Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles
Particle and Fibre Toxicology
Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles
Wan-Seob Cho 0
Rodger Duffin 0
Ian L Megson
William MacNee 0
Jong Kwon Lee
Ken Donaldson 0
0 ELEGI/Colt Laboratory, Centre for Inflammation Research, University of Edinburgh , 47 Little France Crescent, Edinburgh EH16 4TJ , UK
Background: Hazard identification for risk assessment of nanoparticles (NPs) is mainly composed of in vitro cellbased assays and in vivo animal experimentation. The rapidly increasing number and functionalizations of NPs makes in vivo toxicity tests undesirable on both ethical and financial grounds, creating an urgent need for development of in vitro cell-based assays that accurately predict in vivo toxicity and facilitate safe nanotechnology. Methods: In this study, we used 9 different NPs (CeO2, TiO2, carbon black, SiO2, NiO, Co3O4, Cr2O3, CuO, and ZnO). As an in vivo toxicity endpoint, the acute lung inflammogenicity in a rat instillation model was compared with the in vitro toxicity endpoints comprising cytotoxicity, pro-inflammatory cytokine expression, or haemolytic potential. For in vitro assays, 8 different cell-based assays were used including epithelial cells, monocytic/macrophage cells, human erythrocytes, and combined culture. Results: ZnO and CuO NPs acting via soluble toxic ions showed positive results in most of assays and were consistent with the lung inflammation data. When compared in in vitro assays at the same surface area dose (30 cm2/mL), NPs that were low solubility and therefore acting via surface reactivity had no convincing activity, except for CeO2 NP. Cytotoxicity in differentiated peripheral blood mononuclear cells was the most accurate showing 89% accuracy and 11% false negativity in predicting acute lung inflammogenicity. However, the haemolysis assay showed 100% consistency with the lung inflammation if any dose, having statistical significance was considered positivity. Other cell-based in vitro assays showed a poorer correlation with in vivo inflammogenicity. Conclusions: Based on the toxicity mechanisms of NPs, two different approaches can be applied for prediction of in vivo lung inflammogenicity. Most in vitro assays were good at detecting NPs that act via soluble ions (i.e., ZnO and CuO NP). However, in vitro assays were limited in detecting NPs acting via surface reactivity as their mechanism of toxicity, except for the haemolysis assay.
In vitro; In vivo; Inflammation; Mechanism; Nanoparticles; Prediction; Toxicity
Metal oxide nanoparticles (NPs) have been used in
various applications including industrial, electrical,
pharmaceutical, and biomedical fields because of their unique
physicochemical properties compared to bulk chemicals
. The high production volume of NPs and increasing
numbers of functionalized versions constitutes a burden
for toxicity testing and risk assessment for NP exposures
. In response to increasing concerns about the safety
of manufactured nanomaterials, OECD (Organisation
for Economic Co-operation and Development) launched
an internationally harmonised programme about hazard,
exposure, and risk assessment of nanomaterials in 2006
. After six years of work, the OECD has come to
conclusion that the current testing approaches are generally
acceptable for nanomaterials although some adoptions
may be necessary for the certain Test Guidelines .
Current hazard identification for risk assessment of
NPs is mainly conducted with the aid of both in vivo
and in vitro toxicity approaches. In vivo animal
experimentation is more informative than in vitro
experimentation, but there are major ethical and financial
limitations to the in vivo approach . Therefore,
in vitro assay have been suggested as an alternative
method to in vivo testing; indeed in vitro experiments
are often used as an initial screen for the toxicity of
substances and evaluation of their toxic mechanisms. There
are several in vitro cell-based testing methods that are
frequently used in this setting, including some assays
which combine several cell types , and others that use
differentiated macrophages from monocytic cells, which
are more sensitive to weak stimuli . However, in
recent studies comparing several cell lines, cells were found
to respond differently to NPs depending on the
physiological functions and activities of cells [8,9] and failed to
predict the in vivo lung inflammogenicity . However,
those studies were performed with a limited number of
particles and cell assays; more profound studies are
essential to correlate in vitro and in vivo toxicity assays.
There are several critical factors that can produce
discrepancies between studies. Dose is one of the most
important factors when comparing several in vitro assays
with in vivo assays, because even NPs with low toxicity
can be toxic at high doses . Surface area has been
suggested as an appropriate dose metric in
nanotoxicology rather than mass . In addition to surface area
metric, the dispersion of NPs is very important because
highly agglomerated NPs showed less toxicity or
inflammogenicity compared to well-dispersed NPs .
Determining the target organ for NP exposure is also
important for selection of appropriate cell types for
in vitro assays.
In this study, we were concerned with inhalation
exposure of metal oxide NPs in occupational or consumer
settings and we therefore used an acute lung
inflammation model by intratracheal instillation into the lungs of
rats. By comparison, we chose to investigate the
nanotoxicity of the same panel of NPs in lung alveolar and
bronchial epithelial cells and monocytic or macrophage
cells to mimic the in vivo lung environment, using a
range of in vitro toxicity tests.
Characterization of NPs
Table 1 summarize the characterization of NPs including
primary size, hydrodynamic size, and surface area. All
NPs in PBS formed agglomerates, which required
sonication to be dispersed. However, inclusion of 5% fetal
bovine serum in the PBS, particles formed smaller
agglomerates that were readily dispersed because of the
protein corona on the surface of NPs. All NPs and FBS
showed endotoxin levels below the detection limit.
Cr2O3, ZnO, and CuO NPs showed significant
cytotoxicity (cell death measured by LDH levels or trypan blue
staining) compared with vehicle control whilst other
NPs had little or no toxic effect in this assay at the doses
tested (30300 cm2/mL for most NPs, 330 cm2/mL for
ZnO and CuO; Figure 1A). The levels of IL-8 proteins in
the supernatant were significantly increased by both
ZnO and CuO NP (Figure 1B). Note that ZnO and CuO
NP were the most cytotoxic and stimulatory for IL-8
although they were used at doses 10-fold less than the
Only ZnO and CuO NP exposure significantly increased
cytotoxicity to 16-HBE cells (Figure 2A). The levels of
IL-8 proteins were significantly increased by CeO2,
carbon black (CB), and ZnO NP and significantly decreased
by TiO2 NP at the high dose (Figure 2B).
Monocytic THP-1 cells
Cr2O3 and CuO NP exposure significantly increased
cytotoxicity, whilst other NPs showed no differences
compared to vehicle control (Figure 3A). The levels of
IL-1 protein significantly increased only with TiO2 NP
treatment (Figure 3B).
Rat alveolar macrophages showed greater sensitivity to
NPs compared to cell lines. All NPs excluding CB and
SiO2 showed significant cytotoxicity compared to vehicle
control (Figure 4A). In comparison with cytotoxicity,
the levels of IL-1 showed marginal responses which
only TiO2 and CuO NP showing significant increases
(Figure 4B), although it should be noted that the TiO2
Table 1 Physicochemical properties of the panel of nanoparticles
Primary size (nm)b
Surface area (m2/g)c
Hydrodynamic size (nm) in 5% FBS
aNPs were provided by Nanostructural and Amorphous Materials Inc. (Houston, TX, USA) except for CB (Evonik Degussa GmbH, Frankfurt, Germany),
ZnO (NanoScale Corporation, Manhattan, KS, USA), and CuO (Sigma-Aldrich).
bPrimary particle sizes were measured by a transmission electron microscopy.
cSurface area was measured by BET (Brunauer-Emmett-Teller) method.
Figure 1 Cytotoxicity and IL-8 expression of A549 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue
exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of A549 cells at 24 h following treatment. Note that the
surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean SD from
minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.
Figure 2 Cytotoxicity and IL-8 expression of 16-HBE cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by trypan blue
exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-8 of 16-HBE cells at 24 h following treatment. Note that the
surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values are mean SD from
minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, ***p < 0.001.
NP effect was produced at 300 cm2/mL whilst the dose
of 10 cm2/mL of CuO NP was approximately equipotent
Peripheral blood monocyte-derived macrophages
PBMDM were differentiated from the peripheral blood
mononuclear cells (PBMC) by culturing for 5 days. All
NPs excluding CeO2, TiO2, and SiO2 NP showed
significant cytotoxicity compared to vehicle control
(Figure 5A). The levels of IL-1 were significantly
increased by TiO2, CB, NiO, Cr2O3, and ZnO NP
(Figure 5B). The levels of TNF- were significantly
increased by TiO2 and ZnO NP whilst other NPs were
comparable with vehicle control (Figure 5C).
compared to monocytic THP-1 cells. CeO2, TiO2, NiO,
Cr2O3, ZnO, and CuO NP showed significantly increased
cytotoxicity compared to vehicle control (Figure 6A). The
use of differential doses, however, again revealed that ZnO
and CuO NP were an order of magnitude or more
cytotoxic than the others. Levels of IL-1 were increased in
CeO2 and TiO2 NP-treated cells and showed modest
increases with CB, SiO2, Co3O4, and Cr2O3 NP (Figure 6B).
In contrast, the levels of TNF- showed marked increases
on treatment with CeO2 and NiO NP and slight increases
by TiO2 and Cr2O3 NP (Figure 6C). Treatment of
cytochalasin D, a well characterized inhibitor of phagocytosis,
dramatically inhibited the expression of IL-1 showing around
1/15 and 1/7 reductions for CeO2 and TiO2 NP compared
to the same treatment without cytochalasin D (Figures 6D).
Differentiated THP-1 cells
Differentiated THP-1 cells by treatment with phorbol
myristate acetate (PMA) showed much greater sensitivity
The levels of IL-8 in the conditioned media from THP-1
cells before addition of A549 cells were significantly
Figure 3 Cytotoxicity and IL-1 expression of monocytic THP-1 cells after exposure to NPs for 24 h. (A) Cytotoxicity was measured by
trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-1 of monocytic THP-1 cells at 24 h following
treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were 3, 10, and 30 cm2/mL. Values
are mean SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05, **p < 0.01, ***p < 0.001.
increased by TiO2, ZnO, and CuO NP treatment
(Figure 7A). Addition of NP-free conditioned media to
A549 cells produced a marked increase in IL-8 levels in
the CeO2, TiO2, Cr2O3, ZnO, and CuO NP treatment
groups (Figure 7B). Compared with single treatment to
A549 cells presented in Figure 1B, conditioned medium
produced more sensitive and higher responses for the
CeO2, NiO, Co3O4, and CuO NP showed significant
haemolytic potential compared to vehicle control (Figure 8)
and all were tested at equal surface area dose. However, it
was notable that there was a real difference in potency with
NiO NP being at least 2 times more potent than the others
in all treatment doses.
Correlation of in vitro assays with in vivo acute lung
Table 2 summarise the number of total granulocytes in the
bronchoalveolar lavage fluid in the lungs of rats treated at
150 cm2/rat after 24 h of instillation. Table 3 summarise
the utility of the various in vitro assays for predicting lung
inflammogenicity of NP panel. Because the gold standard
for comparison between lung inflammation and several
in vitro assays was dosed at equal surface area dose, we
took the 30 cm2/mL dose as the measure of a significant
effect. Cytotoxicity in differentiated PBMC (PBMDM)
showed the best accuracy - 89% (8/9) accuracy, 11% (1/9)
false negativity, and 0% (0/9) false positivity to predict
acute lung inflammogenicity. The second best assay was
cytotoxicity using primary cultured alveolar macrophages
which showed 78% (7/9) accuracy, 11% (1/9) false
negativity, and 11% (1/9) false positivity. Data from other cell
Figure 4 Cytotoxicity and IL-1 expression of primary cultured alveolar macrophages after exposure to NPs for 24 h. (A) Cytotoxicity
was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-1 of primary cultured
alveolar macrophages at 24 h following treatment. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP
which were 3, 10, and 30 cm2/mL. Values are mean SD from minimum four independent experiments. Significance versus vehicle control (VEH):
*p < 0.05, **p < 0.01, ***p < 0.001.
types or pro-inflammatory cytokine assays using PBMDM
and alveolar macrophages showed a poor correlation with
in vivo lung inflammogenicity. In contrast, the in vitro
assay data at any dose that have statistical significance
showed that the haemolysis assay was the most accurate
(Table 4). Table 3 can also suggest the mechanisms of
action of various metal oxide NPs. Among the NP that
caused inflammation, CeO2 NP had a range of activities
causing IL-8 release from 16-HBE, toxicity to alveolar
macrophages, and TNF- release from THP-1 cells. NiO NP
show no persuasive effects whilst Co3O4 NP seemed to kill
macrophages without stimulating them. TiO2, CB, SiO2,
and Cr2O3 NP showed no convincing effects whilst ZnO
and CuO NP caused significant changes in most of assays.
Cell-based in vitro toxicity testing is very important not
only for evaluation of mechanism of action but also for
alternative testing methods to replace animal
experimentation . In nanotoxicology, in vitro toxicity testing for
NPs has a high priority and indeed most of the studies
have been conducted in vitro . However, the responses
to NPs are known to be very variable depending on cell
type because of the diverse physiological functions of cells
and the heterogeneous physicochemical properties of NPs
. In this study, we used 9 predominantly metal oxide
NPs and tested cytotoxicity and pro-inflammatory
cytokine expression using 8 different cell-based toxicity testing
models relevant to the lung. The efficacy of in vitro testing
methods was evaluated against the potential of the panel
to cause acute lung inflammation in rats using data that
we published previously [16,17]. All NPs used in in vivo
and in vitro study were exactly same batch and both
studies were performed contemporaneously.
Differentiated THP-1 cells by PMA treatment were
considerably more sensitive to NPs with respect to both
Figure 5 Cytotoxicity and pro-inflammatory cytokine expression of differentiated PBMC after exposure to NPs for 24 h. (A) Cytotoxicity
was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-1; (C) levels of TNF-. PBMC
were differentiated by 5-day incubation. Note that the surface area doses were 30, 100, and 300 cm2/mL except for ZnO and CuO NP which were
3, 10, and 30 cm2/mL. Values are mean SD from minimum four independent experiments. Significance versus vehicle control (VEH): *p < 0.05,
**p < 0.01, ***p < 0.001.
cytotoxicity and cytokine expression, compared to
undifferentiated THP-1 cells. The pattern of IL-1 expression
was slightly different from that of TNF-. For example,
TiO2 NP induced the greatest response with respect to
IL-1 but only a slight increase in TNF-; NiO NP on
the other hand did not increase IL-1 but caused a
substantial increase in TNF- levels. This might mean that
each metal oxide triggers inflammation by different
pathways, possibly resulting in different modes of
inflammation depending on their physicochemical
properties . Interestingly ZnO and CuO NP failed to
induce any cytokines response but were found to be
highly cytotoxic. The cytotoxicity of ZnO and CuO NP
is likely due to their water soluble ions . In addition
to this, accelerated dissolution of ZnO  and CuO NP
 inside of the acidic lysosomal fluid might be more
effective in macrophages compared to monocytic cells.
Several NPs such as SiO2 and TiO2 NP were recently
known to cause release of IL-1 by activating NLR pyrin
domain containing 3 (Nlrp3) inflammasome [21,22].
However, ZnO NP did not activate the Nlrp3
inflammasome and failed to stimulate release of IL-1 and the
mechanism of inflammasome activation was poorly
understood . The pH-dependent dissolution of ZnO
and CuO NP inside phagolysosomes causes the NPs to
lose the physico-chemical properties that might
influence the phagocytosis-related inflammasome activation
and pro-inflammatory cytokine expression. The
expression of IL-1 by NPs was markedly reduced by inhibition
of phagocytosis using cytochalasin D. Therefore,
phagocytosis is an important mechanism of producing IL-1
by activation of inflammasome. TNF- is another
important pro-inflammatory cytokine driving inflammation
in the lung and CeO2 and NiO NP, the two NPs highly
responsive to TNF-, was consistent with the
inflammogenicity in the lung .
Treatment of A549 cells with NP-free conditioned
medium from THP-1 cells (combined culture) showed
much greater response in IL-8 production compared to
the direct particle exposure. The increases of IL-8 in
A549 cells caused by CeO2, TiO2, and Cr2O3 NP in the
combined culture were mainly due to the TNF- and
IL1 released by THP-1 cells. The increased IL-8 levels
released by A549 cells on treatment with ZnO and CuO
Figure 6 Cytotoxicity and pro-inflammatory cytokine expression of differentiated THP-1 cells after exposure to NPs for 24 h. (A)
Cytotoxicity was measured by trypan blue exclusion for ZnO and CuO NP whilst others were measured by LDH. (B) Levels of IL-1; (C) levels of
TNF-. (D) Treatment of NP with cytochalasin D (0.2 M) showed marked decrease of IL-1 expression compared to NP without cytochalasin D
(B). THP-1 cells were differentiated by treatment with PMA (10 ng/mL) and NPs were treated at 30, 100, and 300 cm2/mL except for ZnO NP
(3, 10, and 30 cm2/mL) and CuO NP (0.3, 1, and 3 cm2/mL). Values are mean SD from minimum four independent experiments. Significance
versus vehicle control (VEH): *p < 0.05, **p < 0.01, ***p < 0.001.
NP may be due to IL-8 produced by THP-1 cells and
the release of water soluble metal ions by THP-1 cells.
Although the combined culture is a good method to
evaluate the action of pro-inflammatory cytokines
released from one cell type on another cell type [23-25],
the toxic ions released from soluble NPs also should be
carefully taken into account in this assay .
In our previous studies we reported that the CeO2,
NiO, and Co3O4 NP had surface charge as their
biologically effective dose (BED) whilst ZnO and CuO NP
had soluble toxic ions as their BED [26,27]. When NPs
were inhaled, NPs gain a protein corona and so the
surface charge might be blocked and the toxicities mitigated
. However, the protein corona may have little role
with NPs which dissolve in acidic conditions . This
might be the reason why the most of in vitro assay data
of ZnO and CuO NP are consistent with the lung
When NPs were dosed at 30 cm2/mL which is the only
overlapping dose between NPs, the cytotoxicity in
differentiated peripheral blood mononuclear cells was
the most consistent with the lung inflammation data. In
addition, haemolysis assay showed the best accuracy
(100%) when haemolysis at any dose was taken to
represent positivity in the assay. The haemolysis assay has
been proposed as a good model for prediction of in vivo
lung inflammation . However, haemolysis assay is
not applicable to NPs like ZnO which have high binding
affinity with haemoglobin and small-sized well-dispersed
NPs which need ultracentrifugation to get rid of NPs. In
addition, the haemolysis assay should use protein-free
saline as a vehicle because protein corona protects the
surface reactivity in cell culture condition [26,30,31].
Although both cell culture and in vivo condition can
produce a corona, the surface reactivity might be
unmasked more fully in vivo  but unmasking might
be variable in in vitro depending on the cell types and
culture conditions. This might be due to the differences
in the enzymatic activity inside of phagolysosomes
between each model  and synergistic effect by
crosstalking between each cell type. Among 8 different
in vitro systems, alveolar macrophages, differentiated
PBMC, and red blood cells were better for prediction of
in vivo lung inflammogenicity of NPs than other
Figure 7 IL-8 expression by NP-conditioned media. A549 cells were exposed to conditioned media from NP-exposed differentiated THP-1
cells for 4 h. THP-1 cells were differentiated by treatment for PMA (10 ng/mL) and NPs were treated at surface area doses of 30, 100, and
300 cm2/mL except for ZnO NP (3, 10, and 30 cm2/mL) and CuO NP (0.3, 1, and 3 cm2/mL). (A) The levels of IL-8 in the conditioned media from
THP-1 cells before addition of A549 cells. (B) The levels of IL-8 after addition of NP-free conditioned media to A549 cells. Values are mean SD
from minimum four independent experiments. Significance versus vehicle control (VEH): ***p < 0.001.
systems. As a minimum set of in vitro endpoints,
cytotoxicity assay can be suggested for better parameters for
correlation with in vivo lung inflammogenicity than
proinflammatory cytokine assay.
Based on the toxicity mechanisms of NPs, two different
approaches can be applied for prediction of in vivo lung
inflammogenicity. Most in vitro assays may detect toxic
or inflammogenic potential of NPs if the NPs act via
soluble ions (i.e., ZnO and CuO NP). However, in vitro
assays appear not to be good at detecting metal oxide NPs
that act via surface charge as their mechanism of
toxicity, with the key exception of haemolysis.
Materials and methods
Nanoparticle and characterization
A total of 9 different predominantly metal oxide NPs
[CeO2, TiO2 (rutile form), carbon black (CB), SiO2, NiO,
Co3O4, Cr2O3, CuO, and ZnO] were purchased from
commercial sources (Table 1). The primary size of NP
was measured by transmission electron microscopy
(TEM) (JEM-1200EX II, JEOL, Tokyo, Japan). The
surface area (BET, Brunauer-Emmett-Teller) of NPs was
measured using a Micromeritics TriStar 3000
(Bedfordshire, UK). Agglomeration status of NP in PBS with/
without dispersion medium (fetal bovine serum, FBS)
was measured by dynamic light scattering (Brookhaven
90 plus; Holtsville, NY, USA). Endotoxin levels in NP
suspensions were evaluated by an endpoint chromogenic
Figure 8 Haemolysis assay of NP. NPs were treated at surface area
doses of 30, 100, and 300 cm2/mL. Values are mean SD from
minimum four independent experiments. Significance versus vehicle
control (VEH): *p < 0.05, ***p < 0.001.
Limulus Amebocyte Lysate (LAL) assay (Cambrex, MD,
USA). The detection limit of LAL kit was 0.1 1.0 EU/ml.
Rationale for dose selection
All experiments were performed using surface area as a
dose metric because surface area is considered to be the
most appropriate metric for assessing NP toxicity in vivo
and in vitro . For in vitro assays, we performed
preliminary dose-ranging studies for all NPs from 0.1 to
300 cm2/mL. Based on their cytotoxicity, all NPs were
used at doses of 30, 100, and 300 cm2/mL, except for
CuO and ZnO NPs, which were treated at 3, 10, 30 cm /
mL because of their greater toxicity. In our previous
studies, NPs were intratracheally instilled into the lungs
of female Wistar rats at 50, 150, 250 cm2/mL [16,17,29].
As a result, CeO2 and NiO NP were inflammogenic
from 150 cm2/mL and ZnO and CuO NP were
inflammogenic from 50 cm2/mL. Treatment of TiO2, CB, SiO2
Table 2 The number of total granulocytes in the BAL at
24 h after instillation of NPs into rats
Total cells (105)
did not show any significant inflammation. Therefore,
we selected 150 cm2/mL for acute lung inflammation
because NiO NP is well known as toxic particles in
human cell lines  and in rats . To evaluate the
correlation of in vitro data against in vivo data, the dose for
in vivo was fixed at 150 cm2/mL and compared with
in vitro data at various doses.
Dispersion of NPs
Because NPs showed some agglomerates which are not
readily dispersed without any stresses (i.e., sonication) in
PBS, NPs were dispersed with 5% FBS which provide a
protein corona on the surface of particles as previously
described method .
When NPs are inhaled, they immediately interact with
epithelial cells and then alveolar macrophages. Red
blood cells have been used in particle toxicology studies
to determine direct membranolytic effects of particle
surfaces and we emphasise that interactions with red
blood cells are not part of the pathophysiological
mechanism by which NP act. Therefore we selected
alveolar epithelial cells A549 cells, bronchial epithelial
cells 16-HBE cells, monocytic/macrophage cells
THP-1 cells, human peripheral blood mononuclear cells
(PBMC), and rat alveolar macrophages and human red
blood cells. A549 cells were obtained from the European
Collection of Animal Cell Cultures and THP-1 cells
were purchased from American Type Culture Collection
(ATCC). 16-HBE cells originated from Dr. Gruenert of
the University of California, San Francisco, USA .
Rat alveolar macrophages underwent primary culture
following whole lung lavage in 7-week old healthy female
Wistar rats. Human peripheral whole blood was
collected from healthy consenting volunteers (University of
Edinburgh) and PBMC were then isolated from buffy
coats according to the previously described method .
A549 cells and 16-HBE cells were cultured in DMEM
medium containing 5% FBS and THP-1 cells, PBMC,
and rat alveolar macrophages were cultured in
RPMI1640 medium containing 10% FBS. Cells were cultured
at 37C with 5% CO2, 2 mM L-glutamine (Life
Technologies, Paisley, UK), 100 IU/mL penicillin, and 100 U/
mL streptomycin (Life Technologies). The number of
cells for seeding to 6-well plate was 2 105 cells/mL for
A549 cells and 16-HBE cells and 1 106 cells/mL for
monocytic/macrophage cells. The monocytic THP-1
cells were differentiated to macrophages with 10 ng/mL
of phorbol myristate acetate (PMA; Sigma-Aldrich,
Gillingham, Dorset, UK) for 2 days and Peripheral blood
monocyte-derived macrophages (PBMDM) were
differentiated from PBMC by culturing for 5 days. After
The acute lung inflammogenicity data were used from our previously
published data [16,17].
Significance versus vehicle control: *p < 0.05, **p < 0.01, ***p < 0.001.
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and NPs were treated.
Inhibition of phagocytosis by cytochalasin D
To evaluate the role of phagocytosis on the
proinflammatory cytokine release, we treated cytochalasin D
which is a well characterized inhibitor of phagocytosis.
After differentiation of THP-1 cells by PMA, 0.2 M of
cytochalasin D (Enzo Life Sciences, Exeter, UK) were
added to NP suspensions and treated for 24 h. The cell
supernatants were then collected for lactate
dehydrogenase (LDH) and cytokine measurement.
NP treatment and measurement of cytotoxicity
Three different doses of NPs were dispersed in FBS first
with the volume of 5% in working solution and cell
culture medium was added as described above. NPs were
then added to cell cultures and incubated for 24 h.
Cytotoxicity was measured using a lactate dehydrogenase
(LDH) assay kit (Roche Diagnostics Ltd., Burgess Hill,
UK) in the NP-free cell culture supernatant collected by
three rounds of centrifugation at 15000 g for 20 min.
Vehicle control and 0.1% Triton-X treatment were used
as a negative and positive control, respectively. The
cytotoxicity of NP was expressed by percentage compared to
complete cytotoxicity (0.1% Triton-X). To evaluate the
adsorption of LDH on the NPs, bare NPs (NPs alone) or
NPs pre-incubated in 5% FBS to give them a protein
corona were incubated with A549 cell extract rich in LDH
and LDH assay was then performed. Briefly, NPs with or
without 5% FBS at the highest doses used in in vitro
study (30 or 300 cm2/mL) were incubated with 0.1%
Triton-X treated A549 cells for 30 min at room
temperature. LDH assay was performed using NP
containing-cell lysate solution according to the
instruction manual (Roche Diagnostics Ltd.). After colour
development, solutions were centrifuged at 15000 g for
20 min to get rid of NPs and measured absorbance at
490 nm. As a result, bare NPs showed variable
adsorption with some NP adsorbing LDH, whilst NPs with a
protein corona showed minimal adsorption (data not
shown). Therefore, the adsorption of LDH on the NP
with a corona is minimal in this setting. Some metal ions
released from NPs are known to inhibit the color
development of LDH assay . In our panel of NPs, trypan
blue exclusion test was applied to ZnO and CuO NP
owing to this interference.
Combined cell culture
Because one of the critical components of the
inflammatory response induced by NPs in the lung is the release
of inflammatory mediators in contact with alveolar
macrophages, we treated conditioned medium of THP-1
cells to A549 cells by modification of the previously
described method . Briefly, differentiated THP-1
cells by PMA as mentioned above were treated with NPs
for 24 h. NP-free supernatant was then prepared by
three rounds of centrifugation at 15000 g for 20 min
and treated to A549 cells for 4 h. After 4 h of
incubation, medium was washed with PBS for three times and
replaced with fresh DMEM medium and incubated for
an additional 20 h.
Measurement of cytokines (IL-1, IL-8, and TNF-)
IL-1, IL-8, and TNF- are pro-inflammatory cytokines
correlated with inflammogenic potential of particles .
We measured IL-8 protein levels in the supernatant of
A549 cells, 16-HBE cells, and combined culture and IL-1
and TNF- in supernatants of monocytes/macrophages
cells (THP-1 cells, PBMDM, and alveolar macrophages)
according to the instruction manual (R&D Systems).
Haemolysis assay was performed according to the
previously described method . Briefly, human red blood
cells were incubated with NPs at 30, 100, and 300 cm2/mL
dispersed in saline without any proteins. Saline and 0.1%
Triton X-100 was used as negative and positive control,
respectively. After 30 min incubation, the amount of
released haemoglobin was determined by absorbance at
= 550 nm. ZnO NP was excluded in this assay owing to
high binding affinity with haemoglobin .
Correlation of in vitro assays with in vivo acute lung
To evaluate the correlation of in vitro assays with in vivo
toxicity, we used some previously published animal
experimental data from our own group [16,17] in order to
avoid unnecessary sacrifice of animals. Although in vivo
data were published earlier, both studies were performed
at the same time with the same batch of NPs. The
number of total granulocytes was selected as a marker for
acute lung inflammogenicity. For in vitro assays,
statistically significant increases in any doses were regarded as
positive and other cases were regarded as negative
(i.e. no effect). For in vivo experiments, statistically
significant increases in the number of total granulocytes
compared to vehicle control were regarded as positive.
Because one of the most important parameter
comparing each result was the treatment dose, we compared
in vitro assays at 30 cm2/mL (the only overlapping dose
used for all the particles except in Figures 6 and 7 where
3 cm2/mL was the highest dose for CuO NP) or any
dose showing statistical significance.
We conducted minimum 4 individual experiments and
all data were expressed as mean standard deviations.
Data were analyzed using the GraphPad Prism 5
(GraphPad Software, Inc., La Jolla, CA, USA). One-way analysis
of variance with post hoc Tukeys pairwise comparisons
test was used to compare the differences between
groups. A p value of < 0.05 was considered to be
The authors declare they have no competing financial interests.
WSC, RD, MB, ILM, WMacN, and KD provided key intellectual input
culminating in the conception and design of these studies and aided in the
writing of this manuscript. The studies were carried out by WSC who also
contributed to the writing of the manuscript. JKL and JJ provided expertise
on interpretation of data and both contributed to the writing of the
manuscript. All authors read and approved the final manuscript.
1. Nel AE , Madler L , Velegol D , Xia T , Hoek EM , Somasundaran P , Klaessig F , Castranova V , Thompson M : Understanding biophysicochemical interactions at the nano-bio interface . Nat Mater 2009 , 8 : 543 - 557 .
2. Borm PJ , Robbins D , Haubold S , Kuhlbusch T , Fissan H , Donaldson K , Schins R , Stone V , Kreyling W , Lademann J , et al: The potential risks of nanomaterials: a review carried out for ECETOC . Part Fibre Toxicol 2006 , 3 : 11 .
3. Organisation for Economic Co-operation and Development: Nanosafety at the OECD: the first five years 2006-2010 . Paris: OECD; 2011 .
4. Organisation for Economic Co-operation and Development: Six years of OECD work on the safety of manufactured nanomaterials: achievements and future opportunities . Paris: OECD ; 2013 .
5. Donaldson K , Borm PJ , Castranova V , Gulumian M : The limits of testing particle-mediated oxidative stress in vitro in predicting diverse pathologies; relevance for testing of nanoparticles . Part Fibre Toxicol 2009 , 6 : 13 .
6. Alfaro-Moreno E , Nawrot TS , Vanaudenaerde BM , Hoylaerts MF , Vanoirbeek JA , Nemery B , Hoet PH : Co-cultures of multiple cell types mimic pulmonary cell communication in response to urban PM10 . Eur Respir J 2008 , 32 : 1184 - 1194 .
7. Park EK , Jung HS , Yang HI , Yoo MC , Kim C , Kim KS : Optimized THP-1 differentiation is required for the detection of responses to weak stimuli . Inflamm Res 2007 , 56 : 45 - 50 .
8. Sohaebuddin SK , Thevenot PT , Baker D , Eaton JW , Tang L : Nanomaterial cytotoxicity is composition, size, and cell type dependent . Part Fibre Toxicol 2010 , 7 : 22 .
9. Kroll A , Dierker C , Rommel C , Hahn D , Wohlleben W , Schulze-Isfort C , Gobbert C , Voetz M , Hardinghaus F , Schnekenburger J : Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays . Part Fibre Toxicol 2011 , 8 : 9 .
10. Sayes CM , Reed KL , Warheit DB : Assessing toxicity of fine and nanoparticles: comparing in vitro measurements to in vivo pulmonary toxicity profiles . Toxicol Sci 2007 , 97 : 163 - 180 .
11. Editorial : The dose makes the poison . Nat Nanotechnol 2011 , 6 : 329 .
12. Duffin R , Tran L , Brown D , Stone V , Donaldson K : Proinflammogenic effects of low-toxicity and metal nanoparticles in vivo and in vitro: highlighting the role of particle surface area and surface reactivity . Inhal Toxicol 2007 , 19 : 849 - 856 .
13. Fubini B , Ghiazza M , Fenoglio I : Physico-chemical features of engineered nanoparticles relevant to their toxicity . Nanotoxicology 2010 , 4 : 347 - 363 .
14. Oberdorster G , Maynard A , Donaldson K , Castranova V , Fitzpatrick J , Ausman K , Carter J , Karn B , Kreyling W , Lai D , et al: Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy . Part Fibre Toxicol 2005 , 2 : 8 .
15. Johnston HJ , Hutchison G , Christensen FM , Peters S , Hankin S , Stone V : A review of the in vivo and in vitro toxicity of silver and gold particulates: particle attributes and biological mechanisms responsible for the observed toxicity . Crit Rev Toxicol 2010 , 40 : 328 - 346 .
16. Cho WS , Duffin R , Poland CA , Howie SE , MacNee W , Bradley M , Megson IL , Donaldson K : Metal oxide nanoparticles induce unique inflammatory footprints in the lung: important implications for nanoparticle testing . Environ Health Perspect 2010 , 118 : 1699 - 1706 .
17. Cho WS , Duffin R , Bradley M , Megson IL , MacNee W , Howie SEM , Donaldson K : NiO and Co3O4 nanoparticles induce lung DTH-like responses and alveolar lipoproteinosis . Eur Respir J 2012 , 39 : 546 - 557 .
18. Cho WS , Duffin R , Poland CA , Duschl A , Oostingh GJ , Macnee W , Bradley M , Megson IL , Donaldson K : 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 2012 , 6 : 22 - 35 .
19. Bian SW , Mudunkotuwa IA , Rupasinghe T , Grassian VH : Aggregation and dissolution of 4 nm ZnO nanoparticles in aqueous environments: influence of pH, ionic strength, size, and adsorption of humic acid . Langmuir 2011 , 27 : 6059 - 6068 .
20. Studer AM , Limbach LK , Van Duc L , Krumeich F , Athanassiou EK , Gerber LC , Moch H , Stark WJ : Nanoparticle cytotoxicity depends on intracellular solubility: comparison of stabilized copper metal and degradable copper oxide nanoparticles . Toxicol Lett 2010 , 197 : 169 - 174 .
21. Yazdi AS , Guarda G , Riteau N , Drexler SK , Tardivel A , Couillin I , Tschopp J : Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1alpha and IL-1beta . Proc Natl Acad Sci USA 2010 , 107 : 19449 - 19454 .
22. Hornung V , Bauernfeind F , Halle A , Samstad EO , Kono H , Rock KL , Fitzgerald KA , Latz E : Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization . Nat Immunol 2008 , 9 : 847 - 856 .
23. Totlandsdal AI , Refsnes M , Skomedal T , Osnes JB , Schwarze PE , Lag M : Particle-induced cytokine responses in cardiac cell cultures-the effect of particles versus soluble mediators released by particle-exposed lung cells . Toxicol Sci 2008 , 106 : 233 - 241 .
24. Shaw CA , Robertson S , Miller MR , Duffin R , Tabor CM , Donaldson K , Newby DE , Hadoke PW : Diesel exhaust particulate-exposed macrophages cause marked endothelial cell activation . Am J Respir Cell Mol Biol 2010 , 44 : 840 - 851 .
25. Jimenez LA , Drost EM , Gilmour PS , Rahman I , Antonicelli F , Ritchie H , MacNee W , Donaldson K : PM(10)-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-alpha . Am J Physiol Lung Cell Mol Physiol 2002 , 282 : L237 - L248 .
26. Cho WS , Duffin R , Thielbeer F , Bradley M , Megson IL , Macnee W , Poland CA , Tran CL , Donaldson K : Zeta potential and solubility to toxic ions as mechanisms of lung inflammation caused by metal/metal oxide nanoparticles . Toxicol Sci 2012 , 126 : 469 - 477 .
27. Donaldson K , Schinwald A , Murphy F , Cho WS , Duffin R , Tran L , Poland C : The biologically effective dose in inhalation nanotoxicology . Acc Chem Res 2013 , 46 ( 3 ): 723 - 732 .
28. Thielbeer F , Donaldson K , Bradley M , Johansson EM , Cho WS , Duffin R , Megson IL , Macnee W : Surface functionalization affects the zeta potential, coronal stability and membranolytic activity of polymeric nanoparticles . Nanotoxicology . in press.
29. Lu S , Duffin R , Poland C , Daly P , Murphy F , Drost E , Macnee W , Stone V , Donaldson K : Efficacy of simple short-term in vitro assays for predicting the potential of metal oxide nanoparticles to cause pulmonary inflammation . Environ Health Perspect 2009 , 117 : 241 - 247 .
30. Casals E , Puntes VF : Inorganic nanoparticle biomolecular corona: formation, evolution and biological impact . Nanomedicine (Lond) 2012 , 7 ( 12 ): 1917 - 1930 .
31. Maiorano G , Sabella S , Sorce B , Brunetti V , Malvindi MA , Cingolani R , Pompa PP : Effects of cell culture media on the dynamic formation of proteinnanoparticle complexes and influence on the cellular response . ACS Nano 2010 , 4 ( 12 ): 7481 - 7491 .
32. Macickova T , Navarova J , Urbancikova M , Horakova K : Comparison of isoproterenol-induced changes in lysosomal enzyme activity in vivo and in vitro. Gen Physiol Biophys 1999 , 18 Spec No: 86 - 91 .
33. Horie M , Nishio K , Fujita K , Kato H , Nakamura A , Kinugasa S , Endoh S , Miyauchi A , Yamamoto K , Murayama H , et al: Ultrafine NiO particles induce cytotoxicity in vitro by cellular uptake and subsequent Ni(II) release . Chem Res Toxicol 2009 , 22 : 1415 - 1426 .
34. Ogami A , Morimoto Y , Myojo T , Oyabu T , Murakami M , Todoroki M , Nishi K , Kadoya C , Yamamoto M , Tanaka I : Pathological features of different sizes of nickel oxide following intratracheal instillation in rats . Inhal Toxicol 2009 , 21 : 812 - 818 .
35. Cozens AL , Yezzi MJ , Kunzelmann K , Ohrui T , Chin L , Eng K , Finkbeiner WE , Widdicombe JH , Gruenert DC : CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells . Am J Respir Cell Mol Biol 1994 , 10 ( 1 ): 38 - 47 .
36. Dransfield I , Buckle AM , Savill JS , McDowall A , Haslett C , Hogg N : Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression . J Immunol 1994 , 153 : 1254 - 1263 .
37. Kroll A , Pillukat MH , Hahn D , Schnekenburger J : Current in vitro methods in nanoparticle risk assessment: limitations and challenges . Eur J Pharm Biopharm 2009 , 72 : 370 - 377 .
38. Monteiller C , Tran L , MacNee W , Faux S , Jones A , Miller B , Donaldson K : The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area . Occup Environ Med 2007 , 64 : 609 - 615 .