TOXICOLOGICAL SCIENCES 124(1), 138–148 (2011)
doi:10.1093/toxsci/kfr206
Advance Access publication August 9, 2011
Bioavailability, Intracellular Mobilization of Nickel, and HIF-1a Activation
in Human Lung Epithelial Cells Exposed to Metallic Nickel and Nickel
Oxide Nanoparticles
Jodie R. Pietruska,* Xinyuan Liu,† Ashley Smith,* Kevin McNeil,* Paula Weston,* Anatoly Zhitkovich,*,‡ Robert Hurt,†,‡,§
and Agnes B. Kane*,‡,1
*Department of Pathology and Laboratory Medicine; †Department of Chemistry; ‡Institute for Molecular and Nanoscale Innovation; and §School of
Engineering, Brown University, Providence, Rhode Island 02912
1
To whom correspondence should be addressed at Department of Pathology & Laboratory Medicine, Warren Alpert Medical School, Brown University, Box G-E5,
Providence, RI 02912. Fax: (401) 863-9008. E-mail: [email protected].
Received May 26, 2011; accepted July 27, 2011
Micron-sized particles of poorly soluble nickel compounds, but
not metallic nickel, are established human and rodent carcinogens. In contrast, little is known about the toxic effects of
a growing number of Ni-containing materials in the nano-sized
range. Here, we performed physicochemical characterization of
NiO and metallic Ni nanoparticles and examined their metal ion
bioavailability and toxicological properties in human lung
epithelial cells. Cellular uptake of metallic Ni and NiO nanoparticles, but not metallic Ni microparticles, was associated with
the release of Ni(II) ions after 24–48 h as determined by Newport
Green fluorescence. Similar to soluble NiCl2, NiO nanoparticles
induced stabilization and nuclear translocation of hypoxiainducible factor 1a (HIF-1a) transcription factor followed by
upregulation of its target NRDG1 (Cap43). In contrast to no
response to metallic Ni microparticles, nickel nanoparticles
caused a rapid and prolonged activation of the HIF-1a pathway
that was stronger than that induced by soluble Ni (II). Soluble
NiCl2 and NiO nanoparticles were equally toxic to H460 human
lung epithelial cells and primary human bronchial epithelial cells;
metallic Ni nanoparticles showed lower toxicity and Ni microparticles were nontoxic. Cytotoxicity induced by all forms of Ni
occurred concomitant with activation of an apoptotic response, as
determined by dose- and time-dependent cleavage of caspases and
poly (ADP-ribose) polymerase. Our results show that metallic Ni
nanoparticles, in contrast to micron-sized Ni particles, activate
a toxicity pathway characteristic of carcinogenic Ni compounds.
Moderate cytotoxicity and sustained activation of the HIF-1a
pathway by metallic Ni nanoparticles could promote cell transformation and tumor progression.
Key Words: nickel; nanoparticles; HIF-1a; cytotoxicity;
apoptosis; cancer.
Inhalation of nickel compounds is an occupational hazard
associated with development of lung, nasal, and paranasal sinus
cancers (IARC, 1990; Straif et al., 2009). Inhalation exposure
to poorly soluble metallic nickel, nickel sulfides, and nickel
oxides occurs in production industries, while exposures in
nickel-use industries are complex and include poorly soluble
and soluble nickel compounds (Zhao et al., 2009). Welding
fumes are a potential source of metallic nickel particles in
a range of sizes (0.11–2 lm mass mean aerodynamic diameter)
with variable chemistry and crystallinity. Mixed exposures to
metallic nickel and its compounds in the workplace complicate
assessment of carcinogenicity of individual components
(Sivulka, 2005; Straif et al., 2009). Metallic nickel particles
and welding fumes are classified as possibly carcinogenic to
humans (IARC, 1990). In rodent inhalation studies, Ni3S2
particles (2.0–2.2 lm) and NiO particles (2.2–2.5 lm) induce
lung tumors in contrast to soluble NiSO4 6H2O aerosols that
did not induce lung tumors (Dunnick et al., 1995). Metallic
nickel particles produce lung tumors in rats following
intratracheal instillation (IARC, 1990) but not after inhalation
of metallic nickel powder (1.8 lm mass mean aerodynamic
diameter) for 24 months (Oller et al., 2008).
Nickel nanoparticles are manufactured for multiple applications including catalysts, sensors, and energy storage devices
(Zhang et al., 1998). Nickel is also used as a growth catalyst for
synthesis of some types of carbon nanotubes (Donaldson et al.,
2006), and although these catalyst residues appear to be
encapsulated inside the carbon shells, a fraction is typically
bioavailable (Liu et al., 2007). Inhalation exposure to nickel
and nickel oxide nanoparticles may emerge as a significant
occupational hazard as the nanotechnology industry expands
(Cho et al., 2010; Lu et al., 2009). Metallic nickel and nickel
oxide nanoparticles have been shown to induce significant lung
toxicity and inflammation following intratracheal instillation in
rats (Ogami et al., 2009; Zhang et al., 1998). In both of these
studies, ultrafine nickel (20 nm) and nickel oxide nanoparticles
(800 nm) were more toxic than fine nickel (5 lm) or fine nickel
oxide (4.8 lm) particles. Lung inflammation induced by nickel
oxide nanoparticles was comparable to the effects of crystalline
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NICKEL NANOPARTICLES INDUCE HIF-1a SIGNALING
silica (Min-U-Sil-5) after 6 months of exposure (Ogami et al.,
2009). Potential carcinogenicity of metallic nickel or nickel
oxide nanoparticles has not yet been assessed.
We have recently shown that nickel (II) ion mobilization
from metallic nickel powders or carbon nanotubes is enhanced
from nickel nanoparticles relative to microparticles (Liu et al.,
2007). Nickel carcinogenicity has been associated with
intracellular mobilization and delivery of nickel (II) ions to
target cells (Goodman et al., 2011). Soluble nickel salts show
minimal uptake, while poorly soluble nickel compounds persist
in particle form and are more readily taken up by phagocytosis
in cultured cells (Costa and Mollenhauer, 1980). Intracellular
nickel (II) ions activate multiple signaling pathways that may
contribute to carcinogenicity, including generation of reactive
oxygen species and activation of calcium-dependent and stressinduced signaling cascades (Barchowsky and O’Hara, 2003).
Epigenetic alterations in histone acetylation, chromatin organization, DNA methylation (Arita and Costa, 2009), and
interference with DNA repair pathways (Beyersmann and
Hartwig, 2008) may contribute to nickel carcinogenicity
(Salnikow et al., 2000; Salnikow and Zhitkovich, 2008).
Nickel (II) ions can deplete intracellular ascorbate (Karaczyn
et al., 2006; Salnikow et al., 2004) and inactivate prolyl
hydroxylases (Costa et al., 2005) leading to induction of
hypoxia-inducible factor 1a (HIF-1a) and expression of
hypoxia-inducible genes (Maxwell and Salnikow, 2004). By
mimicking hypoxia, nickel may provide selective pressure for
growth of cells with altered energy metabolism, altered growth
regulation, and resistance to apoptosis, promoting tumor
development (Salnikow and Zhitkovich, 2008). The ability of
metallic nickel nanoparticles to activate hypoxic signaling
pathways has not yet been assessed.
In this work, we sought to test the hypothesis that a shift to the
nano-size range in nickel-containing particles affects the
toxicological properties that may be associated with Ni(II)
carcinogenesis. We used an in vitro model of human lung
epithelial cells and a panel of well-characterized metallic nickel
and nickel oxide nanoparticles to investigate the cellular
responses to micro-sized versus nano-sized nickel-containing
particles. Specifically, we investigated particle internalization by
transmission electron microscopy, nickel mobilization in both
cell-free and cell-based systems by inductively coupled plasma
atomic emission spectrometer (ICP-AES) and Newport Green
fluorescence, respectively, and intracellular responses to nickelcontaining particles, including HIF-1a pathway activation and
cytotoxic responses. We demonstrate that nickel-containing
nanoparticles are taken up by human lung epithelial cells,
resulting in efficient delivery of intracellular nickel (II) ions,
activation of the HIF-1a pathway, and expression of hypoxiainducible genes. Additionally, exposure to nickel-containing
nanoparticles, but not microparticles, induces a cytotoxic response in human lung epithelial cells, in part due to activation of
an apoptotic program as indicated by immunoblot analysis of
cleaved caspases and poly (ADP-ribose) polymerase (PARP),
139
that is more robust than that induced by equivalent doses of
micro-sized nickel nanoparticles.
MATERIALS AND METHODS
Cell culture and treatments. The human lung epithelial cell line NCIH460 (H460) was obtained from ATCC and cultured in RPMI 1640 (Invitrogen,
Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Atlanta
Biologicals, Atlanta, GA) and 1% penicillin/streptomycin (Invitrogen) at 37C
in a humidified chamber containing 6% CO2/94% air. Normal human bronchial
epithelial (NHBE) cells were obtained from Lonza (Walkersville, MD) and
cultured in bronchial epithelial growth medium (BEGM), supplemented with
SingleQuot growth factors and retinoic acid. NHBE cells were used for
cytotoxicity experiments at passage 3–5. Nickel (II) chloride hexahydrate
(NiCl26H2O), metallic nickel nanoparticles (<100 nm Ni0, 99.9% purity), and
micron-sized particles (~3 lm Ni0, 99.7% purity) were purchased from SigmaAldrich (St Louis, MO). Nickel (II) oxide (green) nanoparticles (<100 nm) were
purchased from NanoAmor (Houston, TX). NiCl2 was diluted in deionized
water and sterile-filtered prior to use, while metallic nickel nano- and micropowders were sterilized at 400C for 15 min in a tube furnace while purging
with nitrogen to prevent oxidation. NiO nanoparticles were baked overnight in
air to inactivate endotoxin. Particles were sonicated for 1 h prior to the start of
each experiment to improve dispersion. H460 cells were exposed to soluble
nickel and insoluble nickel particles in RPMI 1640/2% FBS/1% P/S, while
NHBE cells were treated in BEGM.
Characterization. Materials morphology and electron diffraction (ED)
characterization were carried out on a JEOL JEM-2010 high-resolution
transmission electron microscope (TEM) at 200 kV and a Philips 420 TEM
at 120 kV. Particle crystallinity was determined by X-ray diffraction (XRD) on
a Bruker AXS D8 Advance X-ray Diffractometer. Zeta potentials and particle
size distribution were measured using a Zetasizer Nano-ZS 900 in ultrapure
water (18.3 MX), phosphate-buffered saline (PBS), and treatment medium
(phenol red-free RPMI 1640/1% penicillin/streptomycin/2% FBS) following
a 30-min sonication. Particle size and size distribution of <100-nm nickel
nanoparticles were also measured in the three different solutions by dynamic
light scattering spectroscopy. Surface areas of both nano and micro nickel
particles were determined from the Brunauer-Emmett-Teller (BET) N2 vapor
adsorption isotherms obtained at 77 K with an Autosorb-1 (Quantachrome
Instruments, Boynton Beach, FL), using the Brunauer-Emmett-Teller (BET)
model.
Endotoxin testing. Nano-sized and micron-sized metallic nickel particles
and nickel oxide nanoparticles were suspended in endotoxin-free water,
centrifuged, and the endotoxin levels in the supernatant were determined using
the Limulus Ameobocyte Lysate assay with Escherichia coli O113:H10
(Associates of Cape Cod Inc.) as a standard. Endotoxin levels in the nickel
particle preparations (<0.03 EU/ml) were not significantly different from the
negative control (<0.03 EU/ml).
Particle internalization. Cells were fixed at room temperature for
30 min in 2% glutaraldehyde in 0.1M sodium cacodylate buffer, pH 7.4.
Fixed samples were stored in cacodylate buffer with 8% sucrose at 4C.
Samples were subsequently treated with 1% osmium tetroxide in cacodylate
buffer and dehydrated through a series of graded ethyl alcohols. The
samples were infiltrated overnight with a 1:1 solution of 100% ethanol:Spurr
embedding medium (Electron Microscopy Science, Hatfield, PA) and then
infiltrated with fresh Spurr medium for 4 h, embedded in molds, and
polymerized at 60C. Blocks were sectioned at a thickness of 80 nm on
a Reichert Ultramicrotome with a diamond knife, placed on copper grids,
stained with uranyl acetate and lead, and viewed on a Phillips 410 TEM
equipped with an Advantage HR CCD camera and Advanced Microscopy
Techniques imaging software.
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PIETRUSKA ET AL.
Nickel mobilization. Nickel mobilization assays were carried out by
adaptation of the method of Liu et al. (2007). Briefly, nickel particles
suspended at 250 lg/cm2 were sonicated for 1 h prior to dilution in 10 ml
medium at 5 or 20 lg/cm2 in triplicate and incubated at 37C for 2–72 h, using
identical experimental conditions as for cell culture experiments. Suspensions
were centrifuged for 1 h at 4000 rpm, 4C in 3000 NMWL Amico centrifugal
filter devices (Millipore, MA) to remove any free nickel particles. The
concentration of mobilized nickel was measured using a Jobin Yvon JY2000
Ultrace ICP-AES at a wavelength of 221.647 nm. Intensities were calibrated
using six standards ranging in concentration from 0 to 5 ppm, which were
prepared from a 1000 ppm stock solution in treatment medium. Both nickelfree blanks and Ni salt standards were inserted in the test sequence after every
5–10 samples, and extensive rinsing after each measurement ensured no
carryover from high concentration samples.
Detection of intracellular mobilized ionic nickel. H460 cells were plated
onto glass coverslips at 12,500 cells/cm2 and treated as indicated. Cells were
washed with 1mM EDTA/Hanks’ Balanced Salt Solution (HBSS) to chelate
exogenous ionic nickel and loaded with 5lM Newport Green DCF diacetate
(Invitrogen)/Pluronic F127 (Invitrogen)/2% FBS/HBSS. After a 30-min
recovery period in 2% FBS/HBSS, Newport Green fluorescence was visualized
with a Nikon Eclipse E800 fluorescence microscope. Images were obtained
using SPOT software.
Western blots. Cells were plated at 12,500 cells/cm2, treated as indicated,
and adherent and floating cells were collected for Western blot analysis as
described in Pietruska and Kane (2007). Primary antibodies used were as
follows: HIF-1a (1:500; BD Pharmingen), NDRG1 (1:1000; Cell Signaling),
cleaved caspase-3 (1:1000; Cell Signaling), cleaved caspase-7 (1:1000; Cell
Signaling), PARP (1:1000; Cell Signaling), and b-actin (1:2000, AC-15;
Sigma-Aldrich). Membranes were incubated with horseradish peroxidase
(HRP)-conjugated goat anti mouse IgG (Millipore) or HRP-conjugated goat
anti-rabbit IgG (Cell Signaling) and processed for enhanced
chemiluminescence (Amersham, GE Life Sciences, Piscataway, NJ).
Cytotoxicity. Cell number was evaluated using the PicoGreen dsDNA Kit
(Invitrogen) according to the manufacturer’s instructions. H460 cells and
NHBE cells were plated at densities of 12,500 cells/cm2 or 23,400 cells/cm2,
respectively, in 96-well plates and treated as indicated prior to lysis in 200 lg/
ml proteinase K/13 Tris-ethylenediaminetetraacetic acid (TE) overnight at
37C. For H460 cells, which are hyperdiploid, the lysate was diluted 1:4 in 13
TE to avoid assay saturation. Using these assay conditions, PicoGreen
fluorescence was linear with respect to cell number (R2 ¼ 0.988) and DNA
content (R2 ¼ 0.999) (data not shown). For primary diploid NHBE cells, the
entire lysate was used for viability determination. PicoGreen was added at
a final concentration of 13 and fluorescence was measured in a SpectraMax
M2 fluorescence microtiter plate reader (excitation 480 nm/emission 520 nm).
Data are expressed relative to a mock-treated control at each time point.
Statistical analysis. Statistical significance was determined using one-way
ANOVA in conjunction with the Tukey post hoc test (Prism 5
GraphPad Software, La Jolla, CA), and the data were considered significant
when p < 0.05.
RESULTS
Particle Morphology and Characterization
Morphologies of metallic nickel and nickel oxide particles
are shown in Figure 1. Metallic nickel nanoparticles (<100 nm)
are approximately spherical (Fig. 1A, panels i and ii), while
micron-sized metallic nickel particles (3 lm) are nonuniform in
shape (Fig. 1B, panels i and ii). High-resolution TEM of both
<100-nm and 3-lm particles indicates thin (<5 nm) surface
oxide layers, which were characterized using ED patterns (Fig.
1A, panel iii and Fig. 1B, panel iii). Nano-sized metallic nickel
particles exhibit rings representative of face-centered cubic
(FCC) metallic nickel, including Ni (111), (200), (220), and
(222), with weak FCC nickel (II) oxide (NiO) patterns,
including NiO (200), (220), and (311) (Fig. 1A, panel iv). ED
rings for 3-lm particles were indexed as FCC Ni (111), (220),
and (311) with weak patterns present at NiO (200),(220), and
(311) (Fig. 1B, panel iv). Among these ED patterns, Ni (111)/
NiO (200) and Ni (220)/NiO (311) overlapped and are difficult
to distinguish. However, the presence of NiO patterns suggests
that the particles are slightly oxidized. The XRD patterns of
nano-sized and micron-sized nickel particles are shown in Figure
1D. Both nickel samples exhibit three sharp peaks near 2h ¼
44.6, 51.9, 76.4, which can be readily indexed as the (111),
(200), and (220) crystal planes of cubic phase Ni (JCPDS Card
No. 04-0850). Interestingly, small NiO peaks were visible (Fig.
2D, arrow, 2h ¼ 43.3, which can be indexed as crystalline NiO
[JCPDS 47-1049]). The presence of small NiO peaks in the
metallic nickel samples indicates slight oxidation of metallic Ni
nanoparticles, either during fabrication or during handling,
consistent with TEM and ED analysis.
The physicochemical properties of metallic nickel particles
were characterized in the dry state and in aqueous solutions
(Table 1), including ultrapure water, PBS, and the cell culture
medium (RPMI 1640 with 2% FBS) in which all cell-based
assays were performed. Metallic nickel particles aggregate in
aqueous solutions, resulting in an increase in mean particle
diameter relative to those observed for the dry particles using
TEM (Table 1, Fig. 1E). Zeta potential is seen to be both
particle dependent and medium dependent but similar for all
materials in the medium most relevant to in vitro cellular
toxicology studies (RPMI with 2% FBS).
Internalization of Nickel Particles and Intracellular Release
of Nickel (II) Ions
We used the H460 human lung epithelial cell line to
investigate the effects of nickel particles in a cell type relevant
to nickel carcinogenesis. H460 cells accurately recapitulate the
responses of primary human cells to carcinogenic metals
(Reynolds and Zhitkovich, 2007; Reynolds et al., 2009) and
are appropriate for studying internalization of particles and
fibers (Pietruska et al., 2010). We first verified that H460 cells
were capable of taking up nickel particles since we expect the
toxicity and carcinogenicity of insoluble nickel particles to
depend on their uptake by target cells (Costa and Mollenhauer,
1980). H460 cells internalized both nanoparticles and microparticles, and particles could be visualized within cytoplasmic
vacuoles after a 24-h exposure at a dose of 5 lg/cm2 (Fig. 2). In
order to determine whether ionic nickel was mobilized from
NiO nanoparticles or metallic nickel particles, nickel concentration in cell-free culture medium was measured after 2- to 72-h
incubation with nickel particles using identical experimental
NICKEL NANOPARTICLES INDUCE HIF-1a SIGNALING
141
FIG. 1. Characterization of nickel-containing microparticles and nanoparticles. (A) SEM (i) and TEM (ii and iii) of <100-nm metallic nickel nanoparticles.
Surface oxide films are indicated by the arrows in (iii). ED patterns (iv) indicate nickel oxide as well as zero-valent nickel. (B) SEM (i) and TEM (ii and iii) of 3-lm
metallic nickel particles. A very thin oxide layer is indicated by the arrow in (iii). ED patterns (iv) indicate nickel oxide in addition to zero-valent nickel. (C) SEM
(i) and TEM (ii and iii) of <100-nm nickel oxide nanoparticles. (D) XRD spectra of <100-nm and 3-lm metallic nickel particles (left) and <100-nm nickel oxide
nanoparticles (right). A small NiO (200) diffraction peak (arrow in left panel) indicates the slight oxidation of metallic nickel nanoparticles. The standard XRD
patterns for FCC nickel and nickel oxide are shown. Both nickel particles exhibit sharp diffraction peaks of Ni(111), Ni(200), and Ni(220) consistent with standard
FCC metallic Ni. The nickel oxide diffraction is consistent with FCC crystal structure but with significant peak broadening that suggests some disorder or very
small crystallites.(E) Size distribution of metallic nickel particles and nickel oxide nanoparticles in various suspension media relevant to in vitro studies.
conditions as for cell-based experiments. Nickel was readily
mobilized from NiO particles, and nickel mobilization reached
a plateau at 12–24 h (Fig. 3A). Incubation with equivalent
doses of metallic nickel nanoparticles resulted in mobilization
of over 40-fold less soluble nickel, and there was minimal
nickel mobilization from metallic microparticles. When nickel
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FIG. 2. H460 human lung epithelial cells internalize nickel-containing
microparticles and nanoparticles. TEM of untreated H460 (A) or H460
treated 24 h with 5 lg/cm2 <100 nm NiO (B), 5 lg/cm2 <100-nm metallic
nickel nanoparticles (C) or 5 lg/cm2 3-lm metallic nickel particles (D). Scale
bar ¼ 2 lm.
mobilization was expressed as a percentage of the total
available nickel, we observed that approximately 50% of the
nickel in NiO nanoparticles is mobilized within 24 h, while
only 1–3% of the available nickel in metallic nickel nanoparticles is mobilized, even after 72 h (Fig. 3B).
While these data indicate that ionic nickel can be mobilized
from nickel nanoparticles into the extracellular medium, we
wanted to determine whether exposure to nickel nanoparticles
also resulted in intracellular accumulation of ionic nickel in
H460 cells. In order to evaluate qualitatively the presence of
intracellular mobilized nickel, cells were exposed to NiCl2 or
nickel particles and loaded with Newport Green, a cellpermeant dye that fluoresces in the presence of ionic nickel.
Intracellular ionic nickel release was detected by Newport
Green fluorescence within 24 h of exposure to NiCl2 or NiO
nanoparticles and within 48 h of exposure to metallic nickel
nanoparticles (Fig. 3C).
In order to investigate cellular responses to intracellular
mobilized nickel, we evaluated activation of HIF-1a, which is
stabilized in response to nickel as well as in response to
hypoxia and induces transcription of numerous target genes
involved in hypoxia signaling (Salnikow and Zhitkovich,
2008). Compared to control, NiCl2 induced HIF-1a stabilization as early as 4–6 h, which persisted throughout a 24-h
exposure. NiO nanoparticles also induced robust HIF-1a
stabilization, although HIF-1a expression decreased at later
time points due to cytotoxicity (Figs. 4A and 5B). Exposure to
metallic nickel nanoparticles, but not to microparticles, resulted
in HIF-1a stabilization, albeit it to a lesser extent than mass
equivalent doses of NiO and with a delayed response (Fig. 4A).
In all cases, stabilized HIF-1a localized exclusively to cell
nuclei (Fig. 4B). In order to confirm that stabilized HIF-1a was
functional, we evaluated expression of N-myc downstreamregulated gene 1 (NDRG1), a HIF-1a target that is induced
following nickel exposure (Zhou et al., 1998). NRDG1 was
upregulated within 24 h of HIF-1a stabilization following
exposure to NiCl2, NiO nanoparticles, and metallic nickel
nanoparticles but not to metallic micron-sized particles. After
72 h, increased NDRG1 expression was also induced in control
cultures, consistent with previous reports of increased NDRG1
over time in culture (Karaczyn et al., 2006). Taken together,
these results indicate that ionic nickel mobilized from nickel
oxide and metallic nickel nanoparticles activates the HIF-1a
pathway in H460 cells.
Nickel Nanoparticles Induce Apoptosis in H460 Cells
In order to evaluate the cytotoxic effects of nickel particles,
we evaluated fluorescence of the DNA-binding dye PicoGreen
as a surrogate measure of cell number. We first confirmed that
NiCl2 and nickel particles did not interfere with the
fluorescence of PicoGreen (Fig. 5A). NiCl2 induced a dosedependent and time-dependent reduction in cell number during
TABLE 1
Physicochemical Characteristics of Nickel Particles
Zeta potential(mV)a
Sample
<100 nm Ni
3 lm Ni
<100 nm NiO
a
H2 O
PBS
RPMI
RPMI/2% FBS
Surface areab (m2/g)
Densityc (g/ml)
Crystallinityd
27 ± 2
25 ± 3
þ15 ± 1
29 ± 2
27 ± 4
20 ± 1
18 ± 2
17 ± 4
17 ± 2
12 ± 2
10 ± 1
15 ± 2
4.4
0.8
75
8.9
8.9
6.7
FCC
FCC
FCC, partially crystalline
Zeta potential of particles in solution measured after a 30-min sonication; means ± SE of eight replicates are shown.
Surface area measured at Brown by nitrogen vapor adsorption and BET analysis.
c
Manufacturer’s data.
d
Crystallinity determined by XRD.
b
NICKEL NANOPARTICLES INDUCE HIF-1a SIGNALING
143
FIG. 3. Differential mobilization of soluble nickel from nickel-containing microparticles and nanoparticles. (A) Time course of nickel mobilization from <100
nm NiO, <100-nm metallic nickel nanoparticles, and 3-lm metallic nickel particles into cell culture medium, where centimeter square refers to the area of the cell
culture dish, in order to match units to those used to specify dose in cell-based experiments. Upper panels represent the amount of nickel mobilized (ppm); lower
panels represent the percentage of total nickel that is mobilized. Means ± SE of three replicates are shown. *p < 0.05, **, p < 0.01, ***p < 0.001, <100 nm NiO
versus <100 nm Ni; #p < 0.05, ##p < 0.01, ###p < 0.001, Ni nanoparticles versus Ni microparticles, one-way ANOVA with Tukey Honestly Significant
Difference (HSD) post hoc test. (B) Newport Green fluorescence indicating intracellular ionic nickel after a 24-h exposure to NiCl2 or NiO or after a 48-h exposure
to <100-nm or 3-lm metallic nickel particles. On an equal Ni atom basis, 100lM NiCl2 is approximately equivalent to 5 lg/cm2 NiO nanoparticles or 4 lg/cm2
metallic nickel microparticles or nanoparticles.
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FIG. 4. Exposure to nickel-containing nanoparticles activates the HIF-1a pathway in H460 cells. (A) Western blot analysis of HIF-1a stabilization
and NRDG1 expression induced by NiCl2, <100 nm NiO, <100-nm metallic nickel nanoparticles, and 3-lm metallic nickel particles. (B) Nuclear localization of
HIF-1a in H460 treated 6 h with deferoxamine, 24 h with NiCl2 or <100 nm NiO, or 48 h with metallic nickel particles.
a 72-h exposure (Fig. 5B). Mass equivalent doses of nickel
particles exhibited different cytotoxic potential; NiO nanoparticles and metallic nickel nanoparticles, albeit it to a lesser
extent, exhibited dose-dependent and time-dependent toxicity,
as indicated by reduced cell number. Micron-sized metallic
nickel particles were not cytotoxic at any of the tested doses or
times (Fig. 5B). When normalized by the total amount of nickel
in the assay on a molar basis, we observed that NiCl2 and NiO
were approximately equitoxic, metal nickel nanoparticles had
intermediate toxicity, and metal nickel microparticles induced
no significant change in cell number (Fig. 5C). This differential
toxicity was not limited to the H460 cell line as the relative
toxicity of each particle was conserved in primary human
bronchial epithelial cells (Fig. 5C).
We next investigated whether the decrease in cell number
following nickel exposure was associated with induction of
apoptosis. Similar to soluble NiCl2, NiO nanoparticles induced
cleavage of caspase-3, caspase-7, and PARP in H460 cells
within 24–48 h of exposure (Figs. 6A and 6B). The loss of
PARP and b-actin observed after exposure to 20 lg/cm2 NiO
nanoparticles is likely due to extreme cytotoxicity as this
treatment reduced cell number by 75% (Fig. 4B). Metallic
nickel nanoparticles also induced cleavage of caspase-3,
caspase-7, and PARP following 48–72 h of exposure (Fig.
6C), while micron-sized metallic nickel particles did not induce
cleavage of caspases or PARP during a 72-h exposure (Fig.
6D), in agreement with the differential toxicity observed
following exposure of H460 cells to nickel-containing
particles. Taken together, these results indicate that nickelcontaining nanoparticles induce apoptosis in H460 cells,
consistent with the same mechanism of cell death induced by
ionic nickel.
DISCUSSION
Intracellular mobilization of nickel ions from poorly soluble
nickel particles is hypothesized to contribute to nickel
carcinogenicity (Goodman et al., 2011). In this study, nickel
microparticles and nanoparticles were internalized into cytoplasmic vacuoles by human lung epithelial cells (Fig. 2).
Bioavailability of nickel as assessed in an acellular assay was
minimal from metallic microparticles compared with mobilization of up to 3% of available nickel from metallic nickel
NICKEL NANOPARTICLES INDUCE HIF-1a SIGNALING
145
FIG. 5. Nickel-containing nanoparticles are cytotoxic to H460 and NHBE cells. (A) PicoGreen fluorescence is not quenched by NiCl2 or nickel particles.
k DNA standard was preincubated with NiCl2 and/or nickel particles prior to the addition of PicoGreen. Controls in which DNA (DNA) or PicoGreen (PG)
were omitted are also shown. Means ± SE of four replicates are shown. (B) NiCl2, NiO, and <100-nm nickel nanoparticles, but not 3-lm nickel particles, are
cytotoxic to H460 cells. Means þ SE of eight replicates are shown. *p < 0.05, **p < 0.01, ***p < 0.001, <100 nm NiO versus <100 nm Ni; #p < 0.05, ##p <
0.01, ###p < 0.001, Ni nanoparticles versus Ni microparticles, one-way ANOVA with Tukey HSD post hoc test. (C) Comparative toxicity of NiCl2 and nickel
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PIETRUSKA ET AL.
nanoparticles after 72 h and 50% of available nickel from NiO
nanoparticles after 24 h (Fig. 3B). The kinetics and magnitude
of intracellular nickel mobilization as assessed by Newport
Green fluorescence (Ke et al., 2007) corresponded with this
acellular assay, with increased fluorescence 24 h after exposure
to NiO nanoparticles followed by intracellular mobilization
from metallic nickel after 48 h. Lower Newport Green
fluorescence was observed 48 h after exposure to metallic
nickel microparticles (Fig. 3C). We conclude that intracellular
mobilization of nickel is related to surface area and thus
inversely related to particle diameter (Table 1; Fig. 1E)
confirming our previous acellular bioavailability assays using
metallic nickel particles (Liu et al., 2007). Increased toxicity
and intracellular mobilization of nickel following exposure of
human cells to NiO nanoparticles in comparison with NiO
microparticles was reported by Horie et al. (2009). NiO
nanoparticles have also been shown to have high lung toxicity
in acute in vitro and in vivo and in subchronic in vivo assays
(Cho et al., 2010; Lu et al., 2009; Ogami et al., 2009).
The relative potency and kinetics of intracellular nickel
mobilization from these nickel particles were compared by
stabilization and nuclear translocation of HIF-1a protein and
induction of NRDG1, also known as Cap43, a protein induced
by hypoxia and nickel (Zhou et al., 1998). Soluble nickel
rapidly activated the HIF-1a pathway, while NiO nanoparticles
induced early and sustained activation. Metallic nickel nanoparticles showed slower but also sustained activation, while
metallic nickel microparticles showed no activation after 48–72
h (Fig. 4).
Salnikow et al. (2000) showed that sustained activation of
the HIF-1a pathway is correlated with intracellular delivery of
nickel (II) ions. Davidson et al. (2005) reported that soluble
nickel enters cells by the divalent metal ion transporter and
competes with iron sites on prolyl hydroxylases. Inhibition of
HIF-prolyl-hydroxylase decreases binding of Von HippelLindau protein and stabilizes HIF-1a protein, resulting in
sustained activation of the HIF-1a pathway (Davidson et al.,
2005, 2006). Other members of the iron-dependent and
2-oxyglutarate-dependent dioxygenase family are potential
targets of nickel and may be associated with epigenetic
modifications including decreased methylation, chromatin
condensation, and inhibition of ABH3, a DNA repair enzyme
(Chen and Costa, 2009). These epigenetic effects, in addition to
activation of the HIF-1a hypoxia signaling pathway, are
hypothesized to contribute to the carcinogenicity of soluble
nickel and poorly soluble compounds (Costa et al., 2005). In
this study, metallic nickel and nickel oxide nanoparticles also
caused sustained activation of the HIF-1a hypoxia signaling
pathway in human lung epithelial cells, raising concern about
their potential carcinogenicity. This study shows that nanoparticles are a more efficient delivery vehicle for nickel ions
and are more potent in activating the HIF-1a hypoxia signaling
pathway than metallic nickel microparticles or soluble nickel.
For example, a 24-h exposure to 5 lg/cm2 <100 nm NiO
induced a stronger HIF-1a and NDRG1 response than a 24-h
exposure to 100lM NiCl2 (Fig. 5A) doses which represent
approximately equivalent amounts of nickel on a micromolar
basis. Thus, of the two mechanisms of Ni ion delivery into the
cell (ion transport vs. endocytosis with subsequent intracellular
Ni ion release), these studies suggests that endocytosis is the
more efficient delivery mechanism in this experimental model
system.
After 24–72 h, NiCl2 and NiO nanoparticles were equally
toxic to human lung epithelial cells followed by metallic nickel
nanoparticles, but not by metallic nickel microparticles, at
doses up to 20 lg/cm2 (Fig. 5). Similar ranking of toxicity was
observed in the H460 lung epithelial cell line as well as in
primary human bronchial epithelial cells. In order to investigate
the mechanism responsible for nickel-induced toxicity, we
evaluated markers of apoptosis: cleavage of caspase-3,
caspase-7, and PARP after 24 and 48 h of exposure. NiCl2,
NiO nanoparticles, and metallic nickel nanoparticles showed
a dose- and time-dependent cleavage of caspases and PARP as
markers of apoptosis; however, this does not exclude the
possibility that Ni-containing particles also induce necrosis in
a subset of the population. The signaling pathways responsible
for nickel-induced apoptosis are complex and cell-type specific
(Barchowsky and O’Hara, 2003). Regardless of the specific
signaling pathways involved in nickel-induced apoptosis, high
doses of soluble or poorly soluble nickel compounds are toxic
for human lung epithelial cells and may provide selective
pressure for survival and growth of cells resistant to nickelinduced toxicity (Salnikow and Zhitkovich, 2008).
Activation of the HIF-1a pathway following uptake of
metallic nickel and nickel oxide nanoparticles by human lung
epithelial cells has significant implications for potential
carcinogenicity of nanomaterials containing bioavailable nickel
(Maxwell and Salnikow, 2004). Soluble and poorly soluble
nickel compounds have been shown to activate the HIF-1a
pathway (Davidson et al., 2006; Salnikow and Zhitkovich,
2008) and to transform cells in culture (Biedermann and
Landolph, 1987; Costa and Mollenhauer, 1980; Patierno et al.,
1993). While soluble nickel (II) ions show acute toxicity at
high concentrations, poorly soluble nickel compounds produce
sustained HIF-1a activation leading to selection of transformed
cells resistant to acute nickel-induced toxicity and elevated
expression of HIF-1a responsive genes (Salnikow et al., 1999).
Overexpression of HIF-1a is frequently found in solid human
particles in H460 cells and NHBE cells. Toxicity is compared at equal doses on a total Ni atom basis. Means ± SE of eight replicates are shown. *p < 0.05, **p <
0.01, ***p < 0.001, <100 nm NiO versus <100 nm Ni; #p < 0.05, ##p < 0.01, ###p < 0.001, Ni nanoparticles versus Ni microparticles, one-way ANOVA with
Tukey HSD post hoc test.
147
NICKEL NANOPARTICLES INDUCE HIF-1a SIGNALING
in vivo assays for surface reactivity, lung injury, and
inflammation (Cho et al., 2010; Lu et al., 2009). The results
presented in this paper suggest additional screening assays
including cellular bioavailability of metal ions and activation
of the HIF-1a pathway for toxicological evaluation of
nanoparticles containing nickel. New and existing commercial
nanomaterials of high priority for screening include nickel and
nickel oxide nanoparticles (Cho et al., 2010), nickel nanowires
(Byrne et al., 2009), and Ni-containing carbon nanotubes (Liu
et al., 2007), as well as nickel nanocomposites for medical
implants and drug delivery (Fadeel and Garcia-Bennett, 2010).
FUNDING
National Institutes of Health Superfund Research Program
(P42 ES013660, R01 ES016178); Environmental Protection
Agency STAR (R833862).
ACKNOWLEDGMENTS
We gratefully acknowledge Sara Pacheco for her assistance
with statistical analyses. A preliminary report of this work was
published in the Conference Proceedings, Volume 2—Implications on ‘‘International Perspectives on Environmental
Nanotechnology’’ sponsored by U.S. Environmental Protection
Agency Region 5, Superfund Division held on October 7–9,
2008, Chicago, Illinois. This research does not necessarily
reflect the views of either the National Institutes of Health or
the Environmental Protection Agency.
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