Nanotoxicology, 2011; Early Online, 113

2011 Informa UK, Ltd.

ISSN: 1743-5390 print / 1743-5404 online
DOI: 10.3109/17435390.2011.626535

Length-dependent pathogenic effects of nickel nanowires in the lungs

and the peritoneal cavity
Craig A. Poland1,2, Fiona Byrne3, Wan-Seob Cho2, Adriele Prina-Mello4, Fiona A. Murphy2,
Gemma Louise Davies5, J.M.D. Coey3, Yurii Gounko5, Rodger Dufn2, Yuri Volkov4 & Ken Donaldson2,6

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Safenano, Institute of Occupational Medicine, Research Avenue North, Riccarton, Edinburgh, UK, 2Centre for Inammation
Research, University of Edinburgh, Edinburgh, UK, 3School of Physics and Centre for Research on Adaptive Nanostructures and
Nanodevices (CRANN), Trinity College, Dublin 2, Ireland, 4School of Medicine and CRANN, Trinity College, Dublin 2, Ireland,
5
School of Chemistry and CRANN, Trinity College, Dublin 2, Ireland and 6Institute of Occupational Medicine, University of
Edinburgh, Research Avenue North, Riccarton, Edinburgh, UK

mineral, engendered a general suspicion that industrial bres

are pathogenic and this suspicion has fallen on new forms of
engineered nanobres currently being developed. However,
the large variety of industrial bres display a wide range of
toxicities from, in the majority of cases, harmless bres to those
which cause a variety of diseases including cancer. Knowledge
regarding the toxicity of a wide variety of pathogenic and nonpathogenic bres, such as asbestos, led to the development of a
bre pathogenicity paradigm (FPP) through the work of such
luminaries as Stanton (Stanton et al. 1981) and Pott (Pott et al.
1987) and as discussed recently in relation to the organic bre
para-aramid (Donaldson 2009) and in relation to carbon
nanotubes (CNTs) (Donaldson et al. 2010). The FPP is based
on three essential physicochemical attributes which a bre
must possess to be pathogenic in a bre-specic manner.
These are: diameter less than 3 mm to allow aerodynamic
penetration into the lung; a length greater than approximately
15 mm to frustrate macrophage mediated clearance; and
resistance to dissolution and/or breakage in the biological
environment causing the bre to persist biopersistence
(Donaldson 2009). The suggestion that brous nanomaterials
might conform to the FPP was rst raised in relation to CNTs
(Service 1998; The Royal Society and Royal Academy of Engineering 2004). CNTs by virtue of their nano and graphenic
nature are thin and biopersistent, but can vary considerably in
length. Long CNTs therefore can full all the attributes of a
pathogenic bre, if long, and have been shown to be both
highly inammogenicandbrogenicin theperitoneal cavity in
this form (Poland et al. 2008). This raises the question which
forms the basis of this study: do other nanobres show lengthdependent toxicity?
Through the commercialisation of nanoparticles (NPs)
and their incorporation into an ever more diverse range of
products and applications, engineered NPs are increasingly
becoming part of todays world. This has raised the

Abstract
The use of bre-shaped nanomaterials in commercial
applications has met with concern that they could cause health
effects similar to those seen with pathogenic bres such as
certain forms of asbestos. Of the attributes which form the bre
pathogenicity paradigm, bre length is thought to be a critical
factor in determining bre toxicity. We have previously shown
that carbon nanotubes display such length-dependent
pathogenicity but it remains unclear if other forms of brous
nanomaterials conform to the bre pathogenicity paradigm. As
such, our aim is to determine the generality of this hypothesis by
asking whether a radically different form of brous
nanomaterial, nickel nanowires, show length-dependent
pathogenicity. Our results indicate that nickel nanowires
synthesised to be predominantly long (>20 mm) show the ability
to elicit strong inammation in the mouse peritoneal model in a
dose-dependent manner; inammation or brosis was not seen
with the short (<5 mm) nanowires. This length-dependent
response was also seen after lung aspiration and within a
macrophage in vitro model adding further weight to the
contention that bre length is an important driver of hazard
potential. This may have important implications when
considering the hazard posed by brous nanomaterials and their
regulation in workplaces.
Keywords: Nanobres, bre pathogenicity paradigm,
inammation, structure activity relationship

Introduction
Fibres have long been used as an industrial material due
to commercially advantageous properties such as tensile
strength and anisotropic electrical or thermal conductivity.
However, the experience with asbestos, a brous silicate

Correspondence: Craig Poland, Safenano, Institute of Occupational Medicine, Research Avenue North, Riccarton, Edinburgh EH14 4AP,
Tel: +44(0)131 449 8096. Fax: +44(0)131 449 8084. E-mail: [email protected]
(Received 21 March 2011; accepted 23 August 2011)

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C. A. Poland et al.
increasing likelihood of exposure of those working with NPs
in the occupational setting and also ultimately the endusers of NP containing products as these undergo attrition
and wear. Such exposures have potential to produce adverse
health consequences (Maynard et al. 2006; Donaldson et al.
2006). Whilst CNTs are the most well known of the nanobres, others are also under development for commercial
application. Of the various methods which continue to be
developed for the production of nanowires, the use of
template-based growth methods is becoming increasingly
popular (Figure 1). Part of the reason for the interest in
nanowires is the controllability of the production process
including diameter, length and density, reduced contamination as well as low production costs and easy scalability
(Cao & Liu 2008). Template-based systems make use of
nano-scale templates into which a material of choice is
deposited, leading to the self assembly of a nanowire followed by recovery of the nanobres from the template.
Templates can consist of wide range of substrates, most
often alumina membranes (Prina-Mello et al. 2006) but
even nano-biological structures such as the tobacco mosaic
virus (Knez et al. 2003) or microtubules (Zhou et al. 2008)
can be used. Whilst nanowires differ from CNTs in commercially desirable traits such as tensile strength, the commercial interest in nanowires is likely to increase in the
coming years. The nickel nanowires (NiNWs) used within
this study are very different from CNT or asbestos bres and
represent an ideal candidate to test an alternative form of
high aspect ratio nanoparticles (HARN) against the FPP. As

such, our aim is to ascertain if this different form of HARN

also shows length-dependent pathogenicity in a model of
direct mesothelial exposure.

Materials and methods

Our approach was the development of long nickel nanowires
(L-NiNW) and short nickel nanowires (S-NiNW) to allow the
critical evaluation of the length hypothesis and its contribution to the toxicity of a bre. These samples were evaluated
for their potency in eliciting both inammogenic and brogenic activity in a model of direct mesothelial exposure using
the mouse peritoneal assay. In addition, we explored the
differential toxicities of these materials within the lung and
also if an in vitro model (THP-1 cells) could be used to
differentiate between toxicity driven by bre length.

NiNW fabrication and characterisation

NiNWs were fabricated by electrochemical template synthesis (Figure 1) using alumina membranes (Anodisc 25,
Whatman, UK) with an average pore diameter of 200 nm, as
reported in our previous work (Prina-Mello et al. 2006;
Byrne et al. 2009). Nanowires were removed from the membrane by dissolving it in 1 M NaOH and re-suspending the
solution in deionised water. The metallic NiNWs due to
oxidation possessed a 34 nm layer of nickel oxide over
the surface (Prina-Mello et al. 2006). S-NiNW and L-NiNW
with average lengths of 4.3 1 mm and 24 7 mm were
examined and sized by counting a minimum of 100 separate

Potentiostat
Reference
electrode
I

Electrode
(anode)
Nickel
sulphate bath

Alumina
membrane

Gold electrode
(cathode)

Deposited nickel
(nanowires)

Figure 1. Diagram of nickel nanowire synthesis. The typical template mechanism for the production of nickel (and other electrically conductive
material) nanowires is shown (adapted from Cao & Liu 2008).

Nanobre toxicity
nanowires scanning electron microscopy (Carl Zeiss Ultra
Plus, UK). In order to control for bulk chemical composition,
we utilised a commercially available nickel nanoparticle
(NiNP) (Nanostructured & amorphous materials Inc., TX,
USA). These particles were non-brous and spherical in
shape, with a mean dry particle diameter of 15 nm
(5 nm; manufacturers description) and a hydrodynamic
diameter of 57 (10) nm in experimental dispersant
(0.5% bovine serum albumin (BSA) (Sigma-Aldrich, Poole,
UK)/saline), ascertained by diffraction light scattering
(Brookhaven Instruments Corporation, NY, USA).

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Particle suspension
All particles were suspended in a solution containing 0.5%
w/v BSA. For in vitro experiments, the solution consisted of
Rosewell Park Memorial Institute-1640 (RPMI-1640) cell
culture media (PAA Laboratories Ltd., UK) supplemented
with 100 U/ml penicillin/streptomycin and 2 mM of
L-Glutamine (PAA Laboratories Ltd., UK). For in vivo experiments, 0.5% BSA was added to sterile saline suitable for
injection. Once dissolved, the BSA solution (media/saline)
was sterile ltered using a 0.22 mm lter (Whatman, UK) to
remove any contamination and large globular proteins and
used immediately. Each particle type was made up in the
BSA surfactant and sonicated in a sealed container using an
ultra-sonicating water bath (Fisherbrand FB11002, 40 kHz,
UK) for 2 h to achieve a visually homogenous suspension.
The samples were then diluted to the appropriate test concentrations with the BSA surfactant and briey sonicated to
ensure proper mixing.

Experimental animals
Eight to twelve week old (2025 g) female C57BL/6 strain
mice (Harlan, UK) were group housed in standard caging
with sawdust bedding, environmental enrichment with
free access to sterile water and food within a pathogenfree Home Ofce approved facility. The animals were maintained on a normal 12 h light and dark cycle and were
allowed 7 days to acclimatise prior to study commencement.
Post exposure animals were subject to daily checks for signs
of distress or welfare issues (none identied). All in vivo
work was carried out by staff holding a valid UK Home
Ofce personal licence under a Home Ofce approved
project licence.

Intraperitoneal injection
After 7 days acclimatisation, groups of three animals were
injected intraperitoneally with 0.5 ml of 100 mg/ml (50 mg/
animal) of each particle treatment or 0.5 ml of 0.5% BSA/
saline as a vehicle control. Animals were immediately
placed back into their cage and monitored to ensure
resumption of normal behaviour. The animals were sacriced at 24 h to investigate the acute inammatory effects or
7 days for investigation of the inammatory and brotic
effects.
At each time point, the mice were sacriced by CO2
asphyxiation or cervical dislocation and the peritoneum
lavaged three times using 2 ml washes of sterile icecold saline. Three washes were shown to be sufcient to

allow the removal of 90% of free oating peritoneal cells by

exhaustive lavage (data not shown). The lavages were pooled
together and placed on ice for the entire duration of the
processing.

Pharyngeal aspiration
Animals were anesthetised using isourane (2-chloro2-(diuoromethoxy)-1,1,1-triuoro-ethane) and the tongue
was gently held at full extension and a 50 ml bolus of test
sample pipetted to the base of the tongue. The animals were
stimulated to inhale via covering of the nasal cavities to
induce a gasp reex and held until several breaths had
occurred (Rao et al. 2003). The animals were further
observed until full recovery and group housed for the duration of the experiment.
At each time point, the mice were sacriced by terminal
anaesthesia using an intraperitoneal injection with 0.5 ml of
pentobarbitone (200 mg/ml) followed by exsanguination via
the abdominal aorta. The thoracic cavity was exposed, and
the trachea cannulated using a 21 gauge needle and ligated.
The lungs were lavaged using three 1 ml washes of icecold sterile saline with the rst lavage retained separately
and the subsequent lavages pooled. All lavages were placed
on ice for the entire duration of the processing.

Dissection and xation

Diaphragm
Following sacrice and peritoneal lavage the abdominal wall
of the animal was removed, exposing the peritoneal cavity.
Lateral incisions extending to the veterebral column were
made, which was then severed below the diaphragm and
the diaphragm carefully removed by cutting through the
chest wall approximately 1 cm from the diaphragm. The
diaphragm was rinsed three times by emersion in icecold sterile saline and placed overnight into methacarn
xative (60% methanol, 30% chloroform and 10% glacial
acetic acid) for histological staining. After overnight incubation in xative, the diaphragm was carefully excised from the
surrounding ribs and the same full width section of the upper
(ventral) portion of the diaphragm removed from each
animal sampled. As previously described (Poland et al.
2008), the diaphragm sections were dehydrated through
graded alcohol (ethanol) and imbedded on-edge in parafn,
with 4 mm sections of the diaphragm made and stained with
haematoxylin and eosin (H&E) stain. Serial images were
taken at 100 magnication along the diaphragm length
using QCapture Pro software (Media Cyberbernetics Inc.,
MD, USA) and seamlessly re-aligned using Adobe Photoshop
CS3 Version: 10.0.1 (Adobe systems Inc.) to show the entire
diaphragm section. Using calibrated software (Image-Pro
Plus, Media Cybernetics Inc., MD, USA), the total length
of each diaphragm along the basement membrane was
measured in order to adjust for any differences in size
between diaphragms. Any areas of granulomatous tissue,
identied by histology as lymphocytic aggregates adhering to
the diaphragm surface (excluding areas of Liver, connective
tissue or lymphatic tissue), were measured using the same
software. Granuloma area on each diaphragm was calculated
in mm2 per unit length of diaphragm (in mm) to yield

granuloma area per unit diaphragm length (mm2/mm) as

shown in Figure 5.

10.0.1 (Adobe systems Inc.) to show the entire section of

the lung.

Lung

Differential cell count

At each time point, the mice were sacriced by terminal

anaesthesia, the lungs exposed and the trachea cannulated
as before. The heart and lungs were removed on-block and
xed by instillation of a cold methacarn xative at a hydrostatic pressure of 20 cm H2O. The entire lung was submerged
in xative for a period of 24 h prior to processing. After
xation, the heart was removed and discarded whilst the
individual lobes of the lung were dissected free and placed
at in a tissue cassette. As before, the lung tissue was
dehydrated through graded alcohol (ethanol) and imbedded
in parafn with four sections cut so as to encompass all lobes
of the lung. Sections were stained with H&E stain to show
gross pathology and Pico-Sirius Red to show collagen deposition (red stain) and serial images taken at
100 magnication using QCapture Pro software (Media
Cyberbernetics Inc., MD, USA). The images were seamlessly
re-aligned using Adobe Photoshop CS3 Version:

The lavage uid (both lung and peritoneal) was then centrifuged at 123 g for 5 min at 4 C in a Mistral 3000i centrifuge
(Thermo Fisher Scientic, Inc., MA, USA) and aliquot of the
supernatant retained for total protein and cytokine measurements. The remaining cell pellet was re-suspended in 0.5 ml
of 0.1% BSA/sterile saline solution. A total cell count was
then performed using a NucleoCounter (ChemoMetec, A/S,
Allerd, Denmark). Differential cell counts were performed
on cyto-centrifugation preparations, stained with Diff Quik.
Images of cells were taken using QCapture Pro (Media
Cyberbernetics Inc., MD, USA).

Total protein measurements

Total protein concentration of the peritoneal lavage uid
was measured using the bicinchoninic acid protein assay.
Sample protein concentrations were established by comparison to a BSA standard (Sigma-Aldrich, Poole, UK) curve

Light microscopy (400)

L-NiNW

TEM

10 m
m

10 m
m

10 m
m

10 m
m

500 nm

S-NiNW

10 m
m

NiNP

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C. A. Poland et al.

Figure 2. Morphological structure of nickel nanowires (NiNW) and nickel nanoparticles (NiNP). Brighteld images of nickel based nanoparticles
were taken at 400 magnication in glycerol after dispersion in dH2O (left hand panel). TEM images were taken for NiNP dispersed in dH2O and
deposited onto formvar coated TEM grids (0.5 mg) prior to imaging. (Right hand panel) (Please note the different scale bar between TEM of NiNW
and NiNP).

Nanobre toxicity

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LDH assay
The level of cellular cytotoxicity/cytolysis was established
using a lactate dehydrogenase (LDH) assay (Roche Diagnostics GmbH, Mannheim, Germany). Briey, 100 ml of
lavage uid was added in triplicate to a 96-well plate and
100 ml of the LDH test reagent (diaphorase/NAD+ mixed with
iodotetrazolium chloride and sodium lactate at a ratio of
1:45) added to each well. Following a 30 min incubation
period, the absorbance of each well at 490 nm wavelength
was established using a Synergy HT microplate reader
(BioTek Instruments, Inc. VT, USA).

THP-1 cell culture

The monocytic cell line THP-1 was obtained from the
American Type Culture Collection and maintained at subculture in RPMI-1640 supplemented with 10% foetal calf
serum (PAA Laboratories Ltd., UK) at 37 C (4% CO2). Cells
were differentiated from their monocytic form into
macrophage-like cells via 24 h incubation with 5 mM of
phorbol myristate acetate (PMA; Sigma-Aldrich, Poole,
UK). Briey, THP-1 cells were seeded into 24-well plates
at a density of 1 million cells/ml (0.5 ml total volume) and
incubated with 5 mM of PMA for 24 h triggering differentiation and causing the cells to become adherent. Any nonadherent cells were removed and the media replaced with
fresh 2% FCS containing media for 24 h. The seeded cells
were then exposed for 24 h to the different lengths of NiNPs
at a concentration of 5 mg/ml. After 24 h an aliquot of the
media supernatant retained for cytokine analysis, the
remaining supernatant aspirated, the cells stained with
Diff Quik stain and images of cells were taken using QCapture Pro (Media Cyberbernetics Inc., MD, USA).

Cytokine/chemokine assay
The media levels of interleukin-1b (IL-1b), interlukin-6
(IL-6), tumour necrosis factor-alpha (TNF-a) and the chemokine CCL3 (MIP-1a) were established using ELISA DuoSet kits (R&D systems, Abingdon, UK) specic to each
analyte of interest. Ninety-six well microtitre plates (Corning)
were incubated overnight at 4 C with 100 ml coating antibody
raised against IL-1b, IL-6, TNF-a or CCL3. The plates were
washed three times with 0.05% Tween-20 in phosphate
buffered saline (PBS; pH 7.2) and blocked using reagent
diluent (1% BSA in PBS; R&D systems, Abingdon, UK) for 1 h
(room temperature) prior to further washing and addition of
test samples/standards in triplicate. After 2 h, the plates were
washed and a biotinylated detection antibody added to each
well followed by a further 2 h incubation, followed by
washing and the addition of HRP conjugated streptavidin.
The plates were washed and developed using a TMB

substrate solution (Sigma-Aldrich, Poole, UK). The subsequent reaction was stopped with 0.5 M H2SO4, resulting in a
yellow colour, and read at 450 nm. Sample concentrations of
IL-1b, IL-6, TNF-a and CCL3 were established via extrapolation from the appropriate recombinant protein standard
curve.

Statistical analysis
All data were analysed using GraphPad Prism 5 (Version
5.03; GraphPad Software Inc. USA). Results were expressed
as the mean + standard error mean (s.e.m) and multiple
comparisons were analysed using one-way analysis of variance with a Tukey-HSD method post-test. Two sample
comparisons were made using the Students t-test. In all
cases, values of p < 0.05 were considered signicant.

Results
Particle characterisation
By altering the deposition time in the bre-production
process, the length of the nanowires was altered allowing
the formation of a predominantly long (L-NiNW) and a
predominantly short test sample (S-NiNW) as shown
in Figure 2.
The L-NiNWs were predominantly (73%) above 20 mm in
length (Figure 3) with a mean length ( standard deviation)
of 24 mm (7 mm) and hence given the notation L-NiNW. The
second nanowire sample used was a short bre sample with
100% of the bres less than 10 mm and 77% less than 5 mm in
length (Figure 3) with a mean bre length of 4 1 mm,
denominated S-NiNW. The TEM image shown in the right
hand panel of Figure 2 shows the short bres forming
disjointed end-on chains (dipoledipole interaction between
NiNWs due to their remnant magnetisation); these are

100
S-NiNW
90

L-NiNW

80
% Fibres greater than

(01000 mg/ml). The samples were then incubated at 37 C

for 30 min after the addition of the test reagent (1 part Cu II
sulphate solution (4% w/v) to 50 parts bicinchoninic acid
(Sigma-Aldrich, Poole, UK)). The absorbance was then read
at 570 nm using a Synergy HT microplate reader (BioTek
Instruments, Inc. VT, USA) and the sample protein concentration established via derivation from the BSA standard
curve.

70
60
50
40
30
20
10
0
0

10

15

20

25

30

35

40

Length (m)
Figure 3. Nickel nanowire size distribution. Size distribution was
performed by measurement of a minimum of 100 bres imaged under
scanning electron microscopy (SEM, Carl Zeiss Ultra Plus, UK).

 C. A. Poland et al.
cavity with ice-cold sterile saline. The total number of
inammatory neutrophils (Polymorphonuclear leukocyte;
PMN), a potent marker of acute inammation, and total
protein levels within the lavage uid as a measure of
increased vascular permeability due to inammation was
established (Figure 4A). The results demonstrate a highly
signicant increase with L-NiNW (p < 0.001) compared to
controls, which is signicantly greater than that of S-NiNW
and NiNP (p < 0.001). Figure 4B shows the typical cellular
response post injection with the S-NiNW (top panel) and
NiNP (bottom panel) showing complete unperturbed uptake
of the particles by macrophages. Uptake of the L-NiNW
sample (middle panel) is associated with frustration of the
process of phagocytosis by failure to fully enclose the bre.
This leads to an inammatory cell inux as noted by the
presence of neutrophils in the lavage uid.
Figure 5 shows the response at the diaphragmatic mesothelial surface 7 days post injection with the two NiNWs and
control NPs, the point of bre egress and hence deposition of
the long bres. Treatment with S-NiNW or the compact
NiNP control showed no lesions on the peritoneal aspect
of the diaphragm at the mesothelium. In contrast, treatment

Length-dependent bre toxicity

Twenty-four hours after injection of 50 mg of L-NiNW (~7.9 

106 bres), S-NiNW (~31.25  106 bres) and NiNP into the
peritoneal cavity, the inammatory response to each particle
was evaluated following washing (lavaging) the peritoneal

A
***

Total lavage PMN (106)

12

S-NiNW

20 m

0
NP

L-

Ni

Ni

NW

NW
Ni
S-

VE

L-NiNW

1600
Total protein (mg/ml)

***

1200
20 m
800

NiNP
#

400

NP
Ni

NW
Ni
L-

S-

Ni
NW

0
VE

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readily dispersed into singlet short bres in protein solution

for injection as shown under the light microscopy images
in the left hand panel. All of these bres irrespective of
length had straight morphology and regular diameter of
200 10 nm.
Sonication of the NiNW samples during dispersion for
in vivo/in vitro use did lead to a small degree of shortening.
Subsequent control experiments using a separate batch of
NiNWs demonstrated that a 2 h sonication using an ultrasonicating water bath induced a 7% and a 4% decrease in the
long and short samples, respectively (data not shown). This
therefore suggests that whilst marginal shortening did occur,
the resultant bres still met the minimal and maximal
length requirements of the experimental design for the
long and short bres respectively as reected in Figure 2
(light microscopy image).

Treatment (50 g per mouse)

20 m

Figure 4. Length-dependent inammogenicity of nickel nanowires. (A) Female C57BL/6 mice were intraperitoneally injected with 50 mg of long
(L-NiNW) and short (S-NiNW) nickel nanowires and the peritoneal cavity lavaged 24 h later to assess the level acute inammation. Panel B shows
typical macrophage uptake of NiNW and nanoparticles in the peritoneal cavity 24 h after injection. Scatter plot with mean of three animals s.e.m.
Signicance vs. vehicle control indicated by p < 0.001 and vs. L-NiNW #p < 0.001. All images were taken at 1000 magnication under Brighteld
illumination.

Nanobre toxicity
A S-NiNW

B L-NiNW

C NiNP

G
G

M
M

0.005
0.004
0.003
0.002
0.001

P
iN
N

W
iN
N
L-

S-

iN

0.000
VE
H

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Lesion area (mm2/mm)

Treatment (50 mg)

Figure 5. Granulomatous response post injection with long (L-NiNW)
but not short nickel nanowires (S-NiNW) or nanoparticles. C57BL/
6 mice were intraperitoneally injected with 50 mg of S-NiNW (A) or
L-NiNW (B) nickel nanowires and nickel nanoparticle (NiNP; C). Seven
days post injection the animals were sacriced and the diaphragm
removed and sectioned for histological examination for the presence of
granulomatous lesions. The peritoneal aspect of the diaphragm is
shown with the muscular area (M) and overlying mesothelium (arrow)
shown. Areas of granuloma (G) are shown and were measured and
expressed per millimeter of the total section length (D). Mean of three
animals (for vehicle control, S-NiNW, NiNP exposure) and six animals
(for L-NiNW exposure) s.e.m.

with L-NiNW resulted in granulomas at the mesothelial

surface.
To investigate the dose-relatedness of inammogenicity
of the L-NiNW, mice were injected intraperitoneally at mass
doses of 0.5, 1, 10 and 50 mg per mouse in a 0.5 ml vehicle of
0.5% BSA/saline and the peritoneal cavity lavaged 24 h later.
Injection of L-NiNW into the peritoneal cavity of C57BL/
6 mice led to an induction of a straight-line dose response
relationship for neutrophil inltration (r2 = 0.9863; Figure 6).
This response at 50 mg per mouse was in excess of what
we have previously seen with the same mass of long
multi-walled CNT and long bre amosite asbestos (Poland
et al. 2008); total protein levels in the peritoneal lavage uid
at 24 h supported the PMN data. Injected animals were also
lavaged at 7 days post injection to show chronicity of the
inammation (Figure 6; right hand panel). However, after
7 days the inammatory response was markedly reduced
showing no doseresponse relationship and only a dose of
1 mg of NiNW per mouse produced a signicant increase in
neutrophil inux over vehicle control.
We excluded a role for soluble agents leaching from the
particle surface in these effects by using an aqueous extract
which is a commonly used methodology to collect soluble
metals from particle samples (Brown et al. 2000; Cho et al.
2011; McNeilly et al. 2004) and assessed their role in adverse
effects. This was prepared by 24 h mixing of the highest dose
of L-NiNW (50 mg) in sterile saline without biological

macromolecules as is commonly the case in many studies

by other groups (Hetland et al. 2001; Knaapen et al. 2002)
and forms the basis of a recommended methodology for
studying the soluble bio-available components of particles
(Julien et al. 2011). This was followed by centrifugation to
remove the long nanowires and which would have contained any soluble Ni ions or other soluble components.
Soluble components are known to play a role in the toxicity
of combustion derived NPs (Nel et al. 2001; McNeilly
et al. 2004) and other metallic NPs such as Cu and Zn
(Cho et al. 2011) but the lack of inammation after instillation of the aqueous extract (Appendix Figure 1) conrmed
that nickel ions did not contribute to the inammogenicity
of the L-NiNW sample.
In addition to the mesothelioma hazard, which we studied here by examining the short-term mesothelial inammatory response to the NiNW, bres also pose a hazard to
the lungs in the form of brosis and lung cancer. We assessed
this by aspirating 50 mg of L-NiNW and S-NiNW and NiNP
into the lungs of mice (Figure 7).
As NiNPs are known to be inammogenic in the lung
(Lu et al. 2009), we expected an inammatory reaction with
all forms of nickel aspirated into the lungs but we also
expected an enhanced response and characteristic pathology
due to long bres and the clearance problems that they pose
to macrophages as described above. This was conrmed by
the presence of elevated BAL PMN in all nickel exposed
groups compared to vehicle control group (Appendix
Figure 2). However, there was a dramatic difference in
pathological response between the nickel particle types at
7 days. This was most evident in the comparison between the
NiNP and the long L-NiNW. The presence of NiNP caused a
very mild diffuse alveolitis as would be expected by the NiNP
reaching the alveolar region following instillation. However,
there was no remodelling or brosis of the airways and only
mild alveolar wall thickening at sites of inammation
although there were very occasional instances of small
granulomas with mild collagen staining around larger aggregates of NiNPs (not shown). Deposition of L-NiNW in contrast led only to a moderate inammatory response in the
peripheral airways (Figure 8, Appendix Figure 2) but did lead
to a strong granulomatous response evident as areas of
intense nuclear staining in solid, highly cellular granulomas
(Figure 7C). These granulomas consisted of macrophages
and numerous NiNW bres. Due to the shorter size of the sNiNW, retention did not occur predominantly at the terminal
bronchioles and particles reached the alveolar region with
subsequent alveoli wall thickening rather similar to what
was seen with NiNP. Figure 8 shows representative images
of the alveolar septae taken at equidistance from the
terminal bronchioles and shows that both L-NiNW and
S-NiNW generated thickening of the alveolar septae to a
similar degree.

In vitro toxicity of long bres

An in vitro exposure method was developed to investigate

long and short bre effects. We used the monocytic
leukaemia cell line THP-1, which was further differentiated into macrophages using PMA. The differentiated

 C. A. Poland et al.
24 h post injection
***

15
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0
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10

10
5
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800
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Lavage protein (mg/ml)

***

0.1

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1600

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Total lavage PMN (x106)

Total lavage PMN (x106)

20

1200
800

***
*

400
0

100

L-NiNW dose (mg per mouse)

0.1

10

100

L-NiNW dose (mg per mouse)

Figure 6. Inammatory response post intraperitoneal injection with long nickel nanowires (L-NiNW). Female C57BL/6 mice were intraperitoneally
injected with increasing doses of L-NiNW and 24 h or 7 days post injection were sacriced and the peritoneal cavity lavaged. Total lavage neutrophil
(Polymorphonuclear leukocyte (PMN)) numbers were counted (top graphs) and total lavage protein measured as a general marker of inammation
(bottom graphs). Mean of three animals s.e.m. Signicance vs. vehicle control indicated by *p < 0.05, **p < 0.01 and ***p < 0.001.

THP-1 cells were exposed for 24 h to the different lengths of

NiNPs and their responses analysed. As shown in Figure 9,
secretion of the acute phase response cytokines IL-1b and
IL-6 followed the pattern seen in vivo in the peritoneal
cavity with long and short nanowires and NiNP. In these
cases, a signicant increase in both cytokines was seen after
treatment with L-NiNW (p 0.001) over vehicle control and
the S-NiNW and NiNP. Length-dependent effect of the
L-NiNW was seen with TNF-a and the chemokine CCL3
(MIP-1a) but NiNP also signicantly increased levels of
TNF-a and CCL3.
Cytocentrifugation preparations after treatment with
S-NiNW (Figure 9E) and L-NiNW (Figure 9F) show cellular
uptake of the particles. In the case of L-NiNW, bres can be
seen both within and protruding out of the macrophages
whilst the S-NiNW were completely enclosed (as shown by
arrows).

Discussion
Our intention within this series of experiments was to
ascertain if a radically different form of HARN to CNTs
also show length-dependent pathogenicity in a model of
direct mesothelial exposure and a surrogate for the thoracic
cavity. Using a compositionally and structurally different
form of HARN, we report here that, similar to asbestos
and CNTs (Poland et al. 2008; Donaldson et al. 1989),
NiNW show clear length-dependent inammogenicity in

the peritoneal cavity, with L-NiNW being highly inammogenic and S-NiNW not signicantly inammogenic.
The potential cause of this difference in response to long
and short nanowires, which were otherwise chemically
identical, can be sought in the handling of bres by macrophages. Macrophages removed from the peritoneal cavity
24 h after injection of either the S-NiNW or NiNP showed
complete engulfment of the particles which are localised to
the cytoplasm. However, a proportion of macrophages
removed after intraperitoneal injection of the L-NiNW sample show incomplete phagocytosis with bre protruding
from the cell or two macrophages sharing a single bre
which causes both macrophages to undergo frustrated
phagocytosis. Frustrated phagocytosis is accompanied by
release of cellular components involved in microbiocidal
activity such as NADPH oxidase-dependent release of reactive oxidant species (ROS) (Hansen & Mossman 1987),
cytokines and release of proteases and other components
of cytoplasmic lysosomes. This may cause further recruitment of inammatory cells as well as innocent bystander
injury to the surrounding mesothelium (Kamp et al. 1992;
Ye et al. 1999). Indeed, within our experiments, an increase
in the lavage cytokine IL-6 was noted in the peritoneal cavity
after injection of L-NiNW. In order to provide clearer information of the effect of long bres on macrophages, we used
an in vitro system in which the NiNW were incubated
with macrophages. The results showed internalisation of
the shorter bres and similar protrusions of the long

Nanobre toxicity
A

Vehicle control

S-NiNW

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2 mm
C

2 mm
D

L-NiNW

NiNP

*
*
*
*
*

2 mm

2 mm

Figure 7. Lung pathology 7 days post aspiration with particles. The effect of different lengths of nickel nanowire samples and nickel nanoparticles is
demonstrated 7 days after 50 mg aspiration of each test sample into the lungs of C57BL/6 mice. Each panel shows an entire lung section stained with
haematoxylin & eosin (H&E) stain to demonstrate gross pathology with call-outs showing high magnication (1000) terminal bronchioles. These
call-outs show the same stained with H&E and Pico-Sirius Red (PSR) to show collagen deposition (red stain). Asterisks denote the presence of
representative collagenous granulomas. Lung images taken at 25 and call-outs at 1000 magnication. Treatments performed N = 3 and vehicle
control N = 2.

bres out of the cells as seen in vivo. The same

length-dependent effects for IL-1b and IL-6 were also noted
which suggests that IL-1b and IL-6 are involved in the lengthdependent inammation in the peritoneal cavity in vivo
whilst TNF-a and CCL3 may not. The ndings that NiNP
stimulated the release of TNF-a and CCL3 by NiNP in vitro is
somewhat puzzling but may relate to the release of soluble
nickel from the NINP driving a reaction due to its large
surface area. Indeed, the ability of soluble nickel to drive an
allergic type reaction resulting in high TNF-a levels after
exposure has been shown in humans (Mller et al. 1999) and
in vitro (Cortijo et al. 2010) although in the latter it also
caused an increase in IL-1b in a type II alveolar epithelial cell
line (A549). Indeed, it has previously been reported that
there can be dramatic differences in the ability of particles to

stimulate TNF-a and IL-1b, e.g., with silica there was a

10 times greater IL-1b response than TNF-a in the
NR8383 macrophage cell line (Liu et al. 2007) whilst environmental particles caused more than 10-fold greater TNF-a
than IL-1b response in human alveolar macrophages in
culture (Ishii et al. 2004). Differences in signalling pathways
for the two cytokines may account for these differences
along with differences in particle stress effects on cells in
culture, e.g., oxidative stress versus direct membrane damage. However, the importance of these data lie in their ability
relevance as an indicator for a predictive in vitro assay for
length-dependent effects although clearly further work is
needed.
In relation to the ability of NiNP to stimulate TNF-a
and CCL3 production, it may be the case that such

 C. A. Poland et al.
Vehicle control

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L-NiNW

S-NiNW

NiNP

Figure 8. Lung alveolar septa 7 days post aspiration of nickel particles. The effect of lung aspiration of different lengths of nickel nanowires and
nickel nanoparticles on the alveolar septa is shown 7 days after aspiration of each test sample into the lungs of mice. The images for each are stained
with H&E stain and taken at 1000 magnication at roughly equidistance from the terminal bronchioles; scale bar = 20 mm. Treatments performed
N = 3 and vehicle control N = 2.

pro-inammatory reactivity may drive the airway neutrophilia we saw in the lungs and BAL of mice treated with NiNP.
When considering the mesothelial response as a surrogate
for the pleural response, prolonged contact with nickel
particles of small dimensions is unlikely to happen due to
rapid clearance routes from mesothelial cavities as previously discussed, resulting in a removal of dose. IL-1b activation through cleavage by caspase-1 and subsequent
secretion may occur due to frustration of phagocytosis by
the retained L-NiNW leading to ROS generation via NADPH
oxidase activation. This in turn can lead to activation of the
Nalp3 inammasome resulting in activation of caspase-1 and
secretion of IL-1b (Dostert et al. 2008; Cassel et al. 2008),
which was not seen with the low-aspect ratio nickel nanowires (S-NiNW) and nanoparticles (NiNP) which did not
hinder uptake. The secretion of IL-1b via inammasome
mediated pathway may in turn lead to the corresponding increase in IL-6 which is not inammasomedependent whose expression can be activated by IL-1b
(Zhang et al. 1990); however, this does not explain why
secretion of TNF-a did not also lead to an increase in
IL-6 via nuclear factor kappa-B activation.
Within the lung, normal pulmonary clearance ensures
that most of the particles that reach the distal lung are
removed upwards by macrophages and mucocillary action
and never reach the pleural or peritoneal mesothelium.
However, we recently reviewed the data (Donaldson et al.
2010) supporting the contention that a proportion of all
deposited particles and bres pass through from the lung

into the pleural space (Mitchev et al. 2002; Muller et al.

2002). The normal elutriating effects of passage through the
airways ensures that peripherally-depositing particles are all
small (<5 mm aerodynamic diameter (Dae)) and so those that
do reach the pleura can easily exit through the stomatal
pores in the parietal pleura which are around 312 mm
(Mutsaers 2002) and enter the lymphatic system. We suggest
that long bres, which can still reach the distal lungs and
pleura, because of their uniquely small Dae despite their
length, cannot negotiate these stomata and build up on the
parietal pleura leading to disease (Donaldson et al. 2010).
Thus, the pleural space has a size-selective mechanism of
clearance that has an analogous size-dependent mechanism
in the peritoneal cavity making the mouse peritoneal assay a
realistic model for studying bre length effects that occur
primarily in the pleural cavity, with obvious caveats. These
include the fact that the space between the parietal and
visceral pleura is notional, only around 2050 mm, and uid
lled with a constantly moving apposing surfaces. However,
the bre length-dependent inammatory response in the
peritoneal and pleural spaces is similar with CNT although
the response to L-NiNW waned with time in the peritoneal
cavity, as seen with CNT and long bre amosite asbestos,
whereas over a week the inammation persists in the pleural
space (Murphy et al. 2011).
A length-dependent difference was also seen within the
lung after aspiration of the nickel nanowires and particles as
assessed at the later time point of 7 days for the purpose of
assessing the pathology. This difference in the pathological

2220

***

1480
#
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740

#
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150

100

1500

***
1000

500
#

CCL3 (pg/ml)

***

iN
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iN

4500

W
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iN

iN

VE
H

SN

LN
iN

iN

0
VE
H

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3000

1500
#

S-

Treatment (50 mg/ml)

E

P
iN
N

W
iN
N
L-

VE

P
iN
N

W
iN
N
L-

W
iN
N
S-

VE

0
iN

TNF-a (pg/ml)

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#
**

50
#

***

IL-1b (pg/ml)

IL-6 (pg/ml)

Nanobre toxicity

Treatment (50 mg/ml)

F

50 m

50 m

Figure 9. THP-1 inammatory response to nickel nanowires. Differentiated THP-1 macrophages were treated with 5 mg/ml of long and short nickel
nanowires (S-NiNW and L-NiNW, respectively) and nickel nanoparticles (NiNP). After 24 h the media were aspirated and analysed for secretion of
the acute phase response cytokines (A) interleukin-1b (IL-1b), (B) interleukin-6 (IL-6), (C) tumour necrosis factor-alpha (TNF-a) and (D) the
chemokine CCL3 (MIP-1a). A cytocentrifugation preparation stained with Diff Quik was made of the treated cells to show cellular uptake of
S-NiNW (E) and L-NiNW (F). Scatter plot with mean of four experiments s.e.m. Signicance vs. vehicle control indicated by **p < 0.01,
***p < 0.001 and vs. L-NiNW #p < 0.001. All images were taken at 1000 magnication.

response was not as marked between the long and short

bres as seen in the peritoneal cavity reecting the difference
in structure of these two areas and possibly tempo of
inammatory response. The nature of an instilled particle
dose into the lung associated with aspiration (as opposed to
inhalation) is that the dose rate is substantial, i.e., instantaneous delivery of dose, meaning that the initial response is
intense and then clearance processes come into play and the
inammation wanes. However, in comparison to the peritoneal cavity, the clearance rate in the lung is likely to be
slower resulting in a longer retention of dose of leading to
some associated effects with small particles (e.g., as seen
with the NiNP). Within the peritoneal cavity and most likely
within the pleural cavity where clearance routes are similar,
the outow of small particles via the diaphragmatic stomata

to outlying lymph nodes is very rapid (in contrast to long

bres which cannot negotiate the stomata) meaning a dose is
not retained over a sufcient period of time to elicit any form
of pathology. Indeed, when comparing the pathology of the
two locations, we see that aspiration of spherical NiNP into
the lungs led to alveolar deposition and diffuse alveolitis at
7 days but did not appear to affect terminal bronchioles or
alveolar duct bifurcations. In contrast, the long bres generated numerous granulomas associated with bres and
showed prominent collagen deposition. The frequent occurrence of these granulomas at terminal bronchioles and
alveolar ducts is well described (Brody et al. 1984) and is
the result of interception of the long bres at this narrowing.
The resultant frustrated phagocytosis may mean that macrophages are unable to clear the long bres to the ciliated

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C. A. Poland et al.
epithelium of the terminal bronchiole and so the granuloma
evolves around the retained bres. Within the early granulomas, the pro-inammatory effects of frustrated phagocytosis of the long bres likely leads to further cellular
recruitment at the site of deposition, leading to granuloma
enlargement and blocking of the airspaces. This process was
seen to a much lesser extent after aspiration of S-NiNW but
none the less, occasional granulomas were present at terminal bronchioles and alveolar ducts. To sum up, despite the
same chemical composition, differing morphology of the
nickel samples has led to differential patterns of deposition
and retention (rst alveolar duct bifurcation vs. peripheral
alveoli) and different cellular response (focal granuloma
formation vs. mild diffuse alveolitis). The alveolitis seen
within the L-NiNW treated animals may be an indirect result
of the presence of substantial bronchiolar lesions rather than
necessarily a direct interaction with L-NiNW. This is because
whilst the alveolitis was extensive, the presence of particles
was more focal within the lesions at the terminal bronchioles
(although some bres were noted in the distal alveolar
regions).
In their seminal 1981 paper (Stanton et al. 1981), Stanton
and colleagues discussed the results of 72 experiments
whereby respirable durable minerals of various sizes were
implanted into the pleurae of rats. The resultant malignant
mesenchymal neoplasms were recorded and the data analysed for correlation between the particle dimensions and
the ensuing neoplasms. They found that the probability of
developing such pleural pathology correlated best with bres
that were 8 mm in length and 0.25 mm in diameter, concluding overall that due to the wide variety of compounds
tested, the carcinogenicity of bres was dependent on
dimension and durability. In conclusion, our data presented
herein show that like asbestos and CNTs, NiNW show
length-dependent inammogenicity in the mouse peritoneal
cavity. In addition when instilled into the lung, long bres
were retained in the centri-acinar region following deposition, causing granulomas and inammation, whilst the SNiNW and NiNP had little effect beyond mild alveolar
inammation. The response to L-NiNW was not driven by
the chemical structure or soluble components as nickel in
other, compact forms did not induce a reaction nor did an
aqueous extract. This lends support to the contention that
the FPP is applicable to HARNs generally where they meet
the criteria outlined in the FPP (long, thin biopersistent).
Whilst our experimental data allow further conrmation of
the role of length in bre toxicity based on broad size classes,
further work is required to truly dene at what length a bre
becomes pathogenic.
There is concern that the production and use of NPs on an
industrial scale could lead to exposure and adverse health
effects in workers and the general population (The Royal
Society and Royal Academy of Engineering 2004; Maynard
et al. 2006; The Economist 2007). The success of the nanotechnology industry depends on public acceptance, regulatory compliance and acceptable risk. There is a considerable
need to supply hazard data, both quantitative and qualitative, for the risk assessment process. The data shown here
conrm our ndings with CNTs and suggest the real

possibility that all nanobres will conform to the FPP. The

UK Health and Safety Executive has taken steps to produce
guidelines on the risk management of CNT (The Health and
Safety Executive 2009) highlighting the importance of bre
length and applicability of their guidelines to other biopersistent nano-dimensioned bres (HARN) and the need to
characterise airborne nanobres in order to properly assess
the risk to the workers. Using the FPP as a basis for designing
safe HARN suggests that short or non-biopersistent long
HARN should be the goal.

Acknowledgements
The authors gratefully acknowledge nancial support from
the Colt Foundation, the help of Dr. Robert Morris for
histological assistance and Mr. Steven Mitchell for his electron microscopy expertise. This work was partly supported
by the European Commission FP7 NAMDIATREAM
(NMP-2009-246479) research project (F.B., A.P.M., Y.G.,
Y.V.) and Science Foundation Ireland as part of the MANSE
(SFI-PI grant) and CRANN CSET funded facilities.

Declaration of interest
The authors report no conicts of interest. The authors
alone are responsible for the content and writing of the
paper.

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Supplementary material available online

Appendix.
Supporting Information Available
Within the appendix section the results of the soluble extract of long nickel nanowires injected into the peritoneal
cavity, the total polymorphonuclear neutrophil (PMN) results within the BAL fluid post lung instillation of the
nickel particles and the results of the total lavagable cells and total lavagable PMN can be found.