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Determining the toxicity of rare earth oxide nanomaterials in macrophages and the involvement of membrane damage Jie Gao, Ruibin Li, Fengbang Wang, Xiaolei Liu, Jie Zhang, Ligang Hu, Jianbo Shi, Bin He, Qunfang Zhou, Maoyong Song, Bin Zhang, Guangbo Qu, Sijin Liu, and Guibin Jiang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b04231 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 12, 2017
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Sequestration of membrane phosphates by toxic REE NPs
Cell membrane disruption
Elevated intracellular
Ca2+
Elevated mitochondrial Ca2+
Lysosomal membrane disruption
Released lysosomal enzyme into cytosol
Cell necrosis ACS Paragon Plus Environment
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Determining the cytotoxicity of rare earth element nanoparticles in macrophages
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and the involvement of membrane damage
3
Jie Gao†,‡, Ruibin Li§, Fengbang Wang†,‡, Xiaolei Liu†,‡, Jie Zhang†,‡, Ligang Hu†,‡,
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Jianbo Shi†,‡, Bin He†,‡, Qunfang Zhou†,‡, Maoyong Song†,‡, Bin Zhang#, Guangbo
5
Qu*,†,‡, Sijin Liu†,‡, Guibin Jiang†,‡
6
†
7
Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing
8
100085, P. R. China
9
§
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative
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Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions,
11
Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, Soochow
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University, Suzhou, Jiangsu 215123, P. R. China
13
#
14
250100, China
15
‡
School of Chemistry and Chemical Engineering, Shandong University, Jinan
University of Chinese Academy of Sciences, Beijing 100049, P. R. China
16 17 18 19 20 21 22 23 24 25
Corresponding author:
26
Guangbo Qu, Ph.D, Email:
[email protected] 27
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Abstract
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Rare earth oxide nanomaterials (REE NPs) hold considerable promise, with high
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availability and potential applications as superconductors, imaging agents, glass
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additives, fertilizers additives and feed additives. These results in potential REE NP
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exposure to humans and the environment through different routes and adverse effects
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induced by biological application of these materials are becoming an increasing
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concern. This study investigates the cytotoxicity of REE NPs: nLa2O3, nEu2O3,
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nDy2O3 and nYb2O3 from 2.5 µg/mL to 80 µg/mL, in macrophages. A significant
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difference was observed in the extent of cytotoxicity induced in macrophages by
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differential REE NPs. The high-atomic number materials (i.e. nYb2O3) tending to be
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no toxic while low-atomic number materials (nLa2O3 and nEu2O3 and nDy2O3)
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induces 75.1%, 53.6% and 20.7% dead cells. With nLa2O3 as the representative
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material, we demonstrated that nLa2O3 induced cellular membrane permeabilization,
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through the sequestration of phosphates from membrane. The further mechanistic
42
investigation established that membrane damage induced intracellular calcium
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increased to 3.0 to 7.3 fold compared to control cells. This caused the sustained
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overload of mitochondrial calcium by approximately 2.4 fold, which regulated cell
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necrosis. In addition, the injury of cellular membrane led to the release of cathepsins
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into cytosol which also contributed to cell death. This detailed investigation of
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signaling pathways driving REE NP-induced toxicity to macrophages is essential for
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better understanding of their potential health risks to humans and the environment.
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Introduction
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Owing to their unique physical-chemical properties, rare earth elements (REEs)
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have been extensively applied as superconductors, imaging agents, glass additives,
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fertilizers additives and feed additives, etc.1-3 For instance, addition of Eu2O3 to ZnO
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films could improve the optical and structural properties and intense luminescence.4
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Yb2O3 has been reported as a novel long circulation material as an in vivo CT
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imaging agent.5 Yb2O3 could be applied for novel additive material for concentrator
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solar cells, as a the solar energy converter.6 Dy2O3 nanoparticles mixed
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gadolinium-dysprosium oxide nanoparticles showed promising characteristics as a
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MRI contrast agent for imaging.7 It has been reported that fertilizers contain
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approximate 30% of La2O3. Commercial feed additives for poultry containing 50 %
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REEs oxides earth and La2O3 accounts for 20% of REE oxides.8
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With the increase in application of REEs worldwide, thousands of tons of REEs
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enriched fertilizers released into the agricultural soil, resulting in the large scale of
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contamination of REEs in soil and water near the mining sites REEs.3,
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instance, because of the large REE reserves, China is the largest worldwide producer
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and supplier of REEs.9 It has been evaluated in 2002 that REE containing fertilizers
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discharged into the cultivated soil are above 5000 tons in China.3 Therefore,
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application of REEs has enhanced the large scale contamination of REEs, displaying
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high accumulative in contaminated environmental compartments, and been considered
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persistence in environment and bioaccumulation in biota.15 REEs containing food
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products, industrial products or medicinal agents could be intake through oral,
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intravenous injection and skin exposure.16 The exposed REEs could accumulated in
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blood, brain and bone after introducing to human body,17 leading to a great concern for
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human health.18-21 The inhalation of air-borne particles of REEs oxides is the most
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typical accidental exposure route.22 Because of the chronic ingestion of REEs, the
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population living in high-REE-background showed abnormality in activities of some Page 3 of 34
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10-14
For
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digestive enzymes.23 As various nanomaterials containing REEs have been developed
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as probes for imaging,24-28 these direct administrations of these REEs containing
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nanomaterials, therefore, may also result in a potential risk to human health. It has
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been reported that fibrosis and renal disorders in patients when Gd elements (9-25
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mmol) used as an imaging agent.18, 29
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NPs) present remarkably different biological activities in comparison to their
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larger-scale counterparts, with the higher surface area significantly altering cellular
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uptake and bio-distribution in vivo, resulting in altered biological effects.31 Therefore,
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improved understanding of the impacts on biological systems induced by these
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materials is highly beneficial for the prevention of side effects in biomedical
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applications.32
30, 31
At the nanoscale, REE nanoparticles (REE
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Macrophages are considered a vital sentinel cell, forming a defensive line to
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combat invading pathogens and they also take a prominent role in the clearance of
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nanomaterials from circulatory systems and local organs.26 Because of the instinct
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role of macrophage, the high accumulation level of environmental particles in these
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cells and the resulted negative effects are considered the dominant risk upon particle
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exposure. Disordered macrophages not only lose their defense activity, but also have
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been found to trigger various pathologies such as autoimmune diseases,33
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inflammation and pneumonitis.34 For instance, atmospheric particles could be cleared
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by macrophages located in lung and the accumulation of particle in macrophages
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leads to sustainable inflammation, and the resulted in lung diseases.35,
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environmental exposure of atmospheric particle has been demonstrated to trigger lung
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cancer due to the inflammation initiated by lung macrophages.35, 36 Exposure to REE
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NPs has been associated with nephrogenic systemic fibrosis in patients,18 because
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macrophage internalized REE NPs such as La2O3 and Eu2O3 cause the transfer of
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phosphates from lipids, leading to membrane damage and injury to lysosomal cell
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membranes,
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inflammasome.37-43 However, as a lack of complete understanding of the mechanisms
promoting
activation
of
the
multiprotein
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oligomer
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The
NLRP3
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underlying REE NP effect on macrophages, the impact of REE NPs on cell
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survival
and viability is at present still unclear. Therefore, the investigation on the
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toxicity of REE NPs with macrophage as cell model is of help to evaluate their risks
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under the exposure REE NPs by various routes.
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Physicochemical properties such as elemental composition, shape, size and
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surface groups largely dictate the toxicity profiles of nanomaterials.26 Therefore,
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different REE NPs are therefore assumed to induce distinct biological effects. In the
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present study, we investigated the cytotoxicity of REE NPs on the survival of
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macrophages, establishing potential mechanisms responsible for the cytotoxicity
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induced by these materials. These findings show a safer pathway for the design of
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RE-containing nanomaterials, for application in biomedical systems.
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Materials and Methods
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Chemicals and reagents
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Dulbecco’s Modified Eagle’s Medium (DMEM), penicillin/streptomycin and
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L-glutamine, were all purchased from Invitrogen (Carlsbad, USA). Fetal bovine
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serum (FBS) was obtained from Atlanta Biologicals Inc. (Lawrenceville, USA). All
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horseradish peroxidase-conjugated secondary antibodies were obtained from GE
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Healthcare Life Sciences (Pittsburgh, USA). The mammalian protein extraction
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reagent and ECL reagent were purchased from Thermo Fisher Scientific (Waltham,
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MA, USA). The protein inhibitor cocktail, 2',7'-Dichlorodihydrofluorescein diacetate
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(DCFH-DA), rapamycin, and tetramethylrhodamine methyl ester (TMRM) were
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purchased from Sigma Aldrich (Shanghai, China). The Bradford reagents for protein
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determination were obtained from Bio-Rad (Shanghai, China). The following
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antibodies (Abs) were used: anti-Bid Ab from R&D Systems (Minneapolis, USA),
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anti-LC-3 Ab from Novagen (Medison, USA), while anti-GAPDH and anti-Caspase 3
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Ab were purchased from Cell Signaling Technology (Shanghai, China). Page 5 of 34
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4-(6-Acetoxymethoxy-2,7-dichloro-3-oxo-9-xanthenyl)-4′-methyl-2,2′(ethylenedioxy)
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dianiline-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (Fluo-3/AM),
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9-[4-[Bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-3-[2-[2-[bis[2-[(acetyloxy)meth
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oxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-3,6-bis(dimethylamino)xa
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nthylium bromide(Rhod-2), E-64d, Pepstatin A, CA-074 methyl ester (CA-074Me),
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Cyclosporin A (CsA), Cytochalasin D, ethylenediaminetetraacetic acid (EDTA) and
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Z-VAD-fmk were purchased from MedChem Express (Monmouth Junction, USA).
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Nanomaterials and characterization
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All REE NPs including La2O3 NP (nLa2O3), nEu2O3, nDy2O3, and nYb2O3
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(sources of REE NPs was shown in Table S1) were prepared in endotoxin-free water
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forming stock solutions of 5 mg/mL. Before characterization or exposure, REE NPs
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were sonicated at 32 W for 30 s. High-resolution transmission electron microscopy
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(HRTEM) samples were prepared by depositing of REE NP suspension (20 µg/mL)
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onto a carbon-coated copper electron microscopy grid (Beijing Zhongjingkeyi
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Technology Co., Ltd., China) and dried under ambient conditions overnight before
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analysis using a HRTEM (JEOL-2100F, Japan). Zeta potential and hydrodynamic
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diameters of REE NPs in DMEM medium were determined using a Zetasizer (Malvern
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Nano series, Malvern, U.K.). Fourier transform infrared spectroscopy (FT-IR) was
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performed using a Nicolet 6700 FT-IR spectrophotometer (Thermo Fisher Scientific
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Inc., USA), according to the KBr pellet method. X-ray photoelectron spectroscopy
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(XPS) determination of REE NPs was performed using sample deposits on silicon
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wafers, where samples were air-dried prior to analysis.
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Cell Culture, cellular treatment and toxicity assays
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J774A.1 cells and Hep1-6 cells were purchased from the Shanghai Cell Bank of
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Type Culture Collection of China. Following treatment at 2.5, 10, 20, 40, and 80
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µg/mL,
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phosphate-buffered saline (PBS), with dead cells analyzed by flow cytometric
cells
were
stained
using
50
µg/mL
propidium
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iodide
(PI)
in
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analysis.38,
44-46
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cytotoxicity according to manufacturer instructions (Promega, Shanghai, China).
The CellTox™ Green Cytotoxicity assay was used to determine
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J774A.1 cells were pre-incubated with 1 µM cytochalasin D, 50 µM EDTA or 2
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nM rapamycin for 2 h, respectively, followed by exposure to 20 µg/mL nLa2O3 with
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or without cytochalasin D, EDTA or rapamycin. Cell death was then evaluated using
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PI staining after 24 h.
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RAW-DifluoTM mLC3 cells (invivogen) were cultured in DMEM containing 10%
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(v/v) FBS, 50 U/ml penicillin, 50 mg/ml streptomycin, 100 mg/ml NormocinTM, and 2
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mM L-glutamine. Before the exposure experiment, the culture medium was changed
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with DMEM containing heat-inactivated FBS (30 min at 56 ℃), 50 U/ml penicillin,
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50 mg/ml streptomycin, 2 mM L-glutamine, and 4.5 g/L glucose, 10% (v/v). After 24
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h, RAW-DifluoTM mLC3 cells was treated with nLa2O3 for 6 h followed by the
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observation with confocal microscopy.
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Inductively coupled plasma-mass spectrometry (ICP-MS) analysis for cellular
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uptake of nanomaterials
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Cells were cultured in 10-cm plates and treated with 20 µg/mL REE NPs for 6 h.
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Cells were washed 3 times with PBS. Then we collected and digested the cells by
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combining with RIPA buffer containing 1% protease cocktail (Sigma Aldrich) on ice
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for 30 min. After centrifugation at 15,000 g for 30 min at 4 ºC, the protein
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concentrations of the supernatant of cellular lysates were determined using the
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Bradford method. The suspension of cell lysates post centrifugation ICP-MS or the
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pellets of cellular lysates were digested for ICP-MS determination. In brief, 200 µL of
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supernatants or pellets were digested with 200 µL of nitric acid-H2O2 (3:1) at 75 ºC
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for 12 h, respectively. The digested solutions were diluted to 10 mL with the acid
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concentration below 3% (v/v) and subjected to ICP-MS analysis to determine the
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quantity of particulate REEs (La, Eu, Dy, and Yb). All analyses were performed in
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triplicates using the external calibration method. The standard mixture solution for Page 7 of 34
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REE determination was purchased from Guobiao (Beijing) Testing & Certification
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Co., Lta. (Beijing, China). The calibration curve was shown in Figure S1 (Supporting
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Information). Linear regressions showed that R was above 0.99 (Figure S1). The
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signal intensity repeatability (RSD) of standard was 9.4%. Each group include at least
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3 parallel samples and the average value was calculated. The sum of the dissolved
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REE and particulate REE content is defined as the total intracellular REE
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concentration. The intracellular total REEs concentration was expressed as REE
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content (ng) versus intracellular protein level (mg).
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Transformation of REE NPs in phagolysosomal simulant fluid (PSF)
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Nanoparticles were dissolved in 1 mL PSF solution (142 mg/L Na2HPO4, 6.65
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g/L NaCl, 62 mg/L Na2SO4, 29 mg/L CaCl2∙H2O, 250 mg/L glycine, 8.09 g/L
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potassium phthalate, pH 4.5) at a concentration of 50 µg/mL and sonicated at 32 W
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for 30 s. Samples were incubated at 37 °C for either 0.25, 0.5, 1, 3, or 6 h, prior to
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centrifugation at 15,000 g for 30 min. Pellets were analyzed by inductively coupled
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plasma-optical emission spectrometry (ICP-OES) to assess the concentrations of
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phosphorous and REE ions, while suspensions were digested and REE ion
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concentrations were determined by ICP-MS.38
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Assessment of lysosomal membrane permeabilization (LMP)
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Regarding concentration selection, for the investigation of the cytotoxicity of
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nLa2O3 as the representative material, 20 µg/mL was frequently selected because the
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concentration higher than 20 µg/mL (40 or 80 µg/mL) possess dramatic toxicity and
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could induced cell detached from the tissue culture dish. As a result, some experiments
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such as confocal fluorescence observation could not performed under the exposure
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concentration higher than 20 µg/mL. After exposure to 20 µg/mL nLa2O3 in DMEM
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for 6 h, J774A.1 cells were washed with PBS and then stained with Magic Red™
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Cathepsin B (ImmunoChemistry Technologies, Bloomington, USA) for 2 h according
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to manufacturers’ instructions. The plasma membrane was stained using a membrane Page 8 of 34
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labeling kit (Sigma Aldrich, Shanghai, China) and nuclei were stained with Hoechst
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33342 (Thermo Fisher Scientific, Waltham, MA, USA), at room temperature for 1 h.
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Finally, cells were visualized using a TCS SP5 laser scanning confocal microscope
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(Leica, Germany).
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Calcium flux analysis and mitochondrial potential assessment
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Alterations to concentrations of intracellular Ca2+ and mitochondrial Ca2+ were
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monitored using Fluo-3/AM and Rhod-2 fluorescence probes, respectively. Briefly,
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after treatment nLa2O3 at 20 µg/mL 1 × 105 cells were incubated at 37 °C in the dark
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with 4 µM Fluo-3/AM and 4 µM Rhod-2 for 20 min. Cells were then washed 3 times
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and re-suspended in Hank’s Balanced Salt Solution without Ca2+ and Mg2+. Cells
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treated with REE NPs were additionally stained with 20 nM TMRM, then analysed by
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flow cytometry (FACS) for mitochondrial membrane assessment. FACS analysis was
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performed using an LSR II flow cytometer (Becton Dickinson, Mountain View, CA)
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equipped with a single 488 nm argon laser. Fluo-3/AM fluorescence was assessed
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using the fluorescein isothiocyanate (FITC) channel; while PI, TMRM and Rhod-2
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fluorescence were examined using the PE channel; and both forward and side scatters
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were used to eliminate cellular fragments. Following treatment, a portion of cells
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were also fixed with 10% PBS-buffered formaldehyde with 0.1% Triton X-100 for 10
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min, then stained with 4’ 6-diamidino-2-phenylindole (DAPI) (blue) and Fluo-3/AM
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(green) or Rhod-2 (red). Fluorescent images were visualized through confocal laser
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scanning microscopy as described previously.40
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TEM observation on cell samples
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After treatment with REE NPs at 20 µg/mL for 6 h, cells were harvested and
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fixed in 2.5% glutaraldehyde for 2 h, then washed with PBS buffer. Staining was
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performed using 1% OsO4 for 1 h, prior to dehydration of fixed and stained cells
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using alcohol (40, 50, 70, 80, 90, 95, or 100% ethanol). Cells were then submerged in Page 9 of 34
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propylene oxide for two 30 min cycled and were then embedded in beem capsules
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containing pure resin. Blocks were hardened at 70 °C for a 2-day period, then 70 nm
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sections were cut and stained with 1% lead citrate and 0.5% uranyl acetate, prior to
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examination using a JEOL JEM 2010F high-resolution transmission electron
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microscope (Hitachi Scientific Instruments, Japan).
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Western blot analysis
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The supernatant of cellular lysates was (20 µg) subjected to both sodium
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dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting,
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according to established protocols, as previously described.40 Western blotting signals
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were detected using a BIO-RAD ChemiDoc XRS chemiluminescence system
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(Bio-Rad Inc., CA, USA).
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Statistical Analysis.
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One-way analysis of variance (ANOVA) was utilized for the analysis of mean
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differences between two or more treated groups and the untreated control group,
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while the differences between the two groups were determined using the two-tailed
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Student's t test. Data are shown in mean ± standard deviation (SD) and pYb, with a similar trend to the ability to for P-REEs
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complexity. These results indicate the release of dissolved ions from REE NPs is
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because the interaction between REEs and phosphates. To further evaluate the
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relation between cell death and dissolved ions levels, the regression analysis was
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performed. The positive trend was observed between the cell death percentages and
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the rate of dissolved REEs (Figure S7B, R=0.987, p
