Aerosolized ZnO nanoparticles induce toxicity in alveolar type II epithelial cells at the air-liquid interface.
ABSTRACT The majority of in vitro studies characterizing the impact of engineered nanoparticles (NPs) on cells that line the respiratory tract were conducted in cells exposed to NPs in suspension. This approach introduces processes that are unlikely to occur during inhaled NP exposures in vivo, such as the shedding of toxic doses of dissolved ions. ZnO NPs are used extensively and pose significant sources for human exposure. Exposures to airborne ZnO NPs can induce adverse effects, but the relevance of the dissolved Zn(2+) to the observed effects in vivo is still unclear. Our goal was to mimic in vivo exposures to airborne NPs and decipher the contribution of the intact NP from the contribution of the dissolved ions to airborne ZnO NP toxicity. We established the exposure of alveolar type II epithelial cells to aerosolized NPs at the air-liquid interface (ALI) and compared the impact of aerosolized ZnO NPs and NPs in suspension at the same cellular doses, measured as the number of particles per cell. By evaluating membrane integrity and cell viability 6 and 24 h post-exposure, we found that aerosolized NPs induced toxicity at the ALI at doses that were in the same order of magnitude as doses required to induce toxicity in submersed cultures. In addition, distinct patterns of oxidative stress were observed in the two exposure systems. These observations unravel the ability of airborne ZnO NPs to induce toxicity without the contribution of dissolved Zn(2+) and suggest distinct mechanisms at the ALI and in submersed cultures.
- [show abstract] [hide abstract]
ABSTRACT: Many investigations about the cellular response by metal oxide nanoparticles in vitro have been reported. However, the influence of the adsorption ability of metal oxide nanoparticles toward cells is unknown. The aim of this study is to understand the influence of adsorption by metal oxide nanoparticles on the cell viability in vitro. The adsorption abilities of six kinds of metal oxide nanoparticles, namely, NiO, ZnO, TiO2, CeO2, SiO2, and Fe2O3, to Dulbecco's modified Eagle's medium supplemented with a 10% fetal bovine serum (DMEM-FBS) component such as serum proteins and Ca2) were estimated. All of the metal oxide nanoparticles adsorbed proteins and Ca2+ in the DMEM-FBS; in particular, TiO2, CeO2, and ZnO showed strong adsorption abilities. Furthermore, the influence of the depletion of medium components by adsorption to metal oxide nanoparticles on cell viability and proliferation was examined. The particles were removed from the dispersion by centrifugation, and the supernatant was applied to the cells. Both the cell viability and the proliferation of human keratinocyte HaCaT cells and human lung carcinoma A549 cells were affected by the supernatant. In particular, cell proliferation was strongly inhibited by the supernatant of TiO2 and CeO2 dispersions. The supernatant showed depletion of serum proteins and Ca2+ by adsorption to metal oxide nanoparticles. When the adsorption effect was blocked by the pretreatment of particles with FBS, the inhibitory effect was lost. However, in NiO and ZnO, which showed ion release, a decrease of inhibitory effect by pretreatment was not shown. Furthermore, the association of the primary particle size and adsorption ability was examined in TiO2. The adsorption ability of TiO2 depended on the primary particle size. The TiO2 nanoparticles were size dependently absorbed with proteins and Ca2+, thereby inducing cytotoxicity. In conclusion, the adsorption ability of metal oxide nanoparticles is an important factor for the estimation of cytotoxicity in vitro for low-toxicity materials.Chemical Research in Toxicology 03/2009; 22(3):543-53. · 3.67 Impact Factor
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ABSTRACT: The urgent need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a basis for comparison to existing epidemiological data, we have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human MSTO-211H cells. Non-cytotoxic polyoxyethylene sorbitan monooleate was found to well-disperse CNT. In the present study, the cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference. While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibres at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.Toxicology Letters 02/2007; 168(2):121-31. · 3.15 Impact Factor
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ABSTRACT: The induction of cytokines by airway cells in vitro has been widely used to assess the effects of ambient and occupational particles. This study measured cytotoxicity and the release of the proinflammatory cytokines IL-6 and IL-8 by human bronchial epithelial cells treated with manufactured nano- and micron-sized particles of Al2O3, CeO2, Fe2O3, NiO, SiO2, and TiO2, with soil-derived particles from fugitive dust sources, and with the positive controls LPS, TNF-alpha, and VOSO4. The nano-sized particles were not consistently more potent than an equal mass of micron-sized particles of the same nominal composition for the induction of IL-6 and IL-8 secretion in the in vitro models used in this study. The manufactured pure oxides were much less potent than natural PM2.5 particles derived from soil dust, and the cells were highly responsive to the positive controls. The nano-sized particles in the media caused artifacts in the measurement of IL-6 by ELISA due to adsorption of the cytokine on the high-surface-area particles. The potency for inducing IL-6 secretion by BEAS-2B cells did not correlate with the generation of reactive oxygen species in cell-free media. Direct comparisons of manufactured metal oxide nanoparticles and previously studied types of particles and surrogate proinflammatory agonists showed that the metal oxide particles have low potency to induce IL-6 secretion in BEAS-2B cells. Particle artifacts from non-biological effects need to be considered in experiments of this type, and the limitations inherent in cell culture studies must be considered when interpreting in vitro results. This study suggests that manufactured metal oxide nanoparticles are not highly toxic to lung cells compared to environmental particles.Particle and Fibre Toxicology 02/2007; 4:2. · 9.18 Impact Factor
TOXICOLOGICAL SCIENCES 125(2), 450–461 (2012)
Advance Access publication September 28, 2011
Aerosolized ZnO Nanoparticles Induce Toxicity in Alveolar Type II
Epithelial Cells at the Air-Liquid Interface
Yumei Xie,* Nolann G. Williams,* Ana Tolic,* William B. Chrisler,† Justin G. Teeguarden,† Bettye L.S. Maddux,‡
Joel G. Pounds,† Alexander Laskin,* and Galya Orr*,1
*Environmental Molecular Sciences Laboratory; †Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352; and
‡Materials Science Institute, University of Oregon, Eugene, Oregon 97403
1To whom correspondence should be addressed at Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, PO Box 999 MS:
K8-88, Richland, WA 99352. Fax: (509) 371-6145, E-mail: email@example.com.
Received April 28, 2011; accepted September 18, 2011
The majority of in vitro studies characterizing the impact of
engineered nanoparticles (NPs) on cells that line the respiratory
tract were conducted in cells exposed to NPs in suspension. This
approach introduces processes that are unlikely to occur during
inhaled NP exposures in vivo, such as the shedding of toxic doses
of dissolved ions. ZnO NPs are used extensively and pose
significant sources for human exposure. Exposures to airborne
ZnO NPs can induce adverse effects, but the relevance of the
dissolved Zn21to the observed effects in vivo is still unclear. Our
goal was to mimic in vivo exposures to airborne NPs and decipher
the contribution of the intact NP from the contribution of the
dissolved ions to airborne ZnO NP toxicity. We established the
exposure of alveolar type II epithelial cells to aerosolized NPs at
the air-liquid interface (ALI) and compared the impact of
aerosolized ZnO NPs and NPs in suspension at the same cellular
doses, measured as the number of particles per cell. By evaluating
membrane integrity and cell viability 6 and 24 h post-exposure, we
found that aerosolized NPs induced toxicity at the ALI at doses
that were in the same order of magnitude as doses required to
induce toxicity in submersed cultures. In addition, distinct
patterns of oxidative stress were observed in the two exposure
systems. These observations unravel the ability of airborne ZnO
NPs to induce toxicity without the contribution of dissolved Zn21
and suggest distinct mechanisms at the ALI and in submersed
Key Words: ZnO nanoparticles; Zn21; air-liquid interface;
aerosol exposures; toxicity; alveolar epithelial cells.
Investigation of nanoparticulate toxicity to the respiratory
tract in vitro and in vivo is the most commonly used exposure/
response system. Airborne engineered nanoparticles (NPs) that
enter the respiratory tract are likely to be deposited in the
alveolar region (Donaldson et al., 2008; Mercer et al., 2010;
Oberdo ¨rster et al., 2005), where alveolar epithelial cells present
a vulnerable target for particles that escape scavenging by the
alveolar macrophages (Oberdo ¨rster et al., 2005; Takenaka
et al., 2001). Experimental exposure/response models in vitro
include cultured lung cells that are (1) grown and exposed to
NPs when submersed in diverse cell culture media with
variable supplements, including serum proteins and (2) grown
at the air-liquid interface (ALI) and exposed to aerosolized NPs
or to NPs dispersed in the growth medium. Experimental
exposure/response models in vivo include (1) orapharyngeal
aspiration, (2) intratracheal instillation, and (3) inhalation under
diverse forms of aerosol generation. These key differences in
cell environment and NP exposure limit the ability to extr-
apolate results from in vitro studies to pulmonary toxicity in
animals or humans.
To date, the majority of in vitro studies characterizing the
impact of inhaled NPs on these and other cells that line the
respiratory tract have been carried out in cells exposed to NPs
under submersed culture conditions. Use of routine submersed
culture systems to screen and compare NP toxicities in lung cells
introduces numerous confounding processes under conditions
that may be less relevant to inhaled NP exposures, which occur
at the air-lung lining fluid-cell interface. First, distinct coronas of
proteins and small molecules are formed at the NP surface
depending on the environment. In submersed cultures, the
corona will be composed of serum proteins (if used as medium
supplement or to impair NP agglomeration) and the small
molecules and ions of the individual culture medium. In contrast,
inhaled NPs will be coated by a corona of proteins and lipid
surfactants found in lung lining fluids. These coronas are likely
to modulate the interaction of NPs with cell surfaces and,
ultimately, the cellular fate of the NP (Casey et al., 2008; Dutta
et al., 2007; Horie et al., 2009; Veranth et al., 2007). Second,
when delivered in culture medium to submersed cells, NP
deposition (cellular dose) is a function of gravity and diffusion;
processes modulated by NP density and size, concentration, and
exposure duration (Hinderliter et al., 2010; Teeguarden et al.,
2007). Moreover, formation of large agglomerates in the growth
medium increases gravitational deposition and exposure in
submersed cultures and thereby may alter uptake mechanisms
? The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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and capacity (Donaldson et al., 2001; Porter et al., 2008; Sager
et al., 2007; Wick et al., 2007). These processes create
uncertainties that impair dose comparisons between in vivo
and in vitro studies in submersed cultures. Third, NP stability,
dissolution, and release of potentially toxic ions are dependent,
in part, on fluid pH, composition, and duration of exposure to
the fluid. Furthermore, cells grown at the ALI are polarized with
an apical surface similar to in vivo, which is likely to impact
cellular NP fate and response. These major differences between
the exposures in vivo and in submersed cultures can be
eliminated or minimized in aerosolized NP exposures at the ALI.
ZnO NPs are used extensively in diverse commercial
applications (Ji and Ye, 2008; Wang, 2004). Therefore, they
become a significant source for intended and unintended human
exposure. Intratracheal instillation and inhalation of ZnO NPs in
the rat elicit potent lung inflammatory or cytotoxic responses
that are resolved within days to a month (Cho et al., 2010; Sayes
et al., 2007; Warheit et al., 2009). These responses resemble
‘‘metal fume fever,’’ induced in humans or rodents exposed to
ZnO and other metal oxide fumes (Kuschner et al., 1995;
Wesselkamper et al., 2001). In vitro studies in bronchial and
alveolar epithelial cell lines, exposed to ZnO NPs in solution,
reported that the underlying cellular mechanisms involve
oxidative stress and inflammatory responses, DNA damage,
and cell death (Hsiao and Huang, 2011; Huang et al., 2010;
Karlsson et al., 2008; Wu et al., 2010).
Several in vivo and in vitro studies attempted to decipher the
contribution of the intact NP versus dissolved ions in ZnO NP
toxicity. Conflicting results have led to increasing confusion on
this issue. In vivo oropharyngeal aspiration and intratracheal
instillation studies, as well as an in vitro study, showed that ZnO
NPs that were doped with iron, which slows dissolution, among
other possible changes to the particle properties were less toxic
than undoped particles (George et al., 2010; Xia et al., 2011).
These observations suggest that shedding of dissolved Zn2þmight
play a role in ZnO NP toxicity. However, a recent intratracheal
instillation study in the rat showed that the NPs-induced
eosinophilic inflammation that persisted for a month, whereas
the supernatant, containing only dissolved Zn2þ, elicited a mild
and transient inflammatory response (Cho et al., 2011). This
observation suggests that the more severe responses are likely to
originate from the intact ZnO NPs rather than the dissolved ions.
In vitro studies attempting to distinguish between the
contribution of the intact NP and the dissolved ions to ZnO
NP toxicity reported conflicting results. Studies in alveolar and
bronchial epithelial cell lines and in primary alveolar epithelial
cells showed cytotoxic and inflammatory responses and severe
cellular injury in response to both the NPs and the medium
supernatant, as well as Zn2þsolutions, suggesting that the
toxicity in vitro is likely to be mediated, at least in part, by the
dissolved Zn2þ(Cho et al., 2011; Kim et al., 2010). However,
the exposure of alveolar epithelial cells to ZnSO4in culture
induced cell death at Zn2þconcentrations that were much
higher than the Zn2þconcentrations shed by the NPs at toxic
NP concentrations, suggesting that the dissolved Zn2þis
unlikely to be a major contributor to the oxidative stress
observed in response to the NPs (Lin et al., 2009). In support of
the view that different mechanisms underlie the cellular
responses to the intact or dissolved NPs, a study in alveolar
epithelial cells showed differential induction of interleukin
(IL)-8 and hemeoxygenase-1 (HO-1) messenger RNA expres-
sion in response to ZnO NPs presented to the cells either at the
ALI as aerosolized NPs or in submersed conditions as NPs in
suspension (Lenz et al., 2009).
The confusion that still exists about the origin of airborne
ZnO NP toxicity in vivo is therefore unlikely to be resolved by
in vitro studies of NPs that are presented to the cells in solution.
To more closely mimic in vivo exposures to airborne NPs, we
established the exposure of alveolar type II epithelial cells
(C10) to aerosolized NPs at the ALI. The mouse C10 alveolar
type II epithelial cell line was used because it synthesizes and
secretes lung surfactants, and it would enable comparison to
future inhalation studies in mice. The objective of our study
was to compare the cellular response to ZnO NPs in submersed
cultures with the response to aerosolized NPs at the ALI to
determine the degree of the intact airborne NP toxicity under
conditions that closely mimic inhaled NP exposures in vivo.
We also compared oxidative stress dynamics in response to NP
suspension in submersed cultures and to aerosolized NPs at the
ALI to determine whether differences exist in mechanisms
underlying toxicity in the two exposure systems.
MATERIALS AND METHODS
The exposure of alveolar type II epithelial cells (C10) to aerosolized NPs at
the ALI was established using commercial modules and in-house design to
optimize the efficiency and uniformity of NP delivery while preserving the
health of the cells.
Cells grown on membrane inserts were exposed to aerosolized ZnO NPs at
the ALI and to ZnO NP solution in submersed cultures. Cellular response was
evaluated in the two exposure systems at the same cellular dose, measured as
aggregates per unit area using scanning electron microscopy (SEM) to image
EM grids that were placed randomly over the cells before exposure. This dose
metric enabled accurate comparison of endpoints in the two exposure systems
in response to NP dose range that was commonly reported in the literature. Cell
proliferation, cell viability, membrane integrity, and oxidative stress were used
as endpoints. The MTS assay was used to quantify cell proliferation, propidium
iodide (PI)/Hoechst staining was used to quantify cell viability, and the lactate
dehydrogenase (LDH) assay was used to determine membrane integrity at 6
and 24 h post-exposure. Measurements were averaged across membrane inserts
showing the same number of aggregates per 20 lm2. These endpoints were
used to identify the smallest dose that induced toxicity in each of the exposure
systems for comparing toxicity of airborne NPs with toxicity of NPs in solution
where global dissolution occurs. The comparison enabled to determine the
degree of the intact aerosolized NP toxicity relative to the toxicity of the NPs
and their ions in solution. Dichlorofluorescein (DCF) was used to quantify
reactive oxygen species (ROS) to determine the degree of oxidative stress over
24 h. This endpoint was measured in the two exposure systems and compared
with determine whether distinct mechanisms underlie toxicity at the ALI and in
AEROSOLIZED ZnO NANOPARTICLES AT THE ALI
PI, Hoechst 33258, and RPMI growth medium were purchased from Invitrogen
(Carlsbad, CA). CytoTox 96 Non-Radioactive Cytotoxicity Assay CellTiter 96
AQueous Non-Radioactive Cell Proliferation Assay and 5-(and-6)-chloromethyl-
2#,7#-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were
purchased from Promega (Madison, WI). Tert-butyl hydroperoxide (TBHP) was
purchased from Acros Organics (New Jersey). Fetal bovine serum (FBS) was
purchased from ATCC (Manassas, VA). L-glutamine, penicillin, streptomycin, and
poly-L-lysine hydrobromide were purchased from Sigma (St Louis, MO). Millipore
(Billerica, MA) water was sterilized using 0.22-lm cellulose nitrate filter (Corning,
Corning, NY). Rabbit antibodies against mouse Surfactant Proteins D (AB3434)
and A (AB3420) were purchased from Millipore, and goat Alexa Fluor 488 F(ab)2
fragment anti-rabbit IgG (HþL) (52395A) was purchased from Invitrogen.
C10, a nontumorigenic alveolar type II epithelial cell line derived from
mouse lung, was used in this study. One of the main roles of this cell type is to
produce the surfactants that prevent the alveolar collapse with expiration. Using
immunofluorescence, the expression of surfactant proteins A and D by the C10
cells was confirmed, as demonstrated in Supplementary figure 1, providing
a microenvironment that mimics the environment in vivo. The cells were grown
in RPMI growth medium supplemented with 10% FBS, 2mM L-glutamine, 100
U/ml penicillin, and 100 lg/ml streptomycin. Cells were seeded on cell culture
membrane inserts (BD Falcon, transparent polyethylene terephthalate [PET]
membranes, 4.2 cm2effective growth area, 0.4 lm pore size, 2.0 ± 0.2 3 106
pores/cm2) with 3 ml growth medium on the basal side and 1.3 ml on the apical
side. The cells were kept under submersed conditions until full confluence was
reached. Cultures at the ALI then were achieved by removing the apical growth
medium and incubating at 37?C and 95% relative humidity in a 5% CO2
incubator for an additional 24 h before exposure to the NPs. For exposures in
submersed conditions, fully confluent cells, grown on the same PET
membranes as the cells at the ALI, were covered with 1.3 ml growth media
and exposed to the NPs in solution.
The ALI Exposure System
The exposure system for aerosolized NPs is illustrated in Figure 1. The system
consists of three main components: (1) a vibrating membrane nebulizer (Aeroneb
Lab nebulizer system, Aerogen, Galway, Ireland) that generates aerosolized NPs;
(2) Vitrocell exposure chambers (Waldkirth, Germany), where the aerosolized
NPs are deposited on the cells; and (3) an airflow system that distributes the
aerosolized NPs through the system at a highly controlled rate. Synthetic
breathing air (Air Liquid USA Inc., Houston, TX) was directed at 1 l/min into
a water bubbler for humidification. From there, the air was transported to the
nebulizer for generating the aerosol, which was directed into the three exposure
chambers at 10 ml/min using a vacuum pump and a flow regulator. Each
exposure chamber was designed to expose one membrane insert to the
aerosolized NPs via a ‘‘trumpet,’’ which is visible in the open chambers in the
inset in Figure 1. The exposure chambers were kept at 32?C using circulating
water supplied by a water bath, while the whole system was encased in a heated
incubation chamber to keep the entering airflow at 32?C. Following 10–20 min
exposure sessions, the cells were returned to the incubator until assayed. It was
found that cell death increased with increasing exposure time, and airflow rate
beyond 10 ml/min accelerated this process. Therefore, the minimal exposure time
required to achieve the dose range used in this study, under 10 ml/min flow rate,
Preparation of NP Suspensions
NPs were suspended in sterile water (5 mg/ml) and bath-sonicated (Branson
1210 Ultrasonic Cleaner, Danbury, CT) at room temperature (RT) for 10 min,
followed by 2 3 60 s probe sonication (Misonix Sonicator 3000, Farmingdale,
NY) at 15 watts on ice, with a 15 s rest between pulses. For the ALI exposures, the
NP solution was diluted 1-, 2-, 10-, 50-, and 100-fold, which roughly corresponded
to ? 1000, 700–900, 400–600, and ? 120 aggregates/20 lm2, respectively, during
10–20 min exposure sessions. The final solutions included 5% FBS and 0.02%
saline. FBS was added to minimize differences in particle properties presented to
the cells at the ALI and in submersed cultures. For submersed exposures, NP
concentrations (4, 10, 25, 50, 100, 250, 500, and 1000 lg/ml) were achieved by
diluting the NP solution in RPMI growth media containing 10% FBS. Final probe-
sonication for 2 3 60 s on ice was done before exposure.
NP Characterization and Dose Measurements
ZnO NPs (25 nm primary particle diameter) were kindly provided by Dr
Jeffrey Zink (University of California, Los Angeles) as part of the National
Institute of Environmental Health Sciences Nanotechnology Environmental
Health and Safety consortium effort. Before NP exposure, electron microscopy
(EM) grids (PELCO Carbon Type B support film grids, Ted Pella, Redding,
CA) were placed over the cell monolayer and removed right after exposure for
further analysis using SEM (FEI Quanta 3-D FEG dual beam, Hillsboro,
Oregon) at 10–30 kV. In submersed exposures, poly-L-lysine coated grids were
used, and the grids were dipped once in sterile water to remove salt and other
growth media ingredients before imaging. SEM was used to determine the
relevant size distribution of the particles as seen by the cells and precisely
quantify the exposure dose using Genesis Microanalysis software (EDAX,
Mahwah, NJ). The dose metric used for ZnO NPs in this study was aggregates
per 20 lm2, the size of a typical cell. The number of aggregates per unit area
was measured postexposure for each membrane insert from the SEM images,
Illustration of the system for aerosolized nanoparticle exposure at the ALI (refer to ‘‘Materials and Methods’’ for details).
XIE ET AL.
taken in both exposure systems. Hydrodynamic size distribution and zeta
potential were measured using Zeta PALS (Brookhaven Instruments, Holtsville,
NY). Size distribution measurements were done in 5% FBS, 0.2% saline for the
ALI, and complete RPMI culture media for submersed conditions.
Cell proliferation. CellTiter 96 AQueous Non-Radioactive Cell Prolifera-
tion Assay was used to determine cell viability 24 h post-exposure through the
reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium, inner salt) into aqueous soluble formazan by
dehydrogenase enzymes found in metabolically active cells. The MTS solution, in
RPMI growth media (1:10 dilution), was added to the apical side of the cells for
1 h incubation at 37?C. Formazan absorbance was measured at 490 nm using
a microplate reader (Spectra Max Plus 384, Molecular Devices, Sunnyvale, CA)
with multiple reads per well. The mean absorbance of negative control, where
cells were exposed to solution or aerosol containing no NPs, was established as
100% cellular viability. The absorbance of cells exposed to the NPs was measured
in at least five membrane inserts for each dose, and the values were normalized to
negative control. Significant change in the normalized values relative to negative
control, determined using the two-tailed Student’s t-test, indicated a significant
change in cell proliferation or viability.
Cell death. A double stain of PI and Hoechst was used to quantify dead
cells at 6 and 24 h post-exposure. PI was added to RPMI growth media at
1:1000 dilution, and the solution was placed on the apical side of the cell
culture. After 10 min incubation at 37?C, the cells were counterstained with
Hoechst to quantify total cell number. The cells were visualized using
fluorescence microscopy (Axio Observer, Carl Zeiss, Germany), where the
Hoechst emission was collected at 480/40 nm and the PI emission was collected
at 629/62 nm. PI- and Hoechst-positive cells were counted in images taken
from three membrane inserts for each dose, and the percent of dead cells was
quantified as the percent of PI positive cells from the total Hoechst-stained
cells. Significant change in cell death, an indicator for toxic response, was
determined relative to negative control using the two-tailed Student’s t-test.
Membrane integrity. CytoTox 96 Non-Radioactive Cytotoxicity Assay was
used to quantify LDH release at 6 and 24 h post-exposure through conversion of
a tetrazolium salt (INT) into a red formazan product. For positive control, lysis
solution (0.9% triton X-100) in RPMI growth media was added to the apical side
of control cells for 60 min of incubation at 37?C. Before NP exposure, 50 ll from
the apical and basal media were taken as well as 6 and 24 h post-exposure and
placed in 96-multiwell plates to which substrate mix was added (50 ll). Following
a 30 min incubation at RT under light protection, stop solution was added (50 ll),
and the formazan absorbance was measured at 490 nm using a microplate reader
with multiple reads per well. Absorption values at 6 and 24 h post-exposure were
normalized to the values of the same membrane insert before exposure, and
significance was determined relative to the time-matched negative control using
the two-tailed Student’s t-test. Significant change in the normalized values relative
to negative control indicated a significant change in membrane damage.
ROS generation at the ALI was measured at 2, 4, 6, 8, 14, and 24 h post-
exposures through the oxidation of the cell-permeant compound 5-(and-6)-
chloromethyl-2#,7#-dichlorodihydrofluorescein diacetate, acetyl ester (CM-
H2DCFDA or DCF). Low (106 aggregate/20 lm2) and high (933 aggregate/
20 lm2) exposure doses were used. 25lM CM-H2DCFDA was added to the
apical growth media and incubated for 30 min at 37?C under light protection.
The medium was removed from the apical side, and the cells were trypsinized
until fully detached and resuspended in 1 ml RPMI growth media. Positive
control was treated with 4.7mM TBHP 60 min before the addition of DCF. All
cell samples were centrifuged (IEC Centa MP4R) at 1000 rpm for 2 min and
resuspended in 0.5 ml growth media with PI at 1 lg/ml. Positive control for PI
was prepared by mixing 1 ml of cell suspension with 5 ml of methanol for
30 min at RT followed by centrifugation and resuspension in 0.5 ml of growth
media with 1 lg of PI per ml. DCF and PI fluorescence were measured by flow
cytometry using the Influx (BD Biosciences, Seattle, WA). Forward and side
scatter were used to gate out cellular debris, and a secondary gate, based on PI
emission at 585/29 nm when excited with a 561- nm laser, was generated to
differentiate between the live and dead cell populations. DCF fluorescence was
measured at 520/15 nm when excited with a 488 nm laser. Gating and median
calculations from 30,000 cells were done using Flow Jo software (Tree Star,
Ashland, OR). Median fluorescence values, indicating relative degree of ROS,
were normalized to time-matched negative control and significance was
determined using two-ways ANOVA. A significant change in these values
indicated a significant change in oxidative stress.
Data is presented as mean ± SD. The two-tailed Student’s t-test was used to
compare two group means. ANOVA was performed to compare the significance
of differences between multiple (? 3) groups. For all statistical analyses, p values
of less than 0.05 were considered significant.
Size Distribution and Actual Dose of Aerosolized ZnO NPs
Were Measured in Particles Settled on EM Grids
EM grids were placed randomly on the cell monolayer before
exposures, either at the ALI or in submersed cultures, to enable
accurate measurements of particle size distributions and actual
cellular doses. The grids were removed at the end of the
exposure session and visualized using scanning EM. Figure 2
presents the log-normal size distributions measured from aero-
solized particles that landed on the grids at the ALI (Fig. 2A) or
in submersed cultures (Fig. 2B). In both cases, NP aggregates
were formed. The size distribution at the ALI was measured
under high exposure dose of 930 aggregates per 20 lm2, or per
area of an average cell, showing median diameter of 117 nm with
geometric standard deviation of 2.17. A similar size distribution
was found in submersed conditions (115 nm, r ¼ 2.43) under
low exposure dose of about 54 aggregates per 20 lm2. Unlike the
median diameter of aerosolized NP aggregates, which did not
change with exposure dose, the median diameter of the aggregates
in solution inevitably increased at high NP concentrations.
Transmission EM images of aerosolized NP aggregates that
settled at the ALI are shown in Figure 2C. Dynamic light scatt-
ering measurements, which are dominated by the larger particles,
showed an average size distribution of 288.2 ± 2.4 nm for the
particles in the aerosol solution (5% FBS, 0.2% saline) and 265.7
± 3.6 nm in the RPMI growth medium containing FBS. Zeta
potential of ?22.11 ± 0.47 mV was found for the particles in the
aerosol solution and ?8.78 ± 1.18 mV in the growth medium.
EM was also used to quantify the actual cellular dose as
number of landed aggregates per unit area. EM grids were placed
randomly over the cells in each of the membrane inserts before
exposure and were removed and visualized using scanning EM
at the end of the exposure sessions. Figure 3 demonstrates
the uniform distribution of the aerosolized NP aggregates at the
ALI, which was achieved by the exposure system described
earlier (refer to ‘‘Materials and Methods’’). The scanning EM
image was taken from a high dose of 930 aggregates per 20 lm2.
The same approach was taken to quantify the actual cellular dose
AEROSOLIZED ZnO NANOPARTICLES AT THE ALI
in submersed cultures, where the NP doses added to the growth
medium were measured in lg/ml. EM grids, coated with poly-L-
lysine to ensure NP adherence, were placed on the cell before
adding the NPs to the growth medium. The number of aggregates
per 20 lm2found for each of the doses used in submersed
cultures is listed in Table 1.
Responses to Aerosolized NPs at the ALI Were Compared
with Responses in Submersed Cultures at the Same
Number of Settled NP Aggregates per Cell
Toxicity of aerosolized ZnO NPs was assessed in alveolar
type II epithelial cells grown at the ALI. These cells, which
produce surfactants that prevent the alveolar collapse with
expiration (Weaver and Whitsett, 1991; Wikenheiser et al.,
1993), present a target cell type for inhaled NPs in vivo.
Membrane integrity was measured by the release of LDH into
the basal medium at 6 and 24 h post-exposure. As shown in
Figure 4A, a significant deterioration in membrane integrity was
detected 24 h post-exposure when the dose reached about 500
aggregates per 20 lm2(p ¼ 0.0020). A higher dose of 800
aggregates per 20 lm2was required to detect significant
membrane damage at 6 h post-exposure (p ¼ 0.0207). Exposure
to aerosolized supernatant, taken from a toxic particle dose of
about 800 aggregates per 20 lm2and carrying Zn2þ, failed to
impose membrane damage, indicating that little or no aerosolized
supernatant reached the cells, and the membrane’s integrity was
damaged by exposures to the intact NPs. The conversion of the
doses from aggregates/20 lm2to mass (lg/cm2) and surface area
(lg/cm2) is provided in Supplementary table 1.
Cell viability was evaluated in response to aerosolized NPs at
the ALI 24 h post-exposure by quantifying the degree of cell
proliferation using the MTS assay. As shown in Figure 4B,
a significant decrease in cell proliferation was observed when the
dose reached about 800 aggregates per 20 lm2(p ¼ 0.0499).
Exposure to aerosolized supernatant obtained from a toxic
particle dose of about 800 aggregates per 20 lm2elicited no
toxic response. As mentioned, this observation indicates that
little or no aerosolized supernatant reached the cells, and the
toxicity originated from the exposure to the intact NP.
particles that settled on EM grids placed randomly on the cell monolayer
either at the ALI (A) or under submersed conditions (B) and imaged using
scanning EM. (A) 117 nm (r ¼ 2.17) median diameter is found for
aerosolized particles measured from high exposure dose of about 900
aggregates per 20 lm2or per area of an average cell. (B) A similar median
diameter (115 nm, r ¼ 2.43) is found for particles in solution when measured
from low exposure dose of about 54 aggregates per 20 lm2. (C) Selected
transmission electron micrographs of settled aerosolized ZnO aggregates at
Size distribution of ZnO NP aggregates as measured from
distribution achieved using the system for aerosolized NP exposure at the
ALI. The image was taken from a high exposure dose, where 930 aggregates
per 20 lm2were formed. Such images were used to determine exposure dose
for each membrane insert as the number of settled aggregates per cell.
Scanning electron micrograph demonstrating the uniform
XIE ET AL.
Exposures to NPs in solution were done in cells grown on the
same transwell membrane inserts as the cells at the ALI but
instead were submersed under growth media. The particle
solution was added to the apical medium and replaced with fresh
medium after 1 h to achieve a defined dose of settled aggregates
in a short time period. This approach enables the comparison of
exposures in submersed cultures with aerosolized NP exposures
at the ALI, where a defined number of settled aggregates was
achieved within 10–20 min. Ideally, the particle solution in
submersed exposures would be removed after 10–20 min as
well. However, to achieve high numbers of settled particles in
solution in such a short time, it would be necessary to use a very
high NP concentration, which leads to larger agglomerates.
As shown in Figure 5A, a significant increase in LDH
release into the basal medium was detected 24 h post-exposure
when the exposure dose reaches about 300 aggregates per 20 lm2
(p ¼ 0.0353). This number of aggregates per 20 lm2, which was
quantified using EM and is listed in parenthesis, was achieved by
adding 250 lg/ml NPs to the apical growth medium and replacing
the medium 1 h later. When LDH release was quantified in the
apical medium (Fig. 5B), a significant increase was detected 24 h
post-exposure in response to an even lower dose of 167 agg-
regates per 20 lm2(p ¼ 0.0223). This cellular dose corresponds
to 100 lg/ml NPs added to the apical growth medium and
replaced with fresh medium 1 h later.
Cell viability was assessed 24 h post-exposure to NPs in
decrease in cell proliferation was detected when the cellular
dose reached about 300 aggregates per 20 lm2(p ¼ 0.0205), or
250 lg/ml/1 h, as measured 24 h post-exposure.
Cell viability was also assessed using PI to detect dead cells.
Hoechst was used to stain both live and dead cells and enable the
calculation of percent PI positive cells from the total cell
population. In aerosolized NP exposures at the ALI, a significant
decreasein livecells was observed after 24h inresponseto doses
as shown in Figure 6A. This observation is in agreement with the
was observed at about 800 aggregates per 20 lm2. Exposures in
submersed cultures led to a significant decrease in live cells at
agreement with the MTS assay (Fig. 5C), where a significant
decreaseincellviabilitywas detectedatabout 300 aggregatesper
20 lm2. Examples of fluorescence images used to quantify the
Hoechst, respectively, are shown in Figure 6C.
The data presented indicates that the numbers of aggregates per
20 lm2, or per cell, required to achieve a significant deterioration
in membrane integrity and cell viability in response to aerosolized
NP exposures at the ALI and in submersed conditions were in the
same order of magnitude.
Oxidative Stress Was Quantified in Response to Aerosolized
ZnO NPs at the ALI and to NP Suspension in Submersed
Cultures Over Time
Using DCF, ROS generation was detected and quantified over
time in response to aerosolized NP exposures at the ALI and to NP
suspension in submersed cultures using flow cytometry. Due to its
overall oxidative stress (Wang and Joseph, 1999). The cells were
lg/ml, which is equivalent to about 1000 aggregates per 20 lm2,
and a low dose of 50 lg/ml, which is equivalent to about 50
aggregates per 20 lm2. As shown in Figure 7A, a significant
at the ALI 6 h post-exposure (p < 0.0001), which decayed back to
baseline 8–10h post-exposure.Nosignificant increase inoxidative
stress was detected at the ALI in response to the low dose. LDH
measurements in these samples (Fig. 7B) showed a significant
correlated with the peak in oxidative stress. These observations
indicate that the mechanism underlying aerosolized ZnO NP
toxicity at the ALI involves a transient increase in oxidative stress
that subsides as cell damage takes place.
In contrast to the oxidative stress pattern at the ALI, a robust
increase in oxidative stress of more than 10-fold was observed in
submersed cultures as early as 2 h post-exposure (p < 0.01) (Fig.
7C), which decayed back to baseline 8–10 h post-exposure.
and in submersed conditions, the pattern of LDH release was
the low dose—as early as 6 h post-exposure.
The main new finding to emerge from this work is the ability
of aerosolized ZnO NPs to induce toxicity at the ALI at doses
that are in the same order of magnitude as those required to
The Number of Aggregates per 20 mm2 as Quantified by EM for
Each of the Doses Used in Submersed Cultures After 1 h From
the Addition of the NPs to the Growth Medium
Exposure dose lg/ml
aAggregates/20 lm2± SD
8 ± 4
54 ± 15
167 ± 25
326 ± 51
1165 ± 145
aAveraged values from three experiments for each exposure dose.
AEROSOLIZED ZnO NANOPARTICLES AT THE ALI
induce toxicity in submersed cultures, where both the NPs and
the dissolved ions contribute to the observed toxicity. The
significance of this finding is that the toxicity of aerosolized ZnO
NPs must originate from direct interactions of cellular structures
with the intact NP or with locally dissolved Zn2þat the contact
site of the NP with the cell rather than from global dissolution.
The work clears the confusion created by in vitro exposures in
submersed cultures by showing that ZnO NP toxicity is not
dependent on the massive dissolution of the particles and the
production of toxic doses of Zn2þ, unraveling the potency of
exposures to the intact NPs. Our observations indicate that
inhaled ZnO NPs have the potential to induce toxicity by local
interactions at the contact site of the NP with cellular structures.
The ALI system that we have established prevented the
exposure of the cells to toxic doses of dissolved ions and the
formation of large agglomerates, enabling the assessment of
airborne NP toxicity at actual cellular dose under conditions
that closely mimic the exposure to inhaled NPs in vivo. Our
approach can also avoid the formation of protein coronas that
are formed around NPs in growth media, which mask the
original surface properties of the NPs and introduce variability
across systems. However, the goal of our study was to compare
the impact of aerosolized NPs and NPs in growth medium.
Thus, FBS was added to the aerosol solution. By quantifying
the number of settled particles per cell, we were able to provide
accurate relationships between the actual cellular dose and
cellular response. This approach also enabled the comparison
of cellular responses at the ALI and in submersed cultures
using the same dose metric, measured as the number of settled
aggregates per cell. However, an inevitable inaccuracy in the
the exposure dose reached about 500 aggregates per 20 lm2, as quantified 24 h post-exposure by LDH released into the basal medium (p ¼ 0.0020). At 6 h post-
exposure, significant compromise in membrane integrity was detected starting at about 800 aggregates per 20 lm2(p ¼ 0.0207). Exposure to aerosolized
supernatant, taken from a toxic particle dose, failed to impose membrane damage, indicating that little or no aerosolized supernatant reached the cells and
membrane integrity was compromised by exposures to the intact NPs. LDH measurements for each dose range at each time point were taken from at least three
different membrane inserts. The absorbance values were normalized to preexposure measurements, taken for each of the membrane inserts, to correct for normal
deterioration that occurs over time. (B) Aerosolized ZnO NP exposures imposed a significant decrease in cell proliferation at the ALI when the exposure dose
reached about 800 aggregates per 20 lm2, as measured by the MTS assay at 24 h post-exposure (p ¼ 0.0499). Exposure to aerosolized supernatant obtained from
a toxic particle dose showed no decrease in cell proliferation. MTS measurements from at least five different membrane inserts were used for each exposure dose
range. These values were normalized to the negative (?) control, where cells were exposed to aerosol containing no particles. Significance was determined for both
LDH and MTS measurements using the two-tailed Student’s t-test with 95% confidence when compared with negative (?) control at the same time point.
(A) Aerosolized ZnO NP exposures of alveolar type II epithelial cells at the ALI significantly compromised the integrity of the cell membrane when
XIE ET AL.
conversion of lg/ml to aggregates/20 lm2might have been
introduced by coating the grids placed in submersed cultures
with poly-L-lysine to avoid the loss of the NPs back to the
growth medium. The positively charged poly-L-lysine could
attract more NPs, leading to larger number of aggregates per
unit area than the number of aggregates that were settled on the
cells. In addition, an increase in the size of the aggregates
occurred in submersed cultures at high NP concentrations.
Such increase in aggregate size was not observed in aerosolized
NP exposures at the ALI. Therefore, while similar numbers of
settled aggregates per cell were compared, larger numbers of
NPs were present in submersed conditions at high exposure
doses. However, this difference in aggregate size only
strengthens our conclusion that the intact aerosolized NP at
the ALI is as potent in inducing toxicity, if not more, as the
NPs and their massively dissolved ions in solution. Other
methods could be used for accurate measurements of the cellular
dose at the ALI, such as a quartz crystal microbalance, which
would report dose in lg/cm2. This approach, however, could not
be used in submersed culture for supporting an accurate com-
parison of toxicity in the ALI and submersed cultures. Other
methods, such as inductively coupled plasma mass spectrometry,
could be used to quantify actual cellular dose in both systems as
well. Using aggregates per unit area as the dose metric, measured
by EM, enabled the characterization of the aggregate size
distribution at the cell surface in both exposure systems.
Our study was conducted over 24 h exposures, showing toxic
responses over short time periods. However, in vivo studies have
shown adverse effects in response to ZnO NPs that were
resolved within days to a month (Cho et al., 2010; Sayes et al.,
2007; Warheit et al., 2009), indicating the need to assess toxicity
over longer time periods. Whether the ALI could provide an
appropriate system for assessing transient toxic responses over
longer time periods is yet to be determined.
The Tox21 paradigm proposes to utilize high throughput
in vitro assays and limited in vivo studies, mainly focused on
pharmacokinetics to rank chemicals and nanomaterial hazards for
further evaluation in more complete model systems such as
whole animals. The ALI system is expected to serve as an inter-
mediate step between high throughput in vitro assay systems,
which have been shown to have a number of dosimetry and
biology related limitations (Teeguarden et al., 2007) and whole
Our work, which identified a clear role for the intact NP in ZnO
NP toxicity, also raises the possibility that different mechanisms
underlie the toxicity induced by the intact NP and the globally
dissolved Zn2þ. While studies in submersed conditions show that
same transwell membrane inserts as the cells at the ALI but submersed under
growth media. A significant increase in LDH release into the basal medium was
detected 24 h post-exposure when the exposure dose reached about 300
aggregates per 20 lm2(p ¼ 0.0353), as quantified by EM and listed in
parentheses. This dose was achieved by adding 250 lg/ml NPs to the apical
growth medium and replacing the medium 1 h later. (B) When LDH release
was quantified in the apical medium, a significant increase was detected 24 h
post-exposure at 167 aggregates per 20 lm2(p ¼ 0.0223). This dose
corresponds to 100 lg/ml NPs added to the apical growth medium and replaced
with fresh medium 1 h later. At least three different membrane inserts were
used for each exposure dose at each time point for both basal and apical LDH
measurements. The absorbance values were normalized to preexposure values
for each membrane insert to correct for normal cell deterioration. (C) A
significant decrease in cell proliferation was detected using the MTS assay
when the exposure dose reached about 300 aggregates per 20 lm2(p ¼
(A) ZnO NP exposures in solution were done in cells grown on the
0.0205), or 250 lg/ml/1 h, as measured 24 h post-exposure. At least five
membrane inserts were used for each exposure dose, and the absorbance values
were normalized to the negative (?) control, where cells were exposed to
solution containing no particles. For both LDH and MTS measurements,
significance was determined using the two-tailed Student’s t-test with 95%
confidence when compared with negative (?) control.
AEROSOLIZED ZnO NANOPARTICLES AT THE ALI
exposures to the NP supernatant or to Zn2þsolutions lead to
inflammatory responses, cellular injury, and toxicity, just as the
exposures to the NPs in solution (Cho et al., 2011; Kim et al.,
2010), these outcomes might originate from molecular processes
that are distinct from the local processes that occur in response to
the intact NP. The dissolved Zn2þin submersed exposures are
likely to interact with molecules at multiple sites along the cell
membrane and enter the cell through multiple and nonspecific
mechanisms. However, the intact NP is likely to interact with
specific molecules that are spatially limited to the contact site and
enter the cell through endocytic pathways specific to the NPs.
This view is supported by the distinct patterns of oxidative stress
that we observed at the ALI and in submersed cultures. The
robust increase, more than 10-fold, observed in oxidative stress as
early as 2 h post-exposure in submersed cultures could be
explained by the global and readily available Zn2þin the
exposure solution. By contrast, the relatively small and transient
increase in oxidative stress at the ALI could be explained by the
local and confined processes at the particle contact site with the
cell. If NP dissolution occurs at the contact site, the interactions
with Zn2þmust also be limited to specific molecules and
organelles around that site. We have shown previously that
specific cell surface molecules mediate the interactions of NPs at
the cell surface and their internalization pathways according to the
particle surface properties (Orr et al., 2007, 2009, 2011). It is
likely that the intact ZnO NP is captured by specific molecules at
the cell surface, which dictate the interactions, internalization
pathways and intracellular fate of the NP, and ultimately, the
response of the cell. In the case of the dissolved ions in submersed
conditions, multiple and nonspecific molecules are likely to
mediate these processes. In support of the possibility that different
mechanisms underlie the toxicity of the intact NP and the globally
dissolved ions are observations made in alveolar epithelial cells
showing distinct changes in HO-1 and IL-8 gene expression
levels when the cells were exposed to aerosolized ZnO NPs at the
ALI or to NPs in solution (Lenz et al., 2009). Similarly,
observations made in alveolar epithelial cells exposed to ZnSO4
in culture showed toxicity at Zn2þconcentrations that were much
higher than the Zn2þconcentrations shed in the presence of toxic
concentrations of the NPs (Lin et al., 2009), unraveling toxicity
that originated solely from the intact NPs. The instillation study in
the rat where severe and long-lasting inflammatory response was
induced by the NPs, but only mild and transient response was
induced by the supernatant (Cho et al., 2011), also supports this
possibility. These observations unravel a distinct and critical role
for the intact NP in ZnO NP toxicity in vivo. It is possible,
however, that locally dissolved Zn2þaround the intact NP
contribute to ZnO NP toxicity in vivo. This possibility is
supported by the aspiration and instillation study, showing
reduced toxicity by ZnO NPs that were doped with iron, which
slows the dissolution rate of the particles (Xia et al., 2011).
However, this study does not exclude the possibility that iron
doping might also decrease the interactive sites available to the
cell at the NP surface.
While studies in marine and freshwater organisms, which are
naturally exposed to NPs in solution, suggest that ZnO NP
toxicity could be attributed mainly to the dissolved Zn2þ
(Aruoja et al., 2009; Franklin et al., 2007; Miao et al., 2010;
Wong et al., 2010), other studies in aquatic and soil organisms
(Manzo et al., 2010; Poynton et al., 2011; Zhu et al., 2009), as
well as in bacteria (Jiang et al., 2009), showed a significant role
for both NP and dissolved Zn2þ, each with distinct underlying
mechanisms (Jiang et al., 2009; Manzo et al., 2010; Poynton
et al., 2011). Differentially regulated genes were observed in
h after aerosolized NP exposure in response to doses within the range of 595–
1390 aggregates per 20 lm2(p < 0.0001). (B) Exposures in submersed
conditions led to a significant decrease in live cells at about 300 aggregates per
20 lm2(p < 0.0001) as early as 6 h post-exposure. (C) Examples of
fluorescence images used to quantify the percent of dead cells (pink) stained
with PI from the total cells (blue) stained with Hoechst.
(A) A significant decrease in live cells was observed at the ALI 24
XIE ET AL.
Daphnia magna, where the genes for multicystatin, ferritin,
and C1q-containing gene were regulated in response to the NPs
but not the ions (Poynton et al., 2011). Physical perturbations
of cellular structures were suggested to underlie the impact of
the intact NP versus Zn2þin bacteria and certain soil organisms
(Jiang et al., 2009; Manzo et al., 2010). Rapid clearance
kinetics of soluble ions was suggested to account for the milder
responses observed in rats exposed to the ions versus the NPs
by instillation (Cho et al., 2011) and could also explain, in part,
differences observed in other organisms. Our study clearly
shows that the intact NP plays a critical role in airborne ZnO
NP toxicity and demonstrates distinct oxidative stress dynam-
ics at the ALI and in submersed cultures. The distinct spatial
and temporal patterns of internalization, molecular interactions,
and intracellular fate that are expected for the NPs and the
dissolved ions are likely to impact distinct cellular functions.
These molecular processes, and whether and how local
dissolution occurs and contributes to toxicity, are currently
Supplementary data are available online at http://toxsci.
(1RC2ES018786-01 to G.O.); Air Force Research Labora-
tory/Oregon Nanoscience and Microtechnologies Institute/
(FA8650-05-1-5041 to B.L.S.M and G.O).
Institute ofEnvironmentalHealth Sciences
providing the ZnO NPs as part of the National Institute of
Environmental Health Science Nanotechnology Environmental
Health and Safety consortium effort. The research was performed
as early as 6 h post-exposure (p < 0.0001). No significant increase in oxidative stress was detected in response to the low dose (106 aggregates per 20 lm2).
Median values from 30,000 cells per time point were normalized to negative control for each time point, and significance was determined using two-ways
ANOVA with 95% confidence when comparing to negative control. (B) LDH measurements in these samples showed a significant decrease in membrane
integrity in response to the high dose, but not low dose, as early as 6 h post-exposure (p ¼ 0.0259), which was correlated with the peak in oxidative stress. LDH
measurements were normalized to the negative (?) control, where cells were exposed to aerosol containing no particles. Significance was determined using the
two-tailed Student’s t-test with 95% confidence when compared with the negative (?) control. (C) A robust increase in oxidative stress was detected in
submersed cultures in response to the high dose (500 lg/ml or about 1000 aggregates per 20 lm2) as early as 2 h post-exposure (p < 0.01). No increase in
oxidative stress was detected in response to the low dose (50 lg/ml or about 50 aggregates per 20 lm2). (D) As in the aerosolized NP exposures, a significant
increase in LDH release was detected in response to the high dose as early as 6 h post-exposure (p ¼ 0.0348). No increase in LDH release was detected in
response to the low dose.
(A) A significant increase in oxidative stress was detected using DCF in response to high dose (933 aggregates per 20 lm2) of aerosolized ZnO NPs
AEROSOLIZED ZnO NANOPARTICLES AT THE ALI
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