Aquatic Toxicology 100 (2010) 178–186
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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis
Sara Tedesco a , Hugh Doyle b , Julian Blasco c , Gareth Redmond b , David Sheehan a,∗
a
Environmental Research Institute of University College Cork, Cork, Ireland
Tyndall National Institute, Cork, Ireland
c
Consejo Superior de Investigaciones Cientificas (CSIC), Marine Science Institute of Andalusia, Cadiz, Spain
b
a r t i c l e
i n f o
Article history:
Received 25 October 2009
Received in revised form 26 February 2010
Accepted 2 March 2010
Keywords:
Gold nanoparticle
Oxidative stress
Toxicity
Thiol
Mytilus
a b s t r a c t
Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy,
food industry and environment remediation. However, little is known about their potential toxicity or fate
in the environment. Mytilus edulis was exposed in tanks to750 ppb AuNP (average diameter 5.3 ± 1 nm) for
24 h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure
selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations
were used to study toxicity and oxidative stress responses. Results were compared to those obtained
for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in
digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE
analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal
membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT)
in both treatments but was greater in AuNP. Oxidative stress occurred within 24 h of AuNP exposure
in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that
nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M.
edulis is a suitable model animal for environmental toxicology studies of nanoparticles.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Nanoparticles have unique properties and are exciting interest across a broad spectrum of potential applications, including
medicine, cosmetics, electronics, innovative food products and
environmental remediation. Nanoparticles intended for imaging or
for use as drug delivery vehicles can be coated with bioconjugates
such as DNA, proteins and monoclonal antibodies to target specific
cells. As these nanoparticles are intended to interact with cells, it is
important to ensure that these modifications do not cause adverse
effects (Lewinski et al., 2008). Gold nanoparticles (AuNP) can easily enter cells (Connor et al., 2005) and the demonstration that
Abbreviations: ANOVA, analysis of variance; a.u., arbitrary units; AuNP, gold
nanoparticles; CCD, charge-coupled device; DDAB, didodecyldimethylammonium
bromide; DDT, dichlorodiphenyltrichloroethane; EST, expressed sequence tag;
ICP-OES, inductively coupled plasma optical emission spectroscopy; IPG, immobilized pH gradient; MDA, malondialdehyde; MPA, mercaptopropanoic acid; NRRT,
neutral red retention time; pI, isoelectric point; PMSF, phenylmethanesulphonylfluoride; ROS, reactive oxygen species; SDS, dodium dodecyl sulfate; TBAB,
tetrabutylammoniumborohydride; TEM, transmission electron microscopy; Tris,
tris(hydroxymethyl)aminomethane; Tris/HCl, (tris/hydroxymethyl) aminomethane
hydrochloride.
∗ Corresponding author at: Proteomic Research Laboratory, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork,
Ireland. Tel.: +353 21 490 4207; fax: +353 21 4274034.
E-mail address: [email protected] (D. Sheehan).
0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2010.03.001
amine and thiol groups bind strongly to AuNP has enabled their
surface modification with amino acids and proteins for biomedical applications (Dani et al., 2008; Shukla et al., 2005; Xu and
Han, 2004). AuNP are also used in biosensors where they markedly
enhance sensitivity and specificity of detection because of their
unique physical, chemical, mechanical, magnetic and optical properties (Zhang et al., 2009). Studies in vitro have demonstrated the
potential of AuNP for targeting, imaging and therapy of breast cancer cells (Li et al., 2009), for enhancing of radiation sensitivity
in prostate cancer cells (Zhang et al., 2008) and for non-invasive
ablation in rat hepatoma cells (Cardinal et al., 2008). The antimicrobial properties of AuNP are also attracting the attention of the
food industry. Recent studies have shown that AuNP enhances the
growth of lettuce seeds (Shah and Belozerova, 2009) and these
and other nanoparticles have been proposed as components of
food containers, due to their anti-bacterial, antiseptic, deodorant
and catalytic properties (Chang, 2008). Nanoscale metal particles such as gold, also represent a new generation of designed
green oxidation technologies for environmental remediation that
could provide cost-effective solutions to remediation of groundwaters contaminated by hazardous compounds such as chlorinated
organic solvents and pesticides (Kamat and Meisel, 2003; Wong et
al., 2009).
Notwithstanding their potential advantages, nanomaterials
may cause undesirable hazardous interactions with biological systems and the environment with potential to generate toxicity.
S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
Recent literature contains conflicting data regarding oxidative
stress (Jia et al., 2009; Lee et al., 2009; Renault et al., 2008; Tedesco
et al., 2008, 2010) and cytotoxicity of AuNP (Cho et al., 2009;
Murphy et al., 2008). Crucial variables seem to be physical dimensions, surface chemistry, shape, method of synthesis, concentration
and time of exposure. The species Mytilus edulis is an intertidal
filter-feeding organism widely used in ecotoxicology studies with
the potential for short-term exposure to nanoparticles even at
extremely low concentrations (Renault et al., 2008; Tedesco et al.,
2008, 2010). In the present study, it was decided to expose M. edulis
for 24 h to AuNP (∼5 nm) and to probe the tissue distribution and
oxidative stress effects of these nanoparticles.
The roles of proteins in signal transduction pathways depend
strongly on the redox properties of cysteine thiol groups
which occur both in proteins and in low-molecular mass thiols
(Winterbourn and Hampton, 2008). Thiols react significantly faster
than other amino acid side-chains with oxidizing species and thus
contribute to antioxidant defence (Hansen et al., 2009). Proteomics
enables broad comparison of dynamic responses to stress in the cell,
by simultaneously examining hundreds to thousands of proteins
with an unbiased and integrative overview of protein abundance
changes (Kultz et al., 2007). In the present work, cytotoxicity of
AuNP (∼5 nm) and CdCl2 was determined in haemolymph, using
the neutral red retention time (NRRT) assay to measure lysosomal
membrane stability. A previous investigation with slightly larger
AuNP (∼13 nm diameter) did not cause a decrease of NRRT (Tedesco
et al., 2008); although a dramatic decrease of lysosomal stability
was reported after 12 h exposure of glass wool nanoparticles in M.
edulis (Koehler et al., 2008) and significant reduction in lysosomal
integrity found in rainbow trout cells exposed to TiO2 engineered
nanoparticles (Vevers and Jha, 2008). An in vitro study showed
AuNP (2 and 10 nm diameter) caused intracellular damage, but it
was not clear if the severe damage seen by TEM within these cells
was totally due to lipid peroxidation or to disrupted membranes
leaking lytic enzymes that, in turn, may have damaged organelles
(Panessa-Warren et al., 2008). In the present work, for the first time,
we detected lipid peroxidation by measuring malondialdehyde levels in digestive gland, gill and mantle of M. edulis exposed to AuNP
(∼5 nm) and CdCl2 for 24 h.
2. Materials and methods
2.1. Exposures
M. edulis (5.5 cm shell length; approximately 20.3 g total weight
and 5.7 g soft tissue wet weight, respectively) were sampled from
a clean site in Cork Harbour, Ireland (Lyons et al., 2003; Tedesco et
al., 2008, 2010). Four groups (n = 50) were held in tanks (1 week,
12 h light/dark cycle, 34–36‰ salinity and 15–16 ◦ C) with feeding
and water changing (48 h intervals; 1 ml per tank of PhytoplexTM
phytoplankton feed, Kent Marine Inc., Acworth, GA, USA). They
were exposed (24 h) to 750 ppb gold nanoparticles (AuNP ∼5 nm)
and 0.2 mM CdCl2 . Controls were treated identically. Both 0.2 mM
CdCl2 (McDonagh and Sheehan, 2006) and 750 ppb AuNP (at larger
size, Tedesco et al., 2008, 2010) had previously shown mild oxidative stress responses. Digestive gland, gill and mantle tissues were
dissected, pooled (groups of five animals), frozen (liquid nitrogen) and stored (−70 ◦ C). They were homogenized (10 mM Tris/HCl,
pH 7.2, 500 mM sucrose, 1 mM EDTA, 1 mM PMSF), centrifuged at
20,000 × g (1 h, 4 ◦ C). These were used for all analyses except measurement of lysosomal membrane stability.
2.2. Synthesis of AuNP
AuNP were prepared using the method described by Jana
and Peng (2003). All reagents and solvents were purchased from
179
Sigma–Aldrich Ltd. and used as received. In a typical synthesis,
172 mg of decanoic acid was dissolved in 10 ml of toluene. Then
100 mg of tetrabutylammonium borohydride (TBAB), dissolved in
4 ml of a stock solution (100 mM) of didodecyldimethylammonium bromide (DDAB) in toluene, was mixed with the decanoic
acid solution. The resulting clear solution was transferred to a
50 ml three-neck flask and stirred under nitrogen at room temperature. Finally, 30 mg of gold chloride (AuCl3 ) in 4 ml of DDAB
solution was rapidly injected under vigorous stirring. The solution
changed colour to a deep red within 1 min, indicating NP formation. The stirring was continued for a further 30 min to complete
the reaction. A 0.1 M solution of mercaptopropanoic acid (MPA)
was prepared in methanol and the pH of the solution adjusted to
approximately 10 by addition of tetramethylammonium hydroxide. The as-prepared AuNP were precipitated from toluene by the
addition of minimal ethanol and separated from the supernatant
by centrifugation (4020 × g; 10 min). The MPA solution was then
slowly added until the precipitated nanoparticles had been fully
re-dispersed in methanol. Finally, the AuNP were precipitated from
methanol by addition of ethyl acetate, separated from the supernatant by centrifugation (4020 × g; 10 min), and re-dispersed in
water.
2.3. Transmission electron microscopy
Transmission electron microscopy (TEM) images were recorded
using a Jeol JEM-2011 TEM equipped with a Gatan DualVision
600 CCD. Samples were prepared by drop casting a 2.5 ␮l aliquot
of the nanoparticle dispersion onto a 300 mesh carbon-coated
copper grid, which was allowed to evaporate under ambient
conditions. Fig. 1A shows a representative TEM image of the
MPA-stabilised AuNP, showing good size monodispersity. Fig. 1B
shows the histogram of AuNP diameters, determined by analysis of TEM images of approximately 500 nanoparticles located
at different regions of the grid, with an average diameter of
5.3 ± 1.0 nm.
2.4. Chemical analyses by ICP-OES
Metal determination was performed on gill, digestive gland
and mantle tissues using inductively coupled plasma optical emission spectroscopy (ICP-OES). The procedure is briefly described:
wet sample tissues were digested according to the procedure
described by Amiard et al. (1987) slightly modified. Wet sample tissues were digested with nitric acid (10.3 M), 2 ml for
60 min at 95 ◦ C and later with hydrogen peroxide (8.8 M) 0.5 ml
for 30 min at the same temperature. Samples were made up
to 5 ml with Milli-Q water. All reagents were Suprapur quality.
The results were expressed as ␮g/g wet weight. Quality data
were ensured by performing metal analysis on reference material of mussel tissue (CRM 278). Good agreement was obtained
between certified and analyzed values for copper, zinc and cadmium.
2.5. Thiol-containing proteins
Protein extract (1 mg for 1DE and 2.5 mg for 2 DE) were first
reacted with 5 M urea (final concentration) for 10 min at room
temperature to unfold proteins. 20 mg of activated thiol sepharose
(GE Healthcare, Little Chalfont, Bucks, UK), and 200 ␮l binding
buffer solution (0.1 M Tris–HCl pH 7.5, 0.5 M NaCl, 1 mM EDTA)
were added followed by incubation on ice for 1.5 h and shaking
every 15 min to ensure complete binding. After incubation, samples
were washed with 500 ␮l binding buffer 8 times and centrifuged
(11,000 × g for 3 min) after each wash; supernatants were discarded to remove any unbounded proteins. 200 ␮l elution buffer
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S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
Fig. 1. (A) TEM image of MPA-stabilised AuNP. (B) Histogram of AuNP diameters determined by analysis of approximately 500 nanoparticles located at different regions of
the grid.
(binding buffer containing 25 mM DTT) was added to sample after
the final washing step and incubated on ice for 1 h with gentle shaking every 15 min to release all thiol-containing proteins bound to
activated thiol sepharose. The sample was centrifuged (11, 000 × g,
3 min) and thiol-containing proteins collected in the supernatant
(Hu et al., 2010).
2.6. Electrophoresis
Protein samples were acetone precipitated and total proteins
were separated in 12% SDS–polyacrylamide gels for 1DE on an Atto
AE-6450 mini PAGE system (Atto, Tokyo, Japan) by the method
of Laemmli (1970). For 2DE, proteins collected on activated thiol
sepharose were precipitated with 10% trichloroacetic acid and then
with acetone. After centrifugation, the resulting pellet was resuspended in rehydration buffer containing 5 M urea, 2 M thiourea,
2% CHAPS, 4% carrier ampholyte (Pharmalyte 3–10, GE Healthcare,
Little Chalfont, Bucks, UK), 1% DeStreak reagent (GE Healthcare)
and a trace amount of bromophenol blue. Final volumes of 125 ␮l
were loaded on 7 cm pH 3–10 non-linear immobilised pH gradient
(IPG) strips (BioRad, Hercules, CA, USA) and rehydrated overnight
for at least 15 hr. IPG strips were focused on a Protean IEF Cell
(BioRad) with linear voltage increases. Following IEF, strips were
equilibrated (20 min) in equilibration buffer (6 M urea, 0.375 M
Tris, pH 8.8, 2% SDS, 20% glycerol) containing 2% DTT, and then
for 20 min in equilibration buffer containing 2.5% iodoacetamide.
Equilibrated strips were embedded in molten agarose (0.5%) containing trace bromophenol blue atop 12% SDS–polyacrylamide gels,
and electrophoresed at a constant voltage (150 V) at 4 ◦ C using an
Atto AE-6450 mini PAGE system (Atto, Tokyo, Japan) until the dye
front reached the end of the gel. After separation, gels were silverstained by the method of Rabilloud (1992) followed by scanning
with densitometry.
analysis software as described above. An average of at least three
replicates from three different extracts for each treatment and tissue studied was determined.
Scanned 2DE gel images were sent to the Ludesi Analysis Center (LUDESI AB, Ideon Science Park, Lund, Sweden;
www.ludesi.com) for image analysis using Ludesi’s proprietary
image analysis software, following a protocol that adjusted the
sensitivity in order to detect all true spots in the images.
(http://www.ludesi.com/analysis center). Protein spots were automatically detected and the results were manually verified and
edited where needed with an average of 210 spots resolved.
Gels were matched using all-to-all spot matching, avoiding introduction of bias caused by the use of a reference gel. Matching
was iteratively evaluated and parameters refined to optimize
match quality. Integrated intensities were measured for each spot,
background corrected, and then normalized. This removed systematic gel intensity differences originating from variations in
staining, scanning time and protein loading by mathematically
minimizing the median expression difference between matched
spots.
2.8. Lysosomal membrane stability
Haemolymph was withdrawn from adductor muscle of specimens while alive and immediately analyzed for lysosomal
membrane stability as Neutral Red Retention Time (NRRT; Regoli et
al., 2004). Haemolymph collected from the adductor muscle of 12
specimens was incubated on a glass slide with a freshly prepared
neutral red working solution (2 ␮l/ml saline from a stock solution of
20 mg neutral red dye dissolved in 1 ml DMSO); haemocytes were
microscopically examined (100×) at 20 min intervals to determine
the time at which 50% of cells had lost into the cytosol the dye
previously taken up by lysosomes.
2.7. Quantification of proteins
2.9. Lipid peroxidation
Protein content was calculated by the method of Bradford (1976)
using bovine serum albumin (BSA) as standard. For each 1DE separation, all bands were subsequently analyzed by Quantity One
image analysis software (BioRad, Hercules, CA, USA) measuring the
total intensity for each lane quantified as arbitrary units (a.u.). All
1DE gels were stained with coomassie brilliant blue G250, scanned
in a GS-800 calibrated densitometer (BioRad Laboratories) and the
optical density from each lane measured by Quantity One image
Lipid peroxidation products (as malondialdehyde content) were
analyzed in digestive gland, gill and mantle tissues. After homogenization in 20 mM Tris–HCl, pH 7.4, samples were centrifuged
(3,000 × g, 20 min) and then derivatized in 1 ml reaction mixtures
containing 10.3 mM 1-methyl-2-phenylindole (dissolved in 3:1
acetonitrile:methanol) with 32% HCl, calibrated against a malondialdehyde standard curve and expressed as nmol/g wet wt (Shaw
et al., 2004).
S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
181
Fig. 2. Representative 2DE separations of thiol-containing proteins from digestive gland of (A) control, (B) AuNP- and (C) CdCl2 -treated. Isoelectric point (pI) with range of
pH units from 3 to 10; marker used is with molecular weight (M.W.) which covers from 14.4 kDa (bottom) to 66 kDa (top). Technical replicates were performed at least in
triplicate.
2.10. Statistical analyses
Values were expressed as means ± standard error (S.E.) performed in triplicate. Tissue samples and haemolymph were
compared by one-way analysis of variance (ANOVA). A post hoc
comparison test (Dunnett) was utilized to discriminate between
groups of means. Statistical analyses of data were performed using
the Software Statistica 7.0 (Stat Soft, Tulsa, USA). Statistical analyses of data from 2DE gels were performed by ANOVA at the Ludesi
Analysis Center, with opportune filtering on volume and presence
in each subset of spots. Our data were considered statistically significant only for those showing p < 0.05.
3. Results
3.1. Chemical analyses
To detect uptake of CdCl2 and AuNP, the content of Au and Cd in
digestive gland, gill and mantle were measured. Our previous studies using larger diameters of AuNP revealed modest oxidative stress
in digestive gland, the tissue where highest accumulation of Au was
found by ICP-OES measurements (Tedesco et al., 2008, 2010). The
present study confirmed that digestive gland is a sensitive target
tissue for AuNP of even smaller diameter. Content of Cd and Au
were determined in digestive gland, gill and mantle by ICP-OES.
This showed 12 ±3.4 ␮g of Au per g of wet weight in digestive gland
whilst negligible values were observed in gill and mantle. Therefore, AuNP predominantly accumulates in digestive gland (95%) and
only slightly in gill (3.9%) and mantle (1.5%). No trace of Au was
found in mussels exposed to CdCl2 or controls. By contrast, when
exposed to 0.2 mM CdCl2 , M. edulis showed distribution of cadmium
within gill and digestive gland (23.2 ± 2.25 ␮g of Cd per g of wet
weight and 21.4 ± 6.78 ␮g of Cd per g of wet weight respectively).
Only 16% of the total amount of Cd detected was found in mantle
and insignificant amounts were found in mussels exposed to AuNP
or controls.
3.2. Thiol-containing proteins
Based on these ICP-OES results and the ability of AuNP to bind
thiol groups strongly, we focused our attention on thiol-containing
proteins in digestive glands of mussels exposed to AuNP. Results
were compared with control and CdCl2 treatment in digestive
gland. Triplicate technical and biological replicates were performed
in 1DE of thiol-containing proteins selected by activated thiol
sepharose (Hu et al., 2010). The overall quantity of thiol-containing
proteins collected was lower in AuNP (p < 0.05) than CdCl2 -treated
samples or controls (data not shown). 2DE separations of thiolcontaining proteins of digestive glands from treated and controls
Fig. 3. Quantification of thiol-containing protein spots from 2DE in digestive gland
of control, AuNP- and CdCl2 -treated. Data are presented as mean ± SD and were
performed in triplicate. Significantly different from control (*p < 0.05).
were analyzed in triplicate to quantify and visualize relative differences in volume and presence of spots. This revealed significant
decrease in the total number of spots evident in AuNP-treated
but not in CdCl2 -treated animals (Figs. 2 and 3; p < 0.05). A total
of 24 spots which were consistently present in control separations disappeared in AuNP separations (Fig. 4A numbered with the
individual identification ID; p < 0.05). Details of analysis of two of
the most significantly different spots (ID: 74 and 176) are shown
in Fig. 4B (p < 0.001). These spots, disappeared in CdCl2 -treated
samples also (p < 0.001). Matching based on spot volume revealed
26 thiol-containing protein spots with higher volume in control
than AuNP-treated mussels (Fig. 5A; p < 0.05), of which 6 spots
were most significant (p < 0.001). Furthermore, 19 spots showed
higher volumes in controls than both AuNP or CdCl2 -treated mussels (Fig. 5B; p < 0.05). Results obtained from both 1DE and 2DE
showed stronger effects on thiol-containing proteins in digestive
gland exposed to AuNP compared to CdCl2 suggesting that –SH
groups were modified in response to AuNP exposure.
3.3. Lysosomal membrane stability measured as neutral red
retention time (NRRT)
Our previous study (Tedesco et al., 2008) found lysosomal
membrane stability measured as NRRT in the haemolymph to
be decreased on exposure to menadione though not to AuNP
(∼13 nm). In the present study, significant decrease of NRRT was
found with both treatments (Fig. 6) but especially on AuNP expo-
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S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
Fig. 4. Thiol-containing proteins of digestive gland of M. edulis present in control but not for AuNP- and CdCl2 -treated samples. (A) Representative 2DE separation of thiolcontaining proteins in control, showing 24 spots marked with the identification (ID) numbers present in control and absent in AuNP-exposed, p < 0.05. (B) Three-dimensional
views from representative thiol-containing protein spots (ID 74 and 176) found in digestive gland control but not in both treatments (AuNP- and CdCl2 -exposed), p < 0.001.
sure (p < 0.01). These data are consistent with pro-oxidant effects
of both compounds and emphasise the role of AuNP particle size in
inducing oxidative stress.
(Fig. 7). Our previous investigation (Tedesco et al., 2010) found no
significant increase of malondialdehyde in tissues exposed to AuNP
at ∼13 nm (Tedesco et al., 2010).
3.4. Lipid peroxidation measured as malondialdehyde levels
4. Discussion
Lipid peroxidation was detected in digestive gland, gill and mantle. Malondialdehyde levels did not show any difference between
control, CdCl2 and AuNP-treated samples in gill and mantle
(data not shown) while a significant increase of malondialdehyde
(p = 0.01) was found in digestive gland of animals exposed to AuNP
Nanoparticles represent an emerging potential environmental
threat but there is difficulty in interpreting nanotoxicology studies
due to the range of nanoparticle types and variation of size, shape
and coating (Lu et al., 2009; Oberdörster et al., 2005; Stoeger et al.,
2006).
S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
183
Fig. 5. Thiol-containing proteins from digestive gland downregulated in both treatments (AuNP- and CdCl2 -exposed). (A) Representative 2DE separation of thiol-containing
proteins in control, showing 26 spots marked with their ID numbers with higher volume in control compared to the other treatments, p < 0.05. (B) Three-dimensional views
from 15 representative thiol-containing protein spots in control (white squared) and downregulated in AuNP-exposed (light grey squared) and CdCl2 (dark grey squared)
with at least p < 0.05.
In this study, AuNP accumulated almost exclusively in digestive
gland confirming the results of our previous study on ∼13 nm AuNP
(Tedesco et al., 2010). By contrast, CdCl2 distributed almost equally
between digestive gland and gill. Mussels filter particulate matter and xenobiotics from water, subsequently moving this to the
mouth for ingestion, extracellular digestion by the crystalline style
during passage through the gut and final sorting and absorption
by digestive gland for intracellular digestion (Ward et al., 1993).
Digestive gland is also known to be a key site of xenobiotic and
especially metal detoxification (Moore et al., 2007; Viarengo et
al., 1989). A recent study showed longer gut retention time in M.
edulis exposed to polystyrene nanoparticles, suggesting that most
of the nanoparticles were directed into the tubules of the digestive
glands and potentially taken up by the digestive cells via endocytosis (Ward and Kach, 2009). Difference in distribution between CdCl2
and AuNP suggests selective handling by the animal of the two toxicants with a strong possibility of oxidative stress effects in digestive
gland in particular (McDonagh and Sheehan, 2006; Renault et al.,
2008, Tedesco et al., 2008, 2010).
Protein thiols play numerous roles in biology including in
antioxidant defence and absorption of ROS resulting in their oxidation to derivatives such as disulphides, sulphinic, sulphonic and
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Fig. 6. Lysosomal membrane stability measured as NRRT in haemolymph of M. edulis
of control, AuNP and CdCl2 treated. Data are presented as mean ± SD with n = 10.
Significantly different from control (*p < 0.05) and (**p < 0.01).
sulphenic acids (Eaton, 2006; Hansen et al., 2009). Some proteins
use ROS-mediated thiol modification specifically to regulate their
function in signalling and transcriptional processes (Brandes et al.,
2009). Activated thiol sepharose affinity-selects thiol-containing
proteins from digestive gland (oxidised variants of thiols do not
bind to this resin) (Hu et al., 2010). M. edulis exposed to AuNP
exhibited a decreased amount of thiol-containing proteins in comparison both to controls and CdCl2 -treated. This is consistent with
direct oxidation of thiols by ROS induced in response to AuNP. Cadmium is a non-redox metal widespread in aquatic sediments which
indirectly enhances formation of ROS (Liu et al., 2009). However,
previous work showed stronger oxidative stress effects in gill of M.
edulis exposed for 24 h to CdCl2 (McDonagh and Sheehan, 2006),
and a strong positive correlation between total non-protein-SH
and Cd has been demonstrated in an aquatic macrophyte, suggesting a central role for thiols in Cd detoxification (Mishra et al.,
2009). The sub-proteome of thiol-containing proteins was further
analyzed by 2DE which revealed changes in spot patterns consistent with greater protein thiol oxidation in response to AuNP
compared to both CdCl2 and control. It has been pointed out that
Fig. 7. Lipid peroxidation of digestive gland of M. edulis measured as malondialdehyde (MDA) levels. Data are presented as mean ± SD, n = 5. Significantly different
from control (*p < 0.05).
some proteins are especially susceptible to ROS depending on factors such as individual ROS rate constants with differing residues,
concentration of targets and lowered pKa of specific protein thiols
(Winterbourn and Hampton, 2008). AuNP themselves can interact
with organic macromolecules present in the environment (Diegoli
et al., 2008) and have especially strong affinity for protein thiol
groups (Cedervall et al., 2007; Krpetic et al., 2009; Aubin-Tam et
al., 2009). Given the low concentration of AuNP used in our in
vivo study, direct binding of thiols to AuNP cannot quantitatively
account for the observed decrease in thiol-containing proteins and
direct production of ROS is a more plausible explanation. This is
further supported by more traditional independent measures of
biological and oxidative stress such as lysosomal membrane stability and lipid peroxidation.
Measurements of NRRT (Reeves et al., 2008; Tedesco et al., 2008)
showed a decrease for both treatments (AuNP and CdCl2 ) confirming significant biological stress. In this study, the effect was stronger
in the case of AuNP than CdCl2 or than that previously reported for
AuNP of larger diameter (Tedesco et al., 2008). Koehler et al. (2008)
reported decreased lysosomal membrane stability in tubules of M.
edulis hepatopancreas after 12 h of exposure to glass wool nanoparticles while AuNP of larger size induced acute inflammation and
apoptosis in the mouse liver and were found to accumulate in lysosomes of liver Kupffer cells and spleen macrophages up to 7 days
(Cho et al., 2009). Our study also showed, for the first time, that
AuNP (∼5 nm) caused significant lipid peroxidation in digestive
gland. A previous in vitro study revealed that AuNP (2 and 10 nm)
caused intracellular damage in human epithelial cells. Cytoplasmic
vacuoles containing aggregates of 10 nm AuNP showed worst vacuolar membrane damage. It was concluded that direct contact of the
Au-core with vacuolar membranes led to membrane lipid peroxidation, producing breaks in the vacuolar membranes. In contrast,
2 nm AuNP became localised within cell nuclei where heterochromatin was no longer visible, suggesting that nuclear condensed
DNA had been altered, or damaged (Panessa-Warren et al., 2008).
Pan et al. (2009) recently demonstrated that oxidative stress triggered by small AuNP exposure is most likely amplified further by
mitochondrial damage.
Different pro-oxidants do not necessarily cause generation
of the same ROS population (Davies, 2005; Winterbourn and
Hampton, 2008) but several instructive points of comparison are
possible between AuNP and CdCl2 treatments used in this study. We
previously observed that treatment with CdCl2 increased antioxidant defences in M. edulis (McDonagh and Sheehan, 2006) but, in
the present study, AuNP was consistently more strongly oxidizing
as evidenced by lipid peroxidation, decreased NRRT and decreased
numbers of thiol-containing proteins evident in electrophoretic
separations. CdCl2 treatment only differed from controls on the
basis of decreased NRRT. It is well known that cadmium may
displace iron or copper from metalloproteins leading to oxidative stress via the Fenton reaction (Ma, 2010). Although cadmium
is highly reactive to protein thiols (Ma, 2010; McDonagh and
Sheehan, 2006), small AuNP seemed to have greater effects on
thiol-containing protein profiles in this study than this well-known
environmental pollutant. ROS production could result from the
proportionately high surface area of AuNP used in this investigation (Nel et al., 2006). AuNP (∼5 nm) are known to catalyze
NO production from endogenous S-nitroso adducts with thiol
groups in blood serum (Jia et al., 2009). NO reacts rapidly with
superoxide producing peroxynitrite (ONOO− ) which can interact
with lipids, DNA, and proteins via direct oxidative reactions or
via indirect, radical-mediated damage (Senaratne et al., 2006). In
particular, our work showed AuNP (∼5 nm) caused significantly
greater oxidative stress and cytotoxicity effects than AuNP of larger
average diameters (Pan et al., 2007, 2009; Tedesco et al., 2008,
2010).
S. Tedesco et al. / Aquatic Toxicology 100 (2010) 178–186
Competing interest
All the authors declare to have no competing financial interests.
Acknowledgments
This work was supported by the Programme for Research in
Third level Institutions of Higher Education Authority of Ireland.
Sara Tedesco is supported by an EMBARK PhD fellowship of the
Irish Research Council for Science Engineering and Technology. Prof
Sheehan’s group is a member of the NeuroNano consortium (EU
Small Collaborative Project NNP4-SL-2008-214547).
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