Progressive Severe Lung Injury by Zinc Oxide

Cho et al.
Particle and Fibre Toxicology 2011, 8:27

http://www.particleandfibretoxicology.com/content/8/1/27
RESEARCH Open Access
Progressive severe lung injury by zinc oxide

nanoparticles; the role of Zn2+ dissolution inside
lysosomes
Wan-Seob Cho1, Rodger Duffin1, Sarah EM Howie2, Chris J Scotton3, William AH Wallace4, William MacNee1,
Mark Bradley5, Ian L Megson6 and Ken Donaldson1*
Abstract
Background: Large production volumes of zinc oxide nanoparticles (ZnONP) might be anticipated to pose risks, of
accidental inhalation in occupational and even in consumer settings. Herein, we further investigated the
pathological changes induced by ZnONP and their possible mechanism of action.
Methods: Two doses of ZnONP (50 and 150 cm2/rat) were intratracheally instilled into the lungs of rats with
assessments made at 24 h, 1 wk, and 4 wks after instillation to evaluate dose- and time-course responses.
Assessments included bronchoalveolar lavage (BAL) fluid analysis, histological analysis, transmission electron
microscopy, and IgE and IgA measurement in the serum and BAL fluid. To evaluate the mechanism, alternative
ZnONP, ZnONP-free bronchoalveolar lavage exudate, and dissolved Zn2+ (92.5 μg/rat) were also instilled to rats.
Acridine orange staining was utilized in macrophages in culture to evaluate the lysosomal membrane
destabilization by NP.
Results: ZnONP induced eosinophilia, proliferation of airway epithelial cells, goblet cell hyperplasia, and pulmonary
fibrosis. Bronchocentric interstitial pulmonary fibrosis at the chronic phase was associated with increased
myofibroblast accumulation and transforming growth factor-b positivity. Serum IgE levels were up-regulated by
ZnONP along with the eosinophilia whilst serum IgA levels were down-regulated by ZnONP. ZnONP are rapidly
dissolved under acidic conditions (pH 4.5) whilst they remained intact around neutrality (pH 7.4). The instillation of
dissolved Zn2+ into rat lungs showed similar pathologies (eg., eosinophilia, bronchocentric interstitial fibrosis) as
were elicited by ZnONP. Lysosomal stability was decreased and cell death resulted following treatment of
macrophages with ZnONP in vitro.
Conclusions: We hypothesise that rapid, pH-dependent dissolution of ZnONP inside of phagosomes is the main
cause of ZnONP-induced diverse progressive severe lung injuries.
Background finishing materials in products and buildings because

Zinc oxide nanoparticles (ZnONP) are utilised in many they provide long-term protection from ultraviolet light
commercial products including cosmetics, paints, textiles, [2]. ZnONP have also been used as a dietary supplement
food additives, and personal hygiene products. Because in human and livestock because Zinc can stimulate
ZnONP are translucent and highly effective in protection immune systems and act in an anti-inflammatory way
against ultraviolet A and B radiation, they are important [3,4]. ZnONP has external uses as antibacterial agents in
ingredients of sunscreens and moisturizers [1]. ZnONP is ointments, lotions, mouthwashes, and surface coatings to
widely used as an ingredient of paints and coating and prevent microorganism growth [5].
There are few toxicity reports on ZnONP despite their
widespread use and potential for use in various applica-
* Correspondence: [email protected] tions. Toxicity studies of ZnONP have mainly focused on
1
ELEGI Group, Centre for Inflammation Research, University of Edinburgh,
Edinburgh, UK dermal toxicity, of relevance due to the inclusion of
Full list of author information is available at the end of the article
© 2011 Cho et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Cho et al. Particle and Fibre Toxicology 2011, 8:27 Page 2 of 16
ZnONP within materials that are directly applied to skin. resuspended in 5 ml of DW. NP suspensions were then
Penetration of ZnONP through normal skin was limited weighed and calculated by subtraction of the weight of the
to the stratum corneum in porcine [6] and human mod- container and the same volume of distilled water. There is
els [7]. Exposure of human skin epithelial cells to potential for some error in this method due a small frac-
ZnONP produced severe cytotoxicity accompanied by tion of small NP that could remain in suspension after
oxidative stress and genotoxicity [8]. Few studies have centrifugation and are lost on washing.
been reported concerning the in vivo toxicity of ZnONP
although intratracheal instillation of ZnONP (50 - 70 Intratracheal instillation of ZnONP
nm) in Sprague-Dawley rats induced cytotoxicity and Female Wistar rats (200 - 250 g) were humanely main-
neutrophilic inflammation at 24 h after instillation [9]. tained and handled in accordance with the UK Home
Previously, we assessed the pulmonary inflammogenicity Office Animals Scientific Procedures Act. Intratracheal
of a large panel of NP, including ZnONP and found that instillation was performed as previously described method
metal oxide NP elicited diverse patterns of inflammation [10]. ZnONP were instilled at a surface area dose of 50 or
with different cellular bases, at both the acute and chronic 150 cm2/rat, and 5% rat serum in saline was used as the
phase [10]. In the present study, we extended these experi- vehicle control (n = 5 - 7 per group). Large agglomerates
ments to specifically evaluate the mechanisms of eosino- of ZnONP (diameter- 4,380 nm) in saline without rat
philic inflammation and pulmonary fibrosis induced by serum also instilled at 150 cm 2 per rat to evaluate the
ZnONP instillation. effects of agglomeration on eosinophilia. We used surface
area as a dose metric rather than mass because surface
Methods area has been known as a better descriptor of potential of
We briefly described materials and methods in the main NP to cause toxicity in vitro and in vivo [14]. To evaluate
text but the detailed methods were described in the the time-course of the consequent inflammation, rats were
Additional file 1. sacrificed at 24 h, 1 wk, and 4 wks after instillation. Pre-
paration of bronchoalveolar lavage (BAL) fluid and analy-
Characterization and dispersion of NP sis for LDH and total protein was performed as previously
ZnONP (10.7 ± 0.7 nm) were purchased from NanoScale described method [10].
Corporation (Manhattan, KS, USA) (Table 1). Surface area
of ZnONP was determined with a Micromeritics TriStar ELISA for pro-inflammatory mediators
3000 (Bedfordshire, UK) by Escubed Ltd. (Leeds, UK). For Measurements of cytokines (TNF-a, IL-1b, IL-13, and
dispersion of ZnONP, 5% of heat-inactivated rat serum TGF-b) and chemokines (MIP-2 and eotaxin) were per-
(collected from the healthy female Wistar rat) was added formed in non-diluted BAL fluid following the manufac-
to saline (Baxter, Deerfield, IL, USA) for final concentra- turer’s instructions [IL-13 ELISA was obtained from
tions. The endotoxin levels of ZnONP at 300 cm2/ml were Invitrogen (Camarillo, CA, USA) and other assays were
determined using a Limulus Amebocyte Lysate assay kit from R&D Systems (Minneapolis, MN, USA)]. The
(Cambrex, Walkersville, MD, USA). The hydrodynamic detection limits of ELISA kit was as follow: TNF-a- 5
size and zeta potential of ZnONP in PBS with 5% pg/ml; IL-1b- 5 pg/ml; TGF-b- 1.7 - 15.4 pg/ml; MIP-2-
rat serum were assessed with a Brookhaven 90 plus 0.5 - 2.7 pg/ml; eotaxin- 3 pg/ml; IL-13- 1.5 pg/ml.
(Holtsville, NY, USA) and Zetasizer-Nano ZS instrument
(Malvern, Malvern Hills, UK), respectively. IgE and IgA ELISA in the serum and BAL fluid
To evaluate the serum immunoglobulin E (IgE) levels,
Durability of ZnONP ZnONP were instilled into rats (n = 4) at 150 cm2 per rat
To evaluate the biopersistence of ZnONP in vivo, ZnONP and blood was taken via the tail vein at day 1 and week 1,
were incubated with artificial lysosomal fluid (ALF) [11] 2, 3, and 4 after instillation. Serum was then collected
and artificial pulmonary interstitial fluid (Gamble’s solu- and diluted 1 in 10 with PBS. Total serum IgE and IgA
tion) [12]. As a control particle, the rutile form of TiO2NP levels were determined using a rat IgE ELISA set and rat
(30.5 ± 1.8 nm) was purchased from Nanostructure and IgA ELISA set, respectively (all from BD Biosciences,
Amorphous Materials Inc. (Houston, TX, USA) (Table 1). Oxford, UK).
ALF (pH 4.5) and Gamble’s solution (pH 7.4) were
prepared as previously described [11,13]. ZnONP and Histological analysis (H&E, PSR, and PAS staining)
TiO2NP were incubated with ALF or Gamble’s solution at At each time point, histological analysis of lung tissues
5 mg/ml for 24 h at 37°C with gentle shaking. After 24 h, and picrosirius red (PSR) staining was performed as pre-
50 mg of suspensions were centrifuged at 13 000 × g for viously describe method [10]. For detection of goblet
30 min and the supernatant discarded. After the final cells which contain mucin, periodic acid-Schiff (PAS)
wash with distilled water (DW), pellets were air dried and (Sigma-Aldrich) staining was performed according to
Table 1 Characterization of nanoparticles

Designated name ZnONP ZnONPalt NiONP TiO2NP
Supplier NanoScale Nanostructure and Nanostructure and Nanostructure and
Corp. Amorphous Materials Inc. Amorphous Materials Inc. Amorphous Materials Inc.
Diameter (nm) examined by TEM 10.7 ± 0.7 137 ± 9.2 5.3 ± 0.4 30.5 ± 1.8
Surface area (m2/g)a 48.2 50 91.8 27.5
Mass (μg) per 150 cm2 (high dose- 310 - 163.5 545
rat)
Mass (μg) per 50 cm2 (low dose- 103 - 54.5 182
rat)
Mass (μg) per 15 cm2 (high dose- 31 - 9.18 -
mouse)
Zn2+ (μg) equivalent to 50 cm2 82.8 - - -
(rat study)
Endotoxin (EU/ml)b ND ND ND ND
Hydrodynamic DW 2855 ± 773 4833 ± 625 1210 ± 471 787 ± 324
size (nm) in
PBS 3925 ± 715 5683 ± 510 2236 ± 407 1166 ± 344
PBS (5% rat 423 ± 24 282 ± 124 93 ± 4 119.1 ± 39.6
serum)
PBS (5% 229 ± 187 - 83 ± 44 -
mouse serum)
Zeta potential PBS (5% rat -27.1 ± 1.4 -25.9 ± 0.6 -26.0 ± 5.0 -28.5 ± 5.2
(mV) in serum)
PBS (5% -18.9 ± 1.5 - -21.4 ± 0.8 -
mouse serum)
Values are mean ± S.D. from four independent experiments.
ND = not detectable.
a
Determined by Escubed Ltd. (Leeds, UK).
b
Lower detection limit 0.1 EU/ml.
DW, distilled water.
PBS, phosphate buffered saline.
standard methods. The quantitative image analysis of instillation were fixed with 1.5% glutaraldehyde in 0.1 M
PAS-positive cells was performed using Image-Pro Plus cacodylate buffer, stained en bloc with uranyl acetate,
(Media Cybernetics, MD, USA). PAS-positive signals in and embedded in epoxy resin. Ultra-thin (60 nm) sec-
the airways were separately evaluated according to air- tions were cut, stained with uranyl acetate and lead
way diameter; airways smaller than 1 mm were consid- citrate, and examined with a TEM (JEM-1200EX II,
ered to be small airways or bronchioles and larger than JEOL, Tokyo, Japan).
1 mm were considered to be a large airway or bronchi
[15]. The total area of PAS-positive cells was divided by Instillation of alternative ZnONP
the total area of epithelial cells including basement To evaluate whether the eosinophilic inflammation was
membrane. The data were expressed as percentage of induced by specific types of ZnONP, we instilled another
PAS-positive area versus total epithelial area. type of ZnONP (designated ZnONPalt) into rats. ZnONPalt
were purchased from Nanostructural and Amorphous
Immunohistochemistry for eotaxin, TGF-b, and a-SMA Materials, Inc. (Houston, TX, USA) (Table 1). ZnONPalt
Immunohistochemical staining for eotaxin, transforming were instilled into female Wistar rats at 310 μg per rat,
growth factor-beta (TGF-b), and alpha-smooth muscle which is the same mass of 150 cm2 per rat as the ZnONP
actin (a-SMA) was performed on lung sections. The from NanoScale Corporation. Four rats were used for each
detailed method for immunohistochemistry was treatment group. After 24 h, rats were euthanized and
described in Additional file 1. BAL collection and analysis was performed as described
above.
Transmission electron microscopy (TEM)
TEM was used to evaluate ultra-structural changes in Instillation of ZnONP-free BAL extract to rats
the lungs induced by instillation of ZnONP. Lungs of To evaluate whether inflammatory mediators produced
vehicle control and ZnONP treated rats 4 wks after by ZnONP instillation can produce similar pathologies
to that seen with ZnONP treatment, we extracted Sigma-Aldrich) for 48 h. After activation, cells were
ZnONP-free BAL fluid and instilled this in rats. The washed three times with PBS and stained with 5 μg/ml
detailed method was described in Additional file 1. acridine orange (Sigma-Aldrich) for 15 min. Cells were
then washed three times with PBS and were treated with
Instillation of dissolved Zn2+ to rats ZnONP (10 cm2 /ml; 20 μg/ml) for 24 h. As a control,
To evaluate the effects of dissolved Zn2+ in the acidic solu- TiO 2 NP (10 cm 2 /ml; 36 μg/ml) were used. Cells were
tion, 1 mg/ml of ZnONP in HCl-acidified saline were dis- examined and photographed in a Leica SP5 confocal
solved at a pH of 4.5. After 1 wk, ZnONP-free supernatant microscope (Leica Microsystems, Buckinghamshire, UK).
was collected by three rounds of centrifugation at 13000 × Cytotoxicity was also measured using a lactated dehydro-
g. The supernatant was filtered three times through a 0.22 genase assay kit according to the manufacturer’s manual
μm filter (Millipore, Cork, Ireland) to exclude possible (Roche Diagnostics Ltd.).
bacterial contamination. The concentration of Zn2+ of
supernatant was measured by inductively coupled plasma- Statistical analysis
atomic emission spectrometry (ICP-OES) (Perkin Elmer Data are expressed as mean ± S.D. and were analyzed with
Optima 5300 DV ICP-OES). The pH of the dissolved Zn2+ GraphPad InStat software (Version 3, GraphPad Software,
was 6.5, which was less acidic than 0.9% saline (pH 5.5). Inc., La Jolla, CA). To compare each treatment group,
Thereafter 92.5 and 277.5 μg of Zn2+ were instilled intra- one-way analysis of variance with Tukey’s post hoc pair-
tracheally into rats and cytological and histological evalua- wise comparisons was applied. Student t-test was applied
tion was performed at 24 h and 4 wks after instillation. for comparison between vehicle control and ZnONP treat-
The small discrepancy between the figure of 92.5 μg that ment group in C57BL/6 and BALB/c mice or ZnONPalt
was used in the experiment and the 82.8 μg that should treatment group. We considered p < 0.05 to be statistically
have been used, was the result of an error where we ori- significant.
ginally calculated the equivalent Zn2+ dose for ZnONP
based on the zinc metal weight rather than zinc oxide Results
weight. Characterization of ZnONP
ZnONP showed a “hard agglomerates” which is not readily
Aspiration of ZnONP into mice dispersed without any stresses (mechanical or sonication)
To evaluate whether the eosinophilic inflammation was a in both PBS and DW (Table 1). However, when ZnONP
species- and strain-specific phenomenon, ZnONP were were dispersed with serum protein, they showed a “soft
aspirated into C57BL/6 and BALB/c mice. ZnONP were agglomerates” which is readily dispersed without any stres-
dispersed in 5% heat-inactivated mouse serum (collected ses (mechanical or sonication) because of the protein cor-
from healthy C57BL/6 mice) to a dose of 150 cm2/ml and ona on the surface of NP. The zeta potential of ZnONP in
100 μl (15 cm2 ZnONP) were aspirated into the lungs of PBS was determined as -27.13 ± 1.36 mV. Endotoxin levels
mice which were sacrificed 24 h later. As a control, of ZnONP suspensions and rat serum were below the
NiONP (Table 1) known to cause acute neutrophilic lower detection limit (0.1 EU/ml) whilst 5% mouse serum
inflammation [10] were aspirated at the same surface area was calculated to contain 0.09 ± 0.02 EU/ml.
dose. Four mice per each group were used for cytological
evaluation. Eotaxin (R&D systems) and IL-13 (Invitrogen) Durability of ZnONP
levels were measured in the BAL samples as described Around 90% of ZnONP mass was dissolved within 24 h by
above. TEM was also applied to evaluate the ultra-struc- incubation with artificial lysosomal fluid at pH 4.5, whilst
tural changes in the lung with the same method described ZnONP in artificial interstitial fluid (Gamble’s solution,
above. pH 7.4) showed no dissolution (Figure 1). TiO2NP as a
control showed no dissolution or loss of mass either in
Acridine orange staining ALF or in Gamble’s solution (data not shown). The pre-
To evaluate the lysosomal membrane destabilization by sence of proteins/serum did not influence the durability of
NP, acridine orange staining was applied to THP-1 cells. ZnONP (data not shown).
Human monocytic cell line THP-1 was obtained from
American Type Culture Collection (ATCC) and cultured Differential cell counts in the BAL fluid
at 37°C with 5% CO2 in RPMI containing 10% FBS, 2 mM Instillation of ZnONP produced significant increases in
L-glutamine (Life Technologies, Paisley, UK), 100 IU/ml the total cell number at 1 wk and 4 wks, whilst at 24 h
penicillin, and 100 U/ml streptomycin (Life Technologies). there was no significant change compared to control
THP-1 cells (1 × 106 cells/ml) were seeded to a μ-Dish35 (Figure 2A). The number of polymorphonuclear leuko-
mm, high
(Thistle Scientific Ltd., Glasgow, UK) and differen- cytes (PMN) was significantly increased at 24 h and
tiated using 10 ng/ml of phorbol myristate acetate (PMA; returned to control levels thereafter (Figure 2B). The
Figure 1 Durability of ZnONP in artificial lysosomal fluid (ALF, pH 4.5) and artificial interstitial fluid (Gamble’s solution, pH 7.4). ZnONP
were incubated at 37°C for 24 h with gentle shaking. (A) Gross picture taken by digital camera. The photographs were taken before
centrifugation. (B) Percentage of mass compared to initial mass (50 mg). Values are mean ± S.D. n = 3.
number of eosinophils was significantly increased at all showed no significant changes compared to vehicle con-
time points and peaked at 1 wk after instillation (Figure trol (Figure 3C).
2C). The number of lymphocytes showed no significant
changes in any treatment group. Representative images Lung histopathology
of BAL cells show that some giant cells were found at 4 ZnONP induced diverse pathological lung lesions both
wks after instillation (Figure 2G). at the acute and chronic phase. The representative
pathological lesions could be classified as eosinophilic
Total protein and LDH in the BAL fluid inflammation, airway epithelial cell injury, regenerative
The levels of total protein and LDH in the BAL were proliferation, goblet cell hyperplasia, and pulmonary
significantly increased at 24 h after instillation and fibrosis with atelectasis (collapse of lung tissue affecting
were comparable to controls thereafter except for total part or all of a lung).
protein at 1 wk with low-dose ZnONP (Figures 2H Eosinophilic inflammation
and 2I). ZnONP induced severe eosinophilic inflammation in the
lung tissues which was consistent with BAL fluid analysis
Pro-inflammatory cytokine levels in BAL fluid (Figures 4C and 4D). Eosinophils were mainly present in
The level of IL-1b was significantly increased 24 h and 1 interstitial areas including alveolar septum, peribronchial,
wk after instillation of 150 cm 2 ZnONP (Figure 2J). peribronchiolar, and perivascular interstitium at all time
Eotaxin expression was significantly increased only at points. Eosinophils in the alveoli were greatly increased at
24 h with both doses whilst IL-13 was increased at 24 h 1 wk, consistent with cytological analysis (Figure 4D).
and 1 wk with the 150 cm2 dose only (Figures 2K and Lungs treated with ZnONP showed proliferation of type II
2L). The concentrations of IL-1b, eotaxin, and IL-13 in cells 24 h and 1 wk after instillation (Figures 5B and 5C).
the BAL were dose-related. The levels of TGF-b in the At 4 wks after instillation, foamy macrophages had infil-
BAL peaked at 24 h and were still significantly increased trated into the alveoli and the eosinophilic inflammation
at 1 and 4 wks after instillation. However, levels of TNF- was almost resolved (Figure 4E). Eotaxin, a specific che-
a and MIP-2 showed no significant changes compared moattractant for eosinophils, was strongly expressed at
to controls (data not shown). 24 h after ZnONP instillation (Additional file 2). The cells
that stained most intensely for eotaxin were bronchial/
IgE and IgA levels in the serum and BAL fluid bronchiolar epithelial cells although inflammatory cells
Serum IgE levels were transiently increased at 24 h and were also positive for eotaxin to some extent.
1 wk following instillation of ZnONP instillation and Airway epithelial cell injury and goblet cell hyperplasia
were similar to controls thereafter (Figure 3A). However, One of the most striking pathological effects of exposure
IgE levels in the BAL were not significantly increased by to ZnONP was goblet cell hyperplasia and proliferation
any treatment (data not shown). IgA levels in the serum of airway epithelial cells including bronchial and bronch-
were significantly down-regulated 2, 3, and 4 wks after iolar epithelium. The normal bronchiolar epithelium is
ZnONP instillation (Figure 3B). IgA levels in the BAL mainly composed of ciliated epithelial cells, goblet cells,
Figure 2 Bronchoalveolar lavage (BAL) analysis 24 h, 1 wk, and 4 wks after instillation of ZnONP to rats. (A - G), cytological analysis of
BAL after instillation of ZnONP at 50 and 150 cm2/rat. (A), number of total cells; (B), number of PMN; (C), number of eosinophils. Representative
BAL cell images of vehicle control (D) and ZnONP instillation (150 cm2) at 24 h (E), 1 wk (F), and 4 wks (G). Black arrows indicate alveolar
macrophages; blue arrows indicate PMN; red arrows indicate eosinophils; green arrows indicate giant cells. (H and I), levels of total protein (H)
and LDH (I) in the BAL from rats treated with ZnONP. Levels of LDH in BAL expressed as fold-change compared to vehicle control. (J - M),
expression of inflammatory mediators in the BAL from rats instilled with ZnONP. (J), IL-1b; (K), eotaxin; (L), IL-13; (M), TGF-b. Values are mean ± S.
D. n = 6 for 24 h and 4 wks groups and n = 4 for 1 wk groups. Significance versus vehicle control (VEH): * p < 0.05, ** p < 0.01, # p < 0.001.
Clara cells, and basal cells. In general, goblet cells are proliferation peaked at 24 h, was still apparent at 1 wk
seen occasionally in PAS-stained section in the larger air- and had returned to control levels at 4 wks after instilla-
ways but are sparse in the bronchioles and absent from tion. Interestingly, the proliferation of airway epithelial
the terminal bronchioles where Clara cells predominate. cells was accompanied by striking hyperplasia of goblet
Following ZnONP instillation, ciliated epithelial cells and cells which produce mucus (Figure 6). Goblet cells in the
basal cells became more basophilic and proliferation airway epithelium had undergone florid hyperplasia in
increased compared to vehicle control (Figure 4). The both bronchi and bronchioles at 1 and 4 wks with the
proliferation of airway epithelial cells was also confirmed entire radius of the bronchiole being composed of goblet
by Ki-67 immunohistochemistry (Figure 5) showing that cells in some sections. At 1 wk, goblet cells could be
Figure 4 Representative gross lesion and histology of lungs

after instillation of ZnONP at 150 cm2/rat. (A), lungs were
contracted and collapsed by ZnONP treatment and this caused
puckering of the lung surface. (B - E), each figure is composed of a
low power view (left, 25×), high power view of bronchiolar
epithelium (middle, 400×), and alveoli (right, 400×). (B) vehicle
control at 24 h; (C) ZnONP at 24 h; (D) ZnONP at 1 wk; (E) ZnONP
Figure 3 IgE and IgA levels after instillation of ZnONP at 150 at 4 wks. Red arrows indicate eosinophils and arrowheads indicate
cm2/rat. (A) IgE levels in serum; (B) IgA levels in serum; (C) IgA levels in foamy macrophages. Black arrows indicate goblet cells.
BAL fluid. Values are mean ± S.D. n = 4 - 8 for each treatment group.
Significance versus vehicle control: * p < 0.05, ** p < 0.01, # p < 0.001.
after instillation, using picrosirius red (PSR) staining,
(Figures 7C and 7D). The fibrotic and atelectatic lesions
found even in terminal bronchioles, but were not present ran in bands through the parenchyma and where they
in the transitional region between bronchiolar and alveo- met the pleura, caused puckering of the visceral pleural
lar tissue (Additional file 3). Whilst the goblet cell hyper- surface. Alpha-SMA, a myofibroblast marker, and trans-
plasia was transient at 1 wk, a modest excess of goblet forming growth factor-b (TGF-b) were strongly
cells were still evident at 4 wks. expressed in these bands of fibrosis/atelectasis 1 and 4
Pulmonary fibrosis wks after instillation (Figure 8). TEM examination
Instillation of ZnONP induced interstitial and broncho- showed large bundles of collagen, eosinophils, and neu-
centric patterns of fibrosis, contraction and atelectasis of trophils located in the interstitium and foamy vacuolated
parenchymal lung tissue which was obvious as both macrophages in the alveolar spaces in the lungs of
gross lesions and in histological sections (Figure 4). The ZnONP treated rats 4 wks after instillation (Figure 9).
areas of fibrosis were evident especially at 1 and 4 wks In addition to pulmonary fibrosis, the smooth muscle
Effects of NP-free BAL fluid on inflammation in rat lungs

NP-free BAL fluid collected 24 h after instillation of
ZnONP into rats was instilled into naïve rat lungs.
There was no inflammation at 1 and 4 wks after instilla-
tion of NP-free BAL fluid from ZnONP-exposed lungs
(data not shown).
Inflammatory pattern of dissolved Zn2+ after instillation

into rat lungs
The high dose of soluble Zn2+ (277.5 μg) but not the
low dose of soluble Zn2+ (92.5 μg) caused death of the
rats because of overdose of highly toxic Zn2+ in a single
acute exposure at high dose rate. This is contrasting
with the fact that similar dose of ZnONP was associated
with 100% survival. Instillation of the low dose of solu-
ble Zn2+ caused severe eosinophilic inflammation and
mild neutrophilic inflammation at 24 h after instillation
(Figure 11). The number of eosinophils and levels of
LDH and total protein of the low dose of Zn2+ group
were significantly higher but the number of PMN was
significantly lower than those of ZnONP at 50 cm2 (Fig-
ure 11). At 4 wks after instillation, the lungs showed
very similar pathological lesions of ZnONP treatment
including goblet cell hyperplasia, fibrosis, contraction,
Figure 5 Immunohistochemistry for Ki-67 in the lung tissues and atelectasis (Additional file 5).
treated with ZnONP at 150 cm2/rat. Each figure is composed of a
low power view (left, 25×), high power view of bronchiolar Inflammatory profile in the BAL after ZnONP aspiration
epithelium (middle, 400×), and alveoli (right, 400×). (A) vehicle into mice
control at 24 h; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP
at 4 wks. Note that ZnONP increased the proliferating Ki-67 positive
ZnONP were inflammogenic in the lungs of C57BL/6
cells (arrow) in the bronchiolar epithelium and alveolar epithelium. and BALB/c mice as evidenced by significantly increased
numbers of PMN in the BAL at 24 h. However, neither
strain showed eosinophilic inflammation at 24 h (Addi-
layer in the bronchi and bronchioles was thickened 1 tional file 6). NiONP, a control particle, showed signifi-
and 4 wks after ZnONP instillation (Figure 4). cant PMN recruitment (data not shown). Although no
eosinophils were recruited into the BAL, the concentra-
Instillation of agglomerated ZnONP tion of eotaxin and IL-13 in the BAL was significantly
Large poorly-dispersed agglomerates of ZnONP (dia- increased compared to vehicle control following ZnONP
meter- 4,380 nm) produced around 91,000 eosinophils aspiration (Additional file 7). Although eosinophils were
(1.3%) in the BAL whilst well-dispersed ZnONP (dia- not detected in the BAL fluid, TEM analysis showed
meter- 242.9 nm) produced 595,000 eosinophils (36.7%) that the eosinophils were recruited in the alveolar inter-
in the BAL (Additional file 4). stitium (Figure 12). NiONP, a control particle, showed
neutrophilic inflammation and neither eotaxin nor IL-13
Study with alternative ZnONP showed significant changes compared to vehicle control
To evaluate whether eosinophilic inflammation was a (Additional files 6 and 7).
generic property of ZnONP, we instilled ZnONP
obtained from an alternative commercial source that Destabilization of lysosomes and cytotoxicity in
were slightly larger in size distribution (90 - 210 nm in macrophages exposed to ZnONP in vitro
size) into female Wistar rats. This ZnONP sample Lysosomal staining with acridine orange, showing lyso-
(designated ZnONPalt) significantly increased the num- somal stability, waned following ZnONP treatment com-
ber of PMN and eosinophils in the BAL following instil- pared to vehicle control and this was accompanied by
lation (Figures 10B and 10C). In addition, ZnONP alt loss of viability (Additional file 8). In contrast, vehicle
induced similar levels of LDH and total protein com- control and TiO2NP-exposed macrophages showed high
pared to ZnONP at the same mass dose (Figures 10E acridine orange fluorescence intensity confirming that
and 10F). these cells had intact lysosomes (Figure 13) and this was
Figure 6 PAS staining for goblet cells in the lung tissues treated with ZnONP at 150 cm2/rat. Each panel is composed of low power view
(left, 100×) and high power view of bronchiolar epithelium (right, 400×). (A) Vehicle control; (B) ZnONP at 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4
wks; Percentage of PAS-positive cells in the bronchus (> 1 mm in diameter) (E) and bronchiole (< 1 mm in diameter) (F). Goblet cells (arrow)
peaked at 1 wk and still present 4 wks after ZnONP instillation. Each data point represents an independent bronchus or bronchiole in the lung
tissues. Significance versus vehicle control: * p < 0.05, ** p < 0.01, # p < 0.001.
reflected in lack of cell death in the exposed macro- compared to other metal oxides and control NP [10].
phages (Additional file 8). The aim of the present paper was solely to investigate
the detail of the ZnONP-induced response with a view
Discussion to better understanding the mechanism whereby they
In previous studies we found that zinc oxide nanoparti- cause such severe pathological effects in rat lungs fol-
cles (ZnONP) were highly fibrogenic and caused an lowing a single intratracheal instillation.
eosinophil exudate into the BAL, a finding that is highly In this study, ZnONP were well dispersed with serum
unusual and possibly unique following particle exposure protein. Stability of NP depends on a balance between
[10]. The current paper set out to determine the likely attractive and repulsive forces between particles [16].
mechanism of the effects of ZnONP. This paper is nota- Incubation of NP with serum protein forms a protein
ble by its lack of systematic inclusion of benchmark or corona which acts as steric stabilizer preventing agglom-
control particles. The reason is that we have already eration [17]. When NP deposit in the lung, surfactant
published extensive findings on the ZnONP response proteins and lipids are adsorbed onto the NP forming a
Figure 7 Picrosirius red staining of the lung tissues treated

with ZnONP at 150 cm2/rat. (A) vehicle control; (B) ZnONP at 24
h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks. Note that collagen
deposition (arrow) was increased 1 and 4 wks after instillation of
ZnONP. Bar scale: 50μm.
lipoprotein corona. Therefore, intratracheal instillation

of NP dispersed with serum protein partially mimics the
interaction of NP with the lung surface environment. To
minimize xenogeneic effects, the serum protein from the
same strain animals as those used in the experiments
was used as a dispersion medium.
All NP in this study showed negative charge by zeta
potential measurement because of the negatively
charged protein corona which is the actual charge that Figure 8 Immunohistochemistry for a-SMA, and TGF-b in the
is encountered by cells [18]. The recognition by phago- lung tissues following treatment with ZnONP at 150 cm2/rat. (A
cytes facilitates phagocytosis which ingests NP into pha- - D), a-SMA in the lungs. (A) vehicle control at 24 h; (B) ZnONP at
gosomes which are then acidified by phagosome/ 24 h; (C) ZnONP at 1 wk; (D) ZnONP at 4 wks. Note that a-SMA
positive cells (dark brown staining) were seen in smooth muscle
lysosomes fusion. Because ZnONP showed fast dissolu-
layer of airways and vessels of normal lung (A and B). In addition,
tion in acidic solution, they are not likely to persistent they were seen profusely in the fibrotic lesions (arrow) at 1 (C) and
in the acid milieu of the phagolysosomes. Therefore the 4 wks (D) after ZnONP instillation. (E - H), TGF-b in the lungs. (E)
effect of surface charge is likely to be less important for vehicle control at 24 h; (F) ZnONP at 24 h; (G) ZnONP at 1 wk; (H)
high-solubility NP although insoluble positively charged ZnONP at 4 wks. Note that ‘I’ denotes the area of TGF-b positive
fibrosis and the whole image of (H) was dominated by TGF-b
NP are more toxic than neutral or negatively charged
positivity.
NP [19].
ZnONP induced pulmonary eosinophilia from 24 h to
4 wks after a single intratracheal instillation without any
prior sensitisation process. There are published studies transient neutrophilic inflammation [22]. In addition,
showing eosinophilic inflammation in animals following the Sephadex beads are polymer particles which are fun-
various sensitisation procedures; particles such as damentally different from ZnONP used in this study.
TiO2NP [20] and ambient particulate matter [21] have NP-free BAL exudates collected 24 h after rats were
been implicated in enhancing development of the mur- instilled intratracheally with ZnONP at 150 cm2 did not
ine model of allergic asthma. In addition, instillation or produce any inflammatory reaction either at 1 wk or 4
intravenous injection of Sephadex beads (complex of wks. Considering that eosinophilia peaked 1 wk after
cross-linked dextran polymers; diameter: 20 - 50 μm) instillation and pulmonary fibrosis was mature 4 wks
also reported to cause eosinophilic inflammation in the after instillation, the lack of inflammation caused by
lung [22]. However, the eosinophilia caused by Sephadex NP-free BAL exudate instillation suggest that BAL fluid
beads may be produced by their extremely large size might be too diluted to produce the pathologies caused
because ultrasonication of this particle produced only a by ZnONP instillation.
In mice, ZnONP aspiration caused eosinophilia but

eosinophils were not found in the alveoli (BAL fluid)
but were present in the alveolar interstitium. The inter-
stitial type of eosinophilia was consistent with the ele-
vated levels of eotaxin and IL-13 in the BAL.
Eosinophils in the interstitium are as likely to cause tis-
sue injury as eosinophils in the bronchoalveolar space, if
not more so [23].
We undertook a number of assays to investigate the
mechanism of the complex and severe pathological syn-
drome seen following exposure to ZnONP. In the con-
ventional rodent asthma model, recruited eosinophils
are associated with airway remodelling including peri-
bronchial fibrosis, smooth muscle hyperplasia, and
Figure 9 Transmission electron microscopy images of lungs at
4 wks after instillation of ZnONP at 150 cm2/rat. (A), vehicle mucus secretion with the involvement of eotaxin and
control; (B - D), ZnONP treatment group. Bar scale: B and C = 2 μm; IL-13 [24,25]. In the ZnONP model we found that
A and D = 5 μm. Collagen bundles (arrowhead) were found in the eotaxin and IL-13 were produced early in rats and mice
perivascular region (B) and alveolar interstitium (C) where they co- exposed to ZnONP and these are key mediators of eosi-
localized with eosinophils (black arrow) and PMN. Foamy
nophil recruitment [25,26]. IL-13 is especially involved
macrophages (white arrow) were localized in the alveolar spaces.
in the regulation of eosinophil infiltration, IgE synthesis,
goblet cell hyperplasia, mucus hypersecretion, and sub-
epithelial fibrosis in asthma [25,27]. Therefore, IL-13
provoked by ZnONP might exert an important role on
the wide spectrum of pathological effects seen here with
ZnONP exposure.
Although the main inflammatory cells induced by
ZnONP were eosinophils, PMN were also recruited during
the acute phase. PMN have been regarded as the represen-
tative acute inflammatory cells playing a role in particle
effects and their recruitment is highly correlated with the
surface area dose of low-toxicity, low-solubility particles
[28] and toxic particle such as crystalline silica [14]. The
pro-inflammatory cytokine recruiting PMN into ZnONP-
exposed lungs was most likely IL-1b which is known to
induce neutrophilic inflammation in the lung [29]. How-
ever, PMN recruitment by ZnONP was confined to the 24
h time-point whilst significant IL-1b in BAL continued to
the 1 week time-point at the highest dose.
Intratracheal instillation of ZnONP induced massive
proliferation of airway epithelial cells and goblet cell
hyperplasia. ZnONP were reported to be very cytotoxic to
BEAS-2B cells in vitro by generating reactive oxygen spe-
cies [30]. In the present study, ZnONP dramatically
increased the levels of LDH and total protein in the BAL
at the acute phase indicating cell death and increased vas-
cular permeability, respectively. Therefore, the prolifera-
tion of airway epithelial cells likely represents a
Figure 10 Pulmonary toxicity of ZnONP alt at 24 h after regenerative response to the cytotoxicity induced by
instillation into lungs of rats. (A-C), BAL cell analysis at 24 h after
instillation of ZnONPalt to rats. (A) Number of total cells; (B) number
ZnONP. Proliferation of airway epithelial cells led to large-
of PMN; (C) number of eosinophils. Levels of LDH (D) and total scale goblet cell hyperplasia. Goblet cell hyperplasia was
protein (E) in the BAL fluid at 24 h after instillation of ZnONPalt to most pronounced at 1 wk and had waned by 4 wks after
rats. Values are mean ± S.D. n = 4 for each treatment group. instillation of ZnONP. Goblet cell hyperplasia plays an
Significance versus vehicle control (VEH): * p < 0.05, ** p < 0.01, # p important role in protecting the airway from damage due
< 0.001.
to inhaled particles [31]. The resulting increase in mucus
Figure 11 Pulmonary toxicity of dissolved Zn2+ at 24 h after instillation into lungs of rats. Dissolved Zn2+ in saline was instilled at 92.5
μg/rat. (A - C), cytological analysis of BAL cells. (A), number of total cells; (B), number of PMN; (C), number of eosinophils. Levels of LDH (D) and
total protein (E) in the BAL at 24 h after instillation of ZnONP. Values are mean ± S.D. n = 4 for each treatment group. Significance versus vehicle
control (VEH): * p < 0.05, ** p < 0.01, # p < 0.001. Zn2+ (92.5 μg/rat) was compared with ZnONP at 50 cm2/rat: $ p < 0.05.
flow traps inhaled particles and removes them from the goblet cell hyperplasia is also a feature of airway injury
airways by muco-ciliary clearance. In addition, goblet cells including exposure to cigarette smoke, and sulphur diox-
can be progenitors of ciliated cells to maintain mucus flow ide, and in asthma where it can contribute to obstruction
[32] and hyperplasia of goblet cells is reversible on cessa- of airways [33]. TGF-b is known to enhance goblet cell
tion of administration [31]. Hypersecretion of mucus by hyperplasia and mucus hyper-secretion in mice through
Following ZnONP instillation collagen fibres, deter-

mined by PSR staining, were increased from 1 wk and
were more marked at 4 wks. At 4 wks, TEM images
showed that large swathes of collagen fibres were pri-
marily located in the perivascular and alveolar intersti-
tium co-localized with eosinophils. Myofibroblasts, the
major source of extracellular matrix proteins and con-
tractile forces during fibrogenesis [34], were also seen in
the contracted and fibrotic lung lesions at 1 and 4 wks
after treatment. TGF-b also is known to cause airway
remodelling including peribronchial fibrosis and smooth
muscle hyperplasia [24].
Instillation of ZnONP increased IgE levels in the serum
but not in the BAL. Multi-walled carbon nanotubes [35],
Figure 12 Representative transmission electron microscopy
images of lungs 24 h after instillation of ZnONP at 15 cm2 to diesel exhaust particles [36], and ultrafine carbon black
C57BL/6 mice. (A), vehicle control; (B - D), ZnONP treatment group. [37] have all been reported to increase serum IgE levels
Bar scale: A = 10 μm; B = 5 μm; C and D = 2 μm. Eosinophils by an adjuvant-like mechanism in murine asthma mod-
(arrow) were infiltrated in the alveolar interstitium. Macrophage els. Surprisingly, compared to previous studies, the
(arrowhead) was severely vacuolated.
increase in IgE levels by ZnONP were induced by just a
single instillation without any sensitization process. The
an NF-B-dependent mechanism [24]. In this study, we levels of IgA in the serum were decreased compared to
found strong immunostaining for TGF-b at all time- vehicle control. Although there was no statistical signifi-
points, but particularly at 1 and 4 wks after instillation cance, IgA levels in the BAL showed an increasing trend
when goblet cell hyperplasia was most pronounced. Con- at 1 wk after instillation of ZnONP. Increases in mucosal
sistent with the immunostaining results, total TGF-b con- secretory IgA were present in the BAL, and this might
centration in the BAL was also increased at all time- explain the decrease of IgA in serum if BAL IgA was
points. derived from the vascular space. The increased IgA levels
Massive pulmonary fibrosis with contraction and in the BAL might act be anti-inflammatory in inflamed
atelectasis was also induced by ZnONP instillation. lung [38]. TGF-b is also known to induce IgA isotype
expression by activating Smad3/4 complex translocation
into the nucleus [39].
Our data on ZnONP durability in acid and neutral
conditions suggests a mechanism for pathogenicity of
ZnONP. ZnONP were stable at neutral pH or saline but
very rapidly dissolved in the acidic artificial lysosomal
fluid (pH 4.5). Thus ZnONP in the approximately neu-
tral surfactant fluid or in the cytosol might be persistent.
However, when ZnONP are internalized into the acid
environment of the lysosome, they will be rapidly dis-
solved producing a high local concentration of Zn 2+
ions. Zn2+ is one of the essential elements in cell home-
ostasis and remains in a bound form inside cells because
free Zn2+ is very reactive and cytotoxic [40]. The acute
increase in free Zn 2+ levels may damage lysosomes,
allowing the contents to escape into the cytoplasm
where they may damage other organelles leading to cell
death [41,42]. We suggest therefore that Zn2+ released
from the phagolysosomes of dead or damaged cells is
Figure 13 Lysosomal destabilization by ZnONP in THP-1 cells.
Differentiated THP-1 cells by PMA (10 ng/ml) were stained with
the source of the Zn 2+ after ZnONP uptake in the
acridine orange for 15 min and cells were treated with NP for 24 h. lungs. The interaction of Zn2+ with cells generates oxi-
(A), vehicle control; (B) TiO2NP at 10 cm2/ml (36.4 μg/ml); (C) ZnONP dative stress and finally triggers cell death signalling cas-
at 10 cm2/ml (20.6 μg/ml). Acridine orange dye aggregates inside of cades [41,43]. When ZnONP were added to activated
lysosome which showing red fluorescence. ZnONP showed less THP-1 cells (a differentiated macrophage cell line), lyso-
signals compared to vehicle control or TiO2NP.
somes were destabilised by a mechanism which seems
likely to involve dissolution of ZnONP under the acid be elicited only by exposure to ZnONP well dispersed
condition of the lysosomes. Unlike ZnONP, TiO 2 NP and at high doses.
showed no dissolution in acid or neutral conditions and ZnONP-induced eosinophilia, fibrosis, and goblet cell
when incubated with macrophages the fluorescence hyperplasia mediated by the soluble Zn 2+ is to our
intensity of lysosomes was not reduced. The loss of lyso- knowledge a novel finding in rat lungs. Interestingly,
somal integrity induced by ZnONP was accompanied by one epidemiological study showed that the level of Zn2+
cell death. A role for dissolved Zn 2+ in the toxic in ambient particulate matter was associated with
mechanism of ZnONP is further supported by Muller et asthma morbidity in USA [49]. Therefore, ZnONP pose
al. [44] who reported that ZnO nanowires were rapidly a unique and substantial hazard to the lungs and
dissolved in the acidic pH of lysosomes causing struc- hygiene precautions and control of airborne exposure
tural changes in mitochondria and cell death including should be instituted in any situation with the potential
necrosis and apoptosis [44]. for exposure in order to reduce the risks of the kinds of
Instillation of dissolved Zn 2+ from ZnONP treated lung pathology described here.
under acid conditions in saline, produced eosinophilia 24
h after instillation. Moreover, the toxicity of soluble Zn2+ Conclusion
was much greater than similar mass dose of ZnONP Figure 14 summarises the diverse pathological changes
based on mortality, number of eosinophils, and levels of (eosinophilia, airway epithelial cell injury, regenerative
LDH and total protein. The higher number of eosinophils goblet cell hyperplasia, bronchocentric pulmonary fibro-
by ZnONP than that of ZnONPalt might be due to the sis, and atelectasis) induced by a single installation of
smaller primary particle size and their wider distribution ZnONP. Although more studies are required, our data
inside of the lung. The chronic lung lesions caused by suggest that a single high exposure of ZnONP produced
Zn 2+ instillation were similar to those induced by eosinophil influx as well as severe fibrosis and airway
ZnONP in terms of bronchocentric interstitial pulmonary epithelial injury. The main cause of these effects appears
fibrosis, goblet cell hyperplasia, and atelectasis. to be the dissolution of ZnONP in the acid milieu inside
There are many inhalation/instillation studies using phagosomes. This study suggests that dissolution of
zinc compounds although few papers on nanoparticles. ZnONP to ions in the acid environment of the lyso-
No papers reported eosinophilia on exposure to zinc somes causes lysosomal destabilisation and cell death.
compounds except for our previous papers using ZnONP The resultant widespread cell death along the path fol-
[10,45] and one human case report with zinc oxide [46]. lowed by the instilled dose is likely a key factor in the
The possible reason for this discrepancy is (1) species severe cell death and subsequent pathogenicity seen.
and strain differences, (2) location of eosinophils infiltra- Exposure to ZnONP should be strictly regulated in
tion to the interstitium or airspaces, (3) dose, (4) polydis- occupational/consumer setting to minimise the likeli-
persity of particles, and (5) durability of particles. In hood of such severe adverse effects occurring.
particular the dispersed NP size might be important as
we have shown by comparing well-dispersed and agglom-
erated NP which might specifically address the role of
ZnONP agglomerate size. We noted that large poorly-
dispersed agglomerates of ZnONP recruited much less
number and percent eosinophils (1.3%) compared to
well-dispersed ZnONP (37.7%). Based on this data we
also conclude that micron-sized particles would not
cause substantial eosinophilia in BAL following instilla-
tion. This finding is consistent with our previous study
which showed no eosinophilic inflammation with ZnON-
P alt (137 nm) using same strain of rats and instillation
technique used in here [47]. In addition, other previous
publications also showed no eosinophilic inflammation
by nano-size ZnO (50 -70 nm) or micrometer-size ZnO
(< 1000 nm) [9,48]. These previous studies instilled ZnO Figure 14 Diagram of lung injury caused by ZnONP instillation.
without any dispersion which implies agglomeration into ZnONP induced (1) goblet cell hyperplasia, (2) thickening of
much larger particles whose compartmentation in the bronchiolar smooth muscle layer, (3) eosinophilia, (4) fibrosis with
increased numbers of myofibroblasts (yellow) and collagen (thin
lung might differ from singlet NP or small agglomerates.
blue fibres), (5) perivascular edema, and (6) type II cell hyperplasia.
Therefore, the eosinophilic inflammation by ZnONP may
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Progressive Severe Lung Injury by Zinc Oxide

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Cho et al.

Particle and Fibre Toxicology 2011, 8:27

RESEARCH Open Access

Progressive severe lung injury by zinc oxide

Background finishing materials in products and buildings because

Table 1 Characterization of nanoparticles

Figure 4 Representative gross lesion and histology of lungs

Effects of NP-free BAL fluid on inflammation in rat lungs

Inflammatory pattern of dissolved Zn2+ after instillation

Figure 7 Picrosirius red staining of the lung tissues treated

lipoprotein corona. Therefore, intratracheal instillation

In mice, ZnONP aspiration caused eosinophilia but

Following ZnONP instillation collagen fibres, deter-

Additional material References

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