Kinetics and dissolution of intratracheally administered nickel oxide nanomaterials in rats
Shinohara et al. Particle and Fibre Toxicology
Kinetics and dissolution of intratracheally administered nickel oxide nanomaterials in rats
Naohide Shinohara 0
Guihua Zhang 0
Masashi Gamo 0
0 National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Ibaraki 305-8569 , Japan
Background: The toxicokinetics of nanomaterials are an important factor in toxicity, which may be affected by slow clearance and/or distribution in the body. Methods: Four types of nickel oxide (NiO) nanoparticles were single-administered intratracheally to male F344 rats at three doses of 0.67-6.0 mg/kg body weight. The rats were sacrificed under anesthesia and the lung, thoracic lymph nodes, bronchoalveolar lavage fluid, liver, and other organs were sampled for Ni burden measurement 3, 28, and 91 days post-administration; Ni excretion was measured 6 and 24 h after administration. Solubility of NiO nanoparticles was determined using artificial lysosomal fluid, artificial interstitial fluid, hydrogen peroxide solution, pure water, and saline. In addition, macrophage migration to trachea and phagosome-lysosome-fusion rate constants were estimated using pulmonary clearance and dissolution rate constants. Results: The wire-like NiO nanoparticles were 100% dissolved by 24 h when mixed with artificial lysosomal fluid (dissolution rate coefficient: 0.18/h); spherical NiO nanoparticles were 12% and 35% dissolved after 216 h when mixed with artificial lysosomal fluid (1.4 × 10−3 and 4.9 × 10−3/h). The largest irregular-shaped NiO nanoparticles hardly dissolved in any solution, including artificial lysosomal fluid (7.8 × 10−5/h). Pulmonary clearance rate constants, estimated using a one-compartment model, were much higher for the NiO nanoparticles with a wire-shape (0.069-0.078/ day) than for the spherical and irregular-shaped NiO nanoparticles (0-0.012/day). Pulmonary clearance rate constants of the largest irregular-shaped NiO nanoparticles showed an inverse correlation with dose. Translocation of NiO from the lungs to the thoracic lymph nodes increased in a time- and dose-dependent manner for three spherical and irregularshaped NiO nanoparticles, but not for the wire-like NiO nanoparticles. Thirty-five percent of the wire-like NiO nanoparticles were excreted in the first 24 h after administration; excretion was 0.33-3.6% in that time frame for the spherical and irregular-shaped NiO nanoparticles. Conclusion: These findings suggest that nanomaterial solubility differences can result in variations in their pulmonary clearance. Nanoparticles with moderate lysosomal solubility may induce persistent pulmonary inflammation.
Toxicokinetics; Intratracheal administration; Clearance rate constant; Lymph node; Dissolubility; Artificial biological fluid (Gamble's solution)
particle size is identical [
]. Therefore, the toxicity
and pulmonary clearance of NiO nanoparticles could be
associated with particle size and solubility. In addition, in
vivo nanoparticle dissolution should be considered, as well
as water solubility.
This study evaluated the relationship between the
toxicokinetics and biological solubility of NiO using an
artificial biological fluid, as biological solubility is
difficult to determine in situ. Further, we measured
pulmonary clearance kinetics, extrapulmonary
translocation, and excretion of four NiO nanomaterials,
which differed in particle size and biological solubility,
after intratracheal administration in rats. Finally, the
pulmonary clearance and lung-to-lymph translocation
rate constants of these four nanomaterials were
compared for a range of doses.
Preparation and characterization of NiO suspensions
Four NiO particles (A, B, C, and D) were used in the
present study. The names and manufacturers of these
NiO particles, as well as the suspension characteristics,
including primary particle size, surface area, and size of
the agglomerates in suspension, are shown in Table 1.
NiO particles (2 g) were sonicated in 50 mL pure
water for 2 h, followed by centrifugation at 1000 g for
30 min at 20°C. The supernatant was collected as a stock
suspension to remove the large aggregate of particles.
Suspensions of 0.67, 2.0, and 6.0 mg/mL for animal tests
and 2.0 mg/mL for biological solubility tests were
prepared by diluting the stock suspension with pure water.
There are growing concerns about the toxicity of
nanomaterials owing to the lack of information on their
potential risks in workers and the general population.
Nanomaterials with the same chemical formula may
exert different toxicities, depending on physicochemical
characteristics such as size, shape, and crystalline
structure. Studies have compared the toxicities of
nanomaterials with different physicochemical properties [
Pulmonary clearance and translocation to
extrapulmonary organs offer valuable insights into the inhalation
toxicity of nanomaterials. Recently, we showed that six
types of TiO2 nanoparticles of different sizes and shapes
had similar pulmonary clearance rate constants, while
TiO2 nanoparticles with an Al(OH)3 coating had much
lower pulmonary clearance rate constants [
displayed higher toxicity [
]. TiO2 nanoparticle toxicity
increases with decreasing particle size [
inflammation was observed in rats intratracheally instilled
with nickel oxide (NiO) nanoparticles, but was minimal
in rats intratracheally instilled with NiO
submicronsized particles [
Although NiO is often considered a poorly soluble Ni
compound, some NiO can be dissolved [
soluble Ni compounds are rapidly cleared from the lung and
exhibit higher toxicity, while inhaled insoluble NiO is
slowly cleared from the lungs and exhibits lower toxicity
]. Ni-containing compounds can induce pulmonary
], which could be related to the
dissolution rate . Solubility and cytotoxicity are higher
for black NiO than for green NiO, even though the
aDetermined by SEM (scanning electron microscopy, S4800, Hitachi High-Technologies Co., Tokyo, Japan) or TEM (transmission electron microscopy, JEM-2010,
JEOL, Tokyo, Japan) of 500 particles for each material
bDetermined by Brunauer-Emmett-Teller (BET) surface area analysis (GEMINI VII, Shimadzu Co., Kyoto, Japan) after drying
cDetermined by dynamic light scattering (DLS) (Zetasizer nano-ZS; Malvern Instruments Ltd., Worcestershire, UK)
d The small NiO D particle size, caused the particles to aggregate when the suspension was dried. Consequently, particle dimensions were difficult to ascertain
even with suspension dilution and spraying
e Reproducibiliy of dynamic light scattering (DLS) measurement for NiO B was not feasible
Primary Specific Converted spherical Number-based
particle sizea surface areab primary particle size agglomerate particle
[nm] [m2/g] based on the specific sizec (DLS measurement)
surface area [nm]
51 18 49
The concentration of the stock suspension was
determined by a standard weight analysis, where the
weightloss of the suspension was measured using a balance
(AUW220D; Shimadzu Co., Kyoto, Japan) after drying at
200 °C in a thermostatic chamber (ON-300S; Asone Co.,
Biological solubility of NiO nanoparticles
For these experiments, six solutions were used as artificial
biological fluids: Saline; hydrogen peroxide (266.7 and
13.3 μmol/L); artificial interstitium solution (Gamble’s
solution); artificial lysosomal solution; pure water. The
final concentrations of the hydrogen peroxide solution
(200 and 10 μmol/L) were selected based on previous
]. The artificial interstitium and artificial
lysosomal fluids were prepared using the method shown
in Additional file 1.
The nanoparticle suspension (10 mL of 2.0 mg/mL)
and artificial biological fluids (30 mL) were mixed and
shaken at 200 rpm. At 0.5–216 h after the mixing period
(12 time points), 2.5 mL fluid were sampled and filtered
at 6000 g for 30 min with an ultrafilter (50,000
molecular weight cut off [MWCO]; VS2032, Sartorius Stedim
Biotech, Aubagne, France) to separate the dissolved Ni
ions; a molecular weight of 50,000 is equivalent to
6–7 nm. Ultrapure water (1 mL) was then added to the
ultrafiltrate, and the samples were centrifuged at 6000 g
for 15 min; these steps were performed twice. The Ni
ion concentration (ng/mL) was then analyzed, fitted to
the curve, and the solution equilibrium was expressed
using the following equation:
Solid k⇄dis ion
The dissolution and solidification rate coefficients, kdis
(/h) and k’ (/h), respectively, were estimated using the
leastsquares approach with the solver function in Excel 2007.
Experimental procedure for animal testing
Two hundred and forty male F344/DuCrlCrlj rats (12 weeks
old; mean body weight 256 ± 11 g) obtained from Charles
River Laboratories Japan, Inc. (Kanagawa, Japan) were
anesthetized by isoflurane inhalation and intratracheally
administered pure water (as the negative control) or NiO
suspensions (0.67, 2.0, or 6.0 mg/kg body weight [BW]) at
1 mL/kg BW using a MicroSprayer® Aerosolizer (model
IA1B-R for Rat; Penn-Century, Inc., Wyndmoor, PA, USA).
Five rats in each group were euthanized by exsanguination
from the abdominal aorta under intraperitoneal
pentobarbital anesthesia (50 mg/kg BW) on days 3, 28, and 91
postNiO particle administration. Thereafter, the lungs were
lavaged twice with 7 mL physiological saline, as previously
]. After the bronchoalveolar lavage fluid (BALF)
sampling, the trachea, lungs, right and left posterior
mediastinal lymph nodes, parathymic lymph nodes, liver,
kidneys, spleen, and brain of each animal were dissected,
rinsed with saline, and weighed. The observation period of
91 days was chosen because the fast clearance pathway
could become less attributed to pulmonary clearance more
than 91 days after administration. The effect of slow
clearance can be evaluated in 91 days according to previous
studies that reported the half time of fast clearance as 25–
27 days [
] and 23–50 days [
], and half time of slow
clearance as 224 days [
] and 75–845 days [
found the differences in fast clearance rates, between doses
and mammalian species, to be small.
Additional tests were conducted using metabolism
cages to evaluate excretion in five rats from each group
administered NiO particles (2.0 mg/kg BW). The feces
and urine were collected between 0 and 6 h and 6–24 h
post-NiO administration. The rats were euthanized and
dissected 6 h and 24 h post-NiO administration. In
addition to the organs listed above, the small intestine,
large intestine, esophagus, and stomach were dissected
after collection of BALF and gastrointestinal contents.
Extracellular Ni ion fractions ([extracellular Ni ions]/
[extracellular Ni particles + extracellular Ni ions +
intracellular Ni particles + intracellular Ni ions]) in BALF
were determined after ultrafiltration.
All animals were treated in accordance with the
guidelines for animal experiments in our laboratory, which
adhere to the guidelines of the Ministry of the Environment,
Ministry of Health, Labour and Welfare, Ministry of
Agriculture, Forestry and Fisheries, and Ministry of
Education, Culture, Sports, Science and Technology. The
experiments were approved by the Animal Care and Use
Committee, Chemicals Evaluation and Research Institute,
and the Institutional Animal Care and Use Committee of
the National Institute of Advanced Industrial Science and
Pulmonary NiO clearance and translocation rate coefficients
We used the previously described [
] model shown in
Fig. 1 to express the pulmonary clearance and
translocation to thoracic lymph nodes. The rate constants (/day) for
pulmonary clearance (kLung) and translocation from the
lung to thoracic lymph node (kLung → Lym) were estimated
using eqs. (2) and (3), according to Shinohara et al. [
t ¼ 0 : BLung ¼ rD
t ¼ 0 : BLym ¼ 0
where BLung and BLym were the lung and total
thoracic lymph node burdens of NiO, respectively; D
was the administered dose of NiO (μg); t was the
time elapsed after administration (days), and r was
the fraction that reached the alveolar region following
NiO administration. The least-squares approach was
used for curve-fitting.
Samples were homogenized in ultrapure water and
then acid-treated prior to determining the Ni
content using inductively coupled plasma-mass
spectrometry (ICP-MS), under the conditions shown in
Table 2. We altered the acid-treatment methods for
NiO C and D because the recovery efficiency for
NiO C was not high when using the method for
NiO A and B.
Good linearity of the calibration curves for Ni using
ICP-MS was observed in a 0–20 ng/mL standard
solution (R2 > 0.999). Recovery efficiencies from Ni standard
solution-spiked samples (5 ng/mL) were >90% for most
of the samples (Additional file 2). Five nanograms per
milliliter of Ni standard solution were added to organ
samples every 10–20 samples, and the measured value
were corrected by the recovery efficiency. Based on the
operation blank for the organ tissue samples, the limit of
quantification was 2 ng/g for most organ tissues, 1 ng/
mL for BALF, 0.5 ng/mL for blood, and 0.5 ng for
trachea and lymph nodes.
Two-way repeated analysis of variance (ANOVA) with
Scheffe’s test was used to compare the study group
NiO concentrations following an F-test with SPSS
20.0 (IMD SPSS, Armonk, NY, USA). Since the
administered doses differed depending on BW, the
organ burdens are shown as normalized values where
the organ burden was divided by the BW at the time
of NiO administration. Percentages of administered
doses are also shown for organ burdens.
Biological solubility of NiO nanoparticles
The Ni ion concentrations increased over time for each
NiO nanoparticle tested in each solution (Fig. 2). NiO B
was 100% dissolved 24 h after mixing in lysosomal fluid,
while only 3.5–6.5% of NiO B had dissolved after 216 h in
the other five solutions. In contrast, approximately 11, 0.70,
and 33% of NiO A, C, and D, respectively, had dissolved
after 216 h in lysosomal fluid, while 2.3–3.7%, 0.14–0.51%,
and 3.9–6.3% of NiO A, C, and D, respectively, had
dissolved at 216 h in the other five fluids.
The dissolution rate coefficients in lysosomal fluid, kdis,
were 2–3 orders of magnitude higher for NiO B than for
NiO A, C, and D (Table 3). With the exception of artificial
interstitium fluid, the solidification rate coefficients, k’, did
not differ much between the NiO particles (approximately
1 × 10−2/h).
Organ NiO burdens after intratracheal administration
NiO burdens in BALF and in the lung (after BALF
sampling) were significantly higher (P < 0.01) in the
NiO-treated rats than in the control group from days 3
to 91, except for rats treated with NiO B on day 91
(Fig. 3, Table 4, Additional files 3 and 4). These NiO
burdens were dose-dependent. The lung burdens for NiO
A, C, and D decreased slowly over time, whereas
burdens for NiO B decreased rapidly to <1% of the
administered dose by day 28 post-administration. Higher
BALFto-total lung burden ratios were observed at lower doses
and with longer observation periods in rats treated with
NiO B; this trend was not observed for NiO A, C, or D
The NiO burdens in most of the thoracic lymph nodes
(total burden in the right and left posterior mediastinal
lymph nodes and parathymic lymph nodes) were
significantly higher in the groups dosed with NiO particles
than in the control group (Fig. 3, Table 4,
Additional files 3 and 4). Except for NiO B, the NiO burden
in the thoracic lymph nodes increased in a dose- and
time-dependent manner from day 3 to day 91, with
percentage burdens (total burden in the thoracic lymph
nodes relative to the administered dose) of 0.015–0.18%
(day 3) to 0.12–12% (day 91). In most rats, a higher NiO
burden was detected in the right and left mediastinal
lymph nodes than in the parathymic lymph node
(Additional file 5).
Although the liver NiO burdens in some rats were
higher than those in the negative control group, no clear
dose- or time-dependency was observed (Fig. 3). No
significant differences were observed in the NiO levels of
the kidney, spleen, and brain in the NiO-treated and
Pulmonary NiO clearance and translocation rate coefficients
The pulmonary NiO clearance rate coefficients, kLung, in
animals treated with NiO B were much higher than
those of animals treated with NiO A, C, or D (Fig. 4 and
Table 6). kLung showed an inverse correlation with dose
for NiO C, but this relationship was not observed for
NiO A, B, or D. The translocation rate coefficients from
lung to lymph nodes, kLung → Lym, increased in a
dosedependent manner for NiO A, C, and D (Fig. 4 and
Table 6). kLung → Lym was highest for NiO D, followed by
NiO A, C, and B.
Evaluation of short-term excretion
The total organ, feces, urine, and gastrointestinal Ni
content 6 h after administration of NiO A, B, C, and
D was 96, 74, 44, and 92%, respectively. The
distribution and excretion per initial (6-h) total NiO burden
are shown in Fig. 5. Ni excretion in urine for up to
24 h was higher for NiO B (35%) than for NiO A
(3.6%), while the Ni excretion of NiO D (2.1%) was
much higher than that of NiO C (0.33%). Ni
excretion levels in the feces for up to 24 h and in the
gastrointestinal contents at 24 h were higher for
animals treated with NiO B or C than for those exposed
to NiO A or D. Although the Ni levels in the
esophagus, stomach, small intestine, large intestine, liver,
and blood were low (< 1%), the burdens in the
kidneys and blood were significantly higher for NiO B
than for the other NiO particles. In addition, the
extracellular ion fractions in BALF were 8.3 and 9.7%
for NiO B at 6 and 24 h after administration,
respectively, whereas those for the other three NiO particles
were below the detection limit.
Representative images of H&E-stained lung tissue
sections are shown in Fig. 6. Sustained pulmonary
inflammation up to 13 weeks post-administration was
observed in NiO A and D, and inflammatory cells
(neutrophils and alveolar macrophages) tended to
increase over time. For NiO B, inflammatory cells were
observed 3 days post-administration, but decreased over
time with almost no inflammation after 13 weeks. NiO
C showed very mild lung effects at 3 days
postadministration, and pulmonary inflammation
disappeared 4 to 13 weeks post-administration. Neutrophil
counts in BALF supported these trends in pulmonary
inflammation for NiO A, B, C, and D (Table 7).
In the dissolution test, only NiO B dissolved rapidly in
artificial lysosomal fluid and more slowly in water, saline,
and artificial interstitium fluid; this finding suggested that
dissolution may occur in macrophage lysosomes following
phagocytosis, and would occur more rarely outside
macrophages. Pulmonary clearance rates were much faster in
rats treated with NiO B than in those treated with NiO A,
C, or D. The clearance rates of NiO B were much faster
than previously reported fast clearance rate constants for
insoluble particles [
]. This suggested that the
clearance pathway of NiO B was different from the clearance
pathways of other insoluble particles. For NiO B, the
relative percentage of the Ni found in BALF to total lung
burden correlated inversely with the dose and increased
over time, whereas those values were stable for the other
three NiO nanoparticles. NiO B was excreted in the urine
within 24 h of administration to a greater extent than the
other NiO particles tested. In addition, the Ni ion/total Ni
in BALF fraction for NiO B was higher than the
corresponding values for NiO A, C, and D. These data suggest
that the Ni ions dissolved from NiO B translocated from
the lungs to the blood and kidneys, and were excreted in
the urine as excretion of intravenously administered Ni
ions has been shown to be rapid .
The pulmonary half-life (= ln2/kLung) of NiO B was
calculated as 4.4–4.5 days, and 310–410, 59–170, and
82–110 days for NiO A, C, and D, respectively. The
halflife of NiO A, C, and D might be found to be longer if the
observation period was set to be longer than that in the
present study. This is because the effects of fast clearance
decrease comparing to slow clearance over time.
Previously, the pulmonary half-life of micron-sized, soluble
NiSO4 particles was determined to be 1.6 h, while that of
micron-sized, insoluble NiO and Ni3S2 particles was 92
and 90 days, respectively [
]. Differences observed
between NiSO4 and NiO B might be attributable to the
91 days after i.t.
1.2% ± 0.24%
0.60% ± 0.041%
high solubility of NiSO4 in water, whereas NiO B is highly
soluble in lysosomal fluid but only slightly soluble in
water, saline, and interstitium fluid. The pulmonary
halflife of six types of insoluble TiO2 particles has been
reported as 34–44 days and 52–94 days for <2 mg/kg and
6 mg/kg, respectively, while that of Al(OH)3-coated TiO2
particles was 64, 141, and 907 days for 0.67, 2, and 6 mg/
kg, respectively [
]. In addition, the slow clearance of NiO
C and Al(OH)3-coated TiO2 particles may be explained by
the toxicity of slightly dissolvable Ni or Al ions.
Particles in the alveolar region can be cleared by the
following two routes: Route 1: Lung → phagocytosis by
macrophage → macrophage transfer to the end of bronchi
→ tracheal ciliary motility after phagocytosis; route 2:
Lung → phagocytosis by macrophage →
phagosomelysosome fusion in macrophage → dissolution in
macrophage. NiO uptake via the alveoli into the pulmonary
circulation was not assessed in the present study because
intravenously administered insoluble TiO2 nanoparticles
have been reported to be trapped in the liver [
], and the
NiO burden in blood and liver was not high in the present
study. Routes 1 and 2 operate in parallel, and events
within each route occur in tandem. Therefore, kLung (/h)
can be expressed using the phagocytosis rate constant,
kphar (/h), the macrophage migration to the end of bronchi
rate constant, kmig (/h), the phagosome-lysosome fusion
rate constant, kfusion (/h), and the dissolution rate constant
in lysosomes, kdis (/h), using the following equation:
kLung ¼ kphar þ
1=kdisþ1=kfusion þ kmig
In the present study, as NiO nanoparticles were not
observed outside macrophages in the microscopic
Fig. 5 Ni content in organs and excretion 6 h and 24 h post-administration. The total recovery 6 h post-administration was 94, 74, 41, and 90%
for NiO A, B, C, and D, respectively
observation on day 3, kphar was estimated to be
approximately 0.05/h. Therefore, assuming that kphar
is 0.01, 0.05, or 0.1/h (52, 98, and >99% of particles
are estimated to be ingested by macrophages 3 days
after exposure), kfusion and kmig were estimated using
the determined values of kLung and kdis. Since kmig
was estimated to be constant with 1 × 10−1–1 × 10−5/h
of kfusion (Fig. 7) for NiO C only, the migration of
macrophages after phagocytosis was suggested to be
the rate-determining step (rate constant: 1 × 10−4–5 ×
10−4/h). In other words, fusion and dissolution (Route
2) do not contribute to the pulmonary clearance of
NiO C because of its low solubility in lysosomal fluid.
This suggestion is in accordance with the
dosedependent overload observed for NiO C (only) in the
animal experiment; this overload may reflect an
inhibition of macrophage migration. For NiO A, B, and
D, kfusion or kmig were estimated to increase in parallel with
increasing solubility in lysosomal fluid (B > D > A) (Fig. 7).
However, since overload was not observed in the
animal experiment, particles located within macrophages
did not affect kmig. Therefore, increases in kmig with
solubility cannot be explained, whereas increases in
kfusion with solubility appear to be reasonable,
indicating that phagosome-lysosome fusion represents the
rate-determining step. The kfusion values were
estimated to be 1 × 10−4, 1 × 10−2, and 2 × 10−4 for any
concentration of NiO A, B, and D, respectively.
Inhibition and/or promotion of phagosome-lysosome fusion
depends on the proteins around the particles [
The adsorbed proteins on the particle can differ
depending on the particle solubility.
For NiO B, the dissolution rate constant in lysosomal
fluid (0.18/h) was much higher than that in interstitium
solution (3.6 × 10−3/h), suggesting that NiO B dissolved in
macrophages immediately following fusion. This is
consistent with the findings that extracellular ion fractions in
BALF were much higher for NiO B than for NiO A, C, or
D. The elevated Ni concentrations in blood, kidney, and
urine of animals treated with NiO B, compared with those
receiving NiO A, C, or D, suggested that dissolved Ni was
cleared via blood to the kidney and excreted in urine. The
fairly low blood Ni level (< 1% 6 h after administration)
indicated that this process occurred rapidly.
NiO translocation to thoracic lymph nodes increased in
a time- and dose-dependent manner for NiO A, C, and D;
this observation has also been reported for other insoluble
particles (TiO2) [
], but not for NiO B. These data
suggest that dissolved Ni ions are distributed in the blood,
but are not transferred to the lymph or immediately
cleared from the lymph nodes.
The translocation of particles to lymph nodes indicates
an overload due to damaged epithelial cells, as previously
]; particles that are cleared more slowly are
transferred to the lymph nodes. In the present study,
however, translocation to the lymph nodes was faster for NiO
D than for NiO A, although pulmonary clearance rates
were faster for NiO D than for NiO A. These phenomena
are likely attributable to the greater solubility of NiO D in
lysosomal fluid. Since Ni ions were not transferred to the
lymph or immediately cleared from the lymph nodes, a
possible mechanism for translocation to lymph nodes
includes damage to epithelial cells, attributable to the
Ni ion from NiO A and D, as well as from
overloaded particles dissolving in the lysosome.
Particles that are soluble outside macrophages (e.g.
NiSO4) have been shown to induce greater acute
pulmonary inflammation than Ni compounds with lower
solubility (e.g. Ni3S2, NiO, and Ni(OH)2) [
]. Ag ions are
rapidly and directly toxic to organs, while nanoparticles
induce toxicity more slowly, after dissolution in the
Fig. 7 Estimated macrophage migration to end of the bronchi rate constant and phagosome-lysosome fusion rate constant. The estimate was
calculated for 0.01, 0.05, and 0.1 /h dissolution rate constants in the lysosome
]. Nanoparticles that are located within the
macrophage and are rapidly soluble, such as NiO B, may
induce acute pulmonary inflammation if the ion is toxic.
Soluble nanoparticles located outside the macrophage can
induce more severe inflammation than those within
macrophages because the ion concentrations are much higher
for the nanoparticles located outside the macrophage.
However, this acute inflammation would decrease rapidly
over time, because the dissolved Ni ions are rapidly
cleared from the lungs [
7, 8, 31
]. In contrast, moderately
soluble nanoparticles located within the macrophage, such
as NiO A and D, may induce persistent pulmonary
inflammation because of their longer retention and continual
release of Ni ions in the lysosome. NiO particles that
showed low solubility accumulated and induced chronic
pulmonary effects in rats and mice, while water-soluble
NiSO4 did not produce these effects [
]. In addition,
nanoparticles such as NiO D, which dissolve slowly within
macrophages induced only mild acute and persistent
pulmonary inflammation. These results suggest that
dissolution tests in biological fluids and ion toxicity data
can provide valuable information on the acute and chronic
toxicity of nanoparticles. However, nanoparticles with
moderate solubility and highly toxic ions (such as NiO A
and D) require animal testing using a relevant exposure
route such as intratracheal administration to obtain
toxicity and toxicokinetic data.
In the present study, we conducted intratracheal
administration of nanomaterials on rats for the evaluation
of inhalation exposure. There are some differences
between rats and humans and between inhalation exposure
and intratracheal administration. The fast clearance rate
was not different between humans and rats, while the slow
clearance rate was 2 or 3 times higher for rats than for
] and the fast clearance rate was 1-order higher
than the slow clearance rate [
]. Therefore, at least the
magnitude relationship of the clearance rate did not switch
under overload-level doses. In a previous study, there were
no significant differences in multi-wall carbon nanotube
retention between intratracheal administration and
] and the treatment groups had the same ranking
whether measured after intratracheal inhalation or after
instillation of tracer particles [
]. Therefore, we can
compare the clearance rate constants between different
nanoparticles using intratracheal instillation test on rats.
The present study measured the dissolution of
nanoparticles in six different solutions, including artificial lysosomal
fluid and artificial interstitium fluid. The tissue
distribution and clearance of four types of NiO nanoparticles with
different characteristics were determined 3, 28, and 91 days
after intratracheal administration in rats. In particular,
NiO nanoparticles that dissolve rapidly in artificial
lysosomal fluid can be easily cleared from the lungs. With
the exception of submicron NiO particles, pulmonary
clearance overload was not observed, suggesting that the
clearance mechanisms are not associated with
macrophage migration to the end of bronchi, but involve
dissolution in macrophage lysosomes.
Additional file 1: Contents of artificial lung interstitium fluid and
artificial lysosomal fluid. The reagents were dissolved in pure water one
by one beginning at the top (DOCX 20 kb)
Additional file 2: Recovery efficiencies from Ni-spiked samples (DOCX 18 kb)
Additional file 3: NiO burdens per initial body weight at the time of
administration. Values for (A) lung, (B) bronchoalveolar lavage fluid (BALF),
(C) trachea, and (D) lymph nodes are shown (DOCX 33 kb)
Additional file 4: NiO burdens per organ tissue weight. Values for (A)
lung, (B) bronchoalveolar lavage fluid (BALF), (C) trachea, and (D) lymph
nodes are shown (DOCX 33 kb)
Additional file 5: Percentage of Ni in each lymph node out of the total
Ni in all lymph nodes (DOCX 19 kb)
ANOVA: Analysis of variance; BALF: Bronchoalveolar lavage fluid; BW: Body
weight; ICP-MS: Inductively coupled plasma-mass spectrometry; NiO: Nickel
This work is part of the research program “Development of innovative
methodology for safety assessment of industrial nanomaterials”, supported
by the Ministry of Economy, Trade and Industry (METI) of Japan.
Ministry of Economy, Trade and Industry (METI) of Japan.
Availability of data and materials
The datasets supporting the conclusion of this article are included within the
article and its additional files.
NS and MG conceived the hypothesis and designed the study. SM, YO, TK,
NI, and MN performed the animal experiments. TS and KK prepared and
characterized the NiO suspension. NS and GZ analyzed the samples using
ICP-MS. NS analyzed the data. NS and MG wrote the manuscript. All authors
have read and approved the final manuscript.
Ethics approval and consent to participate
All animal were treated in accordance with the guidelines for animal
experiments of our laboratory, which adhere to the guidelines of the
Ministry of the Environment, Ministry of Health, Labour and Welfare, Ministry
of Agriculture, Forestry and Fisheries, and Ministry of Education, Culture,
Sports, Science and Technology, Japan. The present study was approved by
the Animal Care and Use Committee, Chemicals Evaluation and Research
Institute, Japan, and by the Institutional Animal Care and Use Committee,
National Institute of Advanced Industrial Science and Technology.
Consent for publication
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