Particulate nature of inhaled zinc oxide nanoparticles determines systemic effects and
mechanisms of pulmonary inflammation in mice
Jen-Kun Chen1, Chia-Chi Ho2, Han Chang3,4, Jing-Fang Lin2, Chung Shi Yang1,
Ming-Hsien Tsai5, Hui-Ti Tsai5, and Pinpin Lin2,5
1Center for Nanomedicine Research and 2National Environmental Health Research
Center, National Health Research Institutes, Zhunan, Taiwan,
3Department of Pathology, School of Medicine, China Medical University, Taichung,
Taiwan, 4Department of Pathology, China Medical University Hospital, Taichung,
Taiwan, and 5Division of Environmental Health and Occupational Medicine, National
Health Research Institutes, Zhunan, Taiwan
Correspondence: Dr Pinpin Lin. Tel: +886-37-246-166 (ext. 36508).
Fax: +886-37-587-406. E-mail: [email protected]
Dr. Jen-Kun Chen. Tel: 246166 (ext. 38117). Fax:
886-37-586447. E-mail: [email protected]
Abstract
Inhalation of zinc oxide nanoparticles (ZnONP) has potential health impact. Because
zinc ion may involve in the toxicity of ZnONP, we compared adverse effects of
inhaled aerosolized ZnONP and zinc nitrate in mice. Aerosolized ZnONP and zinc
nitrate were well-dispersed in the inhalation chamber. Inhalation of 0.86 mg/m3
ZnONP or 1.98 mg/m3 zinc nitrate for 5 h caused acute inflammation mainly at
bronchioloalveolar junctions of lungs at 24-h postexposure. Inhalation of ZnONP or
zinc nitrate increased metallothionein expression in the epithelial cells of
brochioloalveolar junction. While the effects on cytokines secretion in
bronchoalveolar lavage were similar between ZnONP and zinc nitrate, only ZnONP
increased lactate dehydrogenase activity. However, repeated exposure to 0.86 mg/m3
ZnONP 5 h/day for 5 days failed to cause a similar adverse effect. Either single or
repeated exposure to 0.86 mg/m3 ZnONP increased activities of glutamate
oxaloacetate transaminase, glutamate pyruvate transaminase and creatine
phosphokinase in blood. In contrast, exposure to zinc nitrate had no similar systemic
effects. In human bronchial epithelial cells, ZnONP-induced interleukin-8 secretion
was partially prevented by co-treatment with the Toll-like receptor 4 (TLR4)
inhibitor. Furthermore, ZnONP-induced pulmonary inflammation was greater in
wild-type mice than in TLR4-deficent mice. It appears that ZnONP-induced acute
pulmonary inflammation partially depended on TLR4. In summary, we demonstrated
the dose-responsive effects for inhalation of ZnONP and zinc nitrate in mice. The
threshold of cytokines induction for inhalation of ZnONP for 5 h was 0.43 mg/m3.
The particulate characters of ZnONP might contribute to the systemic adverse effects
and shall be evaluated for assessing its health impact in humans.
Keywords: Inflammation, inhalation, nanoparticles, systemic effect, zinc oxide
Introduction
Engineered ZnO nanoparticles (ZnONP) are widely used, such as in solar cells and in
optoelectronic and electronic nanodevices (Ambadea et al., 2009; Kumara & Chena,
2008; Liu et al., 2004). Because ZnONP can reflect ultraviolet light, ZnONP is used
as an ingredient in sunscreens (Nohynek et al., 2007). ZnONP also has antibacterial
activity (Jones et al., 2008) and is used as an external antibacterial agent. For its
application and manufacturing process, the skin, lung and gastrointestinal tract are
major routes of exposure in humans; and the potential health hazard of ZnONP is of
great concern (Kim et al., 2010). Several studies have reported the health effects of
inhalation of ultrafine ZnO particles generated with a furnace system in humans (Fine
et al., 1997) as well as in rodents (Warheit et al., 2009; Wesselkamper et al., 2001a).
While inhalation of 2.5 mg/m3 ultrafine ZnO particles for 2 h causes metal fume fever
(Fine et al., 1997), inhalation of 0.5 mg/m3 ultrafine ZnO particles for 2 h had no
adverse effects in healthy subjects (Beckett et al., 2005). Repeated exposure to
ultrafine ZnO particles for days has induced clinical tolerance accompanied by
reduced pulmonary inflammation in humans (Fine et al., 2000). Similarly, inhalation
of 1 mg/m3 ultrafine ZnO particles increased the percentage of polymorphonuclear
neutrophil (PMN) and proteins in bronchioalveolar lavage fluid (BALF) of mice at
24-h post-exposure. It appears that inhalation of ultrafine ZnO particles caused
transient pulmonary inflammation. More recently, the mechanisms for commercial
ZnONPinduced pulmonary inflammation were explored in different experimental
models. Warheit et al. (2009) reported that inhalation of 25 mg/m3 ZnONP (with
12.1m2/g of specific surface area and a diameter of 90 nm) for 3 h increased
cytotoxicity biomarker [lactate dehydrogenase (LDH) activity] and inflammatory
proteins in BALF of rats. But atmospheric ZnONP in the exposure chamber severely
aggregated to the average size of 2.8 mm (Warheit et al., 2009), and the particle size
might modulate the severity of toxic responses (Nel et al., 2006). It is believed that
Zn2+ ion participated in ZnONP-induced pulmonary inflammation. Instillation of
ZnONP and dissolved Zn2+ in rat lungs caused similar pathological changes, except
eosinophils recruitment in the lungs (Cho et al., 2011, 2012). Many in vitro studies
have
shown that ZnONP release Zn2+ ion in culture media and have suggested that thebiological effects of ZnONP in submerged cell cultures were caused by Zn2+ ion (Kao et
al., 2012; Kim et al., 2010; Xia et al., 2008). However, Xie et al. (2012) suggested that the cytotoxic mechanisms of aerosolized ZnONP and Zn2+ ion might be different in an
air–liquid interface culture of mouse type II lung cells. It appears that the potential difference between ZnONP and zinc ion-induced adverse effects shall be considered. Lung inflammation induced by ZnONP has showed the association with accumulation of neutrophils, eosinophils or macrophages and production of cytokines. Neutrophils, eosinophils and macrophages are the important innate immune cells. The innate immune response relies on recognition of specific stimuli through Toll-like receptors (TLRs) and MyD88 is their adaptor molecules for the induction of inflammatory cytokines (Takeda & Akira, 2001, 2004). Previously, we reported that ZnONP-induced proinflammatory cytokines expression via the MyD-dependent TLR pathways (Chang et al., 2013). The TLR family now consists of 11 members (TLR1–TLR11) in human and 13 members (TLR1–TLR13) in mice (Takeda & Akira, 2004). TLR4 has been reported to play a role in quantum dots and titanium dioxide nanoparticles-induced inflammation (Cui et al., 2011; Ho et al., 2013). It remains unclear whether TLR4 involves in ZnONP-induced inflammation. In our present study, we generated well-dispersed aerosolized ZnONP and zinc nitrate with a nanoaerosol generator for inhalation exposure in mice. We evaluated dose-responsive pulmonary and systemic adverse effects of inhaled ZnONP and zinc nitrate in mice. Here, we also further investigated the role of TLR4 in ZnONP-induced pulmonary effects. We believe that our data may be greatly useful for future safety assessments of ZnONP.
Methods
Physicochemical characterization of nanoparticles
ZnONP were purchased from NanoScale Corporation (Manhattan, KS). ZnONP was characterized for shape, size with distribution, and zeta potential. The shape and size of the nanoparticles were determined by transmission electron microscopy (TEM) (H-7650, Hitachi, Tokyo, Japan). The ZnONP suspended in aqueous samples were dripped and dried on copper grids prior to TEM inspection. All of the copper grids were
preserved in a dry cabinet. Sizes of nanoparticles were counted using SigmaScan_ Pro
5 software (Systat Software Inc., San Jose, CA) and averaged for at least 300
nanoparticles. The hydrodynamic diameters in water or media and zeta potentials of the ZnONP were measured with the Zetasizer (Zetasizer Nano ZS, Malvern
Instruments, Worcestershire, UK). Dynamic light scattering (DLS) measurements were performed in a single-scattering regime with He–Ne laser (633 nm) at an angle of 173_. The suspension was put into a cuvette at 25 _C to enable particle size and zeta
chambers system was measured by a scanning mobility particle sizer spectrometer (SMPS, Series 3936, TSI Inc., Shoreview, MN). Endotoxin concentrations were
quantified with kinetic chromogenic Limulus amebocyte lysate (LAL) reagents (Charles River Laboratories International, Inc., Wilmington, MA). The assay was performed in pyrogen-free test tubes to which 0.2 ml ZnONP and 0.2 ml LAL reagent were added. Synthetic chromogenic substrate, the pro-clotting enzyme (a serine protease) in the LAL, was activated by endotoxin and cleaved the substrate’s peptide, thereby releasing a marker compound that was easily detected by spectrophotometry. LPS standard (0.125 EU/ml) and pyrogen-free LAL reagent water, both provided by the manufacturer, were used as a control.
Animal studies
Six-weeks old female Balb/c, male C3H/NeH and male C3H/NeJ mice were purchased from BioLASCO (Taipei, Taiwan) and acclimated for 2 weeks in the animal facilities at the National Health Research Institutes (NHRI). All animal treatments and
experimental protocols for this study were reviewed and approved by the Institutional Animal Care and Use Committee at NHRI. All mice were housed under a 12 h light/dark cycle, at 23±1 _C, with a relative humidity of 39–43%. Water and food were provided
ad libitum. Animals were ranked and divided into four groups by body weight. Mice in each group were randomly assigned into four experimental groups. Female Balb/c mice were divided into seven mice per group. After exposure and sacrifice at indicated time, biochemical biomarkers in serum or plasma were quantified with a clinical chemical analyzer, DRI-CHEM 3500 (Fujifilm Corp., Tokyo, Japan).
Inhalation exposure of ZnONP and zinc nitrate in mice
Animals were ranked and divided into four groups by body weight. Mice in each group were randomly assigned into four experimental groups. The exposure system contains two animal cages in a chamber, one for control group and the other for exposure group. Control and exposed animals were respectively placed in the control cage and exposure cage side by side. Aerosol containing nanoparticles was delivered into exposure cage and filtered clean air was delivered into the control cage, respectively. Female Balb/c mice were exposed to aerosolized ZnONP (without restraint) or zinc nitrate in a nanoparticle exposure chamber (Mai et al., 2010; Yang et al., 2011). Suspension of ZnONP (1.0, 2.0 and 4.0 mg/mL) or zinc nitrate solution (1.5, 2.0 and 2.5 mg/mL) were prepared and filled in the constant output atomizer (Model 3076; TSI Inc., Shoreview, MN). Compressed clean air was delivered into the constant output atomizer to produce nanoaerosol and subsequently release it into the exposure cage placed in the nanoparticle exposure chamber. In detail, two housing cages (31_19_21 cm for length, width and height) for exposure and control groups were placed in the nanoparticles exposure chamber (175_70_175 cm for length, depth and height) to
establish a double-isolated environment. SMPS spectrometer consisting electrostatic particle classifier (Model 3080N; TSI Inc., Shoreview, MN) with dynamic mass analyzer (DMA, Model 3085; TSI Inc., Shoreview, MN) and condensation particle counter (CPC, Model 3776; TSI Inc., Shoreview, MN) were equipped with the chamber for real-time monitoring of atmospheric nanoparticles in exposure cage. Control animals and exposed animals were placed in control cage and exposure cage side by side. Aerosol containing ZnONP or zinc nitrate generated from constant output atomizer were delivered into exposure cage and filtered clean air was delivered into control cage, respectively. The concentration of airborne ZnONP or zinc nitrate could be controlled by regulating the pressure of the nebulizing gas and adjusting the ventilation rate using the mass flow controller. A SMPS spectrometer was connected to the exposure chamber for real-time monitoring. Eighty four female Balb/c mice (6–8 weeks old) were randomly divided into six groups (n¼7 for each group). For the acute exposure groups, animals were exposed to filtered fresh air (as control), 0.21±0.01, 0.43±0.02,
0.86±0.13 mg/m3 of aerosolized ZnONP or 0.53±0.03, 0.79±0.16, 1.98±0.11 mg/m3
zinc nitrate for 5 h and were inspected 24-h post-exposure. Aerosol of ZnONP or zinc nitrate generated from constant output atomizer was delivered into the exposure cage at flow rate of 3.0 L/min. The air was withdrawn from four corners of the exposure cage at flow rate of 0.5 L/min, which was collected using four simultaneous sampling apparatus throughout the entire aerosol generation in each day. Analytical replicates in three successive days (4 corners/day_3 days), starting from 2 days before animal exposure experiments, were employed to determine the average concentration of ZnONP with standard deviation. For the repeated exposure group, animals were exposed to filtered fresh air (as control) or 0.86 mg/m3 of aerosolized ZnONP 5 h/day
and were inspected 24 h after 5-days exposure. For the control groups, animals were housed under clean and filtered air without administration with ZnONP for 5 h or 5 h/day for 5 days.
Intratracheal instillation of ZnONP in mice
C3H/NeH mice are wild-type and C3H/NeJ mice are TLR4 deficient (Hoshino et al., 1999). Male C3H/NeH and C3H/NeJ mice were divided into control and ZnONP-treated groups. Six (n¼6) from each group per time point were randomly selected for
experiments. Mice were anesthetized by isoflurane inhalation. While under anesthesia, mice were secured on its back on an inclined plane, and the head was elevated. The mouth was secured open with a rubber band, the tongue was held to one side with forceps, and the epiglottis was visualized. A syringe fitted with a blunted, polished needle (19 gauge, 3-inch long, angled at 45_) was inserted into the mouth down to
larynx. The suspended sample was then expelled rapidly. In the treatment group, each mouse was administered with a single dose of 80 mg of ZnONP suspended in 30 mL of
distilled water. Before administration, the ZnONP in water were sonicated with a probe sonicator (Sonics & Materials Inc., Newtown, CT) for 60 s each time with 10 s intervals in a total of 5 min. This procedure was used to minimize agglomeration but showed minimal effects on the solubility of the ZnONP demonstrated by our previous study that499% of ZnONP in water was insoluble after 30-min sonication (Yeh et al., 2012). Mice were anesthetized with 3% isoflurane vapor for 20–30 s at 48 h following dosing. Preparation and evaluations of BALF
Animals were sacrificed via isoflurane inhalation to ensure there was no undue suffering. The whole lung was dissected out surgically and was lavaged with 1mL saline. The recovered amount of lavagate was recorded and saved in individually labeled bottles. The total cell numbers in the BALF from the animals were determined with a cell counter (Coulter Inc., Miami, FL). The BALF was centrifuged at 800 g for 15 min using a Shandon Cytospin 4 (Thermo Scientific, Waltham, MA). The cytospin smear was then prepared and the cell types were discerned from Liu’s staining (Tonyar Biotech Inc., Taoyuan, Taiwan). Neutrophils were stained as a neutral pink and contain a nucleus divided into 2–5 lobes. Lymphocyte is approximately the size of a red blood cell (6 mm) and has a very high nucleusto-cytoplasm ratio. The nucleus of lymphocyte was stained as dark purple. The macrophage is usually the largest leukocyte present (15–20 mm). The cytoplasm of macrophage was stained as clear or light purple and the nucleus of macrophages stained as dark purple.
Total protein and LDH activity in the BALF
Quantification of total protein in the BALF supernatant was performed by Bradford assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard. LDH activity was spectrophotometrically assayed using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega Corporation, Madison, WI) at 490 nm in the presence of lactate. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and creatine phosphokinase (CPK) activities
Serum levels of GOT, GPT and CPK were measured using commercial assay kits. Serum was spotted onto respective Fujifilm Dri-Chem slides (Fujifilm, Kanagawa, Japan) and each biochemical indicator was determined using a blood biochemical analyzer (Fujifilm Dri-Chem 3500s; Fujifilm, Kanagawa, Japan) according to the manufacturer’s instructions. GOT, GPT and CPK are enzymes. An enzyme unit is the amount (micromole) of substrate converted to product per unit time.
Tissue preparation, histopathology and immunohistochemistry
Lung tissues were fixed with 10% neutral buffered formalin for 48 h prior to tissue processing, which included dehydration, and embedding in paraffin. Hematoxylin and eosin stain was used for general histopathological examinations. Metallothionein-1/2 (MT) immunohistochemistry has been described as previous study (Lin et al., 2009).
Briefly, heating 10 min in autoclave for antigen retrieval was essential. The anti-MT was a specific antibody for MT and used in 1:100 dilution. Next, a standard
immunostaining assay was performed according to the reagent manufacturer’s instructions (LSAB, DakoCytomation, Carpinteria, CA). The MTpositive cells exhibited a brown color developed by diaminobenzidine (DakoCytomation, Glostrup, Denmark). The slides were finally counterstained with hematoxylin.
Cell cultures
Human bronchial epithelial cell line BEAS-2B cells (American Type Culture Collection, Manassas, VA) were maintained in LHC-9 (GIBCO, Grand Island, NY). Cells were incubated in a 37 _C incubator with a humidified mixture of 5% CO2 and 95% air. The
medium was changed twice a week; and cells were passaged by trypsinization every week. Cells were seeded in 24-well dishes for 24 h prior to treatment. To prepare stock suspension of ZnONP (10 mM) with the least aggregation/agglomeration, ZnONP were freshly suspended in 10mL of double deionized water followed by water bath
sonication at room temperature for 3 min. The stock suspension was then 100 times diluted by LHC-9 cell culture medium and sonicated at room temperature for 3 min for preparing working solution (100 mM) for in vitro experiments. After ZnONP were suspended in culture medium for 1 or 24 h, only 2.34%, and 3.19% (w/w) of zinc ion was detected in the supernatant after centrifugation at 21 000 g for 15 min. We further checked the stability of ZnONPs in LHC-9 (100 mM) with DLS at 0.2, 0.5, 1, 2, 3, 4 and 24 h after suspension procedures. ZnONP was very stable and the size was between 144.7 and 170.0 nm (with the average of 156.8 nm) within 72 h
(Supplementary Figure 1). Cell viability
Cell viability was determined with dimethylthiazoldiphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates for 24 h and then incubated with 0.2mL of vehicle (LHC-9 medium), ZnONP, or zinc nitrate for 48 h. Subsequently, 1 mg/ml MTT was added to the medium, and cells were incubated for an additional 2 h. Precipitated formazan was dissolved in 0.2 ml DMSO and the absorbance was measured at 535 nm. The data are presented as the percentage of controls.
Enzyme-linked immunosorbent assay (ELISA)
BALF collected from mice at indicated times was subjected for quantifying secreted monocyte chemotactic protein-1 (MCP-1) DOI: 10.3109/17435390.2014.886740 Inhalation of
ZnONP and zinc nitrate aerosol in mice 3 Nanotoxicology Downloaded from informahealthcare.com by National Health Research Institutes on 02/24/14 For personal use only. and chemokine (C-X-C motif) ligand 1 (CXCL1) with ELISA. BEAS-2B cells were seeded in 24-well plates for 24 h and then incubated with 0.5mL of vehicle (LHC-9 medium), ZnONP or zinc nitrate for 48 h. Conditioned media collected from BEAS-2B cell cultures were subjected for quantifying
secreted interleukin-8 (IL-8). Cytokines concentration was determined using the MCP-1 and CXCL1 ELISA kits for mouse, or IL-8 ELISA kit for human (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer’s instructions.
Quantification of atmospheric ZnONP concentration with inductively coupled plasma mass spectrometry (ICP-MS)
ICP-MS (7500cx, Agilent Technologies Inc., Tokyo, Japan) was used to determine the ZnONP concentration (calculated from zinc concentrations) of the air sample for exposure experiments. In detail, air samples were sampled (0.5 L/min) at four corners of exposure cage for entire exposure procedure (5 h) and ZnONP were absorbed and captured by tandem sampling devices. A Midget Impinger (SKC Inc., Eighty Four, PA) was connected with two glass fiber filters (25mm diameter, SKC Inc., Eighty Four, PA) in a series (Supplementary Figure 2). The impinger contained 10mL of 10% nitric acid as absorbing solution to help capturing ZnONP and dissolving ZnO into zinc ions in acidified solution. The first glass fiber filter behind impinger was employed to capture re-aerosolized mist from impinger. Zinc content in the impinger and the first glass fiber filter was combined to calculate the concentration of ZnONP in the exposure
atmosphere. Additionally, the Zn content in the second glass fiber filter was used to evaluate the percentage of breakthrough, presenting effective capture efficiency (6.5% of breakthrough for the highest concentration of ZnONP exposure). Aqueous solution (0.05 mL) in the impinger was mixed with nitric acid (0.02 mL) and then diluted to 10mL with double deionized water before ICP-MS analysis. Glass fiber filters were immersed into 10mL of 0.2% nitric acid to dissolve ZnONP for ICP-MS analysis. These solutions were then directly analyzed for 66Zn. Standard solutions, 1.0, 5.0, 10,
100 and 1000 mg/L were used for sample quantification. All steps in sample preparation and measurements were carried out in a chemical hood.
Statistical analysis
Comparison of the results between various experimentally treated groups (in vitro studies) and their corresponding controls was carried out by one-way analysis of variance (ANOVA). Dosedependent effects in in vivo studies were carried out by trend analysis with linear regression and one-way ANOVA. All comparisons were considered significant when p50.05.
Results
Physicochemical properties of ZnONPs
The physicochemical properties of ZnONP were summarized in Table 1 and Figure 1(A). The appearance of ZnONP shows irregular pillar shape, a kind of rod-like nanostructure but not a cylinder or hexagonal column. Primary particle size of ZnONP described in this study was 9.1±1.9 nm by TEM measurements (Table 1). The ZnONP were suspended in aqueous solution (concentration: 1.0, 2.0 and 4.0 mg/mL in double
deionized water) prior to be nebulized into the atmosphere of exposure cage placed in exposure chamber system. The hydrodynamic sizes of ZnONP (100 mM) were 62.3 nm in water and 156.8 nm in LHC-9 culture medium. The size of airborne ZnONP was 66.9±1.9 nm while be nebulized in the air of inhalation exposure chamber, showing ZnONP aggregated/agglomerated from 9.1 to 66.9 nm while nebulization. ZnONP was not contaminated by endotoxin. The zeta potential of ZnONP was _27.1mV. The specific surface area of ZnONP was 88m2/g. When ZnONP were suspended in water,
phosphate-buffered saline or culture medium (LHC-9) for 1 h, only 0.27, 0.19 or 2.36% (w/w) of zinc ion was detected in the supernatant.
Characterization of aerosolized ZnONP and zinc nitrate in the inhalation exposure chamber
Aerosolized ZnONP and zinc nitrate were well-dispersed in the air of the inhalation exposure chamber (Chen et al., 2011; Yang et al., 2011). The average diameter of ZnONP was 46.4±1.6 nm at the beginning of exposure then slightly aggregated to 66.9±1.9 nm at the steady state (Table 1 and Figure 1B). The size of aerosolized zinc nitrate was 88.9±1.8 nm (Figure 1B). Concentrations of ZnONP in the air reached steady-state from 1 to 5 h (Figure 1C), showing ZnONP concentration varying with relative standard deviation (RSD)510% by real-time monitoring of airborne ZnONP. Three concentrations of ZnONP (0.21±0.01, 0.43±0.02 and 0.86±0.13 mg/m3),
determined with ICP-MS analysis, were set for the following animal studies (Figure 1C). Similarly, three concentrations of zinc nitrate (0.53±0.03, 0.79±0.16 and 1.98±0.11 mg/m3) were set for the following animal studies.
Effects of inhalation of ZnONP or zinc nitrate on cell population and inflammatory biomarkers in BALF of mice
Mice inhaled fresh air as control, 0.21, 0.43 and 0.86 mg/m3 aerosolized ZnONP or
0.53, 0.79 and 1.98 mg/m3 zinc nitrate for 5 h and the biological effects were observed
at 24-h postexposure. To compare the data of ZnONP and zinc nitrate exposure, the doses were presented as both molarity and weight. Inhalation of the highest dose of ZnONP for 5 h significantly increased the numbers of PMN and total protein
concentrations, but decreased macrophage numbers in BALF at 24-h postexposure (Figure 2). It is possible that exposure to ZnONP and zinc nitrate were selectively toxic to macrophage. Similar effects were found in zinc nitrate-exposed mice, except that the middle dose of zinc nitrate also increased the numbers of PMN (Figure 2). Acute inflammation was confirmed by the elevated secretion of pro-inflammatory cytokines, MCP-1 and CXCL1, in BALF (Figure 3). Inhalation of the highest dose of ZnONP
significantly increased MCP-1 and CXCL1 secretion. Inhalation of the middle and highest doses of zinc nitrate both increased MCP-1 and CXCL1 secretion (Figure 3C). But inhalation of the lowest dose of zinc nitrate reduced CXCL1 secretion.
Interestingly, LDH activity was significantly elevated at all chemokine (C-X-C motif) ligand 1 (CXCL1) with ELISA. BEAS-2B cells were seeded in 24-well plates for 24 h and then incubated with 0.5mL of vehicle (LHC-9 medium), ZnONP or zinc nitrate for 48 h. Conditioned media collected from BEAS-2B cell cultures were subjected for quantifying secreted interleukin-8 (IL-8). Cytokines concentration was determined using the MCP-1 and CXCL1 ELISA kits for mouse, or IL-8 ELISA kit for human (R&D Systems, Inc. Minneapolis, MN) according to the manufacturer’s instructions.
Quantification of atmospheric ZnONP concentration with inductively coupled plasma mass spectrometry (ICP-MS)
ICP-MS (7500cx, Agilent Technologies Inc., Tokyo, Japan) was used to determine the ZnONP concentration (calculated from zinc concentrations) of the air sample for exposure experiments. In detail, air samples were sampled (0.5 L/min) at four corners of exposure cage for entire exposure procedure (5 h) and ZnONP were absorbed and captured by tandem sampling devices. A Midget Impinger (SKC Inc., Eighty Four, PA) was connected with two glass fiber filters (25mm diameter, SKC Inc., Eighty Four, PA) in a series (Supplementary Figure 2). The impinger contained 10mL of 10% nitric acid as absorbing solution to help capturing ZnONP and dissolving ZnO into zinc ions in acidified solution. The first glass fiber filter behind impinger was employed to capture re-aerosolized mist from impinger. Zinc content in the impinger and the first glass fiber filter was combined to calculate the concentration of ZnONP in the exposure
atmosphere. Additionally, the Zn content in the second glass fiber filter was used to evaluate the percentage of breakthrough, presenting effective capture efficiency (6.5% of breakthrough for the highest concentration of ZnONP exposure). Aqueous solution (0.05 mL) in the impinger was mixed with nitric acid (0.02 mL) and then diluted to 10mL with double deionized water before ICP-MS analysis. Glass fiber filters were immersed into 10mL of 0.2% nitric acid to dissolve ZnONP for ICP-MS analysis. These solutions were then directly analyzed for 66Zn. Standard solutions, 1.0, 5.0, 10,
100 and 1000 mg/L were used for sample quantification.
All steps in sample preparation and measurements were carried out in a chemical hood.
Effect of inhalation of ZnONP or zinc nitrate on histopathology and MT expression in the lung of mice
ZnONP-induced lung inflammation or epithelial injury mainly at the bronchioloalveolar (BA) junctions (Figure 4). Such destruction was characterized by some regenerative epithelial cells laid on the dilated BA junctions. The inflammatory infiltrate was mild to moderate, and composed of lymphocytes and macrophages. Neutrophilia was not obvious. The destruction of BA junctions was identified only at mice with 0.86 mg/m3
ZnONP exposure. Immunohistochemically, MT expressed at bronchiolar epithelial cells located at the BA junctions, and increased MT-positive cell number with the increasing dosages of ZnONP exposure (Figure 5). In other words, the highest level of MT
expression was detected in exposed lungs at the dosage of 0.86 mg/m3 ZnONP. Some
alveolar macrophages also exhibited MT expression. Inhalation of zinc nitrate caused similar pathological changes and MT expression in lung tissues (data not shown). MT is a zinc responsive protein and its expression is inducible by zinc ion (Haq et al., 2003). The localization of MT-positive cells suggests that inhaled aerosolized ZnONP and zinc nitrate generated in our chamber was small enough to deposit at the BA junctions of the lung.
Effects of inhalation of ZnONP or zinc nitrate on biochemical biomarkers in blood Systemic adverse effects are one of health concerns for inhalation of nanoparticles. Inhalation of the highest dose of ZnONP for 5 h significantly increased GOT, GPT and CPK activities in blood at 24-h post-exposure (Table 2). GOT and CPK activities
remained elevated after inhalation of the highest dose of ZnONP for 5 h/day for 5 days (Table 2). However, inhalation of all doses of zinc nitrate for 5 h had no significant effects on these enzyme activities. These results indicated that inhaled ZnONP, but not soluble zinc nitrate, might distribute to extra-pulmonary tissues and caused systemic adverse effects.
Investigation for the role of TLR4 in ZnONP-induced effects in vitro and in vivo To understand the mechanism of pulmonary inflammation, we further compared effects of ZnONP and zinc nitrate in human bronchial epithelial cells BEAS-2B. The dose-dependent cytotoxicity was similar between ZnONP and zinc nitrate (Figure 6A). Treatment with 50 or 100 mM of ZnONP or zinc nitrate both significantly increased IL-8 secretion (Figure 6B). MyD is an adaptor protein for most TLRs (Takeda & Akira, 2001). Previously, we demonstrated that silencing MyD expression partially abolished ZnONP-induced cytokines expression in mouse lung epithelial cells (Chang et al., 2013). Here, cotreatment with TLR4 inhibitor, CL-0953, more effectively prevented
lipopolysaccharide (LPS) and ZnONP-induced than zinc nitrate-induced IL-8 secretion (Figure 6C). Nanoparticles are frequently contaminated with LPS, which also induces the production of inflammatory mediators such as cytokines and chemokines (Dobrovolskaia et al., 2009). We demonstrated that the LPS contents in ZnONP were low (Table 1). We further confirmed that the LPS didn’t involve in IL-8 induction by ZnONP. Polymyxin B blocks the biological effects of Gram negative LPS through binding to lipid A (Palmer & Rifkind, 1974). Polymyxin B is widely used to eliminate the effects of endotoxin contamination, both in vitro and in vivo. While co-treatment with polymyxin B, a LPS inhibitor (Cooperstock, 1974), significantly abolished LPS induced IL-8 secretion, polymyxin B failed to prevent ZnONP induced IL-8 production (Figure
6D). Consistent with results of in vitro studies, intratracheal instillation of ZnONP-induced greater pulmonary inflammatory responses in wild-type mice than in TLR4-deficient mice (Table 3). C3H/HeJ strain is used as a model of a TLR4-defective mouse strain. TLR4 in C3H/HeJ mouse has a single point mutation of the amino acid, which failed to activate NF-kB in response to LPS (Hoshino et al., 1999). These results suggested that TLR4 at least partially play a role in ZnONP-induced acute pulmonary inflammation.
Discussion
Some studies suggested that zinc ion, which was released from ZnONP, was responsible for ZnONP-induced pulmonary inflammation and cytotoxicity. In our present study, we evaluated two hypotheses: first, inhalation of ZnONP and soluble zinc ion might cause similar pulmonary as well as systemic adverse effects in mice; second, TLR might involve in ZnONP-induced pulmonary effects. Surprisingly, the first hypothesis is only partially correct. Inhalation of ZnONP and soluble zinc induced similar pulmonary effects via different mechanisms. Furthermore, only ZnONP, but not soluble zinc ion, caused systemic effects. Secondly, TLR4 partially contributed to pulmonary effects of ZnONP. Our results suggested that inhalation of ZnONP had systemic adverse effects and these systemic effects might depend on the particulate nature
of ZnONP, but not zinc ion.
In our present study, mice inhaled water droplets with zinc ion or ZnONP. Humidity is present in the atmosphere of environment and of occupational workplaces. Water is the key component leading to zinc ion released from ZnONP. We therefore use zinc nitrate for parallel comparison with ZnONP in this study. Zinc nitrate is a water soluble materials, readily and completely dissociate to zinc ion while encounter a humid environment. Although ZnONP shows gradually dissolution in particular buffer in previous reports, biological responses caused by zinc nitrate exposure can help to differentiate biological responses caused by particulate ZnONP and/or zinc ions releasing from ZnONP. It is believed that Zn2+ ion participated in ZnONP-induced
pulmonary inflammation (Cho et al., 2011, 2012). Indeed, inhalation of aerosolized ZnONP and zinc nitrate caused similar pulmonary inflammatory responses. To our surprise, only ZnONP, but not zinc nitrate, increased activities of biochemical biomarkers in blood. Inhalation of ZnONP caused acute hepatic toxicity at 24 h, indicated by the increased GOT and GPT activities. Persistent elevation of GOT and CPK activities after 5 days exposure also implied muscle injury. Our preliminary study with intratracheal instillation of radioactive 65ZnONP in mice showed that radioactivity
was detected in variety of tissues and mainly excreted in the feces (data not shown). Previously, we reported that 10 nm 65ZnONPs and 65Zn(NO3)2 distributed to variety of
tissues, including the liver, heart and carcass, at 24 h after intravenous injection (Yeh et al., 2012), although 65ZnONPs showed greater tissue accumulation than 65Zn(NO3)2.
It is plausible that ZnONP may cause liver and muscle injury. But it is not clear why zinc nitrate failed to cause liver and/or muscle injury. Either less amounts of zinc nitrate distributed to these organs, or particulate characters of ZnONP are required for causing injuries in these organs. More investigation is needed to clarify the
mechanism. Some studies have shown that inhalation of ZnO fume or ZnONP caused acute pulmonary inflammation. However, these studies did not demonstrate the dose-responsive effects and the threshold of adverse effects was not determined.
Wesselkamper et al. (2001a,b) demonstrated that inhalation of 1 mg/m3 ultrafine ZnO
particles for 3 h induced acute pulmonary inflammation in mice at 24-h post-exposure. Warheit et al. (2009) reported that inhalation exposure of 25 mg/m3 aerosolized
ZnONP (90 nm) and fine ZnO particles (111 nm) for 3 h in rats induced comparable acute pulmonary inflammation. In our present study, aerosolized ZnONP-induced acute inflammation at the dose of 0.86 mg/m3 for 5 h, which was similar to the dose of
ultrafine ZnO particles (Wesselkamper et al., 2001a). The threshold of cytokines induction for inhalation of ZnONP for 5 h in mice (0.43 mg/m3) is one-fifth of the
threshold limit values (2 mg/m3) of ZnO fumes for an 8-h workday and 40-h workweek
[American Conference of Governmental Industrial Hygienists (ACGIH), 2008]. It is not surprising that the dose-responsive effects of inhaled ZnONP and zinc nitrate were comparable, especially when the doses were presented as molarity. Both treatments increased MT expression at the BA junctions. It is consistent with the hypothesis that zinc ion released from ZnONP may be responsible for ZnONP-induced pulmonary inflammation. However, it is also true that ZnONP and zinc nitrate might cause
inflammation via different mechanisms. For example, only ZnONP caused cytotoxicity, as indicated by the increased LDH activity in BALF, in mice. Similar difference was also reported by Cho et al. (2012) via intratracheal instillation in rats. We observed that TLR4 played a more important role for IL-8 induction by ZnONP than by zinc nitrate in human bronchial epithelial cells. We have confirmed that ZnONP was not
contaminated by endotoxin with the LAL assay (Table 1); and an endotoxin inhibitor failed to prevent ZnONP-induced cytokine expression (Figure 6D). The participation of TLR4 was confirmed by comparing ZnONP induced inflammation in wild-type versus in TLR4-deficient
mice. Previously we demonstrated that TLR4 played a more important role in cytokine induction by polyethylene glycol (PEG)-coated quantum dots than by bare quantum dots (Ho et al., 2013). quantum dots hardly entered cells. It is plausible that PEG-quantum dots interacted with TLR4 located on the cell
and subsequently increased IL-8 expression. The particulate characters of ZnONP might play a role in the interaction with TLR4.
Acknowledgements
The authors thank Eva Yu-Ching Chen for TEM analysis (NM-101-PP-04, Electron Microscopy Core Facility, NHRI), Jei-Ping Li for operation of nanoparticle exposure chamber and Nai-Chun Huang for ICP-MS analysis (NM-101-PP-11).
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
This work was supported by research grants (NM-101-PP-07 and NM-101-PP-08) from the Center of Nanomedicine Research at the National Health Research Institutes, Taiwan.
References
American Conference of Governmental Industrial Hygienists (ACGIH). 2008. Threshold limit values for chemical substances and physical agents and biological exposure indices. 2013 TLVs and BEIs, 1 February 2013, Cincinnati, OH. 61 p.
Ambadea SB, Maneb RS, Ghuleb AV, Takwalec MG, Abhyankara A, Chod B, Han S. 2009. Contact angle measurement. A preliminary diagnostic method for evaluating the performance of ZnO plateletbased dye-sensitized solar cells. Scr Mater 61:12–15.
Beckett WS, Chalupa DF, Pauly-Brown A, Speers DM, Stewart JC,Frampton MW, et al. 2005. Comparing inhaled ultrafine versus fine zinc oxide particles in healthy adults: a human inhalation study. Am J Respir Crit Care Med 171:1129–35.
Chang H, Ho CC, Yang CS, Chang WH, Tsai MH, Tsai HT, Lin P. 2013. Involvement of MyD88 in zinc oxide nanoparticle-induced lung inflammation. Exp Toxicol Pathol 65:887–96.
Chen CH, Li JP, Huang NC, Yang CS, Chen JK. 2011. Establishment of airborne nanoparticle exposure chamber system to assess nanoTiO2 induced mice lung effects. Proceedings of the 4th
Nanoscience and Nanotechnology Symposium, 23–25 September 2011, Bali, Indonesia.
Cho WS, Duffin R, Howie SE, Scotton CJ, Wallace WA, Macnee W, et al. 2011. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Part Fibre
Toxicol 8:27.
Cho WS, Duffin R, Poland CA, Duschl A, Oostingh GJ, Macnee W, et al. 2012. Differential pro-inflammatory effects of metal oxide nanoparticles and their soluble ions in vitro and in vivo; zinc and copper nanoparticles, but not their ions, recruit eosinophils to the lungs. Nanotoxicology 6:22–35.
Cooperstock MS. 1974. Inactivation of endotoxin by polymyxin B. Antimicrob Agents Chemother 6:422–5.
Cui Y, Liu H, Zhou M, Duan Y, Li N, Gong X, et al. 2011. Signaling pathway of inflammatory responses in the mouse liver caused by TiO2 nanoparticles. J Biomed Mater Res A 96:221–9.
Nat Nanotechnol 4:411–14. Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Beckett WS. 1997. Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. J Occup Environ Med 39:722–6.
Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Sparer J, Beckett WS. 2000. Characterization of clinical tolerance to inhaled zinc oxide in naive subjects and sheet metal workers. J Occup Environ Med 42: 1085–91.
Haq F, Mahoney M, Koropatnick J. 2003. Signaling events for metallothionein induction. Mutat Res 533:211–26.
Ho CC, Luo YH, Chuang TH, Yang CS, Ling YC, Lin P. 2013. Quantum dots induced monocyte chemotactic protein-1 expression via MyD88-dependent Toll-like receptor signaling pathways in macrophages. Toxicology 308:1–9.
Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al.1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps
gene product. J Immunol 162:3749–52.
Jones N, Ray B, Ranjit KT, Manna AC. 2008. Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett 279:71–6.
Kao YY, Chen YC, Cheng TJ, Chiung YM, Liu PS. 2012. Zinc oxide nanoparticles interfere with zinc ion homeostasis to cause cytotoxicity. Toxicol Sci 125:462–72.
Kim YH, Fazlollahi F, Kennedy IM, Yacobi NR, Hamm-Alvarez SF, Borok Z, et al. 2010. Alveolar epithelial cell injury due to zinc oxide nanoparticle exposure. Am J Respir Crit Care Med 182: 1398–409.
Kumara SA, Chena S. 2008. Nanostructured zinc oxide particles in chemically modified electrodes for biosensor applications. Anal Lett 41:141–58.
Lin CH, Chang LW, Chang H, Yang MH, Yang CS, Lai WH, et al. 2009. The chemical fate of the Cd/Se/Te-based quantum dot 705 in the biological system: toxicity implications. Nanotechnology 20:215101.
Liu X, Wu X, Cao H, Chang RPH. 2004. Growth mechanism and properities of ZnO nanorods synthesized by plasma-enhanced chemical vapor deposition. J Appl Phys 95:3141–7. Mai FD, Li JP, Liu PC, Li FY, Ling YC, Chen JK, Yang CS. 2010. Controllable electrosprayed
nanoparticles for quantitative characterization and exposure protocol. Proceedings of 10th IEEE International Conference on Nanotechnology Joint Symposium with Nano Korea 17–20 August 2010, KINTEX, Korea, pp. 197–200.
Nel A, Xia T, Madler L, Li N. 2006. Toxic potential of materials at the nanolevel. Science 311:622– 7.
Nohynek GJ, Lademann J, Ribaud C, Roberts MS. 2007. Grey goo on the skin? Nanotechnology, cosmetic and sunscreen safety. Crit Rev Toxicol 37:251–77.
Palmer JD, Rifkind D. 1974. Neutralization of the hemodynamic effects of endotoxin by polymyxin B. Surg Gynecol Obstet 138:755–9.
Takeda K, Akira S. 2001. Regulation of innate immune responses by Tolllike receptors. Jpn J Infect Dis 54:209–19.
Takeda K, Akira S. 2004. TLR signaling pathways. Semin Immunol 16: 3–9.
Warheit DB, Sayes CM, Reed KL. 2009. Nanoscale and fine zinc oxide particles: can in vitro assays accurately forecast lung hazards following inhalation exposures? Environ Sci Technol 43:7939–45.
Wesselkamper SC, Chen LC, Gordon T. 2001a. Development of pulmonary tolerance in mice exposed to zinc oxide fumes. Toxicol Sci 60:144–51.
Wesselkamper SC, Chen LC, Kleeberger SR, Gordon T. 2001b. Genetic variability in the
development of pulmonary tolerance to inhaled pollutants in inbred mice. Am J Physiol Lung Cell Mol Physiol 281: L1200–9.
Xia T, Kovochich M, Liong M, Madler L, Gilbert B, Shi H, et al. 2008. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2:2121–34.
Xie Y, Williams NG, Tolic A, Chrisler WB, Teeguarden JG, Maddux BL, et al. 2012. Aerosolized ZnO nanoparticles induce toxicity in alveolar type II epithelial cells at the air-liquid interface. Toxicol Sci 125: 450–61.
Yang CS, Mai FD, Liu B, Chen JK, Lin YC, Li FY. 2011. Nano particle accommodation apparatus, nano particle detection system and method thereof. USPTO US Patent No. 7,886,623 B2. Yeh TK, Chen JK, Lin CH, Yang MH, Yang CS, Chou FI, et al. 2012. Kinetics and tissue distribution of neutron-activated zinc oxide nanoparticles and zinc nitrate in mice: effects of size and particulate
