Temperature-dependence of mitochondrial function and production of reactive oxygen species in the intertidal mud clam Mya arenaria
1 Alfred Wegener Institut for Polar and Marine Research,
Columbusstraße, 27568 Bremerhaven, Germany
2 Physical Chemistry, School of Pharmacy and Biochemistry, University of
Buenos Aires, Buenos Aires, Argentina
* Author for correspondence (e-mail: abele{at}awi-bremerhaven.de )
Accepted 3 April 2002
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Summary |
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Key words: mitochondrial function, reactive oxygen species, heat stress, antioxidant, mud clam, Mya arenaria
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Introduction |
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Thermal stress is also accompanied by oxidative stress in various marine
mollusc species (Abele et al.,
1998,
2001
). These findings were
based on indirect measurements of oxidative stress, such as malonedialdehyde
and lipofuscin accumulation in the tissues
(Abele et al., 1998
), or
changes in antioxidant enzyme activities upon heating (Abele et al.,
1998
,
2001
). Reactive oxygen species
(ROS) are normal byproducts of cellular respiration
(Boveris et al., 1972
), but
little is known about oxygen radical formation in invertebrates and virtually
nothing about marine invertebrate mitochondria. Mitochondria, respiring in
state 3 and in state 4, generate ROS at respiratory chain complexes I
(exogenous NADH dehydrogenase; Turrens and
Boveris, 1980
) and III (ubiquinone, UQ/cytochrome b
complex; Boveris and Chance,
1973
; Cadenas et al.,
1977
; Han et al.,
2001
). Elevated rates of ROS production by ubisemiquinone radicals
(UQ-) are promoted in resting state 4 of isolated
mitochondria, following complete phosphorylation of ADP
(Loschen et al., 1971
;
Boveris and Chance, 1973
) (for
a review see Skulachev, 1996
).
Under these conditions, intracellular oxygen concentrations increase, because
cytochrome c oxidase activity is low, while the proton potential
across the inner mitochondrial membrane (
µH+) is maximal.
Mitochondrial ROS production is described to be a direct function of
µH+ (Skulachev,
1998
) and to be controlled by `mild uncoupling' via
proton leakage through the inner mitochondrial membrane. Moreover, increased
production of reactive oxygen species was detected after the addition of the
respiratory chain blocker antimycin a
(Boveris and Chance, 1973
;
Loschen et al., 1973
;
Boveris and Cadenas, 1975
) and,
generally, when respiratory chain components are in a maximally reduced state,
e.g. under hypoxic conditions.
According to Brookes et al.
(1998), the mitochondria of
ectotherms generally exhibit a lower membrane potential, lower proton leakage
and lower rates of substrate oxidation and, therefore, an overall reduced rate
of state 4 respiration compared to endotherms. This goes hand in hand with
reduced rates of state 3 respiration. Indeed, the rate of mitochondrial oxygen
consumption of invertebrates and ectothermic vertebrates in all functional
states is low compared with that of mammals and birds on a mitochondrial
protein basis. This may not, however, necessarily be true with respect to ROS
production in vertebrate and invertebrate mitochondria.
ROS are not only byproducts of mitochondrial respiration, but can also be detrimental to the generating cell itself if not extinguished or expelled, so it is interesting to study mitochondrial localization in ROS-generating cell types. Superoxide anion radicals (O2-) are converted to membrane-permeable H2O2 by mitochondrial superoxide dismutase, enabling diffusion of peroxide to the extracellular space and the body fluids and, eventually, release to the outer environment through gill diffusion. Clustering of mitochondria at the cellular border would keep diffusion distances short and limit H2O2-related damage in the centre of the cell.
The present study was designed to examine mitochondrial ROS production in a
marine invertebrate species, the intertidal mud clam Mya arenaria.
ROS production was measured in intact and well-coupled isolated mitochondria
in respiratory states 3 and 4 and related to mitochondrial respiration, the
respiratory coupling ratio (RCR) and the efficiency of phosphorylation (ADP:O
ratio) with malate as respiratory substrate. Measurements were conducted over
the temperature range 5-25 °C, covering the habitat temperature range
(5-18 °C) as well as higher temperatures that pose a heat stress to these
animals. Correlated changes in antioxidative defence were investigated by
testing the effects of temperature on the antioxidant enzymes catalase and
superoxide dismutase in vitro, and in an experiment with intact
animals exposed to habitat and higher temperatures. Specifically, the
following questions were addressed. (i) Does temperature stress imply
significant increase of ROS production in mitochondria of marine ectotherms?
(ii) Is ROS formation primarily related to the functional state of the
mitochondria (state 3 versus state 4 respiration) and does it depend
on the level of proton leak, i.e. mild uncoupling of the proton potential
(µH+) in a eurythermal ectotherm under heat stress? (iii)
Given that the rate of ROS formation increases at high temperatures, does the
organism strive to control it either by increasing the leak or by increasing
the antioxidant defence? (iv) Are mitochondria evenly distributed or clustered
in these cells and does the distribution indicate which cellular structures
might be affected by release of active oxygen species?
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Materials and methods |
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Isolation of mitochondria
Mitochondria were isolated from mantle tissue of freshly killed Mya
arenaria. Approximately 4 g of mantle tissue from 6-8 individuals were
washed and finely chopped in 10 ml of ice-cold homogenization buffer (400 mmol
l-1 sucrose, 100 mmol l-1 KCl, 6 mmol l-1
EGTA, 3 mmol l-1 EDTA, 70 mmol l-1 Hepes, 1 µg
ml-1 aprotinin, 1 % bovine serum albumin, BSA, at pH 7.6) modified
after Moyes et al. (1985).
Extraction of mitochondria was achieved by three passes (200 revs
min-1) in a pre-cooled glass/Teflon homogenizer in a total volume
of 45 ml homogenization buffer. The homogenate was centrifuged at 1300
g and 4 °C for 14 min, and the supernatant collected and
centrifuged again at 10 000 g for 16 min to sediment the
mitochondria. The resulting pellet was resuspended in 4 ml of assay medium
(560 mmol l-1 sucrose, 100 mmol l-1 KCl, 10 mmol
l-1 KH2PO4, 70 mmol l-1 Hepes, 5
mmol l-1 glutamate, 1 µg aprotinin and 1 % BSA at pH 7.6). This
suspension of isolated mitochondria could be kept on ice with no apparent loss
of respiratory activity for up to 7 h.
Measurements of mitochondrial respiration and coupling
The measurements of mitochondrial respiration were carried out in
thermostatted respiration chambers with an adjustable sample volume of 1-2 ml
using Clarke oxygen electrodes and an Eschweiler M200 oxymeter connected to a
Linseis two-channel chart recorder. For each measurement, the chambers were
filled with 680-880 µl of O2-saturated assay medium, omitting
BSA and aprotinin, to which 100-300 µl of mitochondrial suspension and 5
µmol l-1 of the myokinase inhibitor
P1,P5-diadenosine-5'-pentaphosphate
(AP5A) in water were added. Rates of oxygen consumption were
measured at constant temperature while continuously stirring the mitochondrial
suspension at 350 revs min-1. State 2 respiration was initiated by
adding 3.3 mmol l-1 of the respiratory substrate malate. After
approximately 5 min, ADP was added at a final concentration of 0.06 mmol
l-1 to start state 3 respiration under saturating conditions. State
4 respiration was recorded after the ADP had been consumed
(Chance and Williams, 1955).
After addition of 2 µg ml-1 of the ATPase inhibitor oligomycin,
state 4+ respiration was recorded which, according to Brand et al.
(1994
), reflects the rate of
proton leakage. Measurements were carried out at 5, 10, 15, 20 and 25
°C.
The oxygen solubility (ßO2) of the assay medium at
experimental temperatures was taken from Johnston et al.
(1994). Oxygen consumption
(
O2) measurements were
corrected for electrode drift at 100 % PO2 and
at 0 % PO2. The respiratory control ratio (RCR)
was calculated by dividing state 3 by state 4 respiration according to
Estabrook (1967
) or by state
4+ respiration following Pörtner et al.
(1999a
). ADP/O ratios were
calculated by dividing the amount of ADP added by the amount of molecular
oxygen consumed during state 3 respiration
(Chance and Williams,
1955
).
Fluorimetric measurements of mitochondrial radical formation
The formation of mitochondrial reactive oxygen species (ROS) was measured
at the same time as oxygen consumption in the same mitochondrial isolates
using the fluorescent dye dihydrorhodamine-123 (DHR). DHR is non-fluorescent
and is oxidized to the fluorescent rhodamine-123 by various reactive oxygen
species. We tested the fluorophore with and without peroxidase and found that
this enzyme is not required as cofactor. The fluorescence signal was detected
using a thermostatted spectrofluorometer (Perkin & Elmer LS 50B) at an
excitation wavelength of 505 nm and emission wavelength of 534 nm (excitation
slit 2.5 nm, emission slit 3.5 nm). DHR (14.4 mmol l-1) was
dissolved in dimethylsulphoxide (DMSO), which had previously been purged with
nitrogen for 30 min. DHR solution (1.5 µl ml-1) was added to the
assay medium without BSA or aprotinin and gently stirred with a magnetic
stirrer, resulting in a final concentration of approximately 20 µmol
l-1. Subsequently, the assay medium containing the DHR was kept in
the dark and at the different measuring temperatures.
The assay was performed with 640-840 µl of DHR-containing medium in a thermostatted cuvette with constant slow stirring. Recording was started after adding 150-300 µl of the mitochondrial suspension. Radical formation was measured during state 3 respiration after adding 0.06 mmol l-1 ADP. After a 10 min delay, 2 µg ml-1 oligomycin was added to stop ATPase activity and to initiate state 4+ respiration. This procedure determined the start of state 4 respiration, which could not be detected with sufficient precision in the DHR assay. Each state was recorded for at least 10 min.
Calibration of the dihydrorhodamine-123 assay for ROS formation in
mitochondrial isolates
DHR oxidation by ROS was quantified using 50 µmol l-1
xanthine/xanthine oxidase (XOD) to yield a constant rate of formation of
oxygen radicals, which reduce the oxidized form of cytochrome c (100
µmol l-1). Calibration was performed in the assay medium at
temperatures between 5 and 25 °C in a thermostatted spectrophotometer. At
each temperature, xanthine oxidase was added at concentrations of 0.75-16 mU
and the reduction of cytochrome c was measured.
At 25 °C in a 50 mmol l-1 phosphate buffer at pH 8.5, one
unit of XOD converts 1 µmol of xanthine to uric acid, producing
approximately the same amount of superoxide anions for the reduction of
cytochrome c. Since our measurements were performed in the
mitochondrial assay medium at pH 7.6, the rate of oxygen radical production
was calculated directly from the measured rate of reduction of cytochrome
c using the millimolar extinction coefficient (550 nm) of
28.5. A linear correlation (r2=0.995; N=5) was
found between added XOD activity (AXOD) (milliunits
ml-1) and the resulting cytochrome c reduction, from which
the rate of ROS production (RROS) (µmol l-1
min-1) could be calculated:
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In a second step, the rate of DHR oxidation was determined with the same
XOD/xanthine system at 15 °C to obtain the ratio of superoxide anions
(xROS) (nmol ml-1 min-1) produced to
the rate of DHR oxidation (SDHR) under assay conditions.
The data were best fitted by a second-order polynomial function
xROS=0.0183(SDHR)2+0.0685SDHR
(r2=0.9906, P<0.05, N=9). DHR did not
react directly with xanthine oxidase, as observed by Hempel et al.
(1999), and was oxidized
spontaneously at a very slow rate, irrespective of temperature. To correct for
possible variation in the rate of DHR oxidation in individual stock solutions,
we measured the slope of each DHR-medium daily, using a standard ROS formation
system of 10 µl of xanthine oxidase (3 milliunits ml-1) and 50
µmol l-1 xanthine at 15 °C. A mean slope was calculated
based on all measurements, and the individual DHR slopes recorded on each day
were corrected for day-to-day variation (<5 %) from the mean slope.
To identify the reactive oxygen species produced in our mitochondrial assays, we used specific quenchers for O2- (SOD), H2O2 (catalase) and OH (100 mmol l-1 DMSO). Assays were run as described above with intact respiring mitochondria under state 3 conditions (with ADP), as well as with submitochondrial particles (SMPs), derived from mitochondrial suspensions which had been treated for 5 min by ultrasound.
In experiments with intact mitochondria, SOD had no effect on DHR oxidation, whereas catalase abolished 52.5±15 % and DMSO another 10 % of the fluorescence increase (catalase + DMSO: 61.6±13 % total quenching of DHR oxidation; means ± S.D., N=11). In experiments with SMPs, application of SOD led to 31±20 % fluorescence quenching (N=7), whereas SOD and catalase together abolished 68.3±7 % of DHR fluorescence (N=7). Addition of DMSO reduced the fluorescence increment by another 10 % (N=2) (means ± S.D.). The rather small effect of DMSO is presumably due to primary quenching of the OH precursor H2O2.
Thus, in assays with intact mitochondria, hydrogen peroxide is apparently the major component responsible for DHR oxidation. Superoxide anions, which cannot leave intact mitochondria by diffusion, are obviously of minor importance. When SMPs are used in the assays, superoxide anions reduce DHR to a variable extent, perhaps depending on residual mitochondrial SOD activity in the preparation. Residual oxidation of DHR is assumed to result from other forms of reactive oxygen species such as lipid radicals, not detected with the applied quenchers.
In view of the quenching experiments, the molar rates of DHR oxidation recorded in the experimental work and calculated according to the calibration with the xanthine oxidase/cytochrome c system, were halved to account for the 50 % contribution of H2O2 formed during DHR oxidation in the mitochondrial assays. In this way we derived the fraction of the quantitatively most important ROS component produced and exported from the mitochondria, H2O2.
Temperature exposure experiment
In May 2000, a laboratory experiment was performed in which one group of
animals was exposed by stepwise warming to 18 °C and then to 25 °C,
while a control group was kept at constant, low temperatures of 10-12 °C.
Animals for this experiment were collected at the end of April 2000 at a
habitat temperature of around 10 °C. In the laboratory, animals were kept
in aquaria with natural, well-aerated sea water at a salinity of 24-26
and fed phytoplankton once a week. After 2 weeks at 10 °C, one
aquarium with 20 experimental animals was warmed by 1 °C every 2 days,
until a final temperature of 18 °C was reached. This plateau was kept for
3 days, after which a first batch of eight animals was killed and samples of
mantle tissue, digestive gland and gills were taken. On the same day, eight
control animals, maintained at 10 °C, were killed and processed in the
same way. The remaining specimens were further warmed from 18 °C to 25
°C, following the same protocol, and kept at this temperature for 7 days
(long-term heat stress). A third group was transferred directly from 10 °C
to 25 °C and maintained at this temperature for 2 days (short-term heat
stress). Each sample of heat-exposed animals was compared with the sample of
eight control animals maintained for the same time period at 10 °C. Tissue
samples were frozen in liquid nitrogen and kept at -80 °C for up to 2
months prior to analysis.
Measurements of antioxidant enzyme activities and malondialdehyde
tissue concentrations
Catalase (CAT; E.C. 1.11.1.6) was extracted into 50 mmol l-1
potassium phosphate buffer (pH 7.0, 1:19 w/v) and measured according to Aebi
(1985). Superoxide dismutase
(SOD; E.C. 1.15.1.1) activity in crude homogenates was measured using the
xanthine oxidase/cytochrome c assay according to Livingstone et al.
(1992
). Enzyme assays were
generally carried out at 20 °C and at the temperature at which the animals
had been kept. Malondialdehyde (MDA) tissue concentrations were measured as a
marker of lipid peroxidation according to Uchiyama and Mihara
(1978
), as described in Storch
et al. (2001
). The method was
modified only with respect to the heating time of the samples, which was 1 h
at 100 °C. Absolute MDA concentrations were calculated using a 5-point
calibration curve, obtained as follows: a malondialdehyde-(bis)-acetate
standard was diluted 1:4000 with 1.1 % phosphoric acid and left standing for 2
h at room temperature in the dark, to yield a 1.01 mmol l-1 MDA
solution, which was diluted to concentrations between 0.5 and 50 µmol
l-1 and processed in the same way as the tissue samples.
Counts and localization of mitochondria with confocal microscopy
A Leica TCS-NT confocal system with an inverted Leica DM IRBE microscope
equipped with an argon-krypton laser was used for confocal imaging of
mitochondrial density and clustering in cells isolated from Mya
arenaria mantle and gill tissue. Small samples of mantle and gills tissue
(<50 mg) were excised from freshly killed animals on ice. The tissue was
transferred to small vials containing 10 µl of Type IA collagenase solution
(Sigma) in 2 ml of Hepes Ringer (300 mmol l-1 NaCl, 140 mmol
l-1 KCl, 5 mmol l-1 Na2SO4, 10
mmol l-1 CaCl22H2O, 5 mmol
l-1 MgCl26H2O, 5 mmol l-1
Hepes, pH 7.6), cut coarsely with small scissors, and digested at 12 °C
for 2 h. Thereafter, cells were separated from the undigested residues by
filtering through 300 µm gauze and precipitated by 25 min of centrifugation
at 75 g and 12 °C in an Eppendorf centrifuge (5810 R). The cells
were washed once with the same Ringer's solution and subsequently stained with
organelle-specific dye (MitoTracker Green FM, Molecular Probes) at a
concentration of 0.2 nmol ml-1 in Ringer for 15 min at room
temperature (20 °C). After staining, the cells were again precipitated by
centrifugation, resuspended in 50-100 µl of Ringer's solution and kept on
ice for subsequent mitochondrial counts. MitoTracker Green stock solution (100
µmol l-1) was prepared in DMSO and frozen in portions of 10
µl at -30 °C until use.
Approximately 30 µl of cell solution was pipetted into a microscope chamber, which was mounted onto the microscope stage of the Leica IRBE confocal microscope and cooled to 12 °C. Cells were imaged with a 100x/1.40-0.70 oil immersion lens with the following properties: excitation wavelength 488 nm, laser power 145, confocal pinhole 0.8, emission filter 530±30 nm, photomultiplier voltage 920, offset -2. A Leica TCS-NT electronic workstation equipped with Leica scanning software was used for digital imaging.
Cells were scanned at 1.4 or 1.5 µm sections in the
z-direction, and mitochondria were counted visually in each section.
Cell diameters were calculated by multiplying the number of sections by their
thickness, and cell volume was calculated as 3/4r3,
where r is cell radius. Five animals were used for these experiments,
and a total of 49 mantle cells and 46 gill cells were examined.
Statistical analyses
All values are given as means ± S.D. Data were plotted as a function
of temperature. Arrhenius break temperatures (ABTs) were calculated from
two-phase regressions. Two intersecting lines were selected that best fitted
the data according to the method of least sum of squares. Significant
differences between the resulting slopes were determined by Student's
t-test. Values of RCR,
O2 and ADP/O
ratios were tested for homogeneity of variance (Levene test) and normal
distribution (KolmogorovSmirnov test). If these tests resulted in a
statistically significant difference (P<0.05), the data were
log-transformed. Significant changes in RCR,
O2 and ADP/O
ratios with temperature were evaluated by analysis of variance (ANOVA) and
analysis of covariance (ANCOVA) using NewmanKeuls post-hoc
comparisons. A non-parametric test (KruskalWallis ANOVA) was used in
those cases where transformation did not reveal homogeneity of variance and
normal distribution. Differences in enzyme activities and MDA contents of
tissue samples from temperature exposure experiments were analysed by
Student's t-test (using computer programs Super-Anova and
Statistica).
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Results |
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Temperature-dependence of mitochondrial ROS formation (DHR
oxidation)
Mitochondrial ROS formation, measured as equivalents of oxidized DHR,
increased exponentially (state 3, y=0.096exp(0.392x),
r2=0.972, N=6-7; state 4+,
y=0.073exp(0.411x), r2=0.967,
N=6-7) within the investigated temperature range
(Fig. 4). The overall increase
in mitochondrial ROS formation was significant only in state 3
(KruskalWallis ANOVA, P<0.01), but not-significant in state
4+ (ANCOVA, P=0.062). However, NewmanKeuls post-hoc
tests yielded a significant increase in state 4+ rate of ROS production
between 15 and 20°C (P=0.032) and between 20 and 25°C
(P<0.01). Overall ROS formation rates were between
0.16±0.09 nmol min-1 mg-1 (N=6) at
5°C and 0.76±0.24 nmol min-1 mg-1
(N=7) at 25°C in state 3, and between 0.13±0.07 nmol
min-1 mg-1 (N=6) at 5°C and
0.62±0.17 nmol min-1 mg-1 (N=7) at
25°C in state 4+. Analysis of least sum of squares yielded an ABT between
15°C and 20°C for states 3 and 4+. Below this ABT, the
temperature-dependent increase in the rate of mitochondrial ROS formation was
less pronounced and displayed a Q10 value of 1.8 in state 3 and 1.9
in state 4+. Above the ABT, the exponential rise resulted in a
Q10=3.1 for both states. Arrhenius activation energy
(Ea) between 5°C and 15°C was 38.9 kJ
mol-1 in state 3 and 43.4 kJ mol-1 for state 4+ ROS
production rate. Above the break temperature, Ea was
approximately 80 kJ mol-1 in both states.
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Rates of mitochondrial respiration and ROS production increased with temperature. Both variables, moreover, correlated significantly with one another in state 3 (Spearman correlation for non-normalized data: r-Spearman=0.74; P<0.01; N=31), and in state 4+ (r-Spearman=0.77; P<0.01; N=31).
The percentage of total oxygen converted to hydrogen peroxide as the major ROS during respiration was calculated on the basis of catalase quenching of DHR oxidation in the mitochondrial assays (Fig. 5). The proportion of hydrogen peroxide formation was significantly higher in state 4+ than in state 3 (P<0.01). The lowest percentage H2O2 production from total oxygen consumption was measured at 15°C and was 2.7±0.98% (N=5) in state 3 and to 5.12±1.76% (N=5) in state 4+. Higher percentage values were found both at higher and at lower temperatures. Maximal conversion of oxygen to H2O2 was found under state 4 conditions at 25°C, at which approximately 7% of the oxygen consumed formed hydrogen peroxide. The dependency of H2O2 production on temperature was not, however, statistically significant (P=0.16 for state 3 and P=0.72 for state 4+ respiration, Fig. 5). H2O2 production in state 4+ correlated inversely with mitochondrial coupling (RCR+: y=-0.51x+0.6237, r=0.26, P<0.01), indicating that the greater the coupling between respiration and phosphorylation, the lower the production and release of hydrogen peroxide from the mitochondria after inhibition of the ATPase by oligomycin.
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Experimental warming of live animals: effects on antioxidant enzyme
activities
Adult individuals of Mya arenaria with a shell length of 4.5-6 cm
were exposed to warming under experimental conditions
(Fig. 6). Stepwise warming to
18°C, which is the maximal temperature recorded naturally in the vicinity
of the animals, with a subsequent plateau of 2 days at 18°C (t-1,
Fig. 6A), led to a significant
increase in MDA concentrations in mantle tissue compared with controls (c-1)
(P=0.03; N=4 controls, N=7 experimental animals),
whereas no effect was found with respect to catalase or SOD activities in
mantle and gill tissues when all samples were assayed at 20°C
(Fig. 6B,C). When the control
group samples were assayed at 10 °C and compared with the 18 °C
temperature group (assayed at 20 °C), SOD and catalase activities were
significantly higher in the warmed than in the control group (SOD,
P=0.02, N=4 controls, N=6 experimental animals;
catalase, P<0.001, N=6 controls, N=7
experimental animals, Fig.
6D,E).
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Stepwise slow acclimation to 18 °C followed by a 1 week plateau at 25 °C in a second group had no significant effect on antioxidant enzyme activities when assayed at 20 °C (Fig. 6B,C: c-2 and t-2). When assayed at the respective maintenance temperatures (incub-T), only catalase displayed significantly higher enzyme activities as a result of slow warming (Fig. 6D, P<0.01, N=8 controls, N=9 experimental animals), while SOD was unaffected (Fig. 6E, P=0.15, N=7 controls, N=9 experimental animals). MDA concentrations in the mantle tissue increased upon slow warming, but the difference between heated and control animals was not significant (Fig. 6A, P=0.72, N=7 controls, N=8 experimental animals). The third group was transferred directly from 10 to 25 °C and kept at that temperature for 2 days, representing short-term exposure to above habitat temperatures. This treatment resulted in a highly significant increase in catalase activity in the mantle tissue (Fig. 6B, c-3 and t-3, P<0.01, N=6 controls, N=9 experimental animals) and to a non-significant increase in SOD activity in gill tissues (Fig. 6C, P=0.099, N=8 controls, N=10 experimental animals), when assayed at 20 °C. When the enzymes were assayed at the respective exposure temperatures, both antioxidant enzyme activities increased significantly with respect to controls (catalase, P=0.003; SOD, P=0.014, N=6-9, Fig. 6D,E). MDA tissue concentrations did not differ between the control and heated groups (Fig. 6A, N=8 controls, N=9 experimental animals).
Enzyme activity versus temperature in vitro
In vitro measurements of the temperature-dependency of catalase
and SOD activities were performed in the tissue extracts used in the
temperature-exposure experiment (Fig.
7). The temperature/activity curve for catalase in M.
arenaria mantle tissue is depicted in
Fig. 7A. No clearcut
temperature-dependency of in vitro catalase activity was found.
Although tissues of early summer animals, collected in June 2001 from the
Wadden Sea were used, highest activities were found at 5 and 10 °C.
However, the variability among samples was so high that no significant
temperature maximum could be identified. In contrast, SOD activity in gill
tissues was significantly dependent on temperature, with maximum in
vitro activity at 18 and 22 °C compared with all other temperatures
(Fig. 7B).
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Cell size and mitochondrial density and clustering
The mean cell volume of mantle cells was 300 µm3, whereas
gill cells had a volume of only approximately 200 µm3. Mean
mitochondrial density was 39±18 per mantle cell (N=49 cells)
and 34±15 per gill cell (N=46 cells). Mitochondria were
clustered in the periphery of the cell close to the cellular membrane and
around the nucleus (Fig.
8).
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Discussion |
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Like the proton leak, ROS formation in the mitochondrial respiratory chain
will contribute to overall rate of mitochondrial oxygen consumption in state
3, depending on proton-motive force, and especially under state 4 conditions,
when µH+ is high. In contrast to the leak, mitochondrial
ROS production is, to some extent, stimulated by the rate of energy turnover
of the cell, as it depends on electron flux through the critical respiratory
complexes I and III in all functional states. During the transition from state
3 to state 4 respiration, electron flux and also ROS formation decrease. These
decreases lead, however, to a higher proton-motive force and, in consequence,
a higher reduction state of the ubisemiquinol (CoQH) pool,
which will favour the transfer of electrons to O2
(Skulachev, 1998
). High
µH+ inhibits the Q cycle and thereby prolongs the
lifetime of the semiquinole (CoQH). Autoxidation of
CoQH initiates chain reactions producing superoxide anions,
which are converted to H2O2 by SOD and then to the
hazardous OH, thus leading to a relative increase in ROS
production under state 4 conditions. This becomes evident when the percentage
of ROS formation of total
O2 is
considered, which increases upon state 3 to state 4 transition. In M.
arenaria mitochondria, ROS production was 2-4 % in state 3 and increased
to 4-5 % in state 4 at habitat temperatures.
Uncouplers have been found to abolish state 4 ROS formation in mammalian
heart mitochondria (Korshunov et al.,
1997), proving the general dependence of mitochondrial ROS
formation on proton-motive force. According to Skulachev
(1998
),
µH+ has a stronger influence than the electron transport
rate on ROS formation rates in the respiratory chain. Nonetheless, the rate of
state 3 respiration in M. arenaria mitochondria correlated
significantly with the rate of ROS formation, indicating a major dependency of
ROS formation on overall mitochondrial oxygen consumption.
ROS formation and proton leak in mitochondria from endotherms and
ectotherms
Initial studies of ROS production by vertebrate heart mitochondria in
vitro report relatively high rates of 2-3.5 nmol
H2O2 min-1 mg-1 mitochondrial
protein under non-phosphorylating (state 4) conditions
(Boveris and Cadenas, 1975).
Other studies report lower values of 0.01-0.15 nmol H2O2
min-1 mg-1 mitochondrial protein in liver mitochondria
of various mammalian species during state 4 respiration
(Sohal et al., 1990
), whereas
state 3 production rates of 0.3-0.6 nmol H2O2
min-1 mg-1 mitochondrial protein were measured in rat
liver mitochondria (Boveris and Chance,
1973
) and 0.5 nmol H2O2 min-1
mg-1 mitochondrial protein in rat heart mitochondria
(Hansford et al., 1997
). A
single study of H2O2 production rates in invertebrate
mitochondria reports values of 0.8-2 nmol min-1 mg-1
mitochondrial protein under state 4 in vitro conditions in the house
fly, Musca domestica (Sohal,
1991
).
The rate of ROS production in M. arenaria at a habitat temperature
of 15°C (state 3, 0.3 nmol H2O2 min-1
mg-1 protein; state 4+, 0.23 nmol H2O2
min-1 mg-1 protein) was within the range reported for
mammalian mitochondria at assay temperatures above 20°C and up to 37°C
(Boveris et al., 1972;
Nohl and Hegner, 1978
;
Hansford et al., 1997
).
However, even when only the fraction of catalase-sensitive DHR oxidation is
considered, the proportion of total oxygen uptake converted to
H2O2 in Mya arenaria mitochondria under state 4
conditions appears to be substantially higher than in mammalian mitochondria.
As total respiration in the bivalve mitochondria amounts to only about 10 % of
mammalian mitochondrial respiration (e.g. from bovine heart; see
Tschischka et al., 2000
), the
percentage conversion of oxygen to ROS must be comparatively higher. Thus, a
percentage fraction of hydrogen peroxide production of 2-3 %, generally valid
for mammalian mitochondria at 25°C, was found only within the habitat
temperature range under state 3 conditions
(Fig. 5). Under heat stress
conditions, ROS production, calculated from the oxidation of DHR, amounted to
5 % of the mitochondrial oxygen turnover under state 3 and up to 7 % under
state 4 conditions. Thus, the proportion of conversion of oxygen to ROS is
indeed enhanced in the mitochondria of this marine ectothermal species under
temperature stress.
Another 10 % of ROS formation quenchable by DMSO and presumably representing OH radicals, was omitted in this approximation to render our data comparable with the literature reports on H2O2 formation. Moreover, the identification of ROS using chemical quenchers is a very crude measure, and more sophisticated analyses are necessary to obtain a clearer picture. Possibly, OH radicals originate from transition-metal-catalysed decomposition of H2O2 in Fenton-like reactions; however, our current knowledge does not permit us to draw a sufficiently detailed picture of the dynamics of ROS formation in M. arenaria mitochondria.
According to Brand et al.
(1994), the proportion of
oxygen consumption used to drive the proton leak (state 4 respiration) is
33±6.8 % of overall mitochondrial respiration in resting rat
hepatocytes. In isolated Mya arenaria mitochondria, state 4
respiration is 36.8±2.5 % of overall mitochondrial respiration, which
represents 30 % of whole-cell respiration at habitat temperatures of
5-15°C (calculated on a theoretical basis of 20 % surplus cellular
non-mitochondrial oxygen consumption; see
Brand et al., 1994
). These
estimates are valid for similar levels of proton-motive force between state 3
and state 4 respiration. They clearly show that, although the nominal values
of state 4 oxygen consumption were five- to 20-fold lower in ectotherms, the
percentage contribution to
O2, despite the
high interspecific varability (<10 % in Sipunculus nudus;
Buchner et al., 2001
; up to 50
% of state 3 respiration in Laternula elliptica,
Pörtner et al., 1999a
),
is comparable to what is found in endotherms
(Brookes et al., 1998
).
Mitochondrial functioning and ROS production in Mya arenaria under
heat stress
As a eurythermal temperate species, Mya arenaria exhibits a rather
broad window of thermal tolerance
(Anderson, 1978). Anderson
found a strong temperature-dependence of whole-animal metabolic rate between 5
and 15°C, while at higher temperatures respiration rates were constant or
even decreased. A Q10 higher than 3 for mitochondrial state 3
respiration between 10 and 15° C below the ABT and the maintenance of RCR
and ADP/O ratio within this temperature range, document metabolic maintenance
and flexibility within the thermal tolerance window.
Reduced mitochondrial coupling and phosphorylation rates (ADP/O), together with a constant rate of state 3 oxygen consumption and a progressive increase in the rate of non-phosphorylating state 4 respiration above the ABT, are indicative of increased proton leakage across the inner mitochondrial membrane because fewer protons are shunted back via the ATPase. Provided that the proton-motive force is similar for state 3 and state 4, the percentage of state 4+ respiration, i.e. proton conductance and ROS formation, of overall state 3 oxygen consumption increases with temperature from 30.8 % (10°C) to 38 % (15°C) and 47 % (25°C). The significant increase in mitochondrial release of partially reduced oxygen (ROS) indicates increased exposure of the cell to hazardous respiratory byproducts under high temperature stress. Thus, mitochondrial ATP production is stagnant above the ABT, whereas futile proton cycling and ROS formation increase, causing progressive energetic inefficiency and, possibly, oxidative stress at high temperatures (Fig. 3).
Quantitatively, ROS production accounts for only 0.4-0.8 nmol oxygen mg-1 protein min-1 at 20 and 25°C and, thus, for no more than 10 % of the non-phosphorylating respiration (state 4). Still, these toxic oxygen compounds (mainly H2O2) are obviously not detoxified inside the mitochondria, but are released into the cytosol and may have harmful effects on mitochondrial membranes and other cellular components. Mitochondrial distribution in the mantle cells was clearly peripheral, indicating that the cellular membranes and any type of integral protein might also be damaged.
The whole animal responded to temperature-induced ROS formation by increasing the activity of antioxidant enzymes (AOX) only when specimens were exposed to heat stress by rapid warming above habitat temperature. This is a further indication that these cells are unable to suppress ROS formation during heating and that adaptational processes including AOX synthesis are then required to control the damaging effects of the radicals produced. Increased AOX activity was not found under mild experimental warming to 20°C, however, indicating that the antioxidant potential still suffices to prevent ROS damage at high, but close to habitat, temperatures. Indeed, the SOD proteins in the gills (and also catalase in this tissue; data not shown) exhibit maximum catalytic activity between 18 and 22°C. Thus, the animals obviously have no need to invest into AOX synthesis upon mild warming.
Slow warming and acclimation to 25°C over 7 days also failed to induce
elevated tissue AOX levels at the end of the treatment. From the existing
data, it is not clear whether a transient induction of AOX has occurred, or if
there is any tissue damage. MDA levels in the tissues were only
insignificantly increased after the long-term heat exposure, but this might be
because MDA is a transient marker of lipid peroxidation, which is incorporated
into ageing pigments and also possibly released into the incubation water
(McAnulty and Waller, 1999).
Mortality was not significantly higher in these than in the other
temperature-exposed groups, but no further damage markers (lipofuscin,
ROS-induced DNA damage or energetic parameters) were assayed to obtain a more
comprehensive picture of the physiological state of the animals. The
respiration experiments of Anderson
(1978
) indicate the beginning
of respiratory impairment in cold-acclimated low and mid-intertidal specimens
during warming to 25°C. All mechanisms associated with hypoxia or anoxia
adaptation may then come into play, including energy-saving strategies
resulting in a decrease in protein synthesis. Such a strategy might explain
why AOX enzymes were not found to increase during long-term heat exposure in
this species.
Perspectives: the role of intracellular
PO2
These considerations suggest that PO2 in the
body and cellular fluids plays an important role in the oxidative stress
experienced by an organism at extreme temperatures. For practical reasons,
however, this investigation, like many others, studied mitochondrial
respiration and ROS formation at PO2 values
that are high compared with the much lower intracellular in vivo
PO2 (see
Pörtner and Grieshaber,
1993).
In the intact animal, intracellular oxygen concentration, in itself,
represents a major hazard to aerobic cells and has to be kept under tight
control to prevent the formation of reactive oxygen species. The futile
cycling of protons causes mitochondria to be a permanent oxygen sink and,
thereby, contributes to low intracellular oxygen tensions. The proton leak
also limits an increase in µH+ and, thereby,
mitochondrial ROS formation (Skulachev,
1996
).
Compared with animals living in environments with constant oxygen levels,
tight control of cellular oxygen tension is even more difficult in intertidal
animals, such as Mya arenaria, that are exposed to large fluctuations
of ambient oxygen levels. Usually, animals respond to fluctuating oxygen
supply by ventilatory and circulatory control. In oxyconformers,
PO2-dependent respiration of mitochondria also
plays a role (Tschischka et al.,
2000; Buchner et al.,
2001
). In the bivalves studied, ventilatory control is maintained
by the inhalation of oxygenated sea water through the siphon. Oxygen uptake
occurs by diffusion over the small gills and the large surface area of the
mantle that lines the valves (Brusca and
Brusca, 1990
). Diffusive transport through the tissue itself
precludes inhomogeneity of the oxygen supply to the cells in the different
tissue layers (Jones, 1986
).
The relative simplicity of this system is obvious and is further supported by
the absence of any oxygen-binding protein from the haemolymph and tissues.
Moreover, infaunal bivalves such as Mya arenaria, especially, live in
low-oxygen environments and are ecologically adapted to endure very low
environmental PO2 and to survive extended
periods of days and weeks of severe hypoxia
(Theede, 1973
). All of these
considerations suggest that mitochondria of Mya arenaria may
experience a wide range in intracellular oxygen levels and, thus, indicate
that further measurements of ROS formation and oxidative stress should be
carried out at these values of intracellular
PO2 in isolated mitochondria as well as in
intact cells. This will allow further evaluation of the dependence of ROS
formation on mitochondrial respiration in marine invertebrates in
vivo.
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