Cadmium-induced apoptosis in oyster hemocytes involves disturbance of cellular energy balance but no mitochondrial permeability transition
,*
1 Biology Department, University of North Carolina at Charlotte, 9201
University City Boulevard, Charlotte, NC 28223, USA
2 Johnson C. Smith University, 100 Beatties Ford Road, Charlotte, NC 28216,
USA
* Author for correspondence (e-mail: insokolo{at}uncc.edu)
Accepted 18 June 2004
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Summary |
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Key words: cadmium, heavy metal, apoptosis, necrosis, oyster, hemocyte, Crassostrea virginica
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Introduction |
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Cadmium is an important environmental pollutant that is released into the
environment as a result of human activities as well as natural processes such
as leaching from cadmium-rich soils and rocks, volcanic activity or diatom
deposition in marine sediment (GESAMP,
1987; Roesijadi,
1996
; Frew et al.,
1997
). Populations of the eastern oyster, Crassostrea
virginica, are exposed to varied cadmium level in estuaries and coastal
areas. Like all marine bivalves, oysters have an ability to concentrate metals
in soft tissues, accumulating large loads of heavy metals from water solutions
and metal-contaminated sediments (reviewed in
Roesijadi, 1996
;
Crompton, 1997
). Resistance to
the toxic effects of these pollutants is provided primarily by binding to
metallothioneins and deposition of insoluble metal-containing granules
(Roesijadi, 1996
). However,
these detoxification mechanisms are imperfect, and 10-80% of the total heavy
metal pool in the cytosol of bivalves is bound to other proteins and
low-molecular-mass compounds depending on the species and the regime of
exposure to heavy metals (Giguere et al.,
2003
). These `excess' metal ions have the potential to exert toxic
effects, including cellular toxicity, on bivalves.
In vertebrates, it has been shown that cadmium exposure results in
apoptosis, or programmed cell death, in a variety of cell types
(Li et al., 2000;
Pourahmad and O'Brien, 2000
;
Robertson and Orrenius, 2000
;
De La Fuente et al., 2002
;
Wätjen et al., 2002
; Aydi
et al., 2003; Shih et al.,
2004
). In response to this metal, cells activate the classical
intrinsic death pathway, in which mitochondria have a central role. In this
pathway, the mitochondrial inner membrane undergoes a permeability transition
(also known as the MPT), resulting in a dramatic increase in permeability
caused by an apparent opening of a channel known as the mitochondrial
permeability transition pore (PTP) (reviewed in
Leist and Nicotera, 1997
;
Mignotte and Vayssiere, 1998
;
Hüttenbrenner et al.,
2003
). Opening of this pore causes a decrease in the potential
across the mitochondrial membrane (
m) and the release
of cytochrome c (cyt c) into the cytoplasm. Cyt c
interacts with apaf-1 and procaspase-9 to form a complex known as the
apoptosome, which in turn activates effector caspases such as caspase-3.
Activation of caspases eventually leads to degradation of the cell's DNA and
to the cascade of other intracellular reactions that culminate in cell death
by apoptosis. In vertebrates, the MPT is generally considered to be an early
universal event in heavy-metal-induced apoptosis
(Leist and Nicotera, 1997
;
Mignotte and Vayssiere, 1998
;
Hüttenbrenner et al.,
2003
).
Currently, there is very limited knowledge about the effects of cadmium on
apoptosis in any cell types within the bivalves. In the mussel Mytilus
edulis, short-term exposure to low nontoxic cadmium levels (1.8 µmol
l-1) was shown to decrease susceptibility of isolated gill cells to
hydrogen peroxide-induced apoptosis, possibly due to induction of
metallothioneins or other antioxidant molecules
(Pruski and Dixon, 2002).
Exposure to elevated levels of heavy metals such as cadmium, zinc, silver and
mercury was shown to decrease viability of bivalve hemocytes
(Brousseau et al., 2000
; Sauve
et al.,
2002a
,b
),
although the mechanisms of cell death were not elucidated. The molecular and
cellular mechanisms of cadmium cytotoxicity in bivalves are not understood and
nothing is known about the effects of cadmium on apoptosis of the hemocytes of
bivalves. Understanding of the influence of this metal over this process on a
cellular and molecular level is important because excessive apoptosis of
hemocytes may have detrimental effects on the ability of oysters to fight
infectious diseases (Sunila and LaBanca,
2003
).
The aim of the present study was to investigate the effects of cadmium on hemocyte viability and apoptosis and to assess the role of cadmium-induced mitochondrial dysfunction in cell death in the eastern oyster, Crassostrea virginica. Our data show that cadmium induced apoptosis and, at higher levels, necrosis in isolated oyster hemocytes. The onset of apoptosis was associated with a 60-80% reduction of ATP levels in oyster hemocytes, whereas a much stronger depletion of ATP (by >90%) was found after exposure to cadmium in the necrosis-inducing range. Surprisingly, no MPT was apparent during Cd2+-induced apoptosis in intact hemocytes or isolated oyster mitochondria, suggesting that this central apoptotic process is not involved in apoptosis in oysters as it is in vertebrates. Overall, our results contrast the earlier findings in vertebrates in which cadmium-induced apoptosis involved the MPT and activation of caspases and suggest that trace-metal-induced apoptosis may proceed by a different pathway in marine mollusks. This pathway may be novel or, more likely, an evolutionarily ancient route to death that is either no longer functional, is superceded or is masked by the feed-forward cascade activated by the MPT in vertebrates.
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Materials and methods |
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Primary culture and cadmium exposure of oyster hemocytes
Oyster shells were surface-cleaned with ethanol and notched along the
ventral edge enough to insert a needle. Hemolymph (1.5-2 ml) was extracted
from an adductor muscle using a 21-gauge hypodermic needle and a syringe
containing 0.5-1 ml of sterile hemocyte support medium (HSM) consisting of
sterile seawater (SW; 650 mOsm)supplemented with 2 g l-1 glucose.
Hemolymph from 3-5 oysters was pooled to obtain a total of
8-12x106 cells. Pooled samples were placed into wells of
6-well tissue culture plates to obtain a cell density of
1.5-2x106 cells per well and allowed to attach for 20-25 min
at room temperature. Cell attachment was controlled under the inverted
microscope. When the cells attached, hemolymph was removed, and 3 ml of HSM
supplemented with 10 µg ml-1 gentamycin and 25 µg
ml-1 amphotericin was added. CdCl2 in 0.05%
HNO3 was added to different wells to obtain final nominal
concentrations of 10-1000 µmol l-1 Cd2+. Equal
volumes of 0.05% HNO3 were added to the control samples (0 µmol
l-1 nominal Cd2+). Hemocytes were incubated for 72 h at
room temperature. Pilot studies have shown that incubation for at least 60 h
is required to induce apoptosis in oyster hemocytes exposed to cadmium and
that incubations in the range of 2-72 h have no significant effect on the
viability of control hemocytes (data not shown).
Following cadmium exposure, hemocytes were harvested using 0.25% trypsin in modified Hanks balanced salt solution (Fisher Scientific, Suwanee, GA, USA) adjusted to 650 mOsm with sucrose, washed in sterile SW (650 mOsm) and used for further analyses.
Effects of cadmium on oyster cells
Annexin V-FITC assay
Following culture, cells were harvested, washed twice with SW, resuspended
in 100 µl SW and stained for annexin V exposure on the plasma membrane
using the annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, San Diego,
CA, USA) and the manufacturer's recommendations. This staining allows the
determination and quantification of apoptotic, necrotic and viable cells.
Briefly, 100 µl of cell suspension (1.5-2.0x106 cells) was
added to a 12x75 flow tube together with 5 µl each of the annexin V
and propidium iodide (PI) solutions provided. Cells were incubated for 15 min
at room temperature in the dark and then analyzed immediately by flow
cytometry for fluorescence in the FL-1 (annexin V) and FL-2 (PI) channels.
Caspase assay
Caspase-3-like activity was measured using a fluorimetric assay as
described elsewhere (Hughes et al.,
1997; Jablonski et al.,
2004
). Briefly, hemocytes were resuspended in 100 µl of 10 mmol
l-1 MgCl2, 0.25% NP-40 and the extracts centrifuged at
100 000 g for 30 min to remove debris and particulate matter.
Supernatants were harvested, combined with an equal volume of 40 mmol
l-1 Hepes (pH 7.4), 20 mmol l-1 NaCl, 2 mmol
l-1 EDTA, 20% glycerol and frozen at -70°C until assayed (less
than 1 week). For positive apoptotic controls, rat thymocytes incubated for 18
h in the absence of the growth factor were used and extracted as described
above. For analysis, cell extracts corresponding to 10-50 µg of protein, as
measured by Bradford assay (Bradford,
1976
), were incubated in 50 mmol l-1 Hepes (pH 7.5), 10
mmol l-1 dithiothreitol, 10% sucrose, 0.1% CHAPS with 200 µmol
l-1 of the caspase-3 substrate DEVD-afc (Kamiya Biomedical Co.,
Seattle, WA, USA). Samples were incubated for 5 min at room temperature and
their fluorescence at 505 nm measured (excitation at 400 nm) on a fluorescence
spectrophotometer (Hitachi Ltd, Tokyo, Japan). Samples were then incubated for
an additional hour and fluorescence measured again. A standard curve of
fluorescence vs free 7-amino-4-trifluromethylcoumarin (afc) was then
used to calculate the specific activity of caspase-3-like enzymes per mg
protein in each sample. For in vitro activation of caspase-3-like
activity, extracts were preincubated with 10 µg ml-1 cyt
c and 1 mmol l-1 dATP at room temperature for 1 h, and
their caspase activity measured as described above.
Mitochondrial membrane potential
Control and cadmium-exposed hemocytes were washed twice in sterile SW,
centrifuged for 10 min at 200 g and 4°C and resuspended in
1 ml of sterile SW to yield a final concentration of
1x106 cells ml-1. Mitochondrial membrane
potential (
m) was measured using a method modified from
Wong and Cortopassi (2002
).
Briefly, digitonin was added to the cell suspension to a final concentration
of 10 µmol l-1 and incubated for 6 min on ice. Cells were then
centrifuged for 10 min at 200 g and 4°C and washed twice
with 5 ml sterile SW. Pilot studies have shown that this concentration of
digitonin effectively permeabilizes cells without affecting the
m (data not shown). The cells were resuspended in 1 ml
of sterile SW and stained with 100 nmol l-1 tetramethylrhodamine
methyl ester (TMRM; Molecular Probes, Eugene, OR, USA) for 30 min at room
temperature in the dark before analysis by flow cytometry as the fluorescence
in the FL-2 channel. After each analysis, the individual samples were treated
with 25 µmol l-1 CCCP (carbonyl cyanide 3-chloropheryl
hydrazone; Fisher Scientific) to collapse the
m, and
the TMRM fluorescence was analyzed again.
ATP levels
ATP concentrations were measured in hemocytes exposed to 0-200 µmol
l-1 Cd2+ for 72 h using CellTiter-Glo® Luminescent
Assay (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Hemocytes were washed in sterile SW, lysed in a cell lysis buffer (CLB)
containing 5 mmol l-1 Tris (pH 7.4), 20 mmol l-1 EDTA
and 0.5% Triton X-100, and 50 µl of cell lysate was combined with an equal
volume of CellTiter-Glo® Reagent, mixed on an orbital shaker for 2 min and
incubated for 10 min at room temperature to stabilize luminescent signal. CLB
without cells was prepared in the same way and used as a negative control.
Luminescent signal was recorded using a Sirius luminometer (Berthold Detection
Systems GmbH, Pforzheim, Germany) with a 10 s integration of the signal. In
order to convert relative luminescence units (RLUs) into ATP concentrations, a
calibration curve was constructed using 0.025-1 µmol l-1 ATP
dissolved in CLB. The calibration curve was linear in the studied range of ATP
concentrations. ATP levels in hemocyte lysates were determined by
interpolation from the calibration curve. Although the initial number of cells
per sample was similar in all replicates, cell mortality in cadmium-exposed
hemocytes could result in variation in final cell numbers. To account for this
variation, we measured protein concentrations in cell lysates using a modified
Biuret assay (Bergmeyer, 1988)
and expressed ATP concentrations as nmol ATP mg-1 protein.
Effects of cadmium on isolated mitochondria
Respiration
Hemocytes of oysters did not provide a sufficient amount of tissue for
mitochondrial isolation; therefore, mitochondria were isolated from oyster
gills using a standard method modified from Ballantyne and Moyes
(1987). Isolation buffer had an
osmolarity of 730 mOsm and consisted of 300 mmol l-1 sucrose, 50
mmol l-1 KCl, 50 mmol l-1 NaCl, 8 mmol l-1
EGTA, 1% bovine serum albumin (BSA; essentially fatty acid free), 2 µg
ml-1 of the protease inhibitor aprotinin and 30 mmol l-1
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 with iso- or hypoosmotic media
(Ballantyne and Moyes,
1987
).
Gills of six or seven 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 Potter-Elvenhjem 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 and re-suspended in the ice-cold EGTA-free isolation buffer to give a mitochondrial protein content of 5-10 mg ml-1.
Oxygen uptake by mitochondria was measured using Clarke-type oxygen electrodes (YSI, Yellow Springs, OH, USA) in 3 ml water-jacketed glass chambers equilibrated at 25°C. Three to five volumes of assay medium were mixed with one volume of isolation medium containing the mitochondria. The assay medium (AM) had an osmolarity of 650 mOsmand consisted of 150 mmol l-1 KCl, 150 mmol l-1 NaCl, 10 mmol l-1 KH2PO4, 20 mmol l-1 sucrose, 0.1% BSA (essentially fatty acid free), 2 µg ml-1 of aprotinin, 5 µmol l-1 myokinase inhibitor AP5A and 30 mmol l-1 Hepes (pH 7.2). Different concentrations of CdCl2 in the range of 5-50 µmol l-1 were added to the assay medium containing mitochondria and incubated for 5 min. Control mitochondria were incubated without cadmium addition. Addition of the highest concentration of cadmium used in this study (50 µmol l-1) did not noticeably change the pH of the assay buffer (i.e. pH change was less than 0.01 units).
All assays were completed within 2-3 h of isolation of the mitochondria.
Preliminary experiments have shown that there was no change in mitochondrial
respiration or coupling during this period. Succinate was used as a substrate
at saturating amounts (10-15 mmol l-1) in the presence of 5 µmol
l-1 rotenone. Maximal respiration rates of actively phosphorylating
mitochondria (state 3) were achieved by addition of 50 µmol l-1
ADP, and state 4 respiration was determined in ADP-conditioned mitochondria as
described by Chance and Williams
(1955). State 4+ respiration
was determined as oxygen consumption rate after addition of 2.5 µg
ml-1 of the 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
;
Abele et al., 2002
). At the end
of the assay, KCN (100 µmol l-1) and salicylhydroxamic acid
(SHAM; 200 µmol l-1) were added to the mitochondria to inhibit
mitochondrial respiration and to measure non-mitochondrial rate of oxygen
consumption. SHAM was used in order to avoid the overestimation of
nonmitochondrial 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 1-5% of the
state 3 respiration. Respiration rates in state 3, 4 and 4+ were corrected for
non-mitochondrial respiration and oxygen electrode drift. Respiratory control
ratio (RCR), which is a measure of mitochondrial coupling, was determined as
the ratio of state 3 over state 4 respiration as described by Estabrook
(1967
). Protein concentrations
in mitochondrial suspensions were measured using a modified Biuret method with
0.5% Triton X-100 added 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. Oxygen
solubility (ßO) 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 min-1 mg-1 mitochondrial
protein.
Mitochondrial swelling
Mitochondrial swelling was measured as described in Li et al.
(2003). Mitochondria were
isolated as described above, and 100 µl of mitochondrial suspension was
added to 0.9 ml of standard AM containing 20 mmol l-1 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, Mulgrave, 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 5 min at 25°C, and changes in mitochondrial volume were
monitored by measuring absorbance at 520 nm under constant stirring. A
reduction in absorbance of a mitochondrial suspension indicates mitochondrial
swelling and is a hallmark of the MPT (Li
et al., 2003
). As a positive control, mitochondria were incubated
for 5 min in a hypoosmotic assay buffer (375 mOsm).
Mitochondrial membrane potential
m was determined as described in Scaduto and
Grotyohann (1999
). Briefly,
mitochondria were isolated as described above, protein concentration was
determined, and mitochondrial suspension was diluted to 2 mg ml-1
mitochondrial protein in the standard AM containing 0.5 µmol l-1
TMRM, 20 mmol l-1 succinate and 5 µmol l-1 rotenone.
Mitochondria were incubated at 25°C for 10 min with 0-50 µmol
l-1 Cd2+ until TMRM fluorescence stabilized.
Concentrations of cadmium above 50 µmol l-1 were not used
because preliminary experiments showed that they completely abolish
respiration in isolated mitochondria (data not shown). Following cadmium
incubations, TMRM fluorescence in mitochondrial suspension was measured under
constant stirring at two excitation wavelengths (573 nm and 546 nm) and a
single emission wavelength of 590 nm (excitation and emission slits 10 nm)
using a fluorescence spectrophotometer (Hitachi Ltd). The ratio of the
fluorescence at 590 nm when alternately excited by the two excitation
wavelengths is a measure of the
m. It has been shown
that the 573/546 ratio linearly increases with increasing
m (Scaduto and
Grotyohann, 1999
). The mitochondrial uncoupler CCCP (50 µmol
l-1) was added to the mitochondrial suspension to collapse the
membrane potential, and the 573/546 ratio was determined. All ratios were
corrected for the background fluorescence of TMRM in the AM without
mitochondria.
Statistics
Effects of cadmium concentrations were analyzed using split-plot repeated
measures ANOVA after testing the assumptions of normality of data distribution
and homogeneity of variances. Dunnet tests were used for post-hoc
pairwise comparisons of sample means.
Statistical analyses were performed using SAS 8.2 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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According to the mammalian model of cadmium-induced apoptosis,
Cd2+ is known to induce apoptosis by triggering a change in
mitochondrial membrane potential (m) resulting in the
release of cyt c and eventual activation of caspase-3. Thus, we next
examined the
m and caspase-3 activity in
Cd2+-treated oyster hemocytes. In contrast to our expectations,
Cd2+ exposure did not facilitate a drop in
m
in oyster hemocytes (Fig. 3).
On the contrary, exposure of hemocytes to 20-50 µmol l-1
Cd2+ resulted in a slight but significant hyperpolarization of
mitochondrial membranes by 10-15% (ANOVA; F3,19=6.21,
P=0.004). At higher Cd2+ levels (200 µmol
l-1), the level of hyperpolarization tended to decrease but the
m was still significantly higher than in the control
cells. The mitochondrial uncoupler CCCP efficiently collapsed
m to similar low levels independent of the cadmium
treatment of oyster hemocytes. Control samples indicate that digitonin, which
was used to permeabilize cell membranes and to facilitate penetration of TMRM
and CCCP into the cells, had no significant effect on
m
in oyster hemocytes (F1,19=0.55, P=0.466;
Fig. 3, striped bar).
|
Analysis of activity of caspase-3-like enzymes, activated by the cyt c release during mitochondrial permeability transition, indicated that this activity was below the detection limit in freshly isolated oyster hemocytes and did not significantly increase in cells treated with vehicle nor the apoptotic (50 µmol l-1) or necrotic (200 µmol l-1) concentrations of cadmium (F3,18=0.86, P=0.48; Fig. 4). Addition of dATP and cyt c to extracts of control hemocytes in vitro resulted in a dramatic increase in caspase-3-like activity up to levels close to those found in apoptotic rat thymocytes, which served as a positive control. This demonstrates that the inactive pro-form of this enzyme is present in the hemocyte extracts and suggests that, unlike the apoptosis induced in mammals, Cd2+ induction of cell death in oyster hemocytes does not activate this cascade.
|
Although there was no decrease in m due to cadmium
exposure in oyster hemocytes, cellular ATP levels were decreased, indicating
severe disturbance of energy status (Fig.
5). Cadmium exposure resulted in a significant depletion of ATP in
oyster hemocytes (ANOVA; F4,36=4.63, P=0.004),
which was dose-dependent and manifest at cadmium levels (20 µmol
l-1) that did not significantly affect cell viability. Exposure to
20-50 µmol l-1 Cd2+, which was in the range of
apoptosis-inducing concentrations, was associated with a 60-88% decrease in
ATP levels, and exposure to 200 µmol l-1 Cd2+, which
induced significant levels of necrosis, resulted in a depletion of ATP levels
by >90%.
|
Studies of the effect of cadmium on isolated oyster mitochondria in
vitro corroborated our findings in intact hemocytes that cadmium affects
mitochondrial function and ATP production without affecting the
m or causing MPT. Thus, ADP-stimulated (state 3)
respiration, indicative of the maximum phosphorylation rate, decreased in a
dose-dependent manner with increasing cadmium concentrations (ANOVA;
F3,21=23.3, P<0.0001;
Fig. 6). On the other hand,
state 4+ respiration, indicative of proton leak, was significantly affected
only by the highest cadmium concentration (50 µmol l-1; ANOVA;
F3,20=12.1, P=0.0001;
Fig. 6). RCR of isolated
mitochondria decreased with increasing cadmium levels, indicating partial
uncoupling of cadmium-exposed mitochondria P<0.0001; (ANOVA;
Fig. 6).
F3,21=40.4, Incubation with 50 µmol l-1
Cd2+ completely abolished coupling of isolated mitochondria. At
higher cadmium levels (100-200 µmol l-1), state 3 and state 4+
respiration was inhibited to less than 5% of the control levels (data not
shown), indicating a complete loss of functionality of mitochondria.
|
Although respiration rate, and especially ADP-stimulated respiration, of
oyster mitochondria was strongly affected by cadmium, this metal had no effect
on mitochondrial volume or membrane potential. Indeed, no swelling indicative
of the MPT was observed in mitochondria exposed to cadmium in the range of
10-1000 µmol l-1 (ANOVA; F5,20=2.49,
P=0.065; Fig. 7A). On
the contrary, the absorbance at 520 nm ofisolated mitochondria exposed to
10-100 µmol l-1 cadmium showed a marginally significant decrease
compared with the control mitochondria, indicating that cadmium might induce
slight contraction of the mitochondrial volume. Cadmium exposure did not
affect m, as measured by 573/546 fluorescence ratio of
TMRM (ANOVA; F4,16=2.53, P=0.08;
Fig. 7B). Post-hoc
comparisons showed that the
m was slightly below the
control only at the highest tested cadmium concentration (50 µmol
l-1; P=0.03), which also strongly inhibited respiration of
isolated oyster mitochondria (Fig.
6). The mitochondrial uncoupler CCCP, used to collapse
m, reduced 573/546 fluorescence ratios of mitochondria
to similar low levels independent of the cadmium treatment.
|
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Discussion |
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Notably, molecular and cellular mechanisms of apoptosis in cadmium-exposed
oyster hemocytes differ radically from the cadmium-induced cell death pathways
in vertebrate cells, which are the only systems where cadmium-induced
apoptosis has been extensively studied. In vertebrates, cadmium exposure
results in fast depolarization of the mitochondrial membrane, opening of the
mitochondrial permeability pore and the release of cyt c into the
cytoplasm (reviewed in Wolf and Eastman,
1999). In the cytoplasm, cyt c associates with apaf-1 and
procaspase-9 to form a complex known as the apoptosome. Formation of this
complex activates caspase-9, which is then responsible for activation of
caspase-3, the central executioner that facilitates many downstream apoptotic
events. Moreover, cadmium addition to isolated mitochondria results in rapid
mitochondrial depolarization, the PTP opening and cyt c release
(Al-Nasser, 2000
;
Li et al., 2003
), suggesting
that in vertebrates the MPT may be due to a direct effect of cadmium on
mitochondria. This suggestion is further supported by the finding that
ruthenium red, which inhibits cadmium transport into mitochondria, also
prevents PTP opening and the loss of transmembrane potential
(Li et al., 2003
).
Mitochondrial depolarization in response to cadmium, with the concomitant
release of cyt c and activation of caspase-3-like enzymes, has been
demonstrated in all mammalian cell types studied to date, including kidney
cells, hepatocytes, neurons and, most relevant, immune cells, suggesting that
this is a universal mechanism of cadmium-induced apoptosis in mammals
(Habeebu et al., 1998
;
Kim et al., 2000
;
Galan et al., 2001
; Kondoh et
al., 2001
,
2002
;
Wätjen et al., 2002
;
Aydin et al., 2003
;
Shih et al., 2004
). By
contrast, cadmium-induced apoptosis in oyster hemocytes is activated by a
pathway fundamentally different from that of the vertebrates and independent
of MPT. In the present study, there was clearly no loss of the
m in response to cadmium exposure either in intact
oyster hemocytes or in isolated oyster mitochondria, strongly suggesting there
was no opening of the PTP. Moreover, mitochondrial swelling, which is another
indicator of the MPT (Li et al.,
2003
), was not induced in oyster mitochondria by even very high
levels of cadmium (100-1000 µmol l-1), even though they
completely abolished mitochondrial respiration and coupling. In fact, the
m was slightly elevated in cadmium-treated oyster
hemocytes, indicating hyperpolarization of mitochondrial membrane. This
hyperpolarization could be due to the inhibition of mitochondrial ATP
synthesis in cadmium-treated hemocytes, as indicated by the strong
dose-dependent inhibition of the ADP-stimulated respiration in cadmium-treated
mitochondria and by the decreased ATP levels in cadmium-exposed oyster cells.
The mechanisms of this cadmium-induced inhibition of ATP synthesis are unknown
and may be due to the inhibition of the F0/F1-ATPase
and/or reduced ADP transport into the mitochondria. Indeed, the respiration
rate of `resting' mitochondria in the presence of oligomycin was not
significantly affected by cadmium except at the highest test concentration (50
µmol l-1), indicating that the electron transfer chain was not
inhibited by the low cadmium levels that inhibited ADP-dependent respiration.
Proton conductance through the F0/F1-ATPase during ATP
production would dissipate the membrane potential, and, therefore, inhibition
of ATP synthesis can conceivably result in hyperpolarization of the
mitochondrial membrane (Brand,
1995
).
No significant activation of caspase-3-like activity was detected in cadmium-treated apoptotic hemocytes, supporting our conclusion that cadmium-induced apoptosis in oysters does not involve the MPT and formation of the apoptosome. However, caspase-3-like activity could be activated in hemocyte extracts by incubating with dATP and cyt c. Levels of the cyt c-activated caspase-3 activity in oysters were comparable with caspase-3 activity in apoptotic rat thymocytes, indicating that the enzyme was present in oyster cells in significant quantities. The lack of activation of caspase-3 in vivo in cadmium-exposed oyster hemocytes suggests that cyt c was not released from mitochondria during cadmium exposure.
Another notable difference between cadmium-induced apoptosis in oysters and
mammalian models is the change in energy status, particularly ATP levels,
during apoptosis. Apoptosis is an energy-requiring process, and earlier
studies on mammalian models have demonstrated that depletion of cellular ATP
levels resulted in a switch of the mode of death from apoptosis to necrosis
(Lopez et al., 2003;
Nieminen, 2003
). In oyster
hemocytes, we found a decrease in the cellular ATP levels with increasing
cadmium levels. Importantly, a significant drop in ATP levels was detected in
the apoptotic range of cadmium concentrations (20-50 µmol l-1),
indicating that low ATP levels per se do not shift the form of cell
death towards necrosis in these cells. A characteristic feature of the
cellular physiology of intertidal mollusks, including oysters, is their
capability for metabolic rate depression under conditions of environmental
stress (Hochachka and Guppy,
1987
; Hand and Hardewig,
1996
; Storey,
1988
). It has been shown that a variety of stressors, particularly
those that affect ATP production such as hypoxia/anoxia, subfreezing and
freezing temperatures, severe osmotic stress, result in a drastic
downregulation of the use of ATP to 5-20% of the normal levels in marine
mollusks (Storey and Storey,
1990
; Storey and Churchill,
1995
; Sokolova et al.,
2000
; Sokolova and
Pörtner, 2001
). This stress-induced decrease in ATP turnover
and cellular energy demand may be important in allowing some crucial
energy-dependent processes (e.g. apoptosis) to proceed despite a decline in
the overall cellular ATP levels. However, a further decrease in ATP levels
will eventually result in the situation when the free Gibbs energy of ATP
hydrolysis is insufficient to support functioning of cellular ATPases
(Sokolova et al., 2000
), thus
resulting in necrotic cell death. Although no data are currently available
about the effects of cadmium exposure on the ATP turnover rates in oysters,
extrapolation from a wide variety of other stressors that affect mitochondrial
ATP production suggests that metabolic rate depression is also likely to occur
in cadmium-exposed oyster cells. Further studies are required to elucidate the
relationship between metabolic rate regulation and the energy partitioning for
apoptotic processes under conditions of cadmium exposure in oysters.
The absence of mitochondrial involvement in apoptosis in response to
cadmium is a unique feature of oyster cells that sets them apart from other
systems where cadmium-induced apoptosis has been studied. However, alternative
pathways of apoptosis, which do not involve MPT, are not unknown and can be
induced by some stimuli in other organisms. In vertebrates, Mn2+
was shown to induce apoptosis without MPT
(Oubrahim et al., 2001),
whereas other metals such as Cd2+, Cu2+, Zn2+
and arsenic result in the MPT (Robertson
and Orrenius, 2000
; De La
Fuente et al., 2002
;
Wätjen et al., 2002
;
Pulido and Parrish, 2003
). In
Drosophila and Caenorhabditis elegans, mitochondria did not
release cyt c or undergo a decrease in
m
during apoptosis induced by a variety of stimuli including UV radiation and
oxidative stress (Varkey et al.,
1999
; Zimmermann et al.,
2002
; Claveria and Torres,
2003
), although other apoptotic changes, such as translocation of
phosphatidylserine to the outer surface of the cell membrane, were conserved
(Zimmermann et al., 2002
). In
contrast to oysters and vertebrates, cyt c added directly to the
Drosophila or C. elegans cell extracts failed to
significantly activate caspase-3 (Kanuka
et al., 1999
). Our data suggest that, unlike insects and
nematodes, oysters possess the cellular potential for caspase-3 activation and
for the amplification of the apoptotic signal through the
mitochondria-dependent pathways, but these pathways are not activated during
cadmium-induced apoptosis. Further investigation will elucidate what types of
apoptotic stimuli can induce a drop in the
m and
activate caspase activity in oysters.
Regardless of the mechanism, it seems likely that elevated hemocyte
mortality due to cadmium-induced apoptosis and necrosis could significantly
impair the cellular defense ability of oysters in much the same way that
diseases which result in the elimination of human immune cells (such as AIDS)
result in immunodeficiency. Field studies of hemocyte numbers in
heavy-metal-exposed oyster populations have yielded controversial results.
Some researchers have reported decreased numbers of circulating hemocytes in
oysters exposed to pollutants in the field or the lab, whereas others have
shown elevated levels or no differences from control oyster populations
(Suresh and Mohandas, 1990;
Pipe et al., 1999
;
Dyrynda et al., 2000
;
Oliver et al., 2001
).
Interestingly, changes in the DNA profile of hemocytes similar to those seen
during apoptosis have been reported for several bivalve populations from
contaminated sites (Bihari et al.,
2003
), suggesting elevated levels of DNA damage and possibly
apoptosis in situ. It is possible that an increase in cell death is
compensated for by the increased proliferation and differentiation of immune
cells. Compensation for elevated cell mortality in oysters exposed to high
cadmium levels, if it occurs in vivo, would result in elevated
hemocyte turnover and the associated energetic costs. There are no studies to
date on the turnover rates of oyster hemocytes from heavy-metal-polluted and
unpolluted areas.
It is worth noting that the number of circulating hemocytes does not
necessarily reflect the total size of the hemocyte population and can change
over a short time span due to the dynamic association/dissociation between
hemocytes and oyster tissues (Ford et al.,
1993). Therefore, the number of circulating hemocytes in
heavy-metal-exposed oysters may not be a reliable estimate of hemocyte
mortality. Our study suggests that the level of apoptosis in hemocytes may be
a better indicator of the general viability and health of the hemocyte
population in oysters compared with the simple enumeration of hemocytes in
circulation and thus may be useful as a biomarker of immunotoxic and cytotoxic
effects of heavy metals in bivalves. Currently, field studies are underway to
test the applicability of apoptosis as a biomarker of immunotoxic effects of
heavy metal pollution in oyster populations.
In summary, cadmium exposure in oysters results in significant cytotoxicity and considerably elevated levels of apoptosis and necrosis in oyster hemocytes. High rates of apoptosis due to cadmium exposure may have important implications for the immune defense of the oysters, resulting in the weakened immune system due to hemocyte loss. Mitochondria are likely to be a key intracellular target for cadmium cytotoxicity in oysters, as indicated by the inhibition of the phosphorylation rate, decreased mitochondrial coupling and depletion of the intracellular ATP levels. However, unlike vertebrate mitochondria, mitochondria of oysters do not undergo permeability transition in response to cadmium. This emphasizes variability of evolutionary pathways of the programmed cell death in response to the same stimulus in different organisms and cautions against extrapolation of cellular biomarkers of heavy metal toxicity developed for vertebrates to invertebrate species.
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References |
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