Cadmium effects on mitochondrial function are enhanced by elevated temperatures in a marine poikilotherm, Crassostrea virginica Gmelin (Bivalvia: Ostreidae)
Biology Department, University of North Carolina at Charlotte, 9201 University City Boulevard, Charlotte NC 28223, USA
E-mail: insokolo{at}uncc.edu
Accepted 26 April 2004
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Summary |
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Key words: mitochondria, cadmium, temperature, proton leak, phosphorylation rate, P/O ratio, mitochondrial volume, bivalve mollusk, Crassostrea virginica
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Introduction |
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Temperature and heavy metals are common stressors in estuarine and coastal
habitats, and their importance is increasing due to the global climate changes
and continuing pollution of the coastal waters
(Helmuth et al., 2002;
GESAMP, 1987
). Cadmium is a
common inorganic contaminant of coastal sediments and waters, and the levels
of this metal can vary greatly, due to both anthropogenic pollution and to
natural sources such as river run-off from cadmium-rich soils, leaching of the
rocks or diatom deposition in marine sediment
(GESAMP, 1987
;
Roesijadi, 1996
;
Frew et al., 1997
). As are
most trace metals, cadmium is toxic at high concentrations, and increasing
evidence points toward mitochondrial dysfunction as an important mechanism of
cytotoxicity of cadmium (for reviews, see
Brierley, 1977
;
Byczkowski and Sorenson, 1984
;
Miccadei and Floridi, 1993
;
Wallace and Starkov, 2000
).
However, cadmium also affects mitochondrial function at non-toxic
concentrations as low as 106 mol l1
(Skulachev et al., 1967
;
Kesseler and Brand,
1994a
,b
,c
,
1995
;
Ye et al., 2001
). In
terrestrial plants and mammals, cadmium is known as a powerful modulator of
mitochondrial function, inhibiting electron transport chain, increasing
generation of reactive oxygen species
(Miccadei and Floridi, 1993
;
Wallace and Starkov, 2000
),
and stimulating proton leak through the inner mitochondrial membrane (Kesseler
and Brand,
1994a
,b
,c
,
1995
; Korotkov et al.,
1996a
,b
,
1999
;
Al-Nasser, 2000
;
Belyaeva et al., 2001
). These
data strongly suggest that mitochondria are key intracellular targets for
cadmium; however, nothing is known about effects of this metal on
mitochondrial function in marine mollusks.
In poikilotherms, a change in environmental temperature leads to a direct
change in the rate of all physiological and biochemical processes and in the
steady-state levels of metabolic intermediates (for a review, see
Hochachka and Somero, 2002).
Increasing the environmental temperature results not only in elevated
respiration rates of mitochondria, but also in a considerable rise in
mitochondrial proton leak and in progressive mitochondrial uncoupling in
marine fish and bivalves (Pörtner,
2001
). Therefore, cadmium and temperature can interact in their
effects on bioenergetics of marine poikilotherms, because the mitochondrion is
likely to be a common intracellular target for these environmental factors. So
far, however, there are no data about temperature-dependent effects of cadmium
on mitochondrial function in marine poikilotherms, and the mechanisms and
potential effects of these multiple stressors on molluscan mitochondrial
function are not known. This research intends to close this gap by studying
the combined effects of cadmium and temperature on mitochondrial function
using eastern oysters Crassostrea virginica as a model.
Eastern oysters are a useful model for the study of interactive effects of
cadmium and temperature on mitochondrial bioenergetics. Like all intertidal
organisms, they may experience rapid and extreme temperature fluctuations in
their habitats, with a change in body temperature as large as
1020°C within a few minutes during summer low tides
(Sokolova et al., 2000b;
Helmuth et al., 2002
;
Sokolova and Boulding, 2004
).
Oysters are also exposed to varying cadmium concentrations in their habitats,
and have an ability to concentrate cadmium in soft tissues to concentrations
exceeding the environmental levels by orders of magnitude
(Roesijadi, 1996
;
Frew et al., 1997
). Body
levels of cadmium in natural oyster populations range from 0.4 to 40 µg
g1 dry mass (Roesijadi,
1996
; Frew et al.,
1997
), corresponding to the intracellular concentrations of ca.
190 µmol l1. During acute exposure to elevated
cadmium concentrations in water or sediments, oysters can accumulate even
higher loads of this metal, up to 300400 µg g1 dry
mass, which corresponds to intracellular concentrations of ca. 670900
µmol l1 (Roesijadi,
1996
). About 80% of the accumulated cadmium in bivalves is
sequestered by metallothioneins
(Roesijadi, 1996
;
Giguere et al., 2003
), which
were previously believed to remove cadmium from the physiologically active
pool. However, recent studies have shown that both free and
metallothionein-bound cadmium ions have a potential to strongly affect
mitochondrial function (Simpkins et al.,
1994
,
1998
). Therefore, both
temperature and cadmium are potent modulators of mitochondrial function of
oysters in nature.
The aims of the present study were (1) to investigate the combined effects of elevated temperature and cadmium on mitochondrial function in Crassostrea virginica, including respiration rate, phosphorylation efficiency and proton leak in isolated mitochondria, and (2) to study the effects of cadmium on mitochondrial volume, which is essential for the maintenance of controlled mitochondrial function. The results of this study support the hypothesis that cadmium effects on mitochondrial function can be considerably modified by environmental temperature in marine poikilotherms, and that these two environmental stressors can have synergistic effects on oyster mitochondria. This study reports for the first time in a marine poikilotherm the sensitivity of different aspects of mitochondrial metabolism to a trace metal and temperature, and is a first contribution to a series of studies examining the effects of trace metals and temperature on oyster bioenergetics in vitro and in vivo.
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Materials and methods |
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Isolation of mitochondria
Mitochondria were isolated from oyster gills using a method modified from
Ballantyne and Moyes (1987).
Isolation buffer had osmolarity of 730 mOsm and consisted of 300 mmol
l1 sucrose, 50 mmol l1 KCl, 50 mmol
l1 NaCl, 8 mmol l1 EGTA, 1% bovine serum
albumin (BSA, essentially fatty acid free), 2 µg ml1 of a
protease inhibitor aprotinin and 30 mmol l1 Hepes (pH 7.5).
Previous studies have shown that isolation of oyster mitochondria in a
slightly hyperosmotic medium maximizes coupling and yields superior quality
mitochondria as compared to iso- or hypoosmotic media
(Ballantyne and Moyes,
1987
).
Gills of 6 or 7 animals were removed, blotted dry and placed in 15 ml of ice-cold isolation medium. The tissue was homogenized with three passes (200 r.p.m.) of a PotterElvenhjem homogenizer using a loosely fitting Teflon pestle. The homogenate was centrifuged for 10 min at 2000 g and 2°C. The supernatant was collected, and the tissue pellet re-homogenized in 15 ml of ice-cold isolation buffer. The second homogenate was centrifuged at 2000 g, and supernatants from the two centrifugations were pooled. The supernatant was then centrifuged at 10 500 g and 2°C for 12 min. The resulting mitochondrial pellet was washed twice with ice-cold EGTA-free isolation buffer to minimize cadmium binding by the chelator and resuspended in the ice-cold EGTA-free isolation buffer to give a mitochondrial protein content of 510 mg ml1.
Mitochondrial oxidation
Oxygen uptake by mitochondria was measured in 3 mlwater-jacketed glass
chambers using Clarke-type oxygen electrodes (YSI, Yellow Springs OH, USA).
Two-point calibration of electrodes was performed at each experimental
temperature, and continuous data acquisition was made using a BIOPAC Data
acquisition system (BIOPAC, Santa Barbara, CA, USA). Temperature effects on
mitochondrial respiration were determined during acute exposure of
mitochondria of oysters acclimated at 15°C to different temperature
increases (15, 25 and 35°C). This acute exposure to elevated temperatures
is an environmentally realistic situation for intertidal animals including
oysters, which may experience fast and acute rise in body temperature by
1020°C in a few minutes during spring and summer low tides
(Sokolova et al., 2000b;
Helmuth et al., 2002
;
Sokolova and Boulding, 2004
).
Temperature in mitochondrial respiration chambers was maintained constant at
15, 25 or 35°C (+0.1°C) using a Fisher Isotemp (Suwanee, GA, USA)
refrigerated water circulator. 35 volumes of assay medium were mixed
with one volume of isolation medium containing the mitochondria. The assay
medium had an osmolality of 650 mOsm and consisted of 150 mmol
l1 KCl, 150 mmol l1 NaCl, 10 mmol
l1 KH2PO4, 20 mmol
l1 sucrose, 0.1% BSA, 2 µg ml1 of
aprotinin and 30 mmol l1 Hepes (pH 7.2). All assays were
completed within 23 h of isolation of the mitochondria. Preliminary
experiments showed that there was no change in mitochondrial respiration or
coupling during this period. A myokinase inhibitor AP5A (5 µmol
l1) was added to the assay buffer to prevent spontaneous
regeneration of ADP from ATP by mitochondria. Sodium succinate was used as a
substrate at saturating amounts (1015 mmol l1) in the
presence of 5 µmol l1 of rotenone. Maximal respiration
rates (state 3) were achieved by addition of 200300 nmol ADP, and state
4 respiration was determined in ADP-conditioned mitochondria as described by
Chance and Williams (1955
).
State 4+ respiration was determined as the oxygen consumption rate after
addition of 2.5 µg ml1 of an ATPase inhibitor oligomycin.
State 4+ respiration in the presence of oligomycin is considered as a good
upper limit estimate of mitochondrial proton leak measured at high
mitochondrial membrane potential (Brand et
al., 1994
; Kesseler and Brand,
1995
). At the end of the assay, KCN (100 µmol
l1) and salicylhydroxamic acid (SHAM, 200 µmol
l1) were added to the mitochondria to inhibit mitochondrial
respiration and to measure the non-mitochondrial rate of oxygen consumption.
SHAM was used in order to avoid overestimation of non-mitochondrial
respiration rate due to the presence of an alternative oxidase in bivalve
mitochondria (Tschischka et al.,
2000
). In all cases, non-mitochondrial oxygen consumption rate was
less than 15% of the state 3 respiration. Respiration rates in states
3, 4 and 4+ were corrected for non-mitochondrial respiration and oxygen
electrode drift.
Effects of cadmium on mitochondrial respiration in vitro were determined in oysters collected during June 2003. Each mitochondrial suspension was divided into 8 portions, and each portion was incubated for 10 min in the absence of cadmium (control) and at different concentrations of CdCl2 in the range 150 µmol l1. After incubation, the respiration rates of state 3, 4 and 4+ were determined as described above. Addition of the highest concentration of cadmium used in this study (50 µmol l1) did not detectably change the pH of the assay buffer (i.e. the pH change was less than 0.01 units).
Oxygen solubility (ßO2) for the assay medium at
each experimental temperature was calculated as described in Johnston et al.
(1994), and respiration rates
were expressed as natom O min1 mg1
mitochondrial protein. Respiratory control ratio (RCR) was determined as a
ratio of state 3 over state 4 respiration as described by Estabrook
(1967
), and RCR+ was
determined as a ratio of state 3 respiration over state 4+ respiration (in the
presence of oligomycin). P/O ratios were calculated by dividing the amount of
added ADP by the amount of oxygen consumed in state 3 respiration
(Hinkle, 1995
). Inhibition
constants for cadmium (Ki) were calculated assuming a
noncompetitive inhibition model (Segel,
1976
).
Mitochondrial swelling
Mitochondrial swelling was determined using a method modified from Li et
al. (2003). Mitochondria were
isolated as described above, and 100 µl of mitochondrial suspension added
to 0.9 ml of the standard assay medium containing 20 mmol l1
sodium succinate. Absorbance of the mitochondrial suspension was measured at
520 nm and 25°C using a UV/Vis Cary 50 spectrophotometer with a
water-jacketed cuvette holder (Varian, Victoria, Australia). After initial
measurements, different concentrations of cadmium chloride were added to the
cuvette. Mitochondria were incubated with cadmium for 20 min on ice, the
cuvettes were then equilibrated for 3 min at 25°C, and changes in
mitochondrial volume were monitored by measuring absorbance at 520 nm under
constant stirring. Excess ADP (600 µmol) was then added to the cuvettes,
incubated for 1 min at 25°C, and absorbance was measured at 520 nm under
constant stirring. A reduction in absorbance of a mitochondrial suspension
indicates mitochondrial swelling. For a positive control, mitochondria were
incubated for 5 min in a hypoosmotic assay buffer (375 mOsm).
Protein concentrations
Protein concentrations in mitochondrial suspensions were measured using a
modified Biuret method with added 1% Triton-X to solubilize the mitochondria
(Bergmeyer, 1988). BSA was used as the standard. Protein content was measured
for each batch of the isolation medium and subtracted from the total protein
content of the mitochondrial suspension to determine the mitochondrial protein
concentration.
Statistics
Effects of the factors `Temperature' and `Cadmium concentration' and their
interactions were analyzed using split-plot repeated-measures analysis of
variance (ANOVA) after testing the assumptions of normality of data
distribution and homogeneity of variances. Effects of factor interactions were
significant for all studied variables except RCR+ (P<0.01, data
not shown), thus preventing analysis of the effects of single factors.
Therefore, we performed separate repeated-measures ANOVAs for each
experimental temperature to test the effects of cadmium on the studied
mitochondrial parameters. A sequential Bonferroni test was used to adjust
probability levels for multiple ANOVAs. To analyze the effect of season
(spring vs summer) and temperature on respiration rates and
respiratory control ratios in oyster mitochondria, a mixed model ANOVA was
used with `Season' as a random factor and `Temperature' as a fixed one. Dunnet
tests were used for post hoc pairwise comparisons, and least-square
difference test (LSD) for planned comparisons. Statistical analyses were
performed using SAS 8.1 software (SAS Institute, Cary, NC, USA). Differences
were considered significant if the probability for Type II error was less than
0.05.
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Results |
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ADP-stimulated (state 3) respiration of oyster mitochondria was inhibited
by cadmium in a dose-dependent manner (Fig.
3). Effects of cadmium on state 3 respiration were significant at
all temperatures (F6,24=7.71, P=0.0001,
F7,35= 15.30, P<0.0001, and
F5,20=33.70, P<0.0001 for 15°, 25° and
35°C, respectively). Notably, at 15°C only the highest cadmium
concentrations (3550 µmol l1) significantly
inhibited state 3 respiration down to ca. 60% of control. In contrast, at
25° and 35°C a significant inhibition of state 3 respiration occurred
at cadmium levels 25 µmol l1 and
10 µmol
l1, respectively, and at the highest cadmium concentration
(50 µmol l1) state 3 respiration was only 1030% of
control rates. Higher cadmium sensitivity of state 3 respiration at elevated
temperatures was reflected in the lower inhibition constants
(Ki) for cadmium, which were 80, 24 and 3 µmol
l1 [Cd2+] at 15°, 25° and 35°C,
respectively.
|
State 4 respiration was significantly affected by cadmium at 25°C and 35°C but not at 15°C (15°C: F6,23=1.90, P=0.125; 25°C: F7,34=8.83, P<0.0001; 35°C: F5,20=22.11, P<0.0001) (Fig. 4). Inhibition constants (Ki) for state 4 respiration were 172, 78 and 4 µmol l1 [Cd2+] at 15°, 25° and 35°C, respectively, indicating higher sensitivity of state 4 respiration to cadmium exposure at elevated temperatures.
|
State 4+ respiration in the presence of oligomycin, which is indicative of proton leak, was significantly and positively correlated with state 4 respiration while being ca. 1020% lower than the latter. Effects of cadmium on state 4+ respiration were significant at all temperatures (15°C: F6,24=4.71, P=0.003; 25°C: F7,34=9.66, P<0.0001; 35°C: F5,20=20.03, P<0.0001). Low concentrations of cadmium (''10 µmol l1) significantly stimulated state 4+ respiration of oyster mitochondria (Fig. 4). On average, exposure to 15 µmol l1 Cd2+ led to 2030% and 1015% increase in state 4+ respiration at 1525°C and 35°C, respectively, compared to control rates. Similar to states 3 and 4+, state 4 respiration of oyster mitochondria was increasingly more sensitive to cadmium as the temperature increased, as indicated by a decline of Ki values (154, 7 and 10 µmol l1 [Cd2+] at 15°, 25° and 35°C, respectively). ANOVA results and graphical analysis of the dose-dependent cadmium effects (Fig. 4) support the conclusion of higher sensitivity of state 4+ respiration to cadmium levels at elevated temperatures. Thus, at the highest cadmium level (50 µmol l1), state 4+ respiration was 83% of control rate at 15°C, but only 20% of control rate at 2535°C.
Cadmium exposure resulted in partial uncoupling of oyster mitochondria, as indicated by a significant decrease in respiratory control ratios (F6,24=11.3, P<0.0001 and F6,24=5.75, P=0.0007 for RCR and RCR+, respectively; Fig. 5). Uncoupling effects of cadmium on oyster mitochondria were observed at concentrations as low as 1 µmol l1 and increased with increasing cadmium concentrations at all temperatures. Notably, elevated temperatures increased the sensitivity of mitochondria to cadmium-induced uncoupling, and the coupling was essentially abolished by exposure to 50 µmol l1 Cd2+ at 25°C and 35°C, but not at 15°C.
|
In order to test that the observed effects on mitochondria were due to cadmium, 2.5 mmol l1 EGTA was added to the assay medium along with 550 µmol l1 of cadmium. The cadmium effects on respiration of oyster mitochondria were completely abolished by addition of EGTA at all temperatures (data not shown).
The P/O ratio was not significantly affected by cadmium at the concentrations 125 µmol l1 (F2,27=0.94, P=0.484). Determination of P/O ratio at high cadmium concentrations (>25 µmol l1) was not possible due to low coupling of mitochondria and thus high error associated with determination of the transition to state 4.
Mitochondrial volume
Incubation with 51000 µmol l1 of cadmium did
not result in swelling of oyster mitochondria
(Fig. 6). There was a trend to
the increase in absorbance at 520 nm (A520) of
mitochondria in the presence of 5100 µmol l1 of
Cd2+, indicating mitochondrial contraction, although it was
statistically non-significant due to high variation
(F5,20=2.49, P=0.065). Addition of ADP resulted
in a decrease in absorbance at 520 nm,indicating swelling of phosphorylating
mitochondria, which was similar in control and cadmium-incubated mitochondria
(F5,20=1.24, P=0.327). Incubation of mitochondria
in hypoosmotic buffer (375 mOsm) used as a positive control led to a
significant decrease in A520 by 0.155±0.036
absorbance units.
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Discussion |
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Although the general physiological response of oyster mitochondria to
increased cadmium concentrations is similar to that of mammals, unlike
homeotherms the mitochondrial function of poikilotherms is also strongly
affected by the environmental temperature. Indeed, the most important novel
finding of the present study is that environmental temperature strongly
modulates cadmium effects on oyster mitochondria. The results of the present
study clearly demonstrate that sensitivity of mitochondrial function to
cadmium increases with increasing temperatures in the environmentally relevant
range. This is reflected in a dramatic drop of Ki values
for cadmium with increasing temperature, indicating that 50% inhibition of
mitochondrial respiration rate requires 80170 µmol
l1 [Cd2+] at 15°C but only 34 µmol
l1 [Cd2+] at 35°C. The temperature-dependent
increase in cadmium sensitivity was especially prominent for ADP-stimulated
(state 3) respiration and respiratory control ratio (RCR and RCR+), reflecting
the degree of mitochondrial coupling. Thus, the threshold cadmium
concentrations, below which no effect on state 3 respiration was observed,
were progressively shifted to lower values with increasing temperature from
35 µmol l1 to
25 µmol l1 and
5 µmol l1 at 15, 25 and 35°C, respectively.
Similarly, cadmium-induced uncoupling was faster and more pronounced with
increasing temperatures. At 15°C, cadmium exposure in the range 150
µmol l1 led to a similar small decrease in RCR, so that
mitochondria retained coupling with a RCR of ca. 2 at all cadmium levels. In
contrast, at 35°C, cadmium concentrations as low as 10 µmol
l1 resulted in a completely abolished mitochondrial
coupling. In the absence of cadmium, only a small reduction of RCR and RCR+
was observed at the highest temperature (35°C), and mitochondria still
retained coupling. This implies that elevated environmental temperatures may
result in the onset of mitochondrial dysfunction at lower cadmium
concentrations, which are essentially non-toxic at lower temperatures. State
4+ respiration rate in the presence of an ATPase inhibitor oligomycin is
considered to be a good upper limit estimate of proton leak at high
mitochondrial membrane potentials typical of resting mitochondria
(Brand et al., 1994
;
Kesseler and Brand, 1995
).
Proton leak in resting mitochondria is an important determinant of basal
metabolic rate (BMR) accounting for 2030% of BMR both in poikilo- and
homeotherms (Brand et al.,
1991
; Hulbert and Else,
1999
). Our study demonstrated that low concentrations of cadmium
(15 µmol l1) elevated proton leak by 2030%.
High levels of proton leak were also maintained at higher cadmium levels, in
contrast to a considerable inhibition of ADP-stimulated respiration.
Cadmium-induced stimulation of the proton leak may have important
implications for the whole organism BMR, leading to an increased cost of
mitochondrial maintenance and thus to a higher cost of basal metabolism in
cadmium-exposed oysters. Decreased coupling and elevated proton leak are also
known to negatively affect thermal tolerance of poikilotherms, in particular,
the critical temperatures characterized by the transition to partial
anaerobiosis (Pörtner,
2001,
2002
;
Sokolova and Pörtner,
2003
). Cadmium-induced mitochondrial dysfunction may result in an
earlier failure of aerobic scope and the onset of anaerobiosis at
progressively lower temperatures as cadmium levels in the environment
increase. In order to test these hypotheses, further research is required to
determine the intracellular cadmium levels and the effects of trace metals on
BMR and heat tolerance in vivo. To date, studies of the heavy metal
effects on the BMR of poikilotherms are scarce and have yielded controversial
results. Thus, Hopkins and co-authors
(Hopkins et al., 1999
) have
demonstrated that BMR is elevated by 32% in banded water snakes Nerodia
fasciata exposed to heavy metals in their environments as compared to
their conspecifics from the reference site. On the other hand, routine
metabolic rates of various fish and invertebrates, which in addition to BMR
included variable non-quantified contributions from physical activity, were
either unaffected or inhibited by exposure to cadmium
(Coenen-Stass, 1998
;
Leung et al., 2000
;
McGeer et al., 2000
;
Knops et al., 2001
;
Rajotte and Couture, 2002
). In
order to understand the role of trace metals in metabolic costs and regulation
of BMR, carefully designed studies measuring BMR over a wide range of trace
metal concentrations are required. The finding that cadmium may significantly
increase proton leak in mitochondria and thus the cost of mitochondrial
maintenance, also cautions against unverified interpretations of the increased
metabolic rate in response to a toxicant as a cost for detoxification or
transmembrane transport of the toxicant, and emphasizes the importance of
taking into account the direct non-adaptive effects of a toxicant on the
components of BMR, which may lead to an increase in basal metabolism.
Cadmium exposure did not result in swelling of oyster mitochondria in
isoosmotic, K+- and sucrose-containing medium. This contrasts with
the results in mammalian mitochondria, where addition of cadmium resulted in
considerable swelling due to energy-dependent accumulation of K+ in
the matrix and/or opening of the mitochondrial permeability pore
(Brierley, 1977;
Rasheed et al., 1984
;
Li et al., 2003
). In
vivo studies demonstrated an inconsistent response of mitochondrial
volume and morphology to cadmium exposure and both mitochondrial swelling and
contraction were reported depending on the cell type and cadmium concentration
(Hemelraad et al., 1990a
;
Early et al., 1992
;
Al-Nasser, 2000
;
Romero et al., 2003
). This
suggests that mitochondrial volume changes in response to cadmium vary
considerably between species and cell types. In the present study, there was a
notable trend towards cadmium-induced increase in optical density of suspended
oyster mitochondria, suggesting mitochondrial contraction rather than
swelling. This indicates that, unlike mammalian mitochondria, oyster
mitochondria do not undergo a permeability transition, which is typically
associated with considerable mitochondrial swelling, even at high cadmium
concentrations that completely inhibit respiration. This suggestion is
supported by our recent finding that cadmium exposure does not result in
depolarization of the mitochondrial membrane (I. M. Sokolova, S. Evans, and F.
M. Hughes, manuscript in review). This suggests that, despite an impaired
ability for ATP synthesis and increased proton leak, the activity of proton
pumps is sufficient to maintain the mitochondrial membrane potential and
prevent depolarization and swelling in cadmium-exposed oyster
mitochondria.
As a corollary, mitochondrial function in oysters is highly sensitive to cadmium and temperature, which have synergistic effects on mitochondrial respiration (particularly on ADP-stimulated respiration) and coupling. Cadmium exposure results in elevated proton leak, and reduces phosphorylation rate and coupling in oyster mitochondria. Elevated temperatures increased sensitivity of oyster mitochondria to cadmium many-fold, suggesting that onset of mitochondrial dysfunction will occur at lower cadmium levels as the environmental temperature increases. These findings emphasize the importance of taking temperature into account when studying the effects of environmental toxicants on poikilotherms, or developing biomarkers of environmental stress using poikilothermic organisms as models. This also suggests that oyster populations subjected to one of the stressors in their natural environments (e.g. to elevated temperatures due to seasonal warming or global climate change) may become more susceptible to other stressors (such as trace metal pollution) and vice versa, and emphasizes the importance of analysing multiple stressors in estuaries. Currently, further studies are being conducted in order to elucidate the role of mitochondrial mechanisms and temperaturetrace metal interactions on oyster bioenergetics in vivo.
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Acknowledgments |
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References |
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