|
| |
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 1, 2008
Journal of Experimental Biology 211, 577-586 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011262
Effects of cadmium on cellular protein and glutathione synthesis and expression of stress proteins in eastern oysters, Crassostrea virginica Gmelin
Biology Department, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC 28223, USA
Author for correspondence (e-mail:
isokolov{at}uncc.edu)
Accepted 5 December 2007
 |
Summary |
|---|
 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Key words: heat shock protein, metallothionein, protein synthesis, glutathione, cadmium, bivalve
|
INTRODUCTION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Roesijadi, 1996;
Frew et al., 1997). Eastern
oysters, Crassostrea virginica, are common estuarine inhabitants and
a useful model to study the mechanisms of Cd toxicity and tolerance. Oysters
are exposed to varying Cd concentrations in their habitats and have an ability
to accumulate Cd burdens exceeding environmental levels by orders of magnitude
(Roesijadi, 1996;
Frew et al., 1997). According
to some estimates, up to 40% of total Cd load in estuarine ecosystems can be
contained in oyster soft tissues (Pigeot
et al., 2006), making these mollusks an important vector of Cd
transfer to higher levels of the food chain, including humans.
Cadmium is a toxic metal with no known biological function in animals and
can strongly affect organisms' physiology, survival and performance. Earlier
studies have shown that energy misbalance is implicated in Cd-induced stress
and toxicity in aquatic organisms such as oysters. Mitochondrial dysfunction
is an important aspect of Cd toxicity in oysters, leading to impaired ATP
production, reduced aerobic capacity and elevated oxidative stress
(Sokolova, 2004;
Cherkasov et al., 2007). On
the other hand, exposure to Cd results in elevated basal metabolic demand in
oysters, thus resulting in a mismatch between energy demand and supply and
greatly limiting the aerobic scope of an organism
(Lannig et al., 2006). Reduced
aerobic scope due to the toxin exposure may negatively affect the organism's
fitness due to physiological tradeoffs that can divert energy from essential
processes such as growth, reproduction or locomotion towards maintenance
(Sibly and Calow, 1989;
Calow and Forbes, 1998;
Pörtner, 2001;
Roff 2002;
Notten et al., 2006;
Lannig et al., 2006), although
some empirical studies indicate that energetic burden imposed by
detoxification costs may be weak (Van
Straalen and Hoffmann, 2000). Currently, the mechanisms of the
elevated basal energy demand in Cd-exposed oysters are not fully understood,
and further studies are needed to identify the processes contributing to the
Cd-induced energetic burden.
Protein synthesis cost is one of the major components of cellular energy
demand, accounting for 10–20% of standard metabolic rate
(Hand and Hardewig, 1996;
Hulbert et al., 2002).
Exposure to pollutants, including heavy metals, can lead to an increase in
protein synthesis and associated energy costs by switching ribosomes to the
selective translation of protective proteins
(Pytharopoulou et al., 2006).
By contrast, under conditions of acute stress, an organism may limit protein
synthesis by reversible inactivation of ribosomes to conserve energy, which
would result in an overall decline of protein synthesis rates
(Pytharopoulou et al., 2006).
Our earlier studies using indirect methods show that oxygen consumption
associated with protein synthesis increases along with the overall increase in
the basal metabolism during Cd exposure of oysters
(Cherkasov et al., 2006;
Lannig et al., 2006). This
increase was proposed to reflect the cost of the de novo synthesis of
cellular protection proteins and the replacement of the proteins damaged by
Cd. However, in order to test this hypothesis, direct measurements of the
protein synthesis rates and expression of cell protection proteins in response
to Cd exposure are needed.
Metallothioneins and heat shock proteins (HSPs) are among the primary
candidates for cellular protection, which may contribute to the elevated
protein synthesis costs in Cd-exposed oysters. Metallothioneins are
low-molecular-mass (6–7 kDa), cysteine-rich proteins. They have been
shown to participate in free-radical scavenging and antioxidant protection
(Andrews, 2000;
Coyle et al., 2002), but their
primary function is binding of essential and non-essential metal ions in
cytoplasm for storage and/or detoxification and eventually targeting them to
lysosomes for deposition and/or disposal
(Klaassen et al., 1999;
Andrews, 2000;
Tanguy et al., 2001).
Metallothioneins are strongly induced by heavy metals such as Cd, and elevated
expression of these proteins is a hallmark of metal exposure
(Amiard et al., 2006). In
addition to metallothioneins, glutathione (GSH) plays an important role in
metal detoxification. GSH is a short polypeptide containing a cysteine residue
that serves as a primary nucleophile in detoxification reactions, including
metal binding (Meister and Anderson,
1983). Up to 10% of total metals in bivalve cytoplasm may be bound
to GSH (Giguere et al., 2003).
Overexpression of GSH can thus increase metal tolerance of an organism, while
GSH depletion results in an increased susceptibility to heavy metal toxicity
(White et al., 1999;
Conners and Ringwood, 2000;
Ringwood and Conners, 2000;
White and Cappai, 2003).
Heat shock proteins are involved in protection against a wide range of
environmental stressors, and their expression is triggered by oxidatively
modified or partially denatured/misfolded proteins in the cell. Cytosolic
chaperones HSP70 and HSP90 are among the most abundant cellular proteins
protecting against stress-induced damage. HSP70 is the most ubiquitous and
universal cytosolic chaperone family involved in folding/refolding of newly
synthesized and damaged proteins as well as sequestering and degradation of
proteins that are damaged beyond repair
(Mayer and Bukau, 2005). HSP90
is another general chaperone of the eukaryotic cytosol, orchestrating the
folding of many proteins; however, HSP90 alone is insufficient to assist in
refolding of partially denatured proteins and requires other chaperones such
as the HSP70 chaperone family to complete this task
(Csermely et al., 1998;
Mayer and Bukau, 2005). By
contrast, HSP60 is predominantly found in mitochondria and chloroplasts of
eukaryotes, assisting with the protein folding and stress protection in these
organelles (Cechetto et al.,
2000). Given that, in oysters, Cd localizes to mitochondria and
cytosol (Sokolova et al.,
2005), one can expect a strong impact of Cd exposure on expression
of these molecular chaperones.
To date, the effects of Cd exposure on protein synthesis have not been extensively studied in aquatic ectotherms, and the relationship between the global proteosynthetic response and expression of different cytoprotective mechanisms (such as metallothioneins, HSPs and GSH) during Cd stress is poorly understood. It is also not known whether tissue-specific variations in protein synthesis and expression patterns may affect the response of different tissues to Cd exposure. In this study, we used a direct method to determine the effects of Cd exposure on the protein and glutathione synthesis rates in isolated gill and hepatopancreas cells of eastern oysters. We tested a hypothesis that Cd exposure results in elevated rates of protein and/or glutathione synthesis and analyzed relationships between these rates and the expression of key components of cellular protection including GSH, metallothioneins, and cytoplasmic and mitochondrial HSPs.
|
MATERIALS AND METHODS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
.
The study site has very low background concentrations of heavy metals and
organic pollutants [(Mallin et al.,
1999); James Schwarzenberg, J & B Aquafood, personal
communication]. Animals were transported to the University of North Carolina
at Charlotte within 5 h and acclimated at 20±1°C and 30 in
recirculating aerated tanks with artificial seawater (Instant Ocean®; Kent
Marine, Acworth, GA, USA) for at least 3 weeks prior to experimentation. They
were fed ad libitum with a commercial algal blend (1 ml per oyster
every other day) containing live Nannochloropsis oculata, Phaeodactylum
tricornutum and Chlorella spp. (2–20 µm) (DT's Live
Marine Phytoplankton, Premium Reef Blend; DT's Plankton Farm, Sycamore, IL,
USA).
Cell isolation
Gills or hepatopancreas (or digestive gland) tissues from 2–5 oysters
were pooled on ice in 5 ml of digestion buffer containing 24.72 g
l–1 NaCl, 0.68 g l–1 KCl, 1.36 g
l–1 CaCl2.2H2O, 0.18 g
l–1 NaHCO3 and 30 mmol l–1 Hepes
at pH 7.5. Tissues were minced and washed with an additional 10 ml of
digestion buffer. Tissue fragments were digested for 15 min at room
temperature in 0.125% trypsin in balanced Hank's solution (Fisher Scientific,
Suwanee, GA, USA) adjusted to 720 mOsm with sucrose, carefully triturated to
release cells and washed twice with digestion buffer. The suspension was
filtered through 100 µm sterile nylon mesh and centrifuged for 15 min at
900 g to pellet the cells. Cells were washed in cell
suspension medium (CSM) containing 24.72 g l–1 NaCl, 0.68 g
l–1 KCl, 1.36 g l–1
CaCl2.2H2O, 0.18 g l–1
NaHCO3, 4.66 g l–1
MgCl2.6H2O, 6.29 g l–1
MgSO4.7H2O, 30 mmol l–1 glucose and 30
mmol l–1 Hepes at pH 7.5, centrifuged for 10 min at 900
g and re-suspended in 4 ml of the same medium. Cell count was
performed with a Beckman Coulter Z2 cell counter, 100 µm aperture diameter
(Beckman Coulter, Inc., Fullerton, CA, USA). The particle size range window
was set to 10–30 µm, corresponding to the known size of gill and
hepatopancreas cells from oysters (Eble
and Scro, 1996). Viability of isolated cells, determined by the
Trypan Blue exclusion assay, was >90–95% throughout the experiment.
Our earlier studies also show that isolated oyster cells maintain viability
for over 48 h in culture with no appreciable increase in the levels of
apoptosis or necrosis in millimolar concentrations of Cd
(Sokolova et al., 2004).
Sample size (N) reported in the figures and throughout the paper
refers to the number of individual cell isolates, each obtained from 2–5
oysters as described above.
Cadmium determination
Isolated gill and hepatopancreas cells were exposed to different Cd levels
(0–2000 µmol l–1) for 4 h at 20°C. After the
exposures, cell suspensions were centrifuged (2000 g for 15
min), washed twice in Cd-free CSM to remove surface-associated Cd, and
re-suspended in Cd-free CSM. Washed cell suspensions were mixed 1:1 with 70%
nitric acid and digested in a 60°C water bath for 24 h. Cd concentrations
were determined with an atomic absorption spectrometer AAnalyst 800 (Perkin
Elmer, Waltham, MA, USA), equipped with a graphite furnace and Zeeman
background correction. National Institute of Standards and Technology (NIST)
oyster tissue (1566b) was analyzed with the samples to verify the metal
analyses; the percent recoveries over all batches were 94.6±6.6% (mean
± s.d.). Cellular Cd levels are expressed as ng Cd
10–6 cells.
Determination of protein and glutathione synthesis rates
Protein and glutathione synthesis rates were determined by incorporation of
radioactively labeled L-leucine (3H-Leu) or
L-glycine (3H-Gly), respectively
(Bonifacino, 1999), in control
and Cd-exposed cells at 20°C. Briefly, 300 µl of cell suspension
(0.2–1.7x107 cells ml–1) was incubated
with 1 µCi (1 µCi= 37 kBq) of labeled amino acid ([3H]Leu or
[3H]Gly) for 4 h at 20°C with different concentrations of Cd
(0–2000 µmol l–1) and cycloheximide (0 or 2 mmol
l–1). After incubation, 20 µl of sample were placed on
Whatman binder-free glass microfiber filters (type GF/A; Fisher Scientific),
air dried and exposed to 10% ice-cold trichloroacetic acid (TCA) containing 5
mmol l–1 of the respective unlabeled aminoacid
(L-Leu or L-Gly) for 10 min. The filter was then washed
twice with 5% TCA (15 min each wash) and dehydrated in 100% ethanol (three
times 10 min each) at room temperature. After dehydration, filters were air
dried and placed into glass scintillation vials containing 5 ml of ScintiSafe
Econo 1 scintillation cocktail (Fisher Scientific, Suwanee, GA, USA). Pilot
studies with filters spiked with radiolabeled amino acids showed that these
washes were sufficient to remove all unincorporated label. Counting was
performed with a Beckman Coulter LS6000SC scintillation counter. Counts per
minute (c.p.m.) were recalculated to disintegrations per minute (d.p.m.) with
internal counter software. Addition of cycloheximide (2 mmol
l–1) routinely inhibited 95–98% of [3H]Leu
incorporation, confirming that most of the incorporated leucine was due to the
protein synthesis. A similar degree of inhibition of [3H]Leu
incorporation was obtained with another specific inhibitor of protein
synthesis, emetine (300 µmol l–1) (data not shown). The
glutathione synthesis rate in the presence and absence of cadmium was
calculated as [3H]Gly incorporation rate when co-incubated with 2
mmol l–1 cycloheximide under the assumption that most of the
labeled glycine was incorporated into glutathione under these conditions.
Glutathione concentrations
Total glutathione (GSH) concentrations were determined enzymatically by the
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) – glutathione disulfide
(GSSG) reductase recycling assay following the rate of formation of
5-thio-2-nitrobenzoic acid (TNB)
(Griffith, 1980;
Anderson, 1985). Gill and
hepatopancreas cells were incubated at 20°C without Cd (control) or in the
presence of 2000 µmol l–1 Cd (Cd-exposed). After the
predetermined exposure times (0, 4, 8 and 12 h), the cells were homogenized in
equal volumes (1:1) of 10% sulfosalicylic acid (SSA), sonicated for 20 s
(output 30 W; Sonicator 3000; Misonix Inc., Farmingdale, NY, USA) and
centrifuged (16 000 g for 5 min at 4°C). Total glutathione
was measured in the supernatant with a UV-Vis spectrophotometer (VARIAN Cary
50 Bio; Cary, NC, USA) equipped with a temperature-controlled cuvette holder
at 30°C (±0.1°C) at 412 nm. The calibration curve was prepared
using GSH standards dissolved in a 1:1 mixture of CSM and 10% SSA. Results are
expressed as total GSH content in nmol 10–6 cells.
Expression of HSPs determined by immunoblotting
Cell suspensions isolated from gill and hepatopancreas were incubated for 4
h without Cd (controls) or in the presence of 50, 500 and 2000 µmol
l–1 Cd. Cells were collected by centrifugation and
homogenized in ice-cold homogenization buffer [100 mmol l–1
Tris, pH 7.4, 100 mmol l–1 NaCl, 1 mmol l–1
EDTA, 1 mmol l–1 EGTA, 1% Triton-X100, 10% glycerol, 0.1%
sodium dodecylsulfate (SDS), 0.5% deoxycholate, 0.5 µg
ml–1 leupeptin, 0.7 µg ml–1 pepstatin, 40
µg ml–1 phenylmethylsulphonyl fluoride (PMSF) and 0.5
µg ml–1 aprotinin] at a concentration of
4x106 cells ml–1 of buffer. HSPs were
detected using standard immunoblotting methods. Briefly, the homogenate was
sonicated three times for 10 s each (output 69 Watts; Sonicator 3000, Misonix
Inc.) and centrifuged at 14 000 g for 5 min at 4°C.
Protein content was measured in the supernatant using the Bio-Rad Protein
Assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA,
USA). Bovine serum albumin (BSA) was used as a standard. 30 µg of sample
protein per lane was loaded onto 8% polyacrylamide gels and run at 100 mA for
2 h at room temperature. The resolved proteins were transferred onto a
nitrocellulose membrane in 96 mmol l–1 glycine, 12 mmol
l–1 Tris and 20% methanol (v/v) using a Trans-Blot semi-dry
cell (Thermo Fisher Scientific Inc., Portsmouth, NH, USA). To verify equal
protein loading, membranes were stained with Amido Black Stain Solution
(Fisher Scientific; 1 g l–1 Amido Black in 10% methanol, 10%
glacial acetic acid) for 30 s. The membranes were then destained by washing in
several changes of water and blocked overnight in 5% non-fat milk in
Tris-buffered saline, pH 7.6 (TBST). Blots were probed with primary monoclonal
antibodies against HSP70, HSP90 and HSP60, respectively (MA3-007, Affinity
Bioreagents, Golden, CO, USA; SPA-835 and SPA-805, Stressgen Bioreagents, Ann
Arbor, MI, USA). After washing off the primary antibody, membranes were probed
with the respective polyclonal secondary antibodies conjugated with
horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA, USA), and
proteins were detected by enhanced chemiluminescence according to the
manufacturer's instructions (Pierce, Rockford, IL, USA). Densitometric
analysis of the signal was performed by GelDoc 2000TM System with
Quantity One 1-D Analysis Software (Bio-Rad). Each blot included a control
sample as an internal standard, and expression of HSPs in Cd-exposed cells was
calculated as percent of the respective control.
RNA extraction and RT-PCR amplification
Expression of metallothionein mRNA was measured by quantitative real-time
PCR (QRT-PCR). There are no commercially available antibodies specific for
molluskan metallothioneins, and several commercial antibodies developed
against mammalian metallothioneins failed to cross-react with oyster
metallothioneins in our pilot studies (data not shown). Therefore, we have
used mRNA expression as a proxy for metallothionein expression, as it has been
previously shown that it is primarily regulated at the transcription level
(Rose et al., 2006).
RNA was extracted by homogenization in Tri Reagent (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's protocol. RNA concentration and quality were verified by UV spectroscopy. Reverse transcription was performed using 200 U µl–1 SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), 50 µmol l–1 of oligo(dT)18 primers and 5 µg total RNA to obtain single-stranded cDNA.
For QRT-PCR, specific primers were designed to amplify cDNA for
metallothionein I (MTI), total metallothioneins (MTI and MTII) and
β-actin using C. virginica sequences published in GenBank (NCBI
accession numbers AY331695.1, AY331705.1 and X75894.1, respectively). Because
of the high sequence similarity and the duplicated β-domain in MTII
(Tanguy and Moraga, 2001), we
were unable to design specific primers to MTII that would amplify a single
product; therefore, metallothionein II expression could not be measured
separately. Instead, we designed consensus primers that amplified a
single-length PCR product from either MTI or MTII and thus determined
expression of both metallothioneins simultaneously. Expression of β-actin
was used as a housekeeping gene to normalize expression of metallothioneins.
In preliminary studies, several housekeeping genes, including β-actin,
ubiquitin, ribosomal protein L13a and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), were tested because earlier research indicates that some housekeeping
genes may show deviant expression, thus potentially skewing experimental
results if used as a reference (Khimani et
al., 2005). Our pilot study showed that in oysters, the expression
of all four housekeeping genes varied concordantly between samples (data not
shown), suggesting that any of these is a suitable reference gene. Therefore,
in all subsequent analyses, metallothionein expression was normalized to
β-actin. The following primers were used for QRT-PCR. For metallothionein
I: MT I-FW, 5'-cca cct gca aat gtg gat ca-3'; MTI-RV, 5'-atc
aat aca taa aag aaa cat cac tcg-3'. Consensus primers for
metallothioneins I and II: MTI+II-FW, 5'-ggc tgt aaa tgt ggg gag
aa-3'; MTI+II-RV, 5'-gag aac gcc tct cat tgg tc-3'. For
β-actin: Act-Cv-F437, 5'-cac agc cgc ttc ctc atc ctc c-3';
Act-Cv-R571 5'-ccg gcg gat tcc ata cca agg-3'.
Quantitative RT-PCR was performed using a LightCycler® 2.0 Real Time
PCR System (Roche Applied Science, Indianapolis, IN, USA) and QuantiTect SYBR
Green PCR kit (Qiagen, Valencia, CA, USA) according to the manufacturers'
instructions. The reaction mixture consisted of 5 µl of 2x QuantiTect
SYBR Green master mix, 0.3 µmol l–1 of each forward and
reverse primer, 1 µl of 10x diluted cDNA template and water to adjust
to 10 µl. Ten microliters of reaction mixture were loaded into LightCycler
20 µl capillaries (Roche Applied Science) and subjected to the following
cycling: 15 min at 95°C to denature DNA and activate Taq polymerase;
40–55 cycles of 15 s at 94°C, 20 s at 55°C and 15 s at 72°C.
SYBR Green fluorescence (acquisition wavelength 530 nm) was measured at the
end of each cycle for 2 s at the read temperature of 78°C. The read
temperatures of the amplified fragments were determined in the pilot
experiments and set at the value to melt all primer dimers but not the
amplified gene product. At the end of each run, melting temperature profiles
were run between 50 and 99°C with continuous fluorescence acquisition to
confirm the expected melting temperatures for the amplified fragments. In each
run, serial dilutions of a cDNA standard were amplified to determine
amplification efficiency (Pfaffl,
2001), and an internal standard was included to test for
amplification variability between the runs. Dilutions of the experimental cDNA
samples were selected so that their crosspoints for fluorescence fell within
the range of the crosspoints for cDNA standards. Amplification efficiencies
(E) were 2.05+0.04 (N=7), 1.84+0.06 (N=8) and 2.03+0.04
(N=8) for consensus metallothioneins I and II, metallothionein I and
β-actin, respectively. Expression of the target genes (metallothioneins)
were calculated relative to the expression of β-actin and normalized
against the internal standard as proposed by Pfaffl
(Pfaffl, 2001):
 |
CPt and CPref are
differences between crosspoints for fluorescence of the sample and the
internal standard for the target gene and β-actin, respectively.
Chemicals
All other chemicals were purchased from Sigma Aldrich, Fisher Scientific or
GE Healthcare (Buckinghamshire, UK) and were of analytical grade or higher.
Enzymes were purchased from Sigma Aldrich.
Statistical analysis
Repeated-measures ANOVAs were used to test the effects of Cd on the studied
variables after testing the assumptions of normality of data distribution and
homogeneity of variances. Dunnett's tests were used for post-hoc
comparisons of sample means against the respective controls, and one- or
two-tailed LSD (least squared difference) tests were used for planned
comparisons of sample means as appropriate. Individual sample (i.e. cell
isolate) was used as a repeated-measures variable, thus allowing us to
separate the sample-to-sample variability from the factor effect (Cd
exposure). For the sake of clarity, data for protein and glutathione synthesis
are shown as % of the respective controls. However, all statistics for protein
and glutathione synthesis were done on the original data (i.e. the rate of
incorporation of [3H]Leu or [3H]Gly for protein and
glutathione, respectively). Because of high sample-to-sample variation,
statistical tests for mRNA and protein expression were performed on normalized
data (i.e. expression levels in Cd-exposed cells normalized to the respective
controls from the same cell batch). Statistical analyses were performed using
SAS 9.1.3 software (SAS Institute, Cary, NC, USA). Factor effects and
differences between the means were considered significant if the probability
of Type II error was less than 0.05. Values are given as means ±
standard error (s.e.m.).
|
RESULTS |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
|
|
By contrast, the rate of GSH synthesis, estimated by cycloheximide-insensitive incorporation of [3H]Gly, declined in Cd-exposed cells in a dose-dependent manner (Fig. 3). At the highest studied Cd exposure (2000 µmol l–1), GSH synthesis rate in oyster cells declined by 25–35% compared with the respective controls. GSH synthesis accounted for 63 and 52% of the total [3H]Gly incorporation in gill and hepatopancreas cells, respectively, and this percentage did not significantly change with Cd exposure. The rate of GSH synthesis estimated by cycloheximide-insensitive incorporation of [3H]Gly tended to be higher in gill cells (Fig. 3A) but this trend was only marginally significant (P=0.07). Notably, GSH synthesis rate was more strongly suppressed by Cd exposure in hepatopancreas cells than in gills. This was also reflected by a decrease in GSH content in response to Cd exposure, which was significant in hepatopancreas but not in gill cells (ANOVA; P=0.95 and 0.02 for the overall effects of Cd exposure in gills and hepatopancreas, respectively) (Fig. 4). However, GSH depletion was minimal under the experimental conditions of this study, probably reflecting the short-term exposures (4–12 h), and the total GSH concentration dropped by only 4–9% after 4–12 h of exposure of hepatopancreas cells to 2000 µmol l–1 Cd.
|
|
Expression of metallothioneins and HSPs
Metallothionein mRNA was detected in considerable amounts in the control
cells, indicating that these genes are constitutively transcribed in the
absence of a stressor. The constitutively expressed metallothionein (MTI) mRNA
levels normalized to actin tended to be lower in control (non-Cd-exposed)
hepatopancreas cells compared with the gills (1.05±0.42 and
0.28±0.08 in gills and hepatopancreas, respectively,
P<0.05), whereas the differences in total (MTI+II) mRNA expression
were not significant between the two tissues under control conditions
(P>0.05). Cadmium exposure resulted in a significant
dose-dependent increase in metallothionein levels in the hepatopancreas but
not in the gill cells (Fig. 5).
Expression levels of total metallothioneins (MTI and II) in hepatopancreas
cells were 2.5–4-fold elevated after 4 h of exposure to 50–2000
µmol l–1 Cd (Fig.
5A), whereas mRNA levels of MTI alone rose by 3- to nearly 8-fold
at the same exposures (Fig.
5B).
|
Analysis of HSP60, HSP70 and HSP90 expression using western immunoblotting indicated a strong dose-dependent increase in the mitochondrial HSP60 and cytosolic HSP70 protein levels (by up to 1.5–2-fold) in gills but not in hepatopancreas (Fig. 6A,B). No induction of HSP90 in response to Cd exposure was detected in isolated oyster cells (Fig. 6C).
|
|
DISCUSSION |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Overall, oyster hepatopancreas cells accumulated considerably higher
(2–2.5-fold) Cd loads than gill cells, indicating faster uptake and/or
greater retention of Cd. This is not likely to be a simple consequence of
different metabolic rates because our earlier studies indicated that oyster
gill and hepatopancreas cells have similar rates of basal metabolism
(Cherkasov et al., 2006).
Given that the main cellular uptake route for Cd is through membrane ion
carriers for essential metals such as Zn and Ca (reviewed in
Roesijadi and Robinson, 1993;
Bressler et al., 2004), higher
rates of uptake may reflect higher density and/or activity of these carriers
in hepatopancreas cells. Our present data can shed no light on this
hypothesis; however, the fact that bivalve gills are the primary site of metal
ion uptake from aqueous phase and are characterized by a high density of
active and passive ion transport systems
(Prosser, 1973;
Marigomez et al., 2002;
Apeti et al., 2005) makes this
explanation less plausible. Notably, the high rates of Cd accumulation and a
greater Cd burden in hepatopancreas cells go hand-in-hand with higher rates of
overall protein synthesis and a significantly greater expression of Cd-induced
metallothioneins in these cells. In mollusks, the majority of metal ions
(including Cd) in cytoplasm are bound to metallothioneins, although at high
and/or prolonged exposures, metals may `spill over' onto the
high-molecular-mass proteins (Roesijadi,
1982; Roesijadi,
1996; Roesijadi and Klerks,
1989; Roesijadi et al.,
1995; Giguere et al.,
2003). In mammalian cells, high intracellular levels of inducible
metallothioneins enhance Cd uptake into the cells, possibly by maintaining a
transmembrane gradient of Cd through binding of intracellular free
Cd2+ (Blais et al.,
1999). Thus, it is very likely that the higher level of inducible
metallothioneins and a higher overall rate of protein synthesis provide more
Cd binding sites and are responsible for higher Cd accumulation in
hepatopancreas cells.
It is worth noting that the faster Cd accumulation in isolated
hepatopancreas cells found in this study contrasts some earlier reports on
whole-organism Cd exposures, which indicated higher rates of Cd accumulation
in gills (Roesijadi and Klerks, 2005;
Sokolova et al., 2005;
Cherkasov et al., 2006;
Cherkasov et al., 2007). This
may reflect differences in Cd exposure regimens between in vitro and
in vivo studies. During in vivo exposures to waterborne Cd,
gills are likely to be the primary uptake site for this metal, whereas Cd
accumulation in hepatopancreas occurs at a later stage following Cd transport
by the circulatory system from gills to the internal organs
(Cherkasov et al., 2007).
Similarly, earlier studies in other aquatic organisms demonstrated that the
highest accumulation in vivo (at least initially) occurs in the organ
that first comes into contact with Cd and is capable of active ion exchange
– i.e. in the gills during exposure to waterborne Cd and in the gut
during exposure to dietary Cd (Chowdhury
et al., 2005). Therefore, our finding of higher Cd accumulation in
hepatopancreas cells compared with gills indicates differences in intrinsic
cellular capacities of uptake and retention of Cd; by contrast, in
vivo rates of Cd accumulation will depend on exposure routes and Cd
availability to different organs.
Our study indicates that increased protein synthesis rate is among the
earliest cellular responses to Cd and was evident in gill and hepatopancreas
cells already after the first few hours of exposure. This increase was
dose-dependent and paralleled by the elevated expression of stress response
proteins such as metallothioneins or HSPs, suggesting that the observed
response at least partially reflects de novo synthesis of stress
proteins. This agrees with previous findings indicating that acute exposure of
cells to Cd2+ triggers a preferential increase in stress protein
synthesis (Somji et al., 2000;
Coyle et al., 2002;
Madden et al., 2002;
Urani et al., 2007).
Currently, there are very few published data on the quantitative contribution
of stress proteins to the global protein synthesis rates. Earlier studies
showed that synthesis rates of HSPs can be quite high even under non-stress
conditions and are comparable to those of some housekeeping genes such as
actin (Lanks, 1983). According
to some estimates, stress proteins can represent up to 7% of the total protein
pool (Kultz, 2003). Given that
stress protein expression increases several fold during stress exposure, their
contribution to global protein synthesis will also increase. Since protein
synthesis in general (irrespective of the nature of produced proteins) and the
functioning of stress proteins such as HSPs are ATP-dependent processes
(Feder and Hofmann, 1999), a
Cd-induced increase in protein synthesis and stress protein expression may
represent a significant cost to the organism, diverting energy from essential
functions such as growth, reproduction and immunity towards metal
detoxification and damage repair.
Interestingly, stress protein response to Cd greatly differed between gill
and hepatopancreas cells. Basal levels of constitutive MTI expression were
higher in gill cells compared with hepatopancreas. This may reflect the role
of oyster gills as the key sites of gas and ion exchange that form an
interface between the organism and its environment and thus are among the
first organs to be exposed to environmental insults including variations in
oxygen levels, pH, pollutants, etc. Metallothioneins with their high
sulfhydryl content can function as antioxidants by reacting with free radicals
and reactive oxygen species (Basu and Lazo,
1990; Andrews,
2000; Coyle et al.,
2002), and high levels of constitutive metallothioneins may thus
provide protection against this background environmental stress to the gills.
Moreover, constitutive metallothioneins can play an important role in
transport, metabolism and storage of essential metals
(Coyle et al., 2002);
therefore, high levels of constitutive metallothioneins in gill cells may
reflect their role in uptake and handling of essential metals in oysters. Cd
exposure of isolated gill cells did not result in an induction of
metallothionein expression above the background constitutive levels whereas
HSP60 and HSP70 were significantly overexpressed with increasing Cd levels. By
contrast, in hepatopancreas cells, Cd exposure resulted in a dramatic
induction of metallothioneins whereas expression of HSPs was unchanged.
The observed tissue-specific patterns of HSP expression may be explained by
differential inducibility of metallothioneins in gills and hepatopancreas.
Metallothioneins can be considered the first line of defense against
intracellular toxic metals, including Cd, because the metal's binding to
metallothioneins significantly reduces its toxicity, preventing interactions
with critical cellular components such as enzymes, structural proteins, DNA
and membrane lipids. According to different estimates, 75–80% of
cytosolic Cd is bound to metallothioneins in mollusks
(Roesijadi et al., 1995;
Giguere et al., 2003) and only
20–25% is found in non-thionein pools, presumably accounting for
toxicity. At higher exposures, the levels of non-thionein-bound Cd
substantially increase as the metallothionein binding capacity is exhausted
(Roesijadi et al., 1995). High
levels of metallothionein induction by Cd in hepatopancreas appear to provide
sufficient protection to cellular proteins to prevent modifications such as
oxidation, denaturation and/or misfolding, thus alleviating the need for
expression of molecular chaperones. By contrast, gill cells are less protected
from elevated Cd levels because of the absence of a significant
metallothionein induction, which may lead to the spill-over of Cd into the
non-metallothionein pool, proteotoxic stress and expression of HSPs required
for the repair of the damaged proteins and/or their targeting for degradation
(Vayssier and Polla, 1998).
Notably, earlier studies indicate that inducible metallothionein levels in
oyster gills increase during prolonged (days to weeks) exposure to Cd
[Roesijadi (Roesijadi, 1996);
and references therein]. The absence of metallothionein induction in gill
cells during short Cd exposures (4 h) in the present study suggests that the
transcriptional response is slower in these cells, possibly related to their
lower overall biosynthetic activity.
It is also noteworthy that among the studied HSPs, HSP60 and HSP70 but not
HSP90 showed a significant increase in response to Cd exposure in gill cells.
HSP60 is a stress protein constitutively expressed in mitochondria and may
serve as a specific biomarker for mitochondrial injury. Our earlier studies
have shown that mitochondria are target organelles of Cd accumulation and
toxicity in oyster gills (Sokolova,
2004; Sokolova et al.,
2005; Cherkasov et al.,
2006; Cherkasov et al.,
2007). In mitochondria, Cd can affect the electron transport chain
and ATP synthesis, stimulate production of reactive oxygen species and damage
matrix enzymes (Sokolova,
2004; Cherkasov et al.,
2007); thus, HSP60 expression can be expected to be induced early
during Cd exposure. HSP60 is also increased in gill and mantle cells of marine
mussels and in nematodes exposed to copper
(Sanders et al., 1991;
Sanders and Martin, 1993;
Kammenga et al., 1998) and in
rat kidney cell lines exposed to arsenic
(Madden et al., 2002),
indicating that other metals and metalloids targeting mitochondria may have
similar effects on induction of the protective HSP60 response.
Unlike HSP60, HSP70 and HSP90 families belong to cytoplasmic chaperones
that assist in a wide range of protein folding processes including refolding
of misfolded and aggregated proteins in response to cellular stress
(Bensaude et al., 1996;
Söti and Csermely, 2000;
Urani et al., 2005). In
mammalian systems, Cd exposure has been found to induce stress proteins of 72
and 90 kDa in human keratinocytes and in fibroblasts, 70 and 90 kDa in primary
rat hepatocytes, and 32, 72, 90 and 110 kDa in human melanoma cells
(Bauman et al., 1993;
Madden et al., 2002;
Urani et al., 2005). In chick
embryos, exposure to Cd enhanced de novo synthesis of HSP70, whereas
HSP24 and HSP90 were unaffected
(Papaconstantinou et al.,
2003). Data for the Cd-induced chaperone response in aquatic
organisms are limited. However, an earlier study in European oysters,
Ostrea edulis, exposed to 500 µg l–1 Cd for 7
days in vivo showed upregulated HSP70 expression in gills and
hepatopancreas (other HSPs were not tested)
(Piano et al., 2004). By
contrast, expression levels of another general cytosolic chaperone, HSP90, did
not change in response to Cd exposure in oyster cells. Possibly, high levels
of expression of HSP70 in response to Cd provide sufficient protection for
cytoplasmic proteins in these cells, alleviating the need for elevated HSP90
expression. Interestingly, some studies also indicate that divalent cations
greatly suppress the chaperone activity of HSP90, increasing the possibility
that HSP90 can be less effective in Cd-exposed cells
(Jakob et al., 1995). Overall,
it appears that expression of HSP70 is a general feature of the cytosolic
response to Cd across a variety of taxa, reflecting the ubiquitous nature of
this HSP family and their broad specificity towards a wide range of
intracellular proteins.
Elevated synthesis of GSH can serve as an additional cytoprotective
mechanism through direct metal binding, detoxification of reactive oxygen and
the maintenance of cellular redox status
(Meister and Anderson, 1983;
Ringwood and Conners, 2000).
Our earlier study has shown that long-term exposure to Cd resulted in a
significant increase in the GSH level at 20° and 24°C but not 28°C
(Lannig et al., 2006).
However, in the present study, Cd exposure resulted in a significant decrease
in the rates of GSH synthesis in hepatopancreas cells correlated with a small
but consistent depletion of GSH levels. This may reflect higher Cd exposure
levels of isolated cells in our current study compared with the in
vivo exposures of whole oysters in the earlier study
(Lannig et al., 2006). Indeed,
previous research indicates that exposures to high Cd levels typically result
in GSH depletion (Regoli and Principato,
1995; Ringwood et al.,
1998; Mitchelmore et al.,
2003; Toplan et al.,
2003), whereas at lower Cd concentrations no change or an increase
in GSH levels can be observed (Viarengo et
al., 1990; Ringwood et al.,
1999; Mitchelmore et al.,
2003; Regoli et al.,
2004; Lannig et al.,
2006). The present study shows that inhibition of glutathione
synthesis is a likely mechanism for its depletion at high Cd levels, at least
in hepatopancreas cells. It is worth noting that despite a significant (by
nearly 35%) decrease of the rate of GSH synthesis in hepatopancreas cells at
2000 µmol l–1 Cd, a decrease in total GSH levels was much
smaller (less than 10%) and only significant when all exposure times were
taken into account. In gills, Cd exposure had a less pronounced effect on the
rate of GSH synthesis, resulting in an 
25% decrease at the highest Cd
concentration (2000 µmol l–1), and no effect of Cd on
total GSH levels was detected. It suggests that longer, chronic exposures may
be needed for the reduced rates of GSH synthesis to be significantly reflected
in the size of the intracellular GSH pool.
As a corollary, this study directly demonstrates, for the first time, that
Cd exposure results in a strongly elevated protein synthesis in isolated gill
and hepatopancreas cells of oysters associated with elevated expression of
cellular protective proteins. Elevated protein synthesis rates likely reflect
the need for detoxification and cellular protection and may contribute to the
observed increase in the basal maintenance costs of Cd-exposed oysters
(Cherkasov et al., 2006;
Lannig et al., 2006).
Interestingly, stress protein response significantly differed between gill and
hepatopancreas cells despite a similar degree of overall stimulation of
protein synthesis by Cd. In hepatopancreas, overexpression of metallothioneins
appeared to be a major protective response that was also the likely cause of
the observed high levels of Cd accumulation in these cells. No increase in HSP
expression in hepatopancreas indicates that levels of damage to mitochondrial
and cytosolic proteins are not significantly above the background and suggests
that metallothioneins provide adequate protection of critical cellular
proteins against Cd stress. This further emphasizes the high efficiency of
metallothioneins as a protective mechanism against heavy metals. By contrast,
metallothionein induction was not observed in response to Cd in gill cells,
resulting in protein damage and a compensatory increase in molecular
chaperones, HSPs, despite the lower overall levels of Cd accumulation. GSH
synthesis was suppressed in Cd-exposed cells and no compensatory elevation of
GSH levels was observed in response to Cd. These data clearly indicate that
cytoprotective response against Cd stress can vary greatly among different
tissues and may result in their differential sensitivity to metal exposure.
Based on our data, we suggest that gills would be among the most Cd-sensitive
tissues in oysters. High sensitivity of gill cells to Cd stress will have
important implications for whole-organism physiology and may result in
functional hypoxia and energy misbalance due to the elevated metabolic
maintenance cost, on one hand, and the impaired ability for oxygen uptake, on
the other. Indeed, our recent studies show that Cd exposure results in
impaired oxygen uptake in oyster gills whereas oxygen delivery through the
circulation system is not affected (G. Lannig, C. Bock, A. Cherkasov, H.-O.
Poertner and I. Sokolova, in review). This leads to a reduction in the aerobic
scope of the organism, resulting in a lower scope for growth and/or
reproduction, reduced tolerance to other environmental stressors such as
elevated temperatures and thus reduced fitness of an organism in polluted
environments.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Amiard, J.-C., Amiard-Triquet, C., Barka, S., Pellerin, J. and Rainbow, P. S. (2006). Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol. 76,160 -202.[CrossRef][Medline]
Anderson, M. E. (1985). Determination of glutathione and glutathione disulfide in biological samples. Meth. Enzymol. 113,548 -555.[Medline]
Andrews, G. K. (2000). Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59,95 -104.[CrossRef][Medline]
Apeti, D. A., Johnson, E. and Robinson, L. (2005). A model for bioaccumulation of metals in Crassostrea virginica from Apalachicola Bay, Florida. Am. J. Environ. Sci. 1,239 -248.
Basu, A. and Lazo, J. S. (1990). A hypothesis regarding the protective role of metallothioneins against the toxicity of DNA interactive anticancer drugs. Toxicol. Lett. 50,123 -135.[CrossRef][Medline]
Bauman, J. W., Liu, J. and Klaassen, C. D. (1993). Production of metallothionein and heat-shock proteins in response to metals. Fundam. Appl. Toxicol. 21, 15-22.[CrossRef][Medline]
Bensaude, O., Bellier, S., Dubois, M. F., Giannoni, F. and Nguen, V. T. (1996). Heat-shock induced protein modifications and modulation of enzyme activities. In Stress-Inducible Cellular Responses (ed. U. Feige, R. I. Morimoto, I. Yahara and B. S. Polla), pp. 199-219. Basel: Birkhäuser Verlag.
Blais, A., Lecoeur, S., Milhaud, G., Tome, D. and Kolf-Clauw, M. (1999). Cadmium uptake and transepithelial transport in control and long-term exposed Caco-2 cells: the role of metallothionein. Toxicol. Appl. Pharmacol. 160, 76-85.[CrossRef][Medline]
Bonifacino, J. S. (1999). Metabolic labeling with amino acids. Curr. Protoc. Protein Sci. 3.7,3.7.1 -3.7.10.
Bressler, J. P., Olivi, L., Cheong, J. H. and Kim, Y. (2004). Divalent metal transporter 1 in lead and cadmium transport. Ann. N. Y. Acad. Sci. 1012,142 -152.[CrossRef][Medline]
Calow, P. and Forbes, V. E. (1998). How do physiological responses to stress translate into ecological and evolutionary processes? Comp. Biochem. Physiol. 120A,11 -16.
Cechetto, J. D., Soltys, B. J. and Gupta, R. S.
(2000). Localization of mitochondrial 60-kD heat shock chaperonin
protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic
zymogen granules. J. Histochem. Cytochem.
48, 45-56.
Cherkasov, A. S., Biswas, P. K., Ridings, D. M., Ringwood, A. H.
and Sokolova, I. M. (2006). Effects of acclimation
temperature and cadmium exposure on cellular energy budgets in a marine
mollusk Crassostrea virginica: linking cellular and mitochondrial
responses. J. Exp. Biol.
209,1274
-1284.
Cherkasov, A. S., Overton, R. A., Jr, Sokolov, E. P. and
Sokolova, I. M. (2007). Temperature-dependent effects of
cadmium and purine nucleotides on mitochondrial aconitase from a marine
ectotherm, Crassostrea virginica: a role of temperature in oxidative
stress and allosteric enzyme regulation. J. Exp. Biol.
210, 46-55.
Chowdhury, M. J., Baldisserotto, B. and Wood, C. M. (2005). Tissue-specific cadmium and metallothionein levels in rainbow trout chronically acclimated to waterborne or dietary cadmium. Arch. Environ. Contam. Toxicol. 48,381 -390.[CrossRef][Medline]
Conners, D. E. and Ringwood, A. H. (2000). Effects of Glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica). Aquat. Toxicol. 50,341 -349.[CrossRef][Medline]
Coyle, P., Philcox, J. C., Carey, L. C. and Rofe, A. M. (2002). Metallothionein: the multipurpose protein. Cell. Mol. Life Sci. 59,627 -647.[CrossRef][Medline]
Csermely, P., Schneider, T., Soti, C., Prohaszka, Z. and Nardai, G. (1998). The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol. Her. 79,129 -168.
Eble, A. F. and Scro, R. (1996). General anatomy. In The Eastern Oyster Crassostrea virginica (ed. V. S. Kennedy, R. I. E. Newell and A. F. Eble), pp.19 -73. College Park, MD: Maryland Sea Grant.
Feder, M. and Hofmann, G. (1999). Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu. Rev. Physiol. 61,243 -282.[CrossRef][Medline]
Frew, R. D., Hunter, K. A. and Beyer, R. (1997). Cadmium in Oysters and Sediments from Foveaux Strait, New Zealand: Proceedings of the Trace Element Group of New Zealand (ed. R. B. Macaskill), pp. 1-23. Waikato: Waikato University Press.
Giguere, A., Couillard, Y., Campbell, P. G. C., Perceval, O., Hare, L., Pinel-Alloul, B. and Pellerin, J. (2003). Steady-state distribution of metals among metallothioneins and other cytosolic ligands and links to cytotoxicity in bivalves along a polymetallic gradient. Aquat. Toxicol. 64,185 -200.[CrossRef][Medline]
Griffith, O. (1980). Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106,207 -212.[CrossRef][Medline]
GESAMP (1987). Land-sea boundary flux of contaminants: contributions from rivers. GESAMP Rep. Stud. 32,1 -172.
Hand, S. C. and Hardewig, I. (1996). Downregulation of cellular metabolism during environmental stress: mechanisms and implications. Annu. Rev. Physiol. 58,539 -563.[CrossRef][Medline]
Hulbert, A., Else, P., Manolis, S. and Brand, M. (2002). Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms. J. Comp. Physiol. B 172,387 -397.[CrossRef][Medline]
Jakob, U., Meyer, I., Bugl, H., Andre, S., Bardwell, J. C. and
Buchner, J. (1995). Structural organization of prokaryotic
and eukaryotic Hsp90. Influence of divalent cations on structure and function.
J. Biol. Chem. 270,14412
-14419.
Kammenga, J. E. M., Arts, S. J. and Oude-Breuil, W. J. M. (1998). HSP60 as a potential biomarker of toxic stress in the nematode Plectus acuminatus. Arch. Environ. Contam. Toxicol. 34,253 -258.[CrossRef][Medline]
Khimani, A. H., Mhashilkar, A. M., Mikulskis, A., O'Malley, M., Liao, J., Golenko, E. E., Mayer, P., Chada, S., Killian, J. B. and Lott, S. T. (2005). Housekeeping genes in cancer: normalization of array data. BioTechniques 38,739 -745.[Medline]
Klaassen, C. D., Liu, J. and Choudhuri, S. (1999). Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu. Rev. Pharmacol. Toxicol. 39,267 -294.[CrossRef][Medline]
Kultz, D. (2003). Evolution of the cellular
stress proteome: from monophyletic origin to ubiquitous function.
J. Exp. Biol. 206,3119
-3124.
Lanks, K. W. (1983). Metabolite regulation of
heat shock protein levels. Proc. Natl. Acad. Sci. USA
80,5325
-5329.
Lannig, G., Flores, J. F. and Sokolova, I. M. (2006). Temperature-dependent stress response in oysters, Crassostrea virginica: pollution reduces temperature tolerance in oysters. Aquat. Toxicol. 79,278 -287.[CrossRef][Medline]
Madden, E. F., Akkerman, M. and Fowler, B. A. (2002). A comparison of 60, 70, and 90 kDa stress protein expression in normal rat NRK-52 and human HK-2 kidney cell lines following in vitro exposure to arsenite and cadmium alone or in combination. J. Biochem. Mol. Toxicol. 16, 24-32.[CrossRef][Medline]
Mallin, M. A., Ensign, S. H., Parsons, D. C. and Merritt, J. F. (1999). Environmental quality of Wilmington and New Hanover county watersheds 1998-1999. In CMSR Report 99-02, pp. 1-58. Wilmington, NC: Center for Marine Science Research, University of North Carolina at Wilmington.
Marigomez, I., Soto, M., Cajaraville, M. P., Angulo, E. and Giamberini, L. (2002). Cellular and subcellular distribution of metals in molluscs. Microsc. Res. Tech. 56,358 -392.[CrossRef][Medline]
Mayer, M. P. and Bukau, B. (2005). Hsp70 chaperones: cellular functions and molecular mechanism. Cell. Mol. Life Sci. 62,670 -684.[CrossRef][Medline]
Meister, A. and Anderson, M. E. (1983). Glutathione. Annu. Rev. Biochem. 52,711 -760.[CrossRef][Medline]
Mitchelmore, C. L., Ringwood, A. H. and Weis, V. M. (2003). Differential accumulation of cadmium and changes in glutathione levels as a function of symbiotic state in the sea anemone Anthopleura elegantissima. J. Exp. Mar. Biol. Ecol. 284, 71-85.[CrossRef]
Notten, M. J. M., Oosthoek, A. J. P., Rozema, J. and Aerts, R. (2006). Heavy metal pollution affects consumption and reproduction of the landsnail Cepaea nemoralis fed on naturally polluted Urtica dioica leaves. Ecotoxicology 15,295 -304.[CrossRef][Medline]
Papaconstantinou, A. D., Brown, K. M., Noren, B. T., McAlister, T., Fisher, B. R. and Goering, P. L. (2003). Mercury, cadmium and arsenite enhance heat shock protein synthesis in chick embryos prior to embryotoxicity. Birth Defect Res. Part B 68,456 -464.[CrossRef]
Pfaffl, M. W. (2001). A new mathematical model
for relative quantification in real-time RT-PCR. Nucleic Acids
Res. 29,e45
.
Piano, A., Valbonesi, P. and Fabbri, E. (2004). Expression of cytoprotective proteins, heat shock protein 70 and metallothioneins, in tissues of Ostrea edulis exposed to heat and heavy metals. Cell Stress Chaperones 9, 134-142.[CrossRef][Medline]
Pigeot, J., Miramand, P., Guyot, T., Sauriau, P.-G., Fichet, D., Le Moine, O. and Huet, V. (2006). Cadmium pathways in an exploited intertidal ecosystem with chronic cadmium inputs (Marennes-Oleron, Atlantic coast, France). Mar. Ecol. Prog. Ser. 307,101 -114.[CrossRef]
Pörtner, H. O. (2001). Climate change and temperature-dependent biogeography: oxygen limitation of thermal tolerance in animals. Naturwissenschaften 88,137 -146.[CrossRef][Medline]
Prosser, C. L. (1973). Comparative Animal Physiology, Vol. I, Environmental Physiology. Philadelphia, London, Toronto: W. B. Saunders Company.
Pytharopoulou, S., Kouvela, E. C., Sazakli, E., Leotsinidis, M. and Kalpaxis, D. L. (2006). Evaluation of the global protein synthesis in Mytilus galloprovincialis in marine pollution monitoring: seasonal variability and correlations with other biomarkers. Aquat. Toxicol. 80,33 -41.[CrossRef][Medline]
Regoli, F. and Principato, G. (1995). Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals in different field and laboratory conditions: implications for a proper use of biochemical biomarkers. Aquat. Toxicol. 31,143 -164.[CrossRef]
Regoli, F., Frenzilli, G., Bocchetti, R., Annarumma, F., Scarcelli, V., Fattorini, D. and Nigro, M. (2004). Time-course variations of oxyradical metabolism, DNA integrity and lysosomal stability in mussels, Mytilus galloprovincialis, during a field translocation experiment. Aquat. Toxicol. 68,167 -178.[CrossRef][Medline]
Ringwood, A. H. and Conners, D. E. (2000). The effects of glutathione depletion on reproductive success in oysters, Crassostrea virginica. Mar. Environ. Res. 50,207 -211.[CrossRef][Medline]
Ringwood, A. H., Conners, D. E. and DiNovo, A. (1998). The effects of copper exposures on cellular responses in oysters. Mar. Environ. Res. 46,591 -595.[CrossRef]
Ringwood, A. H., Conners, D. E., Keppler, C. J. and Dinovo, A. A. (1999). Biomarker studies with juvenile oysters (Crassostrea virginica) deployed in-situ. Biomarkers 4,400 -414.[CrossRef]
Roesijadi, G. (1982). Uptake and incorporation of mercury into mercury-binding proteins of gills of Mytilus edulis as a function of time. Mar. Biol. 66,151 -157.[CrossRef]
Roesijadi, G. (1996). Environmental factors: response to metals. In The Eastern Oyster Crassostrea virginica (ed. V. S. Kennedy, R. I. E. Newell and A. F. Eble), pp. 515-537. College Park, MD: Maryland Sea Grant.
Roesijadi, G. and Klerks, P. (1989). A kinetic analysis of Cd-binding to metallothionein and other intracellular ligands in oyster gills. J. Exp. Zool. 251, 1-12.[CrossRef]
Roesijadi, G. and Robinson, W. E. (1993). Metal regulation in aquatic animals: mechanisms of uptake, accumulation, and release. In Molecular Mechanisms in Aquatic Toxicology (ed. D. C. Malins and G. Ostrander), pp. 387-420. New York: Lewis Publishers.
Roesijadi, G., Hansen, K. M. and Fuentes, M. E. (1995). Cadmium-induced expression of metallothionein and suppression of RNA to DNA ratios during molluscan development. Toxicol. Appl. Pharmacol. 133,130 -138.[CrossRef][Medline]
Roff, D. A. (2002). Life History Evolution. Sunderland, MA: Sinauer Press.
Rose, W. L., Nisbet, R. M., Green, P. G., Norris, S., Fan, T., Smith, E. H., Cherr, G. N. and Anderson, S. L. (2006). Using an integrated approach to link biomarker responses and physiological stress to growth impairment of cadmium-exposed larval topsmelt. Aquat. Toxicol. 80,298 -308.[CrossRef][Medline]
Sanders, B. M. and Martin, L. S. (1993). Stress proteins as biomarkers of contaminant exposure in archived environmental samples. Sci. Total Environ. 139/140,459 -470.[CrossRef][Medline]
Sanders, B. M., Martin, L. S., Nelson, W. G., Phelps, D. K. and Welch, W. J. (1991). Relationships between accumulation of a 60 kDA stress protein and scope-for-growth in Mytilus edulis exposed to a range of copper concentrations. Mar. Environ. Res. 31,81 -97.[CrossRef]
Sibly, R. M. and Calow, P. (1989). A life-cycle theory of responses to stress. Biol. J. Linn. Soc. 37,101 -116.[Medline]
Sokolova, I. M. (2004). Cadmium effects on
mitochondrial function are enhanced by elevated temperatures in a marine
poikilotherm, Crassostrea virginica Gmelin (Bivalvia: Ostreidae).
J. Exp. Biol. 207,2639
-2648.
Sokolova, I. M., Evans, S. and Hughes, F. M.
(2004). Cadmium-induced apoptosis in oyster hemocytes involves
disturbance of cellular energy balance but no mitochondrial permeability
transition. J. Exp. Biol.
207,3369
-3380.
Sokolova, I. M., Ringwood, A. H. and Johnson, C. (2005). Tissue-specific accumulation of cadmium in subcellular compartments of eastern oysters Crassostrea virginica Gmelin (Bivalvia: Ostreidae). Aquat. Toxicol. 74,218 -228.[CrossRef][Medline]
Somji, S., Todd, J. H., Sens, M. A., Garrett, S. H. and Sens, D. A. (2000). Expression of heat shock protein 60 in human proximal tubule cells exposed to heat, sodium arsenite and CdCl2. Toxicol. Lett. 115,127 -136.[CrossRef][Medline]
Söti, C. and Csermely, P. (2000). Molecular chaperones and the aging process. Biogerontology 1,225 -233.[CrossRef][Medline]
Tanguy, A. and Moraga, D. (2001). Cloning and characterization of a gene coding for a novel metallothionein in the Pacific oyster Crassostrea gigas (CgMT2): a case of adaptive response to metal-induced stress? Gene 273,123 -130.[CrossRef][Medline]
Tanguy, A., Mura, C. and Moraga, D. (2001). Cloning of a metallothionein gene and characterization of two other sequences in the Pacific oyster Crassostrea gigas (CgMT1). Aquat. Toxicol. 55,35 -47.[Medline]
Toplan, S., Ozcelik, D., Dariyerli, N. and Akyolcu, M. C. (2003). Oxidant and antioxidant status of cadmium administered rats. J. Phys. IV France 107, 1309.
Urani, C., Melchioretto, P., Canevali, C. and Crosta, G. F. (2005). Cytotoxity and induction of protective mechanisms in HepG2 cells exposed to cadmium. Toxicol. In Vitro 19,887 -892.[CrossRef][Medline]
Urani, C., Melchioretto, P., Canevali, C., Morazzoni, F. and Gribaldo, L. (2007). Metallothionein and hsp70 expression in HepG2 cells after prolonged cadmium exposure. Toxicol. in Vitro 21,314 -319.[CrossRef][Medline]
Van Straalen, N. M. and Hoffmann, A. A. (2000). Review of experimental evidence for physiological costs of tolerance to toxicants. In Demography in Ecotoxicology (ed. J. E. Kammega and R. Laskowski), pp. 115-124. Chichester: John Wiley.
Vayssier, M. and Polla, B. S. (1998). Heat shock proteins chaperoning life and death. Cell Stress Chaperones 3,221 -227.[CrossRef][Medline]
Viarengo, A., Canesi, L., Pertica, M., Poli, G., Moore, M. N. and Orunesu, M. (1990). Heavy metal effects on lipid peroxidation in the tissues of Mytilus galloprovincialis LAM. Comp. Biochem. Physiol. 97C, 37-42.[CrossRef][Medline]
White, A. R. and Cappai, R. (2003). Neurotoxicity from glutathione depletion is dependent on extracellular trace copper. J. Neurosci. Res. 71,889 -897.[CrossRef][Medline]
White, A. R., Bush, A. I., Beyreuther, K., Masters, C. L. and Cappai, R. (1999). Exacerbation of copper toxicity in primary neuronal cultures depleted of cellular glutathione. J. Neurochem. 72,2092 -2098.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Lannig, A. S. Cherkasov, H.-O. Portner, C. Bock, and I. M. Sokolova Cadmium-dependent oxygen limitation affects temperature tolerance in eastern oysters (Crassostrea virginica Gmelin) Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2008; 294(4): R1338 - R1346. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
