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Multi-hierarchically Profiling the Biological Effects of Various MetalBased Nanoparticles in Macrophages under Low Exposure Doses Jie Zhang, Shunhao Wang, Ming Gao, Ruibin Li, and Sijin Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01744 • Publication Date (Web): 30 Jun 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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ACS Sustainable Chemistry & Engineering

Multi-hierarchically Profiling the Biological Effects of Various Metal-Based Nanoparticles in Macrophages under Low Exposure Doses

Jie Zhang†, ‡, Shunhao Wang†, ‡, Ming Gao†, ‡, Ruibin Li§ ,¶, Sijin Liu*, †, ‡

†

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. ‡

§

University of Chinese Academy of Sciences, Beijing 100049, China.

School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative

Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, 215123, China. ¶

California NanoSystems Institute, University of California, Los Angeles, CA, 90095, USA.

*: Correspondence to Sijin Liu, Ph.D, email: [email protected], Tel: 8610-62849330. Address: No. 18, Shuangqing Road, Haidian District, Beijing, China

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ABSTRACT

Thus far, tremendous efforts have been made to understand the bio-safety of metal-based nanoparticles (MNPs). Nevertheless, most previous studies focused on specific adverse outcomes of MNPs at unrealistically high concentrations with little relevance to the National Institute for Occupational Safety and Health (NIOSH) exposure thresholds, and failed to comprehensively evaluate their toxicity profiles. To address these challenges, we here endeavored to multi-hierarchically profile the hazard effects of various popularly used MNPs in macrophages under low exposure doses. At these doses, no remarkable cell viability drop and cell death were induced. However, cellular anti-oxidant defense system was seen to be initiated in cells by all MNPs even at these low concentrations, albeit to a differential extent and through different pathways, as reflected by differential induction of the anti-oxidant enzymes and Nrf2 signaling. Regarding inflammation, rare earth oxide nanomaterials (REOs) except

nCeO2

greatly

increased

IL-1β

secretion

in

a

NLRP3

inflammasome-dependent manner. By contrast, six REOs, AgNP-5nm, nFe2O3, nFe3O4 and nZnO were found to elevate TNF-α concentration through post-transcriptional regulation. Moreover, all MNPs except nCeO2 drastically altered cellular membrane/cytoskeleton meshwork, but leading to different outcomes, with condensed cellular size and reduced numbers of protrusions by REOs and elongated protrusions by other MNPs. Consequently, REOs (e.g. nDy2O3 and nSm2O3) impaired phagocytosis of macrophages, and other MNPs (such as AgNP-25nm and nZnO) reversely enhanced macrophagic phagocytosis. Alterations of membrane and 2 ACS Paragon Plus Environment

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cytoskeleton meshwork induced by these MNPs also caused disordered membrane potential and calcium ion flux. Collectively, our data profiled the biological effects of different MNPs in macrophages under low exposure doses, and deciphered a complex network that links multi-parallel pathways and processes to differential adverse outcomes.

KEYWORDS: Metal-Based Nanoparticles; Macrophages; Multi-hierarchic Profiles; Adverse Outcome Pathways; Low-Dose Exposure.



INTRODUCTION Metal-based nanoparticles (MNPs) are the most produced nanomaterials because they have a wide variety of compositions and unique properties suitable for biomedicine, catalysis, electronics, food production and package, personal care products, paintings, etc.1,2 According to a report by Nanotechnology Consumer Product Inventory (CPI), 37% of nanoproducts in the global market contain metal-based nanomaterials, which contains several categories including health and fitness, home and garden, automotive, cross-cutting, electronics, food and beverage, appliances and goods for children.3 Under this setting, MNPs may release into the environment and lead to substantial exposure to human beings, especially when they are manufactured in cosmetics, pigments and food additives.4 Our recent study unveiled that massive TiO2 particles in seafood and surimi products, suggesting their exposure to human beings through foods.5 During the past fifteen years, a big array of in vitro and in vivo data have revealed that MNPs may be detrimental to cells and animals.6-9 For example, most MNPs per se have been demonstrated to be reactive to biomolecules (e.g. proteins and lipids),10 resulting in oxidative stress (characterized by marked reactive oxygen species

(ROS) burst) that is believed to be the prominent

mechanism underlying their multi-faceted toxicity profiles.11 Considering the potential biohazard effects of MNPs, the National Institute for Occupational Safety and Health (NIOSH, the US) therefore has proposed approaches to manage the environmental health and safety (EHS) concerns associated with engineered nanomaterials (ENMs).12 Specifically, the NIOSH recommends the thresholds for a 4 ACS Paragon Plus Environment

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few MNPs ranging from 1 to 5 mg/m3 (e.g. 1 mg/m3 for CuO fume, 2.4 mg/m3 for fine TiO2 and 5 mg/m3 for ZnO fume), and the extrapolated in vitro doses are calculated to be at 1.6-8 µg/mL, according to an established calculation method.1 Notably, nTiO2 has recently been classified as a suspected carcinogen by European Chemicals Agency (ECHA).13 Considered together, it would be extremely necessary to perform a comparative toxicity assessment on diverse MNPs at NIOSH recommended exposure doses to better evaluate their EHS impacts. However, most previous studies often performed at high MNP concentrations to date, which may fail to evaluate the effects under realistic exposure scenarios.14 As we summarized in Table S1, rather high concentrations of MNPs (up to 1000 µg /mL) were popularly used to look at various biological effects in different cell types. Moreover, although significant detrimental effects have been observed in cells and organs exposed to some MNPs, these studies examined only one or limited facets of nanotoxicity networks at unrealistically high exposure doses. Therefore, more efforts should be devoted to EHS studies with greater relevance to realistic exposure scenarios, which may reflect distinct toxicity profiles and adverse outcome pathways (AOPs) from those under high-dose exposure. To this end, the primary objective of this study was to multi-hierarchically profile the biological effects of MNPs at low exposure doses. We deliberately selected these MNPs with great EHS impacts considering their production mass, application spectra and exposure likelihood, including silver nanoparticles (AgNPs) with different sizes and shapes,15 diverse rare earth oxide nanomaterials (REOs) 5 ACS Paragon Plus Environment

16

, nTiO217, nZnO18,19,


nFe3O4 and nFe2O320. Given that macrophages are a most cardinal type of sentinel cells against invading nanoparticles,21 we here used macrophages as a cell model to address the challenges raised above. Collectively, we here deciphered the differential biological outcomes and adverse cellular pathways of these representative MNPs in macrophages under low-dose treatment. Our findings thus would open a new path to understand EHS impacts of MNPs under low-dose exposure. EXPERIMENTAL SECTION Characterization of MNPs. REO nanoparticles, purchased from Nanostructured & Amorphous Materials (Houston, USA), were saved in stock solution and sonicated at 32 W for 15 s prior to experiments. Polyvinyl pyrrolidone (PVP) coated AgNPs were purchased from nanocomposix company (San Diego, USA) or Huzheng Nanotechnology Co., Ltd. (Shanghai, China). nZnO and nTiO2 were obtained from Nanostructured and Amorphous Materials (Houston, USA). nFe2O3 and nFe3O4 were synthesized, as previously described.22,23 All MNPs were characterized by TEM (Hitachi H-7500, Japan). A Zetasizer (Malvern Nano series, UK) was used to assess the hydrodynamic diameter and Zeta-potential of MNPs in deionized water and medium supplemented with 10% fetal bovine serum (FBS). Cell culture. J774A.1, HEK293T and THP-1 cells were purchased from Shanghai Cell Bank of Type Culture Collection (Shanghai, China). These cells were cultured in Dulbecco’s modified Eagle medium (DMEM) and RPMI 1640 medium (Hyclone, USA), respectively, supplemented with 10% FBS (Gibco, USA) and 100 U/mL


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penicillin-streptomycin (Hyclone, USA). Cells were cultured at 37 °C in a humidified incubator with 5% CO2. Cell viability assessment. For cell viability assessment, cells were treated with REOs (1, 2, 5, 10, 15 µg/mL), AgNPs (2, 5, 8, 10, 15 µg/mL), iron MNPs (5, 10, 20, 40, 60 µg/mL), nZnO (2, 5, 10, 15, 20 µg/mL) or nTiO2 (10, 20, 50, 100, 200 µg/mL) for

24

h.

Cell

viability

was

evaluated

through

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. MTT assay (Solarbio, China) was performed as described previously.24 Briefly, 10 µL MTT solution was added into the 100 µL cell culture medium in each well. After incubating for 4 h at 37°C, the supernatant was removed and formazan (MTT metabolic product) was resuspend in 100 µL DMSO. The absorbance was measured at a wavelength of 490 nm on a plate reader (Thermo Fisher Scientific, USA). Cell death determination. Cell death was examined through PI staining by flow cytometry. The following doses were used: 5 µg/mL for REOs and AgNPs, 20 µg/mL for iron MNPs and nTiO2 and 10 µg/mL for nZnO. After treatment with MNPs for 24 h, J774A.1 cells were collected and washed with phosphate buffered saline (PBS) for three times. Cells were thereafter re-suspended and stained with PI (50 µg/mL) (BD Biosciences, USA) for 15 min at room temperature. Dead cells were gated on a NovoCyte 1040 flow cytometry (ACEA BIO, China). Cellular localization of MNPs. For cellular localization assessment, J774A.1 cells were seeded in 10 cm plates and treated with several representative MNPs (5 µg/mL AgNP-25nm, AgNP-plate, nSm2O3, nY2O3, nYb2O3 or nDy2O3 and 10 µg/mL nZnO) 7 ACS Paragon Plus Environment


for 24 h. Thereafter, cells were collected, washed and fixed with 2.5% glutaraldehyde. Ultrathin sections (70 nm) were cut and collected onto formvar carbon coated 230-mesh copper grids. Sections were stained and afterwards observed on a high-resolution transmission electron microscope (JEOL JEM 2010F, Hitachi, Japan). ROS production assay. For ROS detection, cells were seeded in 96-well plates. After 1-6 h treatment with MNPs, cells were washed and incubated with 10 µM dichlorofluorescein diacetate (DCFH-DA, Sigma, USA) at 37 °C for 30 min. After washing with PBS for three times, DCF fluorescence was recorded at 525 nm using an excitation wavelength at 488 nm on a microplate reader. Enzyme activity tests. Cells were collected and washed post MNP treatment for 24 h, and were then lysed with RIPA lysis buffer containing protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were assayed by the BCA method (Solarbio, China). For catalase, Gpx and SOD activities, 100 µL lysate from each sample was tested according to the manufacturer’s instruction (Nanjing Jiancheng Bioengineering Institute, China). Enzymatic activities were finally normalized to their according total protein concentrations. Enzyme-linked immuno sorbent assay (ELISA) assay of TNF-α and IL-1β in culture media. After MNP treatment, cell culture media were collected and afterwards detected using mouse TNF-α (R&D, USA) and human IL-1β ELISA kits (NEOBIOSCIENCE, China). In brief, for TNF-α and IL-1β detection, 50 µL and 100 µL cell culture media were added into plates coated with primary antibodies (Abs), respectively. After incubating for 2 h, plates were washed for four times, followed by 8 ACS Paragon Plus Environment

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incubation with 100 µL TNF-α or IL-1β conjugates for another 2 h. Thereafter, plates were washed again and reacted with substrate solution. The absorbance was assayed at 450 nm on a microplate reader. For IL-1β detection, THP-1 cells were first primed with 1 µg/ml phorbol 12-myristate acetate (PMA) overnight, and treated with MNPs, as previously described. 25 RT-qPCR analysis of gene expression. Total RNAs were extracted from cells with Trizol reagent (Invitrogen, USA). Nanodrop (Thermo Fisher Scientific, USA) was used to quantitate RNA concentrations. Thereafter, 2 µg RNAs were reversely transcribed into cDNAs with M-MLV reverse transcriptase (Promega, USA). Expression levels of target genes were assayed with a standard SYBR Green qPCR system on a CFX96TM Real-Time System (Bio-Rad Inc., CA, USA). Primers are listed in Table S2. β-actin was used to normalize the relative expression of other genes. Western blot analysis. Cells treated with MNPs were collected and lysed with RIPA lysis buffer containing protease inhibitor cocktail (Roche, Switzerland). For the assessment of p65 translocation into nucleus, the cytosolic and nuclear proportions of proteins were collected separately by means of nucleoprotein extraction reagents according to the manufacturers’ introductions (Solarbio, China). Equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Abs were as follows, anti-β-actin Ab (1:5000 dilutions, Proteintech, China), anti-Nrf2 Ab (1:1000 dilutions, Proteintech, China), anti-HO-1 Ab (1:1000 dilutions, Proteintech, China), anti-p65 Ab (1:1000 dilutions, Cell Signaling Technology, USA), anti-IκBα Ab (1:1000 dilutions, 9 ACS Paragon Plus Environment


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Cell Signaling Technology, USA), anti-histone 3 (H3) (1:500 dilutions, Proteintech, China), and anti-NLRP3 (1:1000 dilutions, Cell Signaling Technology, USA). β-actin was used as a loading control of total proteins, whereas H3 was used as a loading control of nuclear proteins. Luciferase assays. HEK293T cells were seeded in 24-well plates overnight and co-transfected with 0.5 µg NF-κB luciferase reporter construct and 0.05 µg Renilla luciferase plasmid by Lipofectamine 2000 (Invitrogen, USA). After 4 h incubation, the culture medium was replaced. In the following, 100 µL 1×passive lysis buffer was added to each well of cells treated with MNPs for 24 h. Dual-luciferase reporter assay system (Promega, USA) were then used to assay the luciferase activity of the cellular extracts. The relative luciferase activities were normalized to those of Renilla luciferase. Assessment of cytoskeleton meshwork. J774A.1 cells were seeded in confocal dishes overnight, and were then treated with MNPs for 24 h. After treatment, cells were washed with PBS for three times, followed by fixation with 4% paraformaldehyde in PBS. After Triton X-100 treatment for 5 min, cells were stained with

Rhodamine-phalloidin

for

0.5

h

and

counterstained

with

2-(4-Amadinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for another 10 min. DAPI (blue) and Rhodamine-phalloidin (red) fluorescences were then examined using a TCS SP5 laser scanning confocal microscope (Leica, Germany). Ca2+ flux detection. J774A.1 cells were seeded in 96 well-plates overnight, and then exposed to MNPs for 24 h. Afterwards, intracellular Ca2+ concentrations were 10 ACS Paragon Plus Environment

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monitored by Fluo-3/AM fluorescence probes (Solarbio Biotechnology, China) according to the manufacturer's instructions. Briefly, cells were incubated at 37 °C with 2.5 µM Fluo-3/AM for 30 min in the dark. After that, cells were washed by Hank’s Balanced Salt Solution without Ca2+ and Mg2+ for three times. Fluorescence was recorded at 526 nm with the excitation wavelength at 506 nm on a plate reader. Assay of membrane potential changes. To determine the membrane potential changes, J774A.1 cells were seeded in 96-well plates. 10 µM DIBAC4(3) (AAT bioquest, USA) was pre-incubated with cells for 30 min at 37 °C in the dark. Then, various MNPs were added and incubated with the probes for another 30 min. Fluorescence intensity was monitored at 516 nm with the excitation wavelength at 493 nm on a microplate reader. Phagocytosis of macrophages. J774A.1 cells were seeded in confocal dishes overnight, and treated with materials for 24 h. After washing with PBS, CdSe/ZnO-PEG-COOH QDs (Mesolight, China) were added into fresh culture medium and co-incubated with cells for 6 h at 37 °C in the dark. Then, the excess of QDs was removed by washing with PBS for three times. QD fluorescence was assessed on a TCS SP5 laser scanning confocal microscope. Preparation of bone marrow derived macrophages (BMDMs). BMDMs were prepared according to an established method. 26, 27 Briefly, 6-8 week-old Balb/c mice were sacrificed, and marrow cells were then flushed out from femurs and tibias with PBS. After centrifugation at 1,500 rpm for 5 min, cells were pooled together and cultured in DMEM with addition of high glucose, glutamine, 15% heat-inactivated 11 ACS Paragon Plus Environment


FBS, 25 ng/mL rmCSF-1 (PeproTech, USA), nonessential amino acids (Gibco, USA), penicillin/streptomycin (Invitrogen, USA), and 55 nM β-mercaptoethanol (β-ME, Sigma, USA) for 7 days. Culture medium was changed every day after intact culture for 3 days. Afterwards, BMDMs were used for experiments then. Statistical analysis. All data were presented in mean ± standard deviation (SD). Student's T test was used to compare the difference between two groups, while one-way ANOVA test was used to determine the difference among three or more groups. A P-value less than 0.05 (*) or 0.001 (#) indicated statistically significant differences. RESULTS Overall experimental design and cytotoxicity determination of MNPs. Figure 1a delineates the overall experimental design of the current study. MNPs were first thoroughly characterized through transmission electron microscope (TEM). REOs, including nY2O3, nLa2O3, nCeO2, nNd2O3, nSm2O3, nEu2O3, nGd2O3, nDy2O3, nEr2O3 and nYb2O3, manifested consistent morphology and size (Figure 1b), analogous to previous results.25 Although these particles exhibited poorly controlled morphologies compared to the well-defined materials made in a research laboratory, they are more representative from the perspective of real exposure scenarios. As shown in Figure 1b, sphere-like AgNPs have four different diameters ranging from 5 to 200 nm (here termed as AgNP-5nm, -25nm, -100nm and -200nm). AgNP-plate and AgNP-cube manifested constant morphologies with the technical reports provided by the manufacturer (Figure 1b). Meanwhile, nZnO and nTiO2 showed similar 12 ACS Paragon Plus Environment

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morphology (Figure 1b), as characterized previously.26 And nFe2O3 and nFe3O4 displayed sphere-like shape with the diameter around 20 nm. Figure S1 summarizes the primary size measured by TEM, the hydrodynamic diameter and zeta-potential in water and medium with 10% FBS for these MNPs. All these MNPs showed negative charge in medium containing 10% FBS due to the formation of protein coronas on particles, consistent with previous reports.25, 28-30 To better mimic real exposure scenarios, we deliberately took into account the recommended thresholds by NIOSH for some representative MNPs (1-5 mg/m3) and reported data at MNP-related operation sites (11.3 mg/m3).31 Based on a calculation method used by NIOSH, assuming the sedimentation rate of the MNPs under an in vitro culture system at 70%, the exposure doses for in vitro experiments would be approximately at 1.6-16.6 µg/mL.1 Therefore, in this study, the exposure dose range for cell experiments was set at 0-20 µg/mL. To further explore the sublethal effects of these particles, we customized their concentrations that would not incur dramatic cytotoxicity. Hence, flow cytometry analysis with propidium iodide (PI) staining was used here to define a threshold of

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