional groups and surface modification of MoS2 and BN were characterized by Fourier transform infrared (FTIR) spectra, which were collected on a Thermo Nicolet NEXUS870. Raman spectroscopic analyses were performed with a Renishaw InVia system. Cell Culture and Treatment. The HepG2 cell line obtained from KeyGEN Biotech (Nanjing, China) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum under standard cell culture conditions (37 °C and 5% CO2). Before treatment with MoS2 and BN nanomaterials, cells were seeded in 96-well plates for 24 h. After the cells were exposed to MoS2 or BN nanomaterials for 24 h, toxicity analyses were performed. Stock solutions of MoS2 and BN were prepared in ddH2O at a concentration of 1000 μg/ mL. Before usage, the nanomaterial solutions were ultrasonically disintegrated for 15 min at a power of 100 W. Cell Viability Test. Cell viability after MoS2 and BN nanomaterial exposures was determined using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc.). Ten microliters of CCK-8 solution was added into each well of the 96-well plate after MoS2 and BN exposure. After incubating for 2 h at 37 °C, spectrophotometric measurement of the cells was performed in a microplate reader (Synergy H1, BioTek) at wavelengths of 450 and 690 nm. The absorbance at 690 nm was applied to reduce the influence of the nanomaterials’ light 10835
DOI: 10.1021/acs.est.7b02463 Environ. Sci. Technol. 2017, 51, 10834−10842
Article
Environmental Science & Technology
Figure 1. Microscopy images of MoS2 and BN nanomaterials used in this study. (A) SEM image of MoS2, (B) SEM image of BN, (C) TEM image of MoS2, and (D) TEM image of BN.
Confocal images were acquired with a Leica TCS SP8 confocal microscope. Measurement of Soluble Mo and B Species. The HepG2 cells were seeded in a 6-well microplate. After 24 h of nanomaterial exposure, cells were collected, washed, and homogenized by ultrasonication in ddH2O. The samples were centrifuged at 14 000g for 20 min. Then the supernatants were filtrated with a 0.22 μm membrane and applied to measure the soluble Mo and B species. Protein contents of cell samples were measured by a BCA Protein Assay Kit (Beyotime) to normalize Mo and B concentrations. Levels of Mo and B in cells were determined by ICP-MS (PerkinElmer). Statistical Analysis. For all assays, three independent trials were performed. Results are expressed as means ± standard deviation. Statistical differences were evaluated using the oneway analysis of variance (ANOVA) test followed by Tukey’s post hoc test. A p-value
