Aquatic Toxicology 84 (2007) 153–161
Metallothionein turnover, cytosolic distribution and the
uptake of Cd by the green mussel Perna viridis
T.Y.-T. Ng a , P.S. Rainbow b , C. Amiard-Triquet c , J.C. Amiard c , W.-X. Wang a,∗
a
Department of Biology, The Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon, Hong Kong, China
b Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom
c Université de Nantes, Nantes Atlantique Universités, Faculté de Pharmacie, SMAB, Service d’écotoxicologie, F-44000 Nantes, France
Received 30 October 2006; received in revised form 11 January 2007; accepted 27 January 2007
Abstract
We examined the relationship between Cd kinetics (uptake from solution and diet, and efflux), metallothionein turnover, and changes in the
cytosolic distribution of accumulated Cd between protein fractions in the green mussel Perna viridis. We pre-exposed the mussels to 5, 20, 50
and 200 ␮g l−1 of Cd for 1 week and determined the biokinetics of Cd uptake and efflux in the mussels. The dietary assimilation efficiency
of Cd increased by 2 times following exposure to 20–200 ␮g l−1 Cd, but the dissolved uptake rate was unchanged by pre-exposure to any Cd
concentrations. The efflux rate of Cd was also similar among control and Cd pre-exposed mussels. The cytosolic distribution of Cd in the mussels
that had been exposed to dissolved Cd, showed that besides metallothionein (7000–20,000 Da), high molecular weight proteins (>20,000 Da) were
important for Cd binding and depuration. In general, the Cd pre-exposed mussels had higher metallothionein turnover with a higher metallothionein
synthesis rate, but similar metallothionein breakdown rates as the control mussels. Metallothionein synthesis rate was correlated to the dietary
assimilation of Cd, whereas metallothionein breakdown and Cd efflux rate were independent of each other. This study provides important new
information for the role of metallothionein turnover on Cd kinetics in an aquatic invertebrate.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Cd; Kinetics; Cytosolic distribution; Metallothionein turnover; Perna viridis
1. Introduction
In marine bivalves cytosolic proteins play a vital role in metal
sequestration, toxicity and elimination, albeit with variation
among different species (Amiard et al., 2006). Metallothioneins (MTs) are cysteine-rich, metal-binding, heat-stable low
molecular weight cytosolic proteins which play a role in the
detoxification of certain trace metals including Ag, Cd, Cu, Hg
and Zn. Cd and Cu in the cytosol of the clam Ruditapes decussatus had the greatest degree of binding to MT (Bebianno and
Serafim, 2003). Furthermore, MT concentrations increased in
the clam Ruditapes philippinarum and the green mussel Perna
viridis after they were exposed to increasing concentrations of
Ag (Ng and Wang, 2004) and Cd (Shi and Wang, 2005) respectively in the laboratory. In contrast, metals bound to non-MT
cytosolic proteins such as high molecular weight (HMW) and
low molecular weight (LMW) proteins have often been associ-
∗
Corresponding author. Tel.: +852 2358 7346; fax: +852 2358 1559.
E-mail address: [email protected] (W.-X. Wang).
0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2007.01.010
ated with deleterious effects at different biological levels, e.g.,
lipid peroxidation, growth reduction, reproduction impairment,
behaviour modification and mortality (Couillard et al., 1995;
Baudrimont et al., 1999; Wallace et al., 2000). In many previous
studies, the role of MT has been emphasized, while the roles
of other cytosolic proteins are often neglected. Our recent study
provided preliminary evidence that heat-sensitive or HMW proteins can act as a temporary pool for storing metals when green
mussels first encounter the metals (Ng and Wang, 2005a). Thus,
it is of interest to examine any relationship between differences
in uptake rate of a trace metal such as Cd after metal preexposure and associated differences in the distribution of the
metal between cytosolic proteins.
MT is not only involved in metal detoxification processes
(Amiard et al., 2006), but also plays an essential role in other
metabolic processes (Mason and Jenkins, 1995). MT, like other
proteins, will be degraded in lysosomes. The activation of the
physiological process involving MT in response to a metal challenge might be reflected in increased turnover (synthesis and
breakdown) of the protein, but not necessarily in changes in MT
concentration (Mouneyrac et al., 2002). Such faster turnover
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T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
may represent a dynamic steady state with proteins undergoing continuous and often selective breakdown. Metals released
during the protein breakdown have the potential to bind to
newly synthesized MT, so extending the turnover time of the
metal in relation to the protein (Roesijadi, 1995; Mouneyrac et
al., 2002). Thus in the oyster Crassostrea virginica, the biological half life of Cd bound to MT was 70 days, whereas
the half life of the MT protein itself was only 4 or 20 days
depending whether the estimate was made during or immediately after Cd exposure (Roesijadi and Klerks, 1989; Roesijadi,
1995). As a result of the different affinities of the relevant metals for MT and the differential susceptibilities of MT-bound
metals to lysosomal degradation, the uptake and excretion of
metals may be affected to different degrees by changes in MT
turnover rate. MT turnover has been investigated in the marine
mussel Mytilus edulis (Bebianno and Langston, 1993) and the
gastropod Littorina littorea (Bebianno and Langston, 1998).
These earlier studies emphasized the relationship between MT
breakdown and metal elimination, but no study has examined the relationship between MT synthesis and metal uptake
kinetics.
In this study, we have attempted to investigate the relationships between MT turnover, changes in the cytosolic distribution
of accumulated Cd and Cd kinetics (uptake from solution and
diet, and efflux) in the green mussel P. viridis. Metal exposure
was found to alter the Cd uptake kinetics by the mussels (Wang
and Rainbow, 2005). In order to have a variation of uptake kinetics and MT concentration in the mussels, we pre-exposed the
mussels to dissolved Cd for 1 week and determined the subsequent kinetics of Cd uptake using gamma-emitting 109 Cd as a
radiotracer. MT synthesis and breakdown were determined using
beta-emitting l-[35 S] cysteine as a marker since MT is atypically
rich in the sulphur-bearing amino acid cysteine (Amiard et al.,
2006) and 35 S has been previously applied to determine MT
breakdown in molluscs (Bebianno and Langston, 1993, 1998).
Cd distribution among cytosolic proteins in the mussels was
analyzed using high performance liquid chromatography.
2. Materials and methods
2.1. Collection of mussels and Cd pre-exposure
Green mussels P. viridis (shell length: 2.5–3 cm) were collected from Ma Liu Shui, Tolo Harbour, Hong Kong. The
mussels were allowed to acclimate in aerated seawater (24 ◦ C,
33 psu) in the laboratory and fed with the diatom Thalassiosira
pseudonana at a ration of 2% mussel tissue dry weight per day.
After acclimation for 1 week, 50 mussels were transferred to 10 l
seawater without added Cd (control) or with stable Cd (5, 20, 50,
or 200 ␮g l−1 ) for 1 week. The series of Cd concentrations was
selected for a variation of responses in the mussels. Although
some concentrations (e.g., 50 and 200 ␮g l−1 ) that we used in this
study were 50–100× higher than environmentally realistic concentrations, we used these as an extreme scenario to investigate
how mussels handled or detoxified Cd as the ambient Cd concentration increased. The different concentrations of Cd were
prepared from CdCl2 ·2H2 O (analytical grade, 1000 mg l−1 ).
Mussels were exposed to dissolved Cd for 16 h each day, then
placed in clean seawater and fed with T. pseudonana for 8 h.
2.2. Concentrations of Cd and metallothionein (MT) in the
mussels
Five mussels were taken from each treatment for the analysis
of the accumulated Cd concentrations in the soft tissues after
the week’s exposure. Mussels were rinsed with filtered seawater
to remove the surface adsorbed Cd, then the soft tissue was dissected and dried at 80 ◦ C. Concentrated nitric acid (trace-metal
grade) was added to digest the tissue at 110 ◦ C for 1 day. Then,
the digest was diluted by 2% nitric acid and the Cd concentration
in the samples was measured by inductively coupled plasmamass spectroscopy. Oyster tissue (standard reference material
1566b, National Institute of Standards and Technology) was
similarly digested and used as a quality control of the metal
analysis. Agreement was within 10%. The Cd concentration of
mussels is expressed as ␮g g−1 dry wt.
A silver saturation method (Scheuhammer and Cherian,
1991) is well established for measuring MT concentration in
bivalves and was used to measure the MT (if any) induced
by Cd in the mussel tissue. Five mussels from each treatment
were dissected and the soft tissue weighed. The tissue was
then homogenized in 3 ml Tris–base buffer (20 mM, pH 7) with
10 mM ␤-mercaptoethanol (antioxidant) and 0.1 mM phenylmethylsulfonyl fluoride (protease inhibitor). The homogenate
was centrifuged at 20,000 × g for 20 min at 4 ◦ C and separated into soluble (supernatant) and insoluble (pellet) fractions.
The soluble fraction was used for the MT assay. Briefly, 0.5 ml
20 mg l−1 stable Ag with 20 kBq ml−1 110m Ag in 0.5 M glycine
buffer was added to 0.3 ml of the homogenate soluble fraction to saturate the MT-binding sites for 10 min before analysis.
Then excess Ag was removed by adding 0.1 ml rabbit blood cell
haemolysate prepared as in Scheuhammer and Cherain (1986),
followed by heat treatment at 100 ◦ C for 5 min and centrifugation
at 785 × g for 5 min. The addition of haemolysate and heat treatment was repeated 2 times. Then, the supernatant was analyzed
for 110m Ag. MT concentration (␮g g−1 wet wt.) was calculated
as 3.55× the Ag concentration as in the mammalian tissue.
MT recovery was >70%, using standard MT from rabbit liver
(Sigma). The MT concentration was expressed as ␮g g−1 wet wt.
of tissue.
2.3. Assimilation of Cd from phytoplankton by the
pre-exposed mussels
The dietary uptake of Cd from the phytoplankton was
determined using our established methods for the mussels
(Blackmore and Wang, 2002). The diatoms T. pseudonana were
collected during the exponential phase of growth and added to
250 ml filtered seawater with f/2 nutrient (Guillard and Ryther,
1962) that did not contain EDTA, Cu or Zn. 109 Cd (74 kBq−1 ,
equivalent to 0.6 ␮g l−1 ) was spiked into the medium and the
diatoms were cultured for 4 days in order to attain uniform
labelling of the cells. Cd concentration in the radiolabelled
diatoms was about 1.2 ␮g g−1 . Cells were then filtered by a 3 ␮m
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
polycarbonate membrane and resuspended in filtered seawater
twice to remove the surface loosely adsorbed Cd on the cells.
Five mussels from each treatment were put individually into containers with 500 ml filtered seawater and acclimated for 5 min
before feeding. Radiolabelled diatoms were added at 10 min
intervals for 30 min. After feeding, the mussels were rinsed with
filtered seawater and taken for 109 Cd live counting. These mussels were then left for depuration in a 20 l seawater circulating
tank. The depuration of 109 Cd was monitored at 3, 7, 12, 24, 36
and 48 h. Faeces from the mussels were removed and unlabelled
T. pseudonana was supplied to the mussels regularly. Assimilation efficiency (%, AE) was defined as the percentage of 109 Cd
retained in the mussels at 48 h.
2.4. Dissolved uptake of 109 Cd and subcellular distribution
of 109 Cd and 35 S
Three pre-exposed mussels from each treatment were placed
into each of three replicate beakers with 400 ml filtered seawater spiked with 280 kBq 109 Cd l−1 (equivalent to 2.22 ␮g l−1 Cd)
and 460 kBq 35 S l−1 (in the form of l-[35 S] cysteine, equivalent
to 0.12 ␮M). Water was also dual labelled with l-[35 S] cysteine
for uptake because we were interested in examining the distribution of l-[35 S] cysteine in the cytosolic proteins, especially in the
MT-like protein (MTLP). One mussel and 4 ml water were sampled from each beaker at 0 (water only), 1, 2 and 3 h (water and
mussels) for 109 Cd and 35 S analysis. The soft tissues of the mussels were dissected out, weighed wet and homogenized in 3 ml
20 mM Tris–base buffer with antioxidant and protease inhibitor
(the same buffer used in the silver saturation assay, except with
the addition of 150 mM NaCl). The tissue homogenate of each
mussel was separated into cytosol and insoluble fractions by differential centrifugation. Briefly, the homogenate was centrifuged
at 1450 × g at 4 ◦ C for 15 min, and then the supernatant was
further centrifuged at 100,000 × g at 4 ◦ C for 1 h to separate
the cytosol (supernatant) and organelles (pellet). The organelles
were combined with the pellet collected from the first spin as
the total insoluble fraction.
The cytosol and insoluble fractions were then analyzed for
109 Cd. The cytosol was filtered through a methanol and Tris–
base buffer pre-conditioned Waters Sep-Pak® cartridge (C18
reversed phase sorbent) to remove the lipids or large molecules
(>80 ␮m) before further fractionation by high performance liquid chromatography (HPLC). The Waters Macrosphere GPC
100A size exclusion column (250 mm × 4.6 mm, separation
size: 2500–350,000 Da) had first been calibrated with protein
standards (BioRad Gel Filtration Standard, molecular size of
markers: 1350–670,000 Da), and MT standard from rabbit liver
(Sigma) was used as internal quality control. Aliquots of samples (100 ␮l) were injected into the column and eluted by the
Tris–base buffer (20 mM, pH 7, 150 mM NaCl) at a flow rate
of 0.4 ml min−1 . Although most studies monitor MT at a wavelength of 254 nm, we used 280 nm to detect the total protein
because we were also interested in examining proteins other than
MT, e.g., high molecular weight protein. When a peak appeared,
the fraction was collected for 109 Cd counting, followed by
an addition of 3 ml scintillation cocktail (Wallac OptiPhase
155
‘HiSafe’ 3). The mixture was incubated in the dark overnight
before analysis for 35 S. We defined the cytosolic proteins according to their molecular size—HMW, MTLP and LMW proteins.
Since the molecular size of MTLP is reported to range from 7000
to 20,000 Da in bivalves (Geret and Cosson, 2002; Ciocan and
Rotchell, 2004), we defined the fractions with molecular sizes
7000–20,000 Da as MTLP, <7000 Da as LMW, >20,000 Da as
HMW. Since MTLP is often separated from the heat-treated
cytosol in most studies, we compared the chromatograms of
the heat-treated cytosol and untreated cytosol of the mussels
in our trial experiments. We found no difference between the
two protein profiles, which may support that the protein separated with molecular size 7000–20,000 Da was only MTLP and
thereafter, we did not heat-treat the cytosol for size exclusion
chromatography. Counts of 109 Cd or 35 S in the tissue and cytosolic fractions were normalized to wet wt. of the tissue (cpm g−1 )
and convert to concentrations (pg g−1 ) for Cd and l-[35 S]
cysteine.
2.5. Depuration of dissolved 109 Cd and subcellular
distribution of 109 Cd and 35 S
About 20 mussels from each Cd pre-exposure treatment were
exposed to dissolved 109 Cd and 35 S with an additional spike of
0.46 kBq 35 S ml−1 to ensure a high concentration of 35 S in the
water during the exposure period. Water was dual labelled with
35 S for the similar reason as uptake. Water samples were taken
for 109 Cd and 35 S analysis at the beginning and end of exposure.
After 15 h, the water was replaced and the mussels were left for
depuration in the clean seawater. Three mussels were immediately sampled for 109 Cd and 35 S analysis, with further samples
being taken on days 1, 2, 3, 4 and 6 of depuration. Seawater
was renewed daily. Mussels were dissected and homogenized
as described above for 109 Cd and 35 S analysis in the cytosol,
cytosolic proteins and insoluble fraction.
2.6. Radioactivity measurement and statistical analysis
109 Cd
radioactivity was measured on a Wallac 1480 Wizard 3 in. Perkin-Elmer gamma counter at energy level 88 keV,
whereas the radioactivity of 35 S was measured on a Wallac
WinSpectral 1414 Liquid Scintillation counter (Perkin-Elmer).
Radioactivity was corrected for background, quenching effects
and decay, and the samples were counted for 2–5 min. Since
the samples were dual labelled with 109 Cd and 35 S, we tested
the interference between gamma and beta emissions before
the experiments. Results showed that 35 S increased the 109 Cd
gamma activity by <0.1% and 109 Cd increased 35 S beta activity
by <10%. Therefore, we ignored the interaction and used the
counts directly without any correction.
Data were checked for homogeneity and normal distribution
and the percentage data were arcsine transformed for parametric
statistical analysis. One-way ANOVA was used to test for difference among groups of pre-exposed mussels (P < 0.05). Then,
post hoc tests were applied to identify the differences between
groups (P < 0.05). Regression analysis was used to determine
the regression coefficient (r2 ) and significance (P < 0.05) of
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T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
relationships describing 109 Cd uptake rate in soft tissue, 109 Cd
and l-[35 S] cysteine uptake or efflux rate in cytosolic proteins.
3. Results
3.1. Concentrations of Cd and MT in the pre-exposed
mussels
Cd exposure increased the Cd concentration in the mussel
soft tissues after 1 week. Cd exposure at or above 20 ␮g l−1 for 1
week increased the tissue concentration significantly by 10–100
times (Table 1, P < 0.05). MT concentration in the tissue showed
an increasing trend as the Cd exposure concentration increased,
but there was no statistically significant difference between the
control and other treatments (Table 1, P > 0.05) except that the
MT concentration in the 50 ␮g l−1 exposed mussels was significantly higher than in the 5 ␮g l−1 exposed ones. When MT and
Cd concentration was compared in the same treatment, MT concentrations were similar at Cd tissue levels <10 ␮g g−1 dry wt.,
then increased rapidly and reached a plateau with Cd concentration in the tissue increasing from 20 to 200 ␮g Cd g−1 dry wt.
3.2. Assimilation, dissolved uptake of Cd and subcellular
distribution of 109 Cd and 35 S
Dietary uptake of Cd in the mussels was affected by the preexposure. The AE of Cd was elevated by 2 times after 1 week’s
pre-exposure to 20, 50 and 200 ␮g Cd l−1 (Table 1, P < 0.01).
The 109 Cd and 35 S counts in the water samples were similar
over the period of dissolved uptake, indicating that there was
constant supply of both Cd and cysteine for the mussels. Dissolved uptake of 109 Cd in the whole soft tissues was significantly
correlated to the length of exposure; thus it was possible to calculate the uptake rate (slope constant of the linear regression
in ng g−1 h−1 , Table 1). The controls and Cd pre-exposed mussels had similar Cd uptake rates from the water. The subcellular
distribution of 109 Cd showed that a significantly lower percentage of 109 Cd was associated with the cytosol (44%, P < 0.01)
than with the insoluble fraction. This percentage did not differ significantly among treatments or over the uptake period
(P > 0.05).
109 Cd
did not bind only to MTLP but was also present in
the HMW fraction and to a lesser degree in the LMW fraction (Fig. 1). 109 Cd uptake increased linearly over time in the
HMW protein of most mussels but few significant linear relationships were observed for 109 Cd uptake in the LMW and MTLP
protein fractions. Taking into account the relationship between
109 Cd activity in each fraction and the length of exposure, the
uptake rate in each fraction for each treatment was calculated
from the significant linear regression curves (Table 2). Uptake
rate of 109 Cd into the HMW protein was the highest among all
the cytosolic proteins and furthermore, increase of uptake into
cytosolic proteins was more apparent in the mussels pre-exposed
to Cd. There were positive correlations between total 109 Cd in
the cytosol and 109 Cd associated with individual cytosolic fractions, but the regression for 109 Cd associated with LMW was
not strong (Fig. 2). Thus, MTLP and HMW proteins were both
important for binding Cd when there was an increase of Cd taken
up into the mussels.
l-[35 S] cysteine kinetics were used as a marker of MT
turnover in the mussels. The majority of l-[35 S] cysteine was
incorporated into MTLP and HMW protein (>85%), whereas
very little was associated with LMW protein (5%). There was
higher distribution of l-[35 S] cysteine in the MTLP of the mussels pre-exposed to the higher concentrations of Cd (60% in
MTLP of mussels exposed to 200 ␮g l−1 Cd) (Fig. 1). l-[35 S]
cysteine concentration increased only significantly and linearly
with time in the MTLP of the 20 and 50 ␮g l−1 Cd pre-exposed
mussels, and the HMW protein of the 200 ␮g l−1 Cd pre-exposed
mussels. The uptake rate of l-[35 S] cysteine was similarly calculated as explained above for 109 Cd (Table 2), and was the highest
in the MTLP and HMW fractions, but it was undetectable in the
mussels pre-exposed to low Cd concentrations, e.g., Ctl-LMW
and Ctl, Cd5-HMW fractions (Table 2). The increase of l-[35 S]
cysteine also appeared more apparent in the mussels pre-exposed
to the higher Cd concentrations.
3.3. Cd depuration and subcellular distribution of 109 Cd
and 35 S
Again, 109 Cd and l-[35 S] cysteine concentrations in the water
were similar at the beginning and end of the 15 h exposure. During the 7 days of depuration, Cd pre-exposed mussels had similar
Table 1
Concentrations of Cd and metallothionein (MT) in the tissue (␮g g−1 ) and parameters of Cd kinetics in the mussel Perna viridis pre-exposed to dissolved Cd
Treatment
Cd concentration
(␮g g−1 dry wt.)
Ctl
Cd5
Cd20
Cd50
Cd200
0.6
1.5
10.8
23.0
103.1
±
±
±
±
±
0.4 a
0.9 a
0.9 b
3.9 c
23.2 d
MT concentration
(␮g g−1 wet wt.)
13.0
9.1
13.9
23.7
21.2
±
±
±
±
±
1.7 ab
4.2 a
5.7 ab
8.4 b
6.5 ab
109 Cd uptake rate from
water (ng g−1 h−1 )
1.4
1.2
1.2
1.7
1.2
±
±
±
±
±
0.2**
0.2**
0.2**
0.5*
0.3*
ke (day−1 )
AE (%)
28.9
27.5
50.8
59.9
65.1
±
±
±
±
±
7.5 a
9.1 a
9.2 b
9.0 b
6.2 b
0.043
0.042
0.056
0.033
0.044
Mean ± standard deviation (n = 3–5). AE: Cd dietary assimilation efficiency (%). ke : Cd efflux rate constant from the tissue (day−1 ). 109 Cd uptake rate from water
(ng g−1 h−1 ) was the slope of regression of total 109 Cd uptake in the mussel tissue against time. ke was calculated from the slope of regression of ln(% 109 Cd activity
retained) against time during efflux. Standard error was expressed for Cd uptake rate and ke (slope ± standard error). Asterisk(s) indicate significant fits to linear
regression lines with significance at P < 0.05 (*) and P < 0.01 (**). Different letters indicate significant difference between groups. Ctl: control and numbers after Cd
are the pre-exposed concentrations at ␮g l−1 .
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
157
Fig. 1. Concentrations of 109 Cd and l-[35 S] cysteine (pg g−1 wet wt.) bound to cytosolic proteins in Perna viridis over the uptake period. Mean ± standard deviation
(n = 3). Regression lines were plotted when the regressions were significant (P < 0.05). Low molecular weight (LMW): <7000 Da; metallothionein-like protein
(MTLP): 7000–20,000 Da; high molecular weight (HMW): >20,000 Da. Ctl: control and numbers after Cd are the pre-exposed concentrations at ␮g l−1 .
Table 2
Uptake or efflux rates of 109 Cd and l-[35 S] cysteine (pg g−1 h−1 ) in cytosolic proteins of the mussel P. viridis pre-exposed to dissolved Cd
Treatment
109 Cd
(pg g−1 h−1 )
l-[35 S] cysteine uptake rate
(pg g−1 h−1 )
l-[35 S] cysteine efflux rate
(day−1 )
uptake rate
LMW
Ctl
Cd5
Cd20
Cd50
Cd200
ND
ND
ND
1.5 ± 0.5*
6.7 ± 0.1**
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
MTLP
Ctl
Cd5
Cd20
Cd50
Cd200
ND
3.5 ± 1.0*
ND
ND
ND
ND
ND
32.5 ± 2.5**
48.9 ± 6.3**
ND
−1.3 ± 0.5*
−1.0 ± 0.3**
−1.1 ± 0.4**
−1.4 ± 0.4**
ND
HMW
Ctl
Cd5
Cd20
Cd50
Cd200
4.0 ± 0.8*
ND
5.0 ± 0.3*
6.6 ± 1.8*
4.7 ± 0.1*
ND
ND
ND
ND
31.6 ± 8.9**
ND
ND
ND
ND
ND
109 Cd or l-[35 S] cysteine uptake or efflux rate was the slope of regression of 109 Cd or l-[35 S] cysteine concentrations in each cytosolic protein against time. Standard
error was expressed for 109 Cd, l-[35 S] cysteine uptake and efflux rates (slope ± standard error). Asterisk(s) indicate significant fit to regression lines at P < 0.05 (*)
and P < 0.01 (**). Low molecular weight (LMW): <7000 Da; metallothionein-like protein (MTLP): 7000–20,000 Da; high molecular weight (HMW): >20,000 Da.
Ctl: control and numbers after Cd are the pre-exposed concentrations at ␮g l−1 . ND: not determined due to non-significant regressions or fluctuations in l-[35 S]
cysteine concentration over time, wherever applicable.
158
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
efflux kinetics to each other and to the controls (Table 1). Cd
was depurated at 3–6% each day. After 2 days of depuration,
a relative Cd-enrichment in the cytosol (10–15% increase) was
found in the control and Cd pre-exposed mussels (P < 0.05).
This percentage then remained constant afterwards. 109 Cd was
depurated relatively similarly from the different protein fractions among the mussels, regardless of their pre-exposure history
(Fig. 3). The concentration of 109 Cd in the LMW proteins did
not change significantly whereas the concentration in the MTLP
and HMW proteins fluctuated over time. There was an obvious
increase of 109 Cd in the HMW proteins between day 0 and day
2, and an increase of 109 Cd in MTLP between day 2 and day 4,
especially in the mussels pre-exposed to higher Cd concentrations (50 and 200 ␮g l−1 ). Since there was no consistent trend
of 109 Cd in the different protein fractions over time, we did not
fit the data to any regression lines. When there was a reduction of 109 Cd in the HMW and MTLP, the total 109 Cd in the
cytosol also reduced, but the relationship was weak for MTLP
(Fig. 2).
Release of l-[35 S] cysteine accompanied the depuration of
Cd from the mussels (Fig. 3). l-[35 S] cysteine associated with
MTLP decreased exponentially. Only about 15% l-[35 S] cysteine remained in the MTLP after 1 day, subsequently it became
constant. The loss or efflux rate of l-[35 S] cysteine from MTLP
in general was independent of the exposure history, except for a
non-significant regression relationship for mussels pre-exposed
to 200 ␮g l−1 Cd (Table 2, Fig. 3). The half life of MT was determined by dividing ln 2 over l-[35 S] cysteine efflux rate and it
ranged between 12 and 16 h for control and mussels pre-exposed
to 5, 20 and 50 ␮g l−1 Cd. About 50% of l-[35 S] cysteine was
lost from the LMW and HMW protein fractions over the depuration period. No regression lines were used to fit the data of
LMW and HMW but a trend of decreasing l-[35 S] cysteine was
observed.
3.4. Correlation analysis
MT concentration in the mussels was positively related to l[35 S] cysteine uptake rate into MTLP (Fig. 4). l-[35 S] cysteine
uptake rate was also positively correlated to the Cd AE of the
mussels (Fig. 4). However, l-[35 S] cysteine synthesis rate was
not related to the Cd dissolved uptake rate in the mussels.
4. Discussion
Fig. 2. Correlations between the concentration of 109 Cd (pg g−1 wet wt.) in
individual cytosolic protein and the total 109 Cd concentration in the cytosol
of control and Cd pre-exposed mussels that was put into water labelled with
109 Cd for uptake and depurated for 7 days. Total 109 Cd concentration in the
cytosol was the sum of 109 Cd concentration in the LMW, MTLP and HMW
fractions. Low molecular weight (LMW): <7000 Da; metallothionein-like protein (MTLP): 7000–20,000 Da; high molecular weight (HMW): >20,000 Da.
Asterisk(s) indicate significant regressions at P < 0.05 (*) and P < 0.01 (**).
The accumulation rates of Cd and the effects of Cd preexposure on the green mussels were similar to those reported
previously (Blackmore and Wang, 2002; Shi and Wang, 2005).
The relationship of MT concentration and Cd soft tissue concentration of the mussels implies that MT is important for
detoxifying Cd in green mussels, but the detoxification process
was overwhelmed when the Cd exposure continued to increase
(Mason and Jenkins, 1995). The mussels assimilated more Cd
from ingested phytoplankton after the acute Cd pre-exposure,
but the pre-exposure did not affect the Cd uptake kinetics from
the water. Nevertheless, dissolved Cd was incorporated at different rates into the cytosolic proteins of mussels differentially
pre-exposed to Cd.
Cytosol plays an important role in the binding of accumulated
Cd in the mussels since about half of the labelled Cd was distributed among cytosolic proteins in the dissolved exposure, of
which HMW proteins and MTLP were the dominant subcellular
pools for Cd. Cd was distributed to the greatest extent to HMW
protein, and uptake of Cd into this pool showed the most significant relationships with time. The same pattern of Cd penetrating
HMW proteins at the beginning of the exposure was shown in
different bivalves (Evtushenko et al., 1986 and literature cited
therein). In this study, we used 10 mM mercaptoethanol as reducing agent that protected the structure of MT and prevented the
aggregation of proteins during protein extraction. It has been
suggested that the presence of reducing agents may be responsible for increased metal binding in the LMW proteins of the
bivalves (Roesijadi and Drum, 1982; Lobel, 1989; Bragigand
and Berthet, 2003), thus causing artifacts on the separation of
metal-binding proteins. On the other hand, most authors consider
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
159
Fig. 3. 109 Cd and l-[35 S] cysteine concentrations (pg g−1 wet wt.) in the cytosolic proteins of P. viridis during depuration. Mean ± standard deviation (n = 3).
Regression lines were plotted when the regressions were significant (P < 0.05). No regression was fitted for 109 Cd and l-[35 S] cysteine concentrations in LMW and
HMW proteins due to fluctuation trends over time. Low molecular weight (LMW): <7000 Da; metallothionein-like protein (MTLP): 7000–20,000 Da; high molecular
weight (HMW): >20,000 Da. Ctl: control and numbers after Cd are the pre-exposed concentrations at ␮g l−1 .
Fig. 4. Correlations between uptake rate of l-[35 S] cysteine (pg g−1 h−1 ) in
metallothionein-like protein (MTLP) or MT concentration (␮g g−1 wet wt.) and
Cd assimilation efficiency (%, AE) in P. viridis. Regression lines were plotted when the regressions were significant (P < 0.05). Note that l-[35 S] cysteine
uptake rate was calculated for correlation analysis in the control and mussels
pre-exposed to 5 and 200 ␮g l−1 Cd although the regressions of l-[35 S] cysteine
uptake against time (Fig. 1) were not significant. Asterisk indicates significant
regressions at P < 0.05 (*).
that mercaptoethanol plays a protective role for MT structure,
allowing the preservation of metal binding to MT during chromatography (Minkel et al., 1980; Lobel, 1989; Carpene, 1993;
Bragigand and Berthet, 2003). In the present work, any potential
artifactual effect of mercaptoethanol would have led to an overestimate of Cd binding in the LMW fraction, a bias that would
not have promoted the observed comparatively higher importance of MT and HMW proteins in Cd binding. Cd pre-exposed
mussels had more significant uptake of Cd into cytosolic proteins over time, compared to the control mussels, implying that
the mussels were more efficient in handling the metals following metal pre-exposure. In addition, the significant correlations
between total cytosol Cd concentrations and Cd concentrations
bound to MTLP during uptake agreed with the previous studies on Cd accumulation in aquatic invertebrates (Mason and
Jenkins, 1995; Amiard et al., 2006).
Wallace et al. (2003) defined the organelles and heat-sensitive
proteins (e.g., metalloenzymes) as metal sensitive subcellular
metal-binding fractions, considering these to be the sensitive
components of the cells. The non-specific binding of metals to
non-MT fractions can imply the onset of cellular toxicity (e.g.,
HMW proteins). Such binding of metal to proteins other than
detoxificatory ones (including MT) is often described as “spill
over” (Winge et al., 1974; Couillard et al., 1995; Baudrimont
160
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
et al., 1999). Spill over is often a feature apparent in some
laboratory exposures when organisms are exposed to raised concentrations of metals. The slower uptake rate of Cd into the
cytosolic proteins of the mussels exposed to 200 ␮g l−1 Cd may
be explained by a toxic effect of the acute exposure on metal
subcellular distribution or detoxification processes. This can further be supported by the saturation of MT concentration at high
Cd concentrations in the mussel soft tissues. Under the conditions of our experiments, Cd binding to HMW proteins occurred
without MT-overloading, therefore Cd may bind to some HMW
proteins specifically and similar data were reported for scallops
(Evtushenko et al., 1986). Such proteins are supposed to be analogous to albumin and globulin of blood serum – where metal can
be bound to an amino acid carboxyl group – and can participate
in metal transport (Robinson and Ryan, 1988). Since green mussels distributed a substantial amount of Cd into HMW and the
increase of Cd in the cytosol correlated positively with increase
of Cd in HMW protein, HMW appears to be the chief protein
fraction for binding Cd before toxic effects occur. Our previous
study also found a higher percentage of Cd associated in the
HMW proteins of green mussels with higher dissolved uptake
rates (Ng and Wang, 2005a). Therefore, both studies reinforce
the importance of HMW proteins for short-term metal binding
during uptake in the mussels.
Increased excretion of Cd was apparently not a defensive
response in the mussels since the efflux rates of Cd by the
mussels pre-exposed to dissolved Cd were similar to that of
the control. In general, the efflux rate measured in this study
was slightly faster than previously reported in green mussels (0.007–0.029 day−1 ) (Blackmore and Wang, 2002; Ng and
Wang, 2005b), which may be explained by the shorter radiolabelling period used (15 h as opposed to days of radiolabelling
before depuration). Previous studies have reported the importance of cytosol binding of metals on metal elimination. For
example, Cd in the soluble fraction of the oysters was more easily eliminated than Cd in insoluble form (Roesijadi and Klerks,
1989; Geffard et al., 2002). Our results also found an increase
of Cd in the cytosol of the green mussels during the depuration
period. This apparently was not due to biological variation of the
mussels because the increase was observed in mussels from all
treatments. Therefore Cd was probably shifted from the insoluble fraction to the cytosol as supported by the reduction and
increase of Cd in the insoluble fraction and cytosol respectively
in the first 2 days of depuration, but a relatively constant 109 Cd
concentration in the cytosol. Among the cytosolic proteins, part
of the Cd initially associated with HMW protein, shifted to
MTLP during the later period of depuration (2–4 days), similar to
our earlier studies on the dynamics of metals during depuration
in green mussels (Ng and Wang, 2005a). Metals may first bind
to HMW proteins, but move to the MTLP for detoxification or
long-term storage. Baudrimont et al. (2003) and Bebianno et al.
(1994) suggested that MT may be involved in metal elimination
in bivalves, whereas our study only showed a temporary binding
of Cd to MTLP. This may be explained by the MT turnover cycle
that has been rarely considered in previous studies.
l-[35 S] cysteine is proved to be an excellent marker for MT
turnover in the green mussels because there was a high percent-
age of l-[35 S] cysteine in MTLP and a significant incorporation
of l-[35 S] cysteine into the MTLP of the Cd pre-exposed mussels
over time. In addition, the twofold increase of l-[35 S] cysteine
incorporation rate in MTLP of the Cd exposed mussels (e.g., 50
and 200 ␮g l−1 ) compared to the control mussels corresponded
to almost the same fold increase of MT concentration. Therefore, incorporation of l-[35 S] cysteine into MTLP does appear
to be a good index of the MT synthesis rate of the green mussels. Based on our l-[35 S] cysteine data, the MT breakdown of
the green mussels was extremely fast (12–16 h), compared to
other aquatic invertebrates. Previous studies demonstrated that
MT is degraded more quickly in mammals, e.g., 3–5 days in
rats exposed to Cd (Chen et al., 1975) than in marine molluscs,
e.g., 7 days in C. virginica (Roesijadi et al., 1991), 25 days in
M. edulis (Bebianno and Langston, 1993) and 69–190 days in
L. littorea (Bebianno and Langston, 1998). The short half life
of MT that we obtained may be chiefly explained by the different methods or approaches used to determine MT breakdown
compared to previous studies. Bebianno and Langston (1993,
1998) injected l-[35 S] cysteine into the tissue or mantle cavity
of the mussels and snails, then waited a few days for equilibrium
before examining the depuration of 35 S weekly for a month.
Since their observation interval was longer, the MT measurement may be a net product of MT synthesis and breakdown
within the week (i.e. a steady-state measurement). In this study,
we used a kinetic approach to measure the instant breakdown of
MT within 7 days. Generally, the half-life of MT is shorter than
the half lives of associated metals. Metals bound to MT will be
transported to lysosomes where MT is degraded. Metals would
then be released back to the cytosol and induce MT synthesis
again (Viarengo and Nott, 1993). In this study, such a cycle of
Cd binding and release from the MTLP of green mussels was
also observed during depuration. 35 S in fact has been widely
used to examine the synthesis and breakdown of proteins other
than MT in many organisms (Hawkins et al., 1987; Barnes et
al., 2002). We used 35 S in the form of cysteine in this study, in
order to increase incorporation into MT, but significant incorporation of 35 S was still obtained in other proteins (e.g., HMW),
a potential artifact of using 35 S as a marker for MT turnover in
the bivalves. In addition, 109 Cd present in the insoluble fraction
is at least partly sequestered in the lysosomal vacuolar system
which is eliminated by exocytosis. However, the importance of
this excretion pathway is probably less important for Cd than
for other metals (Viarengo and Nott, 1993 and literature cited
therein).
To conclude, both MTLP and HMW proteins are important
for the binding and depuration of Cd in green mussels. The
Cd pre-exposed mussels had a faster MT synthesis but a similar MT breakdown rate to the control mussels. Therefore, the
resultant MT concentration was also higher in the Cd exposed
mussels. The MT synthesis rate was correlated to the assimilation of Cd from phytoplankton. In addition, the Cd efflux rate was
independent of the MT breakdown rate due to the much faster
degradation of MT and the possible rebinding of Cd to new MT.
Our study has significant implications for further investigations
into MT turnover in aquatic invertebrates. A metal challenge may
increase the rate of the MT turnover processes, e.g., synthesis
T.Y.-T. Ng et al. / Aquatic Toxicology 84 (2007) 153–161
or breakdown, but the role of each process on metal kinetics has
often been neglected in previous studies.
Acknowledgement
This study was supported by a Competitive Earmarked
Research Grant from the Hong Kong Research Grants Council
(HKUST6405/05M) to W.-X. Wang.
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