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18. Translocation and effects of gold nanoparticles after inhalation exposure in rats

Nanotoxicology, September 2007; 1(3): 235Á242

Translocation and effects of gold nanoparticles after inhalation
exposure in rats

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 LIYA E. YU1,6, LIN-YUE LANRY YUNG2, CHOON-NAM ONG3,6, YUEH-LI TAN5,
For personal use only. KUMAR SURESH BALASUBRAMANIAM1, DENY HARTONO2, GUANGHOU SHUI4,
MARKUS R. WENK4,7, & WEI-YI ONG5,6,7

Departments of 1Environmental Science & Engineering, 2Chemical & Biomolecular Engineering, 3Community, Occupational
and Family Medicine, 4Biochemistry, and 5Anatomy, 6Toxicology and 7Neurobiology Research Programmes, National
University of Singapore, Singapore

(Received 20 September 2007; accepted 22 October 2007)

Abstract
This study was carried out to test the hypothesis that nanogold particles can accumulate in the olfactory bulb, and
translocate from the lung to other organs after inhalation exposure. Gold nanoparticles were aerosolized and introduced
through an exposure chamber. The number concentration of airborne nano-sized particles was 2 )106 #NSPs/cm3 with !
75% of particulates between 30 and 110 nm. Exposure for 5 days resulted in significant increase of Au in the lung and
olfactory bulb as detected by ICP-MS, but after 15 days, significant accumulation of gold was detected in the lung,
esophagus, tongue, kidney, aorta, spleen, septum, heart and blood. Microarray analysis showed downregulation of many
genes related to muscle in the nanogold-exposed lung. Lipidomic analysis of the lung showed a specific decrease in
phosphatidylserine 36:1 species. We conclude that nanogold is able to translocate from the lung to other organs with time,
and causes significant effects in exposed tissues.

Keywords: Nanogold, nanoparticles, nanomaterials, inhalation, lung, kidney, smooth muscle, phosphatidylserine,
microarray, lipidomics

Introduction iridium nanoparticles are much less phagocytized
by alveolar macrophages and more effectively re-
The rapid development of nanotechnologies likely moved from the lung surface into the interstitium
results in increased exposure of people and other than large particles (Semmler-Behnke et al. 2007).
living organisms to nano-sized particles (NSPs). In It was proposed that inhaled ultrafine carbon nano-
contrast to ultrafine particles whose chemical and particles may pass through the air-blood barrier via
physical properties are determined by primary large-sized gaps between alveolar epithelial cells
emission sources and secondary reactions in the (Shimada et al. 2006). However, another study
atmospheric environment, NSPs derived from na- showed gold particles in alveolar macrophages and
notechnologies possess unique engineered charac- alveolar epithelial cells after inhalation exposure,
teristics. Compared to larger particles, the greater suggesting uptake by endocytosis (Takenaka et al.
surface-to-volume ratio, and engineered functional- 2006). Inhalation exposure to nano-sized cadmium
ities on the surfaces of NSPs could result in greater oxide (40 nm) results in its accumulation in the liver
biological activity if these are taken into the body, (Takenaka et al. 2004).
making them a potential health concern.
Besides the lung, nano-sized 13C (Â35 nm)
The lung retains large amounts of engineered particles have been detected in the olfactory bulb
materials (5Á8 nm gold particles) (Takenaka et al. after intranasal instillation in rats (Oberdo¨ rster et al.
2006). Some of this is able to enter the bloodstream, 2004). The olfactory bulb can also be an entry point
and translocation has been reported within 30 min of instilled 30-nm poliovirus to the brain (reviewed
after instillation of 30-nm gold particles (Berry et al. in Oberdo¨ rster et al. 2005). These findings are
1977). A recent study showed that radioactive supported by recent findings of an accumulation of

Correspondence: Dr Liya E. Yu, Department of Environmental Science & Engineering, National University of Singapore Singapore
119260. Tel: '65 65166474. E-mail: eseley@nus.edu.sg or Dr Wei-Yi Ong, Department of Anatomy, National University of Singapore,
Singapore 119260. Tel: '65 65163662. Fax: '65 6777 3271. E-mail: antongwy@nus.edu.sg

ISSN 1743-5390 print/ISSN 1743-5404 online # 2007 Informa UK Ltd.
DOI: 10.1080/17435390701763108

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 236 L. E. Yu et al. Synthesis and characterization of gold nanoparticles
For personal use only.
a metallic nanoparticle (ultrafine manganese parti- Gold nanoparticles (nAu) were synthesized through
cles) in the olfactory bulb, after inhalation exposure the reduction of hydrogen tetrachloroaurate (III)
(Elder et al. 2006). Pro-inflammatory effects and trihydrate (HAuCl4) (Sigma-Aldrich, St Louis,
altered macrophage chemotactic response have been Missouri, USA) using trisodium citrate dihydrate
demonstrated after exposure to nanomaterials in (Sigma-Aldrich) (Handley 1989). In brief, 0.05%
vivo and in vitro (Renwick et al. 2004; Duffin et al. (w/v) citrate was added to 0.01% (w/v) stock
2007). Apart from particle chemistry (especially solution of HAuCl4 in a 3-necked round bottom
surface chemistry) and physical properties (e.g., flask at 958C for 10 min with vigorous stirring,
size and shape), dose, duration, and route of expo- followed by 20 min of reflux before the reaction
sure of a nanoparticle are likely to be important was gradually cooled down to room temperature.
factors in determining its potential health effects. To image the size and determine the size distribution
of the resultant nAu, a transmission electron micro-
Systematic study of the biodistribution and biolo- scope (Philips CM300 FEG system operating at
gic effects of an engineered nanomaterial can serve 200 kV) was employed. More than 200 particles
as a basis for assessing its impact on health. Gold were sized from TEM micrographs using Image-
nanoparticles (nAu) are chosen as a model engi- Pro Express software (Media Cybernetics, Bethesda,
neered NSP in this study. nAu is used in clothing, Maryland, USA), and showed a mean diameter of
footwear and plastic containers, and extensively 20 nm (Figure 1).
developed as diagnostic tools for in vivo applications
(Nam et al. 2003; Li & Rothberg 2004; Rosi et al. Inhalation exposure
2006; Gordon et al. 2007). This suggests exposure
to the nanoparticle during the manufacturing pro- A whole body exposure chamber was constructed to
cess or use of products. Gold in bulk form is also house four rats at a time. The chamber was fitted
chemically inert compared to other metallic materi- with an observation port. An aerosol generation
als; hence, any biologic effect of nAu is unlikely due system (TSI Inc. Minnesota, USA) introduced
to intrinsic toxicity of the metal. In addition, gold is aerosolized nAu into the exposure chamber at a
generally insoluble and rarely present in biological flow rate of 1.5 liters per min (lpm) along with make-
tissues. Its presence in tissue therefore likely reflects up air at 8 lpm. The size distribution of particles in
actual translocation of the metal in nanoparticle the exposure chamber was monitored using a scan-
form to that tissue. The purpose of this study is to ning mobility particle sizer (SMPS, TSI Inc. Min-
test the hypothesis that nAu can accumulate in the nesota, USA) and an electric low pressure impactor
olfactory bulb, and translocate from the lung to (ELPI, Dekati Ltd. Tempere, Finland). The com-
other organs after inhalation exposure. Microarray
and lipidomic analyses of the lung/kidney were also
conducted, to examine possible changes in gene
expression and lipid composition in these organs,
after exposure.

Materials and methods Figure 1. Transmission electron micrograph of synthesized nAu
with an average diameter of 20 nm. Scale: 50 nm.
Animals and exposure intervals

Adult male Wistar rats weighing approximately
200 g each were purchased from the Centre for
Animal Resources, Lim Chu Kang, Singapore. The
rats were divided into three groups: (i) The first
set of animals was exposed for 6 h per day for
5 consecutive days. They were sacrificed two days
thereafter; (ii) The second set of animals was
exposed for 6 h per day and for consecutive 5 days,
for 3 weeks (i.e., total of 15 days exposure). They
were sacrificed two days after the last exposure;
and (iii) A set of 4 unexposed animals were used
as controls. These animals were kept away from
the exposure chamber. All procedures involving rats
were approved by the Institutional Animal Care and
Use Committee.

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 pressed air used contained fewer than 100 particles/ Effects of inhaled nanogold 237
For personal use only. cm3. All the outlet air was passed through an
impinger containing calcium chloride (0.1 M) to nAu solutions, which was further diluted (4 mg/l)
enforce the aggregation of nAu, which then pre- was also tested along with the digested tissue
cipitated away from the air stream. Background samples. To correct procedural loss, 200 ml of
interference due to NSPs originating from ultrapure internal standard Cd (1 mg/l) was added to indivi-
water was insignificant. The majority of these dual tissue samples prior to microwave digestion.
particles were smaller than 35 nm, i.e., smaller Based on 345 measurements, the average recovery
than the population of airborne NSPs during efficiency was around 72915%. The detection limit
nAu exposure tests (median 76Á79 nm), and their of our experimental procedures is approximately
concentrations were 1.6 )10597% #NSPs/cm3 0.2 ng Au/g tissue, based on standard tests and
(n !70), accounting for less than 7% of total NSPs overall material recoveries. For all organ tissues,
during the nAu exposure experiments. These values the difference between control and treatment
were used to correct the monitored number con- groups was determined using one-way ANOVA
centration of NSPs. Exposure was carried out during with Bonferroni’s multiple comparison post-hoc test.
the day, and rats appeared quiet and without distress
during exposure, and showed no gross abnormalities To examine potential metal contaminants affect-
in behavior. ing rats, we also analyzed the level of other metals
including aluminium, arsenic, beryllium, chromium,
Perfusion and harvesting of tissues copper, cobalt, manganese, nickel, lead, zinc, and
vanadium in lung samples from rats exposed to nAu
Rats were deeply anaesthetized by intraperitoneal for 15 days, and unexposed controls.
injection of 2.3 ml of 6 mg/ml of Nembutal and
perfused through the left cardiac ventricle with a Microarray analysis
solution of normal saline. Tissues from different
organs of the body were then harvested. These Lung and kidney samples from the 15 day nAu-
included: whole blood, olfactory bulb, septum, exposed rats, and unexposed controls were used for
striatum, cerebral neocortex, hippocampus, entorh- microarray analyses. Samples were collected, im-
inal cortex, thalamus and hypothalamus, cerebel- mersed in RNAlater† (Qiagen, Hilden, Germany)
lum, brainstem, tongue, heart (ventricles), lung, solution, and snap frozen in liquid nitrogen. They
thoracic aorta, esophagus (thoracic portion), liver, were kept in a (808C freezer till analysis. RNA was
pancreas, spleen, adrenal glands, kidney, bladder, extracted using the Trizol method, and cleaned
testis, skeletal muscle (quadriceps femoris), bone using the RNeasy kit (Qiagen). The amount and
(shaft of femur), skin (shaved skin of the back), quality of RNA samples were determined using
stomach, small intestine, cecum, and descending agarose gels and the Agilent RNA 6000 Nano Kit
colon. Care was taken not to let the tissues contact (Agilent Technologies, Santa Clara, USA). Total
the fur and to thoroughly wash instruments and RNA (5 ug) from each sample was then used for
samples (especially portions of the gastrointestinal one-cycle cDNA synthesis, followed by synthesis of
tract) with water to remove any nAu that could be Biotin-labeled cRNA and fragmentation of the
adhering to the surfaces, during harvesting. cRNA using the GeneChip† One-cycle Target
labeling and control kit (Affymetrix, Santa Clara,
Quantification of nAu USA, P/N: 900493). Absorbance at 260 nm and 280
nm was used to determine the concentration and
To quantify accumulated nAu, individual tissue purity of the product using a NanoDrop† apparatus
samples were weighed prior to undergoing a stream- (ND-1000, NanoDrop Technologies, Wilmington,
lined microwave-assisted digestion program and USA). The A260/A280 was between 1.9 and 2.1.
then stored in the dark at (258C before analysis The adjusted cRNA yield was calculated as follows:
using inductively-coupled plasma mass spectrometry
(ICP-MS, Perkin Elmer, Massachusetts, USA). To Adjusted cRNA yield 0RNAm Á (total
conduct ICP-MS measurements, aliquots of di- RNAi)(y)
gested samples were transferred and diluted to 20 RNAm 0Amount of cRNA measured after
ml for triplicate analyses using a dual detector mode IVT (mg)
(analog and pulse counts). To establish the calibra- total RNAi 0Starting amount of total RNA
tion curve, standard gold solutions (Sigma-Aldrich, (mg)
MO, USA) in five concentrations (0.2, 2, 4, 10, and y 0Fraction of cDNA reaction used in in vitro
20 mg/l) were injected. The in-house synthesized transcription

Fragmentized and labeled cRNA (15 ug) from
each sample was hybridized with control oligonu-
cleotides B2 and hybridization controls (bioB, bioC,

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 238 L. E. Yu et al. relevant internal standards and presented as a
For personal use only. normalized concentration (to total lipids in sample).
bioD and cre) on each Rat Genome 230 2.0 array
(Affymetrix, P/N: 900505) at 458C overnight at Results
60 rpm. Washing and staining were carried out
using the GeneChip† Hybridization, wash and Characteristics of NSPs in the exposure chamber
stain kit (Affymetrix, P/N: 900720). The arrays
were scanned using GeneChip Scanner 3000 The majority (!75%) of airborne NSPs were in
(Affymetrix), and data analyzed using GeneSpring 30Á110 nm, with a median size between 76 and
software (Agilent Technologies). The level of gene 79 nm. On average, the NSPs in the chamber
expression was normalized per chip, and scaled exhibited a total number concentration of 2 )106
from 0.01Á100. Genes that showed significance (915%, n !1100) #NSPs/cm3, which is similar
(p B0.05) and greater than 10-fold differences to the concentration of roadside particles under
between the nAu exposed- and unexposed rats are conditions of substantial diesel vehicle emissions
presented. Four microarray chips were used for each (Kittelson et al. 2004).
treatment condition (nAu exposed or unexposed) in
each organ (lung or kidney). Distribution of gold in the body after inhalation exposure
(Table I)
Lipidomic analysis
Five days exposure compared to unexposed animals.
Lung samples from the 15 day nAu exposed rats and Significant accumulation of gold was detected in the
unexposed controls were analyzed by electrospray lung (100 ng Au/g tissue) and olfactory bulb (8 ng
ionization mass spectrometry (ESI-MS) as described Au/g tissue) (Table I).
previously (Guan et al. 2006). Briefly, synthetic
lipids with fatty acyl compositions that are naturally Fifteen days exposure compared to unexposed animals
low in abundance were used as internal standards. (Table I). Significant accumulation of Au was
Lipid standards were obtained either from Alabaster detected in over 20 organs and tissues, with a
(AL, USA) or Echelon Biosciences, Inc. (Salt Lake concentration ranging from 996 ng Au/g (for
City, USA). The internal standards were dissolved in pancreas) to 3879131 ng Au/g (for lung). Of the
chloroform at a stock concentration of 10 mg/ml. above tissues, the highest concentrations of gold
Lipids were extracted from lung tissues using a were observed in (decreasing order of concentra-
modified protocol of Bligh and Dyer (1959). Briefly, tion): lung (3879131 ng/g), esophagus (45922 ng/
400 ml of chloroform-methanol, 1:2 (v/v) and 5 mg of g), tongue (4297 ng/g), kidney (2793 ng/g), aorta
internal standards were spiked to 20 mg of tissue (2599 ng/g), spleen (2592 ng/g), septum (2194),
homogenate. After 10-min incubation on ice, 300 ml heart (1993 ng/g) and blood (1991 ng/g) (Table I).
of chloroform and 200 ml of hydrochloric acid (HCl, Negligible differences were observed between the
1M) were added to the mixture and the lipids were nAu exposed and non-exposed rats, in levels of other
isolated from the organic phase after centrifugation. metals such as aluminum, arsenic, beryllium, chro-
The sample was vacuum dried (Thermo Savant mium, copper, cobalt, manganese, nickel, lead, zinc,
SpeedVac), resuspended in 2 ml of chloroform- and vanadium in the lung (data not shown). This
methanol, (1:1, v/v), followed by ESI-MS analysis. indicates minimum interference from other metals,
in our analyses of the health effects of nAu.
Quantification of individual molecular species was
performed using multiple reaction monitoring Fifteen days exposure compared to five days exposure
(MRM) with an Applied Biosystems 4000 Q-Trap (Table I). Significantly greater levels of Au were
mass spectrometer (Applied Biosystems, Foster detected in many organs after 15 day exposure,
City, USA). Samples were directly infused using a compared to 5 days exposure. The skin showed the
Harvard syringe pump at a flow rate of 5 ml/min. In highest incremental increase (!5 times higher accu-
these experiments, the first quadrupole, Q1, was set mulation), followed by bone (3.5 times more), aorta,
to pass the precursor ion of interest to the collision tongue, and esophagus (!2.5 times more). This
cell, Q2, where it underwent collision-induced dis- shows that incremental uptake of nAu through skin
sociation. The third quadruple, Q3, was set to pass exposure is most sensitive to duration of exposure to
the structure specific ion characteristics of the airborne NSPs, which is expected since skin is in
precursor lipid of interest. Each individual ion direct contact with nAu in the exposure chamber.
dissociation pathway was optimized with regard to The substantial increase in bone is surprising. The
collision energy to minimize variations in relative olfactory bulb showed similar uptake of Au after 5 or
ion abundance due to differences in rates of dis- 15 days exposure.
sociation (Merrill et al. 2005; Guan et al. 2006).
Lipid concentrations were calculated relative to the

Effects of inhaled nanogold 239

Table I. Distribution of gold in rat organs after 15 days of nAu exposure.

System Organ Unexposed Controls 5 days nAu exposure 15 days nAu exposure

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 Respiratory Lung 5.50094.440 125.733925.912* 387.4579130.826* #
For personal use only. Cardiovascular Heart 4.15092.62 4.76191.105 19.33693.171* #
Digestive Aorta 2.67092.62 5.90394.417 25.31499.390* #
Blood 5.83093.020 4.93091.611 18.74790.923* #
Exocrine/endocrine/immune
Musculoskeletal/skin Tongue 9.02991.889 11.22592.397 41.97496.756* #
Nervous Oesophagus 9.450912.570 5.80293.232 44.947921.933* #
Stomach 4.23093.960 6.535912.663
Urinary Small intestine 0.87090.8000 1.57790.673 9.97494.977
Reproductive Cecum 2.66092.200 2.43891.333 4.70890.828* #
Colon 1.26091.260 2.10891.025 4.83991.277
7.18792.841* #
Liver 2.93092.570 3.47592.353
Pancreas 1.49091.000 1.78890.816 7.77593.128* #
Spleen 3.58092.930 4.83191.954 8.77495.756* #
Adrenal gland 7.50097.700 5.91291.694 25.02092.146* #
12.91492.480* #
Bone 0.56990.288 0.81290.543
Skeletal muscle 1.63091.260 2.28990.990 9.44692.459* #
Skin 2.31990.275 2.04290.417 6.27891.410* #
15.04293.289* #
Olfactory bulb 4.44094.430 12.13994.123*
Septum 11.73692.218 14.80696.365 13.15394.704*
Entorhinal cortex 11.80691.257 10.77292.184 21.72094.352*
Neocortex 17.06492.924* #
Hippocampus 7.97095.820 4.50390.680
Striatum 9.22097.480 13.50096.069 9.28292.977
Thalamus/hypothalamus 20.270915.790 20.563912.007 12.94793.129
Brainstem 6.67095.010
Cerebellum 14.670916.250 7.67994.625 9.34790.924
4.67093.630 5.36991.662 11.91092.162
Kidney 5.16892.001
Bladder 6.29094.120 9.00090.713
6.58095.810 10.130912.668 9.59290.492* #
Testis 5.69791.804
4.97094.200 27.25893.213* #
3.30192.115 17.90893.221* #

16.28496.380* #

Values indicate mean concentration and standard deviation (ng/g of tissue) n04 in each case. Analyzed by one-way ANOVA with
Bonferroni’s multiple comparison post-hoc test. p B0.05 was considered significant. *indicates significant increase compared to unexposed
controls. #indicates significant increase in the 15-day nAu-exposed rats compared to the 5-day nAu-exposed rats.

Microarray analysis (Table II) Lipidomic analysis (Figure 2)

Many genes were significantly upregulated or down- Significant decrease in phosphatidylserine 36:1 spe-
regulated by more than 2-fold in the lung or kidney cies was found in the lung of the 15 day nAu-
of the nAu exposed rats (data not shown). However, exposed rats, compared to unexposed animals
genes that showed the greatest changes (10-fold (Figure 2). The decrease in phosphatiylserine was
or greater) were mostly downregulated. In the not accompanied by any significant increase in
lung, these included many genes related to muscle, level of lysophosphatidylserine or other lysopho-
including phosphoglycerate mutase 2, myosin heavy spholipids. No significant changes were detected
chain polypeptide 6 and myozenin 2, and several in other glycerophospholipids and sphingolipids,
genes related to secretion, including palate, lung, or amount of lung surfactant, dipalmitoylphosphati-
and nasal epithelium carcinoma associated protein, dylcholine (Furue et al. 2001), between the nAu-
secretoglobin, family 3A, member 1, chloride chan- exposed and unexposed rats (data not shown).
nel calcium activated 3, and glycoprotein 2 (zymo-
gen granule membrane) (Table II). Discussion

The lung and kidney of nAu-exposed rats both This study aimed to examine the biodistribution and
showed more than 10-fold downregulation in RT1 effects of inhaled nAu in rats. A large increase in Au
class II, locus Bb, and two subunits of cytochrome uptake was observed in the lung after 5 days of
c oxidase. Significantly decreased expression of the exposure. This is expected, since previous study
gene for thyroid hormone responsive protein was (Takenaka et al. 2006) showed that a single inhala-
also found in the kidney of the nAu exposed rats tion exposure of 5Á8 nm gold nanoparticles results in
(Table II).

240 L. E. Yu et al.

Table II. Genes that are changed by 10-fold or more in the lung and kidney, in nAu-exposed rats compared to unexposed rats.

Organ Fold change Standard Deviation Gene symbol Description

Lung (51.3 0.0 Pgam2 phosphoglycerate mutase 2a
Kidney (46.6
(45.4 0.1 Myh6 myosin heavy chain, polypeptide 6a
(45.3
(43.0 0.1 Myoz2_predicted myozenin 2 (predicted)a
(38.0
(33.6 0.1 Plunc palate, lung, and nasal epithelium carcinoma associatedb
(32.2
(30.9 0.1 Myl7_predicted myosin, light polypeptide 7, regulatory (predicted)a
(30.0
(22.6 0.1 Cox6a2 cytochrome c oxidase, subunit VIa, polypeptide 2
(22.2
(21.7 0.3 Scgb3a1 secretoglobin, family 3A, member 1b
(19.0
(18.2 0.0 Clca3_predicted chloride channel calcium activated 3 (predicted)b
(17.4
(16.8 0.1 Myh6; Myh7 myosin heavy chain, polypeptide 6; myosin, heavy polypeptide 7a
(15.4
(15.0 0.1 Gp2 glycoprotein 2 (zymogen granule membrane)b
(15.0
(12.5 0.1 Tnni3 troponin I, cardiac a

11.8 0.1 Mb Myoglobina

(44.6 0.1 Clca3_predicted chloride channel calcium activated 3 (predicted)b
(24.1
(17.3 0.2 Tnnt2 troponin T2, cardiaca
(14.6
(12.1 0.1 Tpm1 Tropomyosin 1, alphaa
(10.5
Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/130.1 Ckmt2 Creatine kinase, mitochondrial 2, sarcomerica
For personal use only.11.3
10.5 0.1 Tna_predicted tetranectin (plasminogen binding protein) (predicted)
LysoPS 16:010.2
LysoPS 18:10.1Sln_predicted sarcolipin (predicted)a
LysoPS 18:0
0.1 Csrp3 cysteine-rich protein 3
PS 34:10.2 Smpx small muscle protein, X-linkeda
PS 36:20.1 RT1-Bb RT1 class II, locus Bbc
PS 36:1
PS 38:44.0 Baat bile acid-Coenzyme A: amino acid N-acyltransferase
PS 38:3
PS 38:10.0 RT1-Bb RT1 class II, locus Bbc
PS 40:60.1 Ucp1uncoupling protein 1d
0.5 Cox8h Cytochrom c oxidase subunit VIII-H (heart/muscle)d
0.9 Thrsp thyroid hormone responsive proteine

1.8 Scd1 stearoyl-Coenzyme A desaturase 1

0.0 Stmn2 stathmin-like 2

13.3 isg12(b) putative ISG12(b) protein

13.0 G1p2_predicted interferon, alpha-inducible protein (clone IFI-15K) (predicted)

15.3 RGD:1359153 similar to ubiquitin specific protease UBP43

Values are fold change, and standard deviation. A negative value indicates downregulation in the exposed organ, and vise versa. aindicate
genes related to muscle; bindicate genes related to secretion; cindicate genes related to antigen presentation; dindicate genes related to
mitochondrial function; eindicate genes related to growth.

greatest accumulation (1945957 ng) in the lung. mulation after 5 days of exposure, including the
A further increase in Au concentration in the lung esophagus, tongue, kidney, aorta, spleen, septum,
was observed after inhalation exposure to nAu for heart, blood, bladder, entorhinal cortex, testis,
15 days, as well as significant increase in other skin and adrenal gland. The results indicate a
organs that previously showed negligible Au accu- cumulative effect of nAu exposure. Other than the
lung, esophagus, and tongue, which are directly
1.5E-03 Ctrl exposed to nAu through inhalation or ingestion,
1.2E-03 the kidney showed the next highest accumulation of
9.0E-04 * Nanogold nAu. This could be related to its function as a filter
6.0E-04 for blood, and provides evidence that nAu could
3.0E-04 translocate from the lung to the bloodstream and
0.0E+00 other organs. In contrast to the kidney and spleen,
the liver showed little accumulation of Au. This
Figure 2. Lipidomic analysis of the lung of nAu exposed rats. may be due to differences in expression of a
Significant decrease in phosphatidylserine was observed in the metal sequestering protein, metallothionein, in these
nAu-exposed rats (asterisk, p B0.01) after 15 days of exposure. organs. The rate of metallothionein gene transcrip-
Ctrl: unexposed rats. Nanogold: 15-day nAu-exposed rats. X axis tion and expression remain high after exposure to
indicates lysophosphatidylserine or phosphatidylserine species. Y metals such as mercury in the kidney, but decrease
axis indicates normalized concentration (to total lipids in sample). rapidly in the liver (Zalups & Koropatnick 2000).

Interestingly, little accumulation of nAu was
detected in most parts of the brain regardless of
exposure duration. This is likely due to the blood
brain barrier. The exceptions were the olfactory

Nanotoxicology Downloaded from informahealthcare.com by University of Sussex Library on 03/13/13 bulb, septum, entorhinal cortex and cerebellum. Effects of inhaled nanogold 241
For personal use only. The finding of increased Au in the olfactory bulb
after 5 days exposure, and in the olfactory bulb, Lipidomics (systems scale analysis of lipids and
septum and entorhinal cortex after 15 days expo- their interacting partners) is a promising area of
sure, supports the view that nAu could, with time, biomedical research with a variety of applications
be transported from the olfactory bulb to other parts in drug and biomarker development. Significant
of the brain (Oberdo¨rster et al. 2004; Elder et al. decrease of phosphatidylserine, but not other glyer-
2006; Zhang et al. 2006). The septum and entorh- ophospholipids or sphingolipids was detected in the
inal cortex receive direct neuronal projections from lung of the nAu-exposed rats. One possibility, in
the olfactory bulb, and are important in attention view of the fact that peroxidized fatty acids are
and new memory formation. The Au measured in particularly good substrates for phospholipase A2
unexposed tissue samples is likely due to noise of the (PLA2) (McLean et al. 1993) is that inhaled nAu
experimental procedures, including microwave-as- could have induced oxidative stress and lipid perox-
sisted extraction and ICP-MS analysis. idation of unsaturated fatty acids on the sn-2
position of glycerophospholipids, thus favoring their
The results of microarray analysis of the nano- removal by the enzyme. No significant increase in
gold-exposed lung were striking, in that it showed lysophosphatidylserine or decrease in other glycer-
significant downregulation of many genes related to ophospholipids was, however, detected in the lung,
muscle. These included proteins related to myofi- suggesting absence of large-scale damage to lung
brils, such as myosin, troponin, tropomyosin, or membranes after nanogold exposure. Phosphatidyl-
muscle metabolism, including myoglobin and crea- serine externalization is an important signal for
tine kinase. These observations suggest changes in recognition and elimination of oxidized cells by
smooth muscle in the conducting portion of the macrophages (Tyurina et al. 2007), and loss of this
respiratory tree and/or pulmonary vessels after nAu phospholipid, together with downregulation of RT1
exposure. Carbon nanotubes affects smooth muscle class II, locus Bb, may affect recognition of oxidized
growth in vitro (Raja et al. 2007), whilst titanium cells and antigen presentation by lung macrophages.
dioxide nanoparticles (NanoTiO2) induce emphy-
sema-like lung injury in mice (Chen et al. 2006), We conclude that inhaled nAu accumulates in the
that might be due to effects on smooth muscle. olfactory bulb, and is able to translocate from the
Further studies are hence necessary to examine lung lung to other organs with time. No gross abnorm-
smooth muscle function after nAu exposure. It may alities were observed in the exposed rats, even
also be important to examine changes in muscle in though changes in muscle-related genes and phos-
other organs that have a high proportion of this phatidylserine species in the lung suggest that there
tissue, such as the esophagus, aorta, and heart. could be health effects after exposure to nAu.
Several genes related to secretion, including palate, Further work is necessary to investigate possible
lung, and nasal epithelium carcinoma associated functional changes in the lung and other organs with
protein, secretoglobulin, and chloride channel cal- high bioaccumulation of nAu after inhalation ex-
cium activated 3 were also downregulated in the posure to nanoparticles.
lung. This might affect mucus secretion in the
airway. Acknowledgements

One gene, RT1 class II, locus Bb, was down- The authors would like to thank the Office of Life
regulated by more than 10-fold, in both lung and Sciences and NUS Environmental Research Insti-
kidney of nAu-exposed rats. This is a rat MHC class tute for seed funding, and the NUS Nanoscience
II analog and functions to present peptides pro- and Nanotechnology Initiative for administrative
cessed from extracellular proteins to helper T cells to support. Ms Li-Yen Lee and Hui-Jen Lye provided
initiate an immune response. Two subunits of expert help in Affymetrix microarray analysis. Prof.
cytochrome C oxidase were also significantly down- Jost O. L. Wendt for stimulating discussion at the
regulated in the lung and kidney of nAu-exposed inception of this work. There are no conflicts of
rats. This might affect mitochondrial respiratory interest.
function. The nAu-exposed kidney also showed
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