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1172
VOLUME 114 |NUMBER 8 |August 2006
•
Environmental Health Perspectives
Research
An important step in assessing the toxicology
of particles is to determine their fate after
inhalation. Of particular interest to us are air-
borne ultrafine particles (UFPs; < 100 nm),
which are abundant in ambient urban air and
are of the same size as engineered nanoparti-
cles. Translocation to extrapulmonary sites
after respiratory tract deposition represents an
important mechanism for these particles to
cause direct effects in secondary target organs
(Oberdörster et al. 2005). The extent to
which this process occurs depends on several
factors including particle solubility, particle or
aggregate size, the site of deposition, and the
integrity of the epithelial lining. UFPs deposit
efficiently in all regions of the respiratory
tract, depending on their size; specifically, as
particle size decreases toward the smallest
UFPs, nasopharyngeal deposition increases
(International Committee on Radiological
Protection 1994).
Studies in rats have shown translocation of
soluble manganese compounds from the nose
along olfactory neuronal pathways to the
olfactory bulb (Dorman et al. 2004;
Henriksson and Tjälve 2000; Tjälve et al.
1996; Tjälve and Henriksson 1999) after
inhalation or intranasal instillation exposures.
Likewise, the few studies that have examined
the fate of UFPs deposited on the nasal
mucosa identified translocation along the neu-
ronal olfactory route as a pathway to the olfac-
tory bulb of the central nervous system
(CNS). These include early studies in non-
human primates, which demonstrated the
translocation of solid nanosized particles
(30 nm poliovirus; 50 nm silver-coated gold
colloids) along the axons of olfactory nerves
into the olfactory bulb (Bodian and Howe
1941a, 1941b; DeLorenzo 1970). We have
also shown that inhaled elemental carbon par-
ticles (
13
C; 35 nm, count median diameter)
accumulate in rat olfactory bulb after whole-
body inhalation (Oberdörster et al. 2004).
Regarding penetration into deeper brain
regions, Tjälve et al. (1995) demonstrated that
soluble ionic Mn instilled into the olfactory
chamber of pike has the ability to pass synap-
tic junctions and migrate from the olfactory
tract to more distal regions, including the
hypothalamus. Dorman et al. (2004) found
Mn in the striatum and cerebellum of rats
after subchronic inhalation exposure to a solu-
ble Mn salt (sulfate); however, this was attrib-
uted to uptake from the blood. Thus,
contributions to brain Mn levels from the
blood need to be considered and may also be
an issue for inhaled solid UFPs.
The effects of translocated particles in the
brain are also important to determine. For
example, preliminary information has
emerged from populations of welders that
some of them may develop parkinsonism
17 years earlier than the general population
(Racette et al. 2001). Welding produces high
amounts of fumes containing Mn UFPs
(Zimmer et al. 2002). Several recent epidemi-
ologic studies describe occupational exposure
ranges of approximately 0.01–5 mg/m
3
Mn
in fumes from various welding processes and
materials (Korczynski 2000; Li et al. 2004;
Sinczuk-Walczak et al. 2001). Conflicting
data emerge from animal studies, however,
regarding effects of inhaled Mn compounds
in the brain. Henriksson and Tjälve (2000)
reported changes in glial fibrillary acidic pro-
tein (GFAP) and S-100b, markers of astro-
cyte activation, in several brain regions from
rats exposed intranasally to Mn chloride.
However, Dorman et al. (2004) did not find
any evidence of changes in GFAP levels in the
brain after exposure to Mn sulfate or phos-
phate. Potential contributing factors to the
lack of concurrence in results include differ-
ences in the solubilities of the Mn salts used,
the doses, and the contribution of olfactory
epithelial damage.
In the present study, we sought to address
the hypothesis that a major translocation route
for inhaled poorly soluble Mn oxide UFPs
Address correspondence to A. Elder, Department of
Environmental Medicine, University of Rochester,
575 Elmwood Ave., Box 850, Rochester, NY 14642
USA. Telephone: (585) 275-2324. Fax: (585) 256-
2631. E-mail: Alison_Elder@urmc.rochester.edu
*Current address: Procter and Gamble Co.,
Cincinnati, Ohio.
Supplemental Material is available online at
http://www.ehponline.org/docs/2006/9030/suppl.pdf
We thank P. Wade-Mercer and N. Corson for
their excellent technical assistance.
This work was supported by the U.S. Environmental
Protection Agency (EPA; PM Center R827354),
National Institute of Environmental Health Sciences
(training grant ESO527873 to T.F.; ESO1247),
Department of Defense (MURI FA9550-04-1-0430),
and Department of Energy (W-31-109-ENG-38).
A.M.’s research was performed while at the National
Institute for Occupational Safety and Health.
The views expressed by the authors are their own
and do not necessarily reflect those of the U.S. EPA.
The authors declare they have no competing
financial interests.
Received 20 January 2006; accepted 20 April 2006.
Translocation of Inhaled Ultrafine Manganese Oxide Particles to the Central
Nervous System
Alison Elder,
1
Robert Gelein,
1
Vanessa Silva,
1
* Tessa Feikert,
1
Lisa Opanashuk,
1
Janet Carter,
2
Russell Potter,
3
Andrew Maynard,
4
Yasuo Ito,
5
Jacob Finkelstein,
6
and Günter Oberdörster
1
1Department of Environmental Medicine, University of Rochester, Rochester, New York, USA; 2Procter and Gamble Co., Cincinnati, Ohio,
USA; 3Owens Corning, Granville, Ohio, USA; 4Woodrow Wilson International Center for Scholars, Washington, DC, USA; 5Department of
Physics, Northern Illinois University, DeKalb, Illinois, USA; 6Department of Pediatrics, University of Rochester, Rochester, New York, USA
BACKGROUND:Studies in monkeys with intranasally instilled gold ultrafine particles (UFPs;
< 100 nm) and in rats with inhaled carbon UFPs suggested that solid UFPs deposited in the nose
travel along the olfactory nerve to the olfactory bulb.
METHODS:To determine if olfactory translocation occurs for other solid metal UFPs and assess
potential health effects, we exposed groups of rats to manganese (Mn) oxide UFPs (30 nm;
~ 500 µg/m3) with either both nostrils patent or the right nostril occluded. We analyzed Mn in
lung, liver, olfactory bulb, and other brain regions, and we performed gene and protein analyses.
RESULTS:After 12 days of exposure with both nostrils patent, Mn concentrations in the olfactory
bulb increased 3.5-fold, whereas lung Mn concentrations doubled; there were also increases in
striatum, frontal cortex, and cerebellum. Lung lavage analysis showed no indications of lung
inflammation, whereas increases in olfactory bulb tumor necrosis factor-αmRNA (~ 8-fold) and
protein (~ 30-fold) were found after 11 days of exposure and, to a lesser degree, in other brain
regions with increased Mn levels. Macrophage inflammatory protein-2, glial fibrillary acidic pro-
tein, and neuronal cell adhesion molecule mRNA were also increased in olfactory bulb. With the
right nostril occluded for a 2-day exposure, Mn accumulated only in the left olfactory bulb.
Solubilization of the Mn oxide UFPs was < 1.5% per day.
CONCLUSIONS:We conclude that the olfactory neuronal pathway is efficient for translocating
inhaled Mn oxide as solid UFPs to the central nervous system and that this can result in inflamma-
tory changes. We suggest that despite differences between human and rodent olfactory systems, this
pathway is relevant in humans.
KEY WORDS: brain, central nervous system, CNS, inhalation, intranasal instillation, manganese,
metals, nose, olfactory bulb, respiratory tract. Environ Health Perspect 114:1172–1178 (2006).
doi:10.1289/ehp.9030 available via http://dx.doi.org/ [Online 20 April 2006]
from deposits in the nose is to the olfactory
bulb in the CNS. We characterized the size,
oxidation state, and in vitro solubility of gas-
phase–generated Mn oxide particles and also
compared the translocation kinetics to the
olfactory bulb of Mn oxide and MnCl
2
that
were applied to the nasal epithelium of rats via
instillation. We then measured the accumula-
tion of Mn in lung, liver, and olfactory bulb
after repeated inhalation exposures with both
nares patent or with one naris occluded. We
show that Mn oxide UFPs are translocated to
and retained in the olfactory bulb (ipsilateral to
the patent naris only) and present evidence of
exposure-induced effects in that region of the
brain. These studies demonstrate the impor-
tance of UFPs size and of solubility in olfactory
translocation processes.
Materials and Methods
Animals. Specific pathogen-free male Fischer
344 rats (200–250 g body weight, 3 months of
age) were obtained from Harlan (Indianapolis,
IN) and housed in filter-top plastic cages in a
facility accredited by the International
Association for Assessment and Accreditation
of Laboratory Animal Care; animals had free
access to Purina rodent chow (5001; Purina
Mills, LLC, St. Louis, MO) and water. The
background concentration of particles in this
facility is extremely low (< 50 particles/cm
3
).
All animals were allowed to acclimate for at
least 1 week before use in experimental proto-
cols, which were approved by the University
Committee on Animal Resources (University
of Rochester). Animals were treated humanely
and with regard to the alleviation of suffering.
Generation of Mn oxide UFPs. We gener-
ated Mn oxide UFPs (~ 500 μg/m
3
; 18 ×
10
6
particles/cm
3
) via electric arc discharge
(Palas GmbH, Karlsruhe, Germany) in an
argon-filled chamber between two opposing
Mn rods (purity, 99.95%; Electronic Space
Products International, Ashland, OR).
Oxygen was introduced into the generator
(20 mL/min) to ensure the formation of the
metal oxide. Particle electrostatic charge was
brought to Boltzman equilibrium (
210
Po
source). Available instrumentation monitored
particle mass and number concentrations con-
tinuously as well as aerosol size distribution at
regular intervals [tapered element oscillating
microbalance (Rupprecht and Patashnik,
Albany, NY); condensation particle counter,
model 3022A, and scanning mobility particle
sizer, model 3080 (TSI Inc., St. Paul, MN)].
Gas-phase–generated Mn oxide UFPs
were collected for electron microscopic analy-
ses immediately after generation using a
point-to-plane electrostatic precipitator for
transmission electron microscopy (TEM) sam-
ples (In-Tox Products, Moriarty, NM). For
scanning electron microscopy (SEM) analysis,
the particles were suspended in methanol, one
drop placed on an SEM stub and desiccated
for 24 hr. Electron micrographs (Figure 1S in
Supplemental Material available online at
http://www.ehponline.org/docs/2006/9030/
suppl.pdf) showed that the aerosol was com-
posed of agglomerates with an equivalent
sphere diameter of approximately 30 nm.
Agreement between scanning mobility particle
sizer–derived and TEM-derived size distribu-
tions was very good. Primary particles varied
between 3 and 8 nm in diameter, depending
on the generation conditions.
Exposure of rats to Mn oxide UFPs. For
whole-body inhalation exposures, rats were
placed in compartmentalized, horizontal
flow chambers (31-L Lucite tank; airflow =
30 L/min). In some exposures, the right naris
was occluded according to methods outlined
by Brenneman et al. (2000). Briefly, a 3-mm
piece of polyethylene tubing [0.024 in. outer
diameter (OD)] was inserted into an 8-mm
piece of Silastic tubing (0.065 in. OD), the
ends of which were sealed to form smooth,
round edges. One day before exposure, rats
were lightly anesthetized with Halothane
(Cardinal Health Pharmaceutical Distribution,
Syracuse, NY), and the plug was inserted into
the right naris; a small amount of Duro Quick
Gel (Ethicon, Inc., Somerville, NJ) ensured
that the plug remained in place. Inhalation
exposures were for 6 hr/day, 5 days/week for
up to 12 days with both nares open (12 days
total for tissue Mn determinations; 11 days
total for gene and protein array analyses). With
one naris occluded, the exposure was for
2 days.
For intranasal instillation exposures, the
particles were collected on a filter and resus-
pended in sterile pyrogen-free saline with soni-
cation. Because nasally instilled material is
readily aspirated into the lower respiratory
tract in rodents, thus making olfactory mucosa
exposure less than optimal, we developed a
simple method to maximize the dose to the
olfactory mucosa. Rats were lightly anes-
thetized with Halothane (3%). The trachea
was visualized with a pediatric otoscope and
cannulated transorally with the plastic sheath
of a 14-gauge catheter. The plane of anesthesia
was maintained with Halothane (1.5%).
Breathing through the tracheal cannula
ensured that aspiration of the nasal instillate
into the lung did not occur. A 30-gauge nee-
dle covered with polyethylene tubing was
attached to a 1-mL syringe and the tubing was
inserted into the left naris (5 mm), after which
5–7 μg (in 30 μL) of the suspended particles
was slowly injected with the rats in a supine
position. The rats were kept supine for 5 min,
after which the Halothane was turned off and
the tracheal cannula was removed once the
animal regained consciousness. These methods
were used for both Mn oxide and MnCl
2
exposures.
Characterization of the oxidation state
and oxide form of Mn oxide UFPs. We
investigated the oxidation state of sampled
Mn oxide particles using electron energy loss
spectroscopy (EELS; GIF 2000 system; Gatan
Inc., Pleasanton, CA) and an analytical
TEM/STEM (Tecnai F20ST; FEI Co.,
Hillsboro, OR) operating in scanning trans-
mission electron microscopy (STEM) mode.
Mn L-electron and oxygen K-electron energy
loss edges were recorded from discrete
primary particles and clusters of particles. We
estimated elemental stoichiometry through
comparison of the integrated area under
energy loss edges after background subtrac-
tion, using Digital Micrograph (Gatan Inc.).
Mn oxidation state was further characterized
by comparing acquired spectra with pub-
lished Mn L-electron X-ray absorption spec-
tra for Mn oxide (MnO), Mn tetroxide
(Mn
3
O
4
), manganese sesquioxide (Mn
2
O
3
),
and Mn dioxide (MnO
2
) (Gilbert et al.
2003). Further information on the calculation
of the relative percentages of each state is
given in Supplemental Material available
online (http://www.ehponline.org/docs/
2006/9030/suppl.pdf).
Dissolution rate of Mn oxide UFPs. We
employed two methods to determine the dis-
solution rate of the gas-phase–generated Mn
oxide UFPs in solution: ultrafiltration and
dialysis. For ultrafiltration, samples (0.5 mg)
were dispersed in Mn-free physiologic saline
(1 mL, pH 7.4) using an ultrasonic bath.
Sample suspensions were injected into the inlet
flow line of the ultrafiltration sample cell
(Molecular/Por stirred cell, stirring system
removed; Spectrum Laboratories Inc., Rancho
Dominguez, CA) fitted with a 1,000-molecu-
lar-weight cutoff membrane (Molecular/Por
cellulose ester ultrafiltration membrane;
Spectrum Laboratories Inc.). The sample was
washed into the cell with an additional 2 mL
saline solution. For dialysis, the sample suspen-
sions were injected into the upper chamber of a
dialysis cell (Spectra/Por MacroDialyzer,
Spectrum Laboratories Inc.) fitted with a
1,000-molecular-weight cutoff membrane
(Molecular/Por cellulose ester asymmetric
membrane; Spectrum Laboratories Inc.). The
sample cells were then swirled to evenly dis-
perse the suspensions; after several hours, the
suspensions settled as an even layer on the
membrane filters. Inlet ports were connected to
a peristaltic pump for optimal flow; the outlet
ports were connected to a waste bottle. Outlet
lines were periodically connected to sample
tubes, and 50 mL of the filtrate or dialysate was
collected and analyzed for Mn by inductively
coupled plasma optical emission spectroscopy
(ICP-OES). For measurements of the dissolu-
tion rate above room temperature, the entire
sample cell was immersed in a water bath at
the desired temperature.
Translocation of inhaled Mn oxides to the CNS
Environmental Health Perspectives
•
VOLUME 114 |NUMBER 8 |August 2006
1173
Cellular and biochemical parameters in
bronchoalveolar lavage fluid. Animals were
euthanized with an overdose of sodium pento-
barbital (intraperitoneal, 50 mg/100 g body
weight) followed by exsanguination. As
described in detail elsewhere (Elder et al.
2000), the lungs, trachea, and heart were
removed en bloc, and the right lungs were
lavaged after weighing with a fixed volume of
sterile, pyrogen-free 0.9% saline (five times,
each with 5 mL), separating the first two
lavages for protein and enzymatic analyses.
Cells were pelleted by centrifugation (400 ×g)
for 10 min. The cells were pooled from all
lavage fractions for viability determination
(trypan blue exclusion), enumeration, and dif-
ferential analysis (Diff-Quik; Baxter Scientific,
Edison, NY). Total protein concentration and
lactate dehydrogenase and β-glucuronidase
activities were measured using commercially
available kits (Pierce Chemical Co., Rockford,
IL; Sigma, St. Louis, MO).
Analyses of metal content in tissue samples.
At the time of sacrifice, we removed the skin
and fur from the rats with a dedicated set of
instruments, and the carcass was thoroughly
washed before the removal of tissues; the car-
casses were then moved to a separate room for
excision of organs to be analyzed for metals
content with different sets of instruments. The
left lungs, 1 g of liver tissue, and several brain
regions (olfactory bulbs, striatum, trigeminal
ganglions, midbrain, frontal cortex, cortex,
cerebellum) were removed to measure their
Mn and iron content. In the intranasal instilla-
tion studies, right and left olfactory bulbs and
mucosa (including turbinates and cribriform
plate) were removed. We did not perfuse the
tissues because preliminary tests showed that
perfusion did not affect Mn levels. The tissues
were placed directly into Teflon digestion vials,
weighed, and wet ashed with ultrapure 70%
nitric acid (Baseline, SeaStar Chemicals Inc.,
Sidney, British Columbia, Canada). After ash-
ing, the tissue residue was resuspended in
HNO
3
and the concentration was adjusted to
2% with 18 MΩdeionized water before
graphite furnace atomic absorption spec-
troscopy analysis. Quantitation was achieved
through comparison to reference standards
(Standard Reference Material 1577b from
bovine liver; National Institute of Standards
and Technology, Gaithersburg, MD).
RNA preparation for array and blot analy-
sis. Tissue samples were prepared as previously
described (Carter and Driscoll 2001) using the
standard RNAzol protocol for array and blot
analysis. RNA was amplified using the primer-
specific Smart Probe Amplification Kit
(Clontech, Palo Alto, CA) designed specifically
for the Atlas array systems. Briefly, 50 ng of
total RNA was amplified using specific primer
sets for reverse-transcriptase polymerase chain
reaction (PCR) amplification to full length
double-stranded cDNA, which ensures ampli-
fication of representative original gene popu-
lation. Double-stranded cDNA generated
from the PCR amplification step was pooled
separately from control and treated groups,
respectively, and cDNAs were prepared with
standard reagents and the Rat 1.2 and Rat 1.2
II Array kits (Clontech) following the manu-
facturer’s instructions. Labeled cDNA was
generated during the first strand synthesis
supplemented with [α-
32
P]-deoxyadenosine
5´-triphosphate (3,000 Ci/mmol, 10 μCi/μL)
using Moloney Murine Leukemia Virus
(MMLV) reverse transcriptase (Clontech) and
purified by column chromatography. Details
of array membrane hybridization and array
analyses are given in Supplemental Material
available online (http://www.ehponline.
org/docs/2006/9030/suppl.pdf)
Protein array analysis. Protein was
extracted from lung and brain samples using
the BD Clontech Protein Extraction and
Labeling Kit (Clontech). Briefly, 1 μg of pro-
tein from each sample was pooled and used to
probe the RayBio Rat Cytokine Array I
according to the manufacturer’s protocol
(RayBioTech, Norcross, CA) (Lin et al.
2003). After 20 min exposure, the mem-
branes were scanned using a phosphorimager
(BioRad Molecular Imager Fx; Bio-Rad
Laboratories, Hercules, CA) and analyzed for
relative intensity of expression. Data are
expressed as fold change differences from the
unexposed controls.
Statistical analyses. We analyzed results
for statistical differences by one-way analysis
of variance with appropriate data transforms
using SigmaStat (Systat Software Inc., Point
Richmond, CA). Data were appropriately
transformed if an analysis of residuals suggested
deviations from the assumptions of normality
and equal variance. Differences between groups
were further analyzed using Tukey multiple
comparisons. Such comparisons were consid-
ered statistically significant when p≤0.05.
Results
Tissue distribution of Mn and effects in the
lungs and brain after repeated inhalation
exposure to ultrafine Mn oxide aerosols. We
exposed rats to either filtered air or to ultra-
fine Mn oxide aerosols (465 ± 94 μg/m
3
) for
6 hr/day for 12 days. There were no indica-
tions in terms of either cellular or biochemical
parameters to indicate that active lung inflam-
mation occurred as a result of exposure
(Table 1); the only statistically significant
changes found were decreases in lavage fluid
total cell number after 6 days of exposure and
β-glucuronidase activity after 12 days of expo-
sure. The Mn content in lung tissue increased
progressively and significantly after 6 and
12 days of ultrafine Mn oxide aerosol expo-
sure (Figure 1A), representing about a dou-
bling of the tissue Mn content. Inhalation of
Mn oxide aerosols for 12 days also resulted in
significant increases (~ 3.5-fold) in olfactory
bulb Mn content. Significant increases in Mn
content were also found in the striatum,
frontal cortex, and cerebellum after 6 and
12 days of exposure and in the cortex after
12 days of exposure (Figure 1A), although the
magnitude of these changes was much lower
than what was observed in olfactory bulb.
The increase in liver Mn content was smaller
than the increase in lung tissue (controls, 2.78
± 0.04 ng Mn/mg tissue; 6 days, 2.61 ±
1.61 ng Mn/mg tissue; 12 days, 2.91 ±
0.09 ng Mn/mg tissue). The tissue concentra-
tion of Fe also increased slightly, but signifi-
cantly, after exposure in lung, olfactory bulb,
and cerebellum (Figure 1B).
Gene microarray analyses were performed
on tissue samples obtained from various brain
regions after a total of 11 days of ultrafine Mn
oxide aerosol exposure (498 ± 69 μg/m
3
).
Figure 2A shows the relative increases in
expression for selected genes. Tumor necrosis
factor-α(TNF-α) gene expression was
increased in olfactory bulb, frontal cortex,
midbrain, and striatum. Protein expression of
TNF-αcorrelated with gene expression
increases (Figure 2B). Several other genes
involved in inflammation (e.g., macrophage
inflammatory protein-2) and stress responses
(e.g., GFAP) also showed 2-fold or greater
increases in expression over controls in the
olfactory bulb. Although most of the changes
occurred in olfactory bulb tissue, neuronal
cell adhesion molecule was also increased in
Elder et al.
1174
VOLUME 114 |NUMBER 8 |August 2006
•
Environmental Health Perspectives
Table 1. Summary of lavage data from 6- and 12-day ultrafine Mn oxide exposures in young male F-344 rats.
Mn exposure
Untreated controls 6 days 12 days
Total cells (× 107) 0.680 ± 0.070 0.505 ± 0.049* 0.651 ± 0.069
Percent AM 98.29 ± 1.22 99.44 ± 0.41 99.42 ± 0.52
Percent PMN 0.30 ± 0.12 0.07 ± 0.12 0.11 ± 0.19
Percent lymphocytes 1.04 ± 0.83 0.34 ± 0.42 0.47 ± 0.41
Percent viable 89.83 ± 3.67 92.79 ± 4.83 92.93 ± 0.51
Protein (mg/mL) 0.11 ± 0.01 0.15 ± 0.04 0.15 ± 0.02
LDH (nmol/min/mL) 69.50 ± 6.37 84.13 ± 25.80 79.66 ± 9.32
β-Glucuronidase (nmol/min/mL) 0.461 ± 0.093 0.497 ± 0.086 0.241 ± 0.048*
Abbreviations: AM, alveolar macrophage; LDH, lactate dehydrogenase; PMN, polymorphonuclear leukocyte. Values are
mean ± SD;
n
= 6/group for controls and 3/group for Mn-exposed rats.
*
Significantly different from control (
p
< 0.05).
the frontal cortex and midbrain (Figure 2A).
Several other genes exhibited increases in
expression that were slightly less than 2-fold
higher compared with controls.
Tissue distribution of Mn after inhalation
of ultrafine Mn oxide aerosols with one naris
occluded. The data presented above show that
Mn accumulation was the highest in the olfac-
tory bulb and lower in other brain regions,
consistent with olfactory translocation after
repeated inhalation exposures; however, the
Mn in those tissues could have arisen from
bloodborne Mn. In a separate set of experi-
ments, rats were exposed with the right nares
occluded for 6 hr on each of 2 consecutive
days. Three animals each were killed for tissue
Mn analyses immediately after the first expo-
sure and 18 hr after the first and second expo-
sures. Control animals with both nares open
were killed 18 hr after a 6 hr exposure; there
were also unexposed controls (0 hr). With both
nares patent, significant increases in lung Mn
content were found 18 hr after a 6-hr ultrafine
Mn oxide aerosol exposure (Figure 2S in
Supplemental Material available online at
http://www.ehponline.org/docs/2006/9030/
suppl.pdf), as was expected from the repeated
inhalation studies; liver Mn content also
increased. When the right naris was occluded
during exposure, lung tissue Mn content
increased 11-fold to 1.9 ± 0.05 ng/mg wet tis-
sue immediately after the 6-hr exposure; liver
Mn content increased from 2.5 ± 0.01 to 2.9 ±
0.08 ng/mg tissue. After 18 hr of recovery, sig-
nificant amounts of Mn remained in the lung
(21% of the amount deposited at the end of
exposure), but most of the deposited material
had been cleared, and no increase was observed
in liver tissue. Two consecutive days of expo-
sure (6 hr each) did not significantly alter the
retention kinetics of the ultrafine Mn oxide
aerosols deposited in the lung (Figure 2S in
Supplemental Material available online at
http://www.ehponline.org/docs/2006/9030/
suppl.pdf). Thus, clearance of Mn from the
lung under these exposure conditions is rapid.
Mn also accumulated in the left and right
olfactory bulbs when both nares were patent
(Figure 3). However, when the right naris
was occluded, Mn accumulated only in the
olfactory bulb on the patent (left) side.
Furthermore, Mn accumulation increased in
left olfactory bulb tissue, unlike the lung, with
time postexposure and exposure duration. A
small, but insignificant, amount of Mn also
accumulated in the right olfactory bulb after
two consecutive 6-hr exposures (1.2-fold
increase), potentially from bloodborne Mn due
to dissolution of the Mn oxide in alveolar
macrophages (Lundborg and Camner 1984;
Lundborg et al. 1985) or due to transport
between the nares via a small perforation in the
rat nasal septum, as has been previously
described (Kelemen and Sargent 1946).
Mn oxide UFP oxidation state and solubil-
ity. EELS analysis of primary particles and
agglomerates indicated a mean stoichiometry
close to MnO. Comparison of Mn L-electron
edge structure with published spectra for differ-
ent oxidation states indicated that the particles
were composed of MnO [61%, Mn(II)] and
Mn
2
O
3
[39%, Mn(III)] (Figure 4). At room
temperature, both the ultrafiltration and dialy-
sis experiments showed that the Mn oxide
UFPs dissolved at a rate of 1–1.5% per day
(i.e., Mn detected in outflow via ICP-OES) at a
neutral pH similar to the nasal mucosal milieu.
Subsequent dialysis experiments also showed
that temperature, solution flow rate, and time
(up to 10 days) did not affect the dissolution
rate. However, acidification to pH 4.5, similar
to the phagolysosomal conditions of alveolar
macrophages (Lundborg et al. 1985), resulted
in rapid dissolution. This soluble fraction most
Translocation of inhaled Mn oxides to the CNS
Environmental Health Perspectives
•
VOLUME 114 |NUMBER 8 |August 2006
1175
Figure 1. Mn and Fe contents in lung and brain tissues after 6 and 12 days of inhalation exposure to ultrafine Mn oxide aerosols. (
A
) Mn content in lung and brain
tissues in controls (
n
= 5) and after 6 (
n
= 3) and 12 (
n
= 3) days of exposure. (
B
) Fe content in lung and brain tissues in controls (
n
= 5) and after 12 days of exposure
(
n
= 3). Values are mean ± SE.
*
p
< 0.05 versus filtered air-exposed controls.
120
100
80
60
40
20
0Lung Olfactory
bulb Striatum Midbrain Frontal
cortex Cortex Cerebellum
Fe (ng/mg tissue)
*
*
A B
2.0
1.5
1.0
0.5
0
Mn (ng/mg tissue)
Controls
6 days
12 days
Trigeminus Midbrain Cerebellum
*
*
Cortex
*
Frontal
cortex
*
*
Striatum
*
*
Lung
*
*
Olfactory
bulb
*
*
*
Trigeminus
8
7
6
5
4
3
2
1
0
Controls
Relative intensity (fold increase)
Relative intensity (fold increase)
Midbrain CerebellumFrontal
cortex StriatumOlfactory
bulb
TNF-
α
MIP-
2
GFAP
Neurogenin
MnSOD
NCAM
Na-K-Cl cotransporter
ATP-related K-channel
A B
40
30
20
10
0Olfactory
bulb Frontal
cortex Midbrain Striatum Cerebellum
Figure 2. Gene and protein expression changes, by brain region, from pooled samples after 11 days of inhalation exposure to ultrafine Mn oxide aerosols.
(
A
) Gene expression changes represented as relative intensities (fold increase over normalized control, dashed line). (
B
) TNF-αprotein expression changes (rela-
tive intensities, fold increase over normalized control). Abbreviations: MIP-2, macrophage inflammatory protein-2; MnSOD, manganese superoxide dismutase;
NCAM, neuronal cell adhesion molecule.
likely contributed to bloodborne Mn that was
then distributed throughout the body.
Olfactory bulb uptake of Mn after intra-
nasal instillation of solid ultrafine Mn oxide or
a soluble Mn salt. The issue of whether or not
the solubilization of ultrafine Mn oxide was a
prerequisite for its translocation along the
olfactory nerve was still outstanding after the
experiments described above. Given the low
solubilization rate (1–1.5% per day) of ultrafine
Mn oxide at neutral pH, one would predict
that only this small fraction of Mn oxide
deposited on the olfactory mucosa would be
translocated to the olfactory bulbs in its soluble
form. In order to test this, we instilled 30 μL
of soluble MnCl
2
(5.3 μg Mn) or saline-sus-
pended Mn oxide UFPs (6.7 μg Mn) into the
left naris of anesthetized rats. Despite the fact
that only 1.5% of the Mn in the oxide form
was soluble at a maximum, similar Mn burdens
(given as a percentage of the instilled Mn) were
found in the left olfactory bulb tissue 24 hr
after intranasal instillation of the chloride (8.2 ±
3.6%) or the oxide (8.2 ± 0.7%). Furthermore,
less Mn from the oxide was found on the cribri-
form plate area and the turbinates (representa-
tive of the olfactory mucosa), indicating greater
retention of the MnCl
2
by those tissues
(Figure 5A). In another group of rats, olfactory
bulb uptake of intranasally instilled Mn oxide
occurred rapidly (within 30 min) after expo-
sure, although to a very small degree (0.2% of
amount found on olfactory mucosa at 30 min);
however, this had increased to 6.8% after 24 hr
(Figure 5B). In another experiment, Mn oxide
UFPs that were instilled into the left naris
translocated primarily to the left olfactory bulb,
although a small amount also appeared in the
right olfactory bulb, possibly due to some expo-
sure of the right side of the nose via the nasal
septal window (Kelemen and Sargent 1946)
(Figure 5C). These data indicate that the
appearance of Mn in olfactory tissues cannot be
due to soluble Mn but, rather, that solid UFP
transport occurs efficiently.
Discussion
Traditionally, the respiratory tract is consid-
ered a target organ for effects of inhaled solid
particles. However, more recent evidence from
epidemiologic, controlled clinical and animal
studies with ambient particulate air pollutants
shows that extrapulmonary organs are also
affected (U.S. EPA 2004). Specifically, it has
been hypothesized that inhaled UFPs accumu-
late and cause effects in extrapulmonary organs,
such as the cardiovascular system and CNS
(Donaldson and Stone 2003; Oberdörster et al.
2005; Oberdörster and Utell 2002) because of
their propensity to translocate across epithelial
barriers. Indeed, in our present study we
demonstrate that 6- to 12-day inhalation expo-
sure of rats for to solid Mn oxide UFPs resulted
in significant increases of Mn in several brain
regions, most notably the olfactory bulb. The
fact that occlusion of the right naris during
inhalation for 2 days of the nanosized Mn oxide
led to accumulation of Mn only in the left
olfactory bulb confirmed that translocation
from nasal deposits along the olfactory nerve
accounts for this increase.
The olfactory translocation route has been
well demonstrated for soluble Mn com-
pounds (Dorman et al. 2004; Tjälve and
Henriksson 1999), and it has been suggested
that solubility is an important determinant of
the efficiency of this process (Dorman et al.
2001). Dorman et al. (2004) measured the
translocation of poorly soluble Mn phosphate
into olfactory bulb, cerebellum, and striatum.
In that study, only the olfactory bulb, and not
the more distal structures, demonstrated a
small increase in Mn content. One important
difference to our study, however, is that the
phosphate particles had a mass median aero-
dynamic diameter of 1.6 μm, whereas the
count median diameter of the Mn oxide par-
ticles used here was 31 nm. The axons of
olfactory neurons narrow to a diameter of
approximately 200 nm and become tightly
packed where they pass through the cribri-
form plate pores (DeLorenzo 1957; Plattig
1989). Thus, in order for solid particles
deposited on the olfactory mucosa to be
transported through the cribriform plate, they
should be < 200 nm in size. We suggest,
therefore, that particle size plays a most
important role and that dissolution is not a
prerequisite for neuronal uptake and trans-
location of solid UFPs in the absence of
mucosal injury. Indeed, this suggestion is
based on earlier studies in nonhuman pri-
mates by Bodian and Howe (1941a, 1941b)
and by DeLorenzo (1970) with intranasally
instilled 30 nm poliovirus and 50 nm silver-
coated gold particles, respectively, and on our
recent study in rats with inhaled carbon-13
particles (median size, ~ 30 nm; Oberdörster
et al. 2004) showing translocation along
olfactory neuronal pathways.
The Mn oxide UFPs used in the present
study dissolved at a rate of about 1.5% over
24 hr in physiologic saline at neutral pH.
However, greater solubilization of Mn oxide
has been demonstrated at approximately pH
4.5, as would be encountered in the phago-
lysosome of alveolar macrophages (Lundborg
and Camner 1984; Lundborg et al. 1985).
Given that the nasal mucosa has a pH close to
neutral (Washington et al. 2000) and free
macrophages are not normally present on the
nasal epithelial surface (Harkema J, personal
communication), rapid solubilization of the
intranasally instilled Mn oxide UFPs in our
study is unlikely. We conclude, therefore, that
the increase in olfactory bulb Mn in our study
Elder et al.
1176
VOLUME 114 |NUMBER 8 |August 2006
•
Environmental Health Perspectives
6 hr/
0 hr
0 hr 6 hr/
18 hr 2 × 6 hr/
18 hr 6 hr/
18 hr
Both nares patent
6 hr/
0 hr
0 hr 6 hr/
18 hr 2 × 6 hr/
18 hr
Exposure/
postexposure 6 hr/
18 hr
*
*
**
Left Right
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Mn (ng/mg wet weight)
Figure 3. Accumulation of Mn in right and left olfactory bulb after inhalation exposure to ultrafine Mn oxide
aerosols with the right naris occluded. Exposure duration and postexposure time are shown on the
x
-axis.
Tissues were obtained from the same rats as in Figure 2S in Supplemental Material available online (http://
www.ehponline.org/docs/2006/9030/suppl.pdf). Values are mean ± SE.
*
p
< 0.05 versus 0 hr.
1.2
1.0
0.8
0.6
0.4
0.2
0635 640 645 650 655 660 665 670
61%
39%
Mn2O3
Mn3O4
MnO
MnO2
Measured
spectrum
Mn L edge
Energy loss (eV)
Intensity (arbitrary)
Figure 4. Approximate identification of component
oxides by comparing measured EELS edge structure
of gas-phase–generated Mn oxide to reference
oxide spectra.
is due to neuronal uptake and translocation of
the solid Mn oxide UFPs. Support for this sug-
gestion comes also from the results of our com-
parison of intranasally instilled Mn oxide to
soluble MnCl
2
. If solubilization of the Mn
oxide UFPs were a prerequisite for neuronal
translocation of Mn, one would expect that
significantly less Mn would accumulate in the
olfactory bulb for instilled Mn oxide compared
with MnCl
2
because of the low solubilization
rate of the oxide. Instead, it was the same for
both compounds. In addition, one would
expect more of the Mn oxide than the chloride
to be retained on the olfactory mucosa; how-
ever, the opposite was found (Figure 5A).
Finally, the accumulation kinetics are consis-
tent with a rapid translocation velocity of solid
particles in axons (up to 6 mm/hr; Adams and
Bray 1983).
Another issue to consider is that Mn oxide
UFPs that deposited in the alveolar compart-
ment of the rat respiratory tract in our study
may have undergone dissolution in alveolar
macrophages (Lundborg and Camner 1984;
Lundborg et al. 1985) followed by diffusion of
soluble ionic Mn into the blood circulation. In
addition, Mn oxide UFPs may translocate
across the alveolar–capillary barrier and the
blood–brain barrier in regions where it is dis-
continuous (e.g., choroid plexus, ventricles,
brain stem, hypothalamus). Dissolution and
absorption of Mn oxide particles deposited in
the respiratory tract and cleared into the GI
tract via mucociliary action could also con-
tribute to bloodborne Mn; however, this will
only be a small fraction, < 5% of the ingested
amount (Oberdörster and Cherian 1988).
Although it is likely that bloodborne ionic Mn
contributed to the increase in cerebellar Mn,
the degree to which this may have contributed
to Mn in other brain regions is not known. It is
also possible that Mn crosses the blood–brain
barrier via competition with Fe (Aschner et al.
2005); both Fe and Mn can complex with
transferrin in the blood and thereby compete
for uptake into the CNS capillary endothelium
via the transferrin receptor (Malecki et al.
1999). In addition, Mn appears to facilitate the
transport of Fe into the brain after chronic
exposure (Zheng et al. 1999). Our finding of
higher Fe levels in olfactory bulb and cerebel-
lum after Mn oxide inhalation (Figure 1) may
be related to competitive or facilitated trans-
port, but this needs to be investigated in fur-
ther studies (Fe was not present in the exposure
atmosphere). Because the tissues were not per-
fused, some of the Fe could have come from
the blood, and future studies should be done
using perfused tissues.
Frontal cortex and striatal Mn increases in
our study may have been due either to a contri-
bution from bloodborne Mn or to neuronal
translocation from olfactory bulb to more dis-
tal brain structures. Large molecules, approach-
ing the size of nanoparticles (wheat germ
agglutinin–horseradish peroxidase complex),
were found to translocate from the nasal cavity
to the olfactory bulb and more distal structures
by crossing synapses (Shipley 1985), as was
also observed with herpes virus (McLean et al.
1989). Thus, although ionic bloodborne Mn
can contribute to increased Mn levels in some
brain regions, translocation of solid Mn oxide
UFPs from nasal deposits to CNS regions via
the olfactory nerve should not be excluded as a
possible mechanism.
Using a predictive particle deposition
model for the rat (multiple path particle
dosimetry model; Asgharian and Anjilvel
et al. 1998), we estimated from our data that
about 11.5% of the amount deposited on the
olfactory mucosa (369 ng) was translocated to
the olfactory bulb (42.5 ng). Model inputs
included the Mn oxide particle size distribu-
tion and exposure concentration; the default
breathing parameters for rats (tidal volume,
2.1 mL; respiratory rate, 102/min) and the
rat-specific nasal parameters given in Table 1S
(in Supplemental Material available online at
http://www.ehponline.org/docs/2006/9030/
suppl.pdf) were also considered. Olfactory
bulb weights and Mn concentrations were
those from experimental results found in
Figure 3; the olfactory bulb Mn background
level in unexposed rats was subtracted from
the level in rats exposed to Mn oxide for 6 hr
with both nares patent.
The results of this study raise a number of
important questions, including extrapolation to
humans, the significance for neurotoxicity,
implications for other inhaled nanosized parti-
cles, and translocation from olfactory bulb to
deeper brain structures. Human exposures to
high concentrations of Mn oxide–containing
UFPs occur under certain occupational settings,
such as arc welding (Zimmer et al. 2002).
Concentrations of UFPs containing Mn oxide
can reach 10
7
particles/cm
3
. The issue of the
neurotoxicity of inhaled Mn-containing parti-
cles is important insofar as welding fumes con-
tain 0.01–5 mg/m
3
Mn, depending on the
welding process and materials used (Korczynski
2000; Li et al. 2004; Sinczuk-Walczak et al.
2001), and preliminary results from a small
cohort of welders show that they may develop
Parkinson-like symptoms (Racette et al. 2001).
Dorman et al. (2004) showed that the inhala-
tion of soluble Mn sulfate aerosols did not lead
to an increase in GFAP protein expression in
olfactory bulb tissue; however, Henriksson and
Tjälve (2000) performed three successive
weekly intranasal instillations of MnCl
2
and
showed an increase in this protein, which is a
marker of astrocyte reactivity, or response to
injury. An important point, however, is that
increased GFAP was noted by Henriksson and
Tjälve (2000) only at high doses that also led to
olfactory epithelial damage. In the present
study, we found evidence for increased GFAP
and TNF-αgene expression, among other
things, in olfactory bulb tissue; although
TNF-αmessage expression was elevated in
deeper brain structures, this was not the case for
GFAP (Figure 2A). TNF-αprotein expression
was also increased in those brain regions where
its gene expression was increased (Figure 2B).
We did not perform histopathologic
assessment of the respiratory or olfactory
epithelium and thus do not know if the Mn
oxide UFPs caused inflammation at the olfac-
tory mucosa, which could have affected neu-
ronal uptake and translocation. A significant
nasal inflammatory response may be unlikely
given that there was no inflammation in the
lung after 12 days of exposure (Table 1). Even
a 1-day exposure—which is far less likely to
induce nasal inflammation—resulted in
significant translocation to the olfactory bulb
(Figure 3). Our observation of a decrease in
lung lavage fluid β-glucuronidase activity after
Translocation of inhaled Mn oxides to the CNS
Environmental Health Perspectives
•
VOLUME 114 |NUMBER 8 |August 2006
1177
MnCl2Mn oxide
40
30
20
10
0
125
100
75
50
25
0
Percent
Mn in olfactory bulb (ng)
1,500
1,000
500
0
0.5 24 0.5 24
Time postinstillation (hr)
Untreated
control Treated
1.5
1.0
0.5
0
*
A B C
Mn in olfactory mucosa (ng)
ng Mn/mg wet tissue
Untreated
control Treated
*
Figure 5. Translocation of instilled Mn to olfactory bulb in separate experiments. (
A
) Percentage of instilled
Mn retained in olfactory bulb (shaded bars) and retained in olfactory mucosa (solid bars) 24 hr after instil-
lation of either MnCl2or Mn oxide into left naris,
n
= 3/group. (
B
) Background corrected amount of Mn in
olfactory bulb (shaded bars) and on olfactory mucosa (solid bars) at 30 min and 24 hr after instillation of
Mn oxide into the left naris,
n
= 3/group. (
C
) Amount of Mn in left (shaded bars) and right (solid bars) olfac-
tory bulb tissue from untreated control rats (
n
= 5) and 24 hr after instillation of Mn oxide into the left naris
(
n
= 6 rats). Values are means ± SE.
*
p
< 0.05 versus untreated controls.
Elder et al.
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•
Environmental Health Perspectives
12 days of exposure is similar to the findings
of Bairati et al. (1997), who reported decreases
in the activity of this enzyme in plasma from
humans exposed occupationally to Mn and
lead. These changes were used as a marker of
heavy metal exposure. Because the physiologic
and pathologic implications of these changes
are unclear, histologic evaluations of lung and
liver after chronic exposures in animals should
be performed in future studies. Our results
with MnCl
2
suggest that it is retained to a
greater extent than the oxide in olfactory
mucosa (Figure 5B), thus possibly explaining
the damage to those tissues by more soluble
forms of Mn delivered at high doses.
The earlier studies of olfactory trans-
location of nanosized particles of different
types (viruses, gold, carbon) together with the
present study of ultrafine Mn oxide may
imply that all nanosized particles deposited on
the olfactory mucosa will translocate to the
brain. However, uptake into sensory nerve
endings and subsequent translocation is likely
to depend not only on size, but also on many
other particle characteristics, such as shape,
chemistry, surface properties (area, porosity,
charge, surface modifications), agglomeration
state, solubility, and dose. Although there are
no data regarding these parameters for sensory
neurons, they affect endo- and transcytosis of
nanosized materials (e.g., Kato et al. 2003;
Kreuter 2004; Rejman et al. 2004). However,
our findings may not be directly applied to
nanoparticles in general until more data are
available on mechanisms controlling neuronal
uptake and translocation.
We conclude from our studies that the
olfactory neuronal pathway represents a
significant exposure route of CNS tissue to
inhaled solid Mn oxide UFPs. In rats, which
are obligatory nose breathers, translocation of
inhaled nanosized particles along neurons
seems to be a more efficient pathway to the
CNS than via the blood circulation across the
blood–brain barrier. Given that this neuronal
translocation pathway was also demonstrated
in nonhuman primates, it is likely to be opera-
tive in humans as well.
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CORRECTION
In “Materials and Methods” in the original
manuscript published online, the authors
incorrectly stated that animals were housed
in wire-bottom cages; this has been corrected
here.
