Effect of ambient oxygen concentration on activities of enzymatic
antioxidant defences and aerobic metabolism in the hydrothermal
vent worm, Paralvinella grasslei
Benjamin MARIE 1, Bertrand GENARD 2, Jean-François REES 2,§
Franck ZAL 1,*, §
1
Equipe Ecophysiologie : Adaptation et Evolution Moléculaires, UPMC – CNRS UMR 7144,
Station Biologique, BP 74, 29682 Roscoff cedex, France
2
Institut des Sciences de la Vie, Université Catholique de Louvain, Animal Biology Unit,
Croix du Sud, 5 B-1348 Louvain-la-Neuve, Belgium
* Please address reply to:
Dr. Franck Zal
Equipe EAEM
Station Biologique de Roscoff
BP 74, 29682 Roscoff cedex
France
Phone : +33 (0)2 98 29 23 09
Fax : +33 (0)2 98 29 23 24
Email: [email protected]
§
These authors equally contributed to this work.
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Abstract
The alvinellid Paralvinella grasslei is a common endemic polychaete from the deep-sea
hydrothermal vent communities located on the East Pacific Rise (EPR). These organisms
colonise a large range of microhabitats around active sites where physico-chemical conditions
are thought to generate reactive oxygen species (ROS). Furthermore, in this aerobic organism,
ROS could also be generated by the activity of the mitochondrial respiratory chain. In this
paper, we investigated the effect of ambient oxygen concentration on the activities of three
essential antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; glutathione
peroxidase, GPX) and their relationships with the activity of enzymes involved in aerobic
metabolism (cytochrome c oxidase, COX; citrate synthase, CS). Results of incubation of P.
grasslei in a high-pressure vessel with circulating seawater at different oxygen partial
pressures indicate that this worm regulates COX and CS activities differently in gills and
body wall. CAT and GPX activities increase in these tissues when animals are maintained in
filtered surface seawater. Moreover, levels of malondialdehyde increase in gills, testifying
that oxidative damage occurs under these conditions. CAT and GPX activities are positively
related to COX and CS activities, but no correlation was detected between SOD and the
metabolic enzyme activities. In comparison with littoral annelids, SOD activities are very
high whereas CAT activities are very low or absent in P. grasslei. The possible reasons for
the occurrence of such differences are discussed.
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Introduction
Fauna inhabiting deep-sea hydrothermal vents are highly endemic and have developed
specific adaptations to cope with atypical and extreme physico-chemical conditions (Childress
and Fisher 1992). In addition to the elevated hydrostatic pressure and the complete absence of
sunlight shared by all deep-sea areas, this environment is characterised by the resurgence of
toxic hydrothermal fluids (Von Damm 1990). The species restricted to deep-sea hydrothermal
vents live in the mixing zone between the hydrothermal fluid and the deep-sea water, and are
distributed according to their physico-chemical tolerance to the fluid (Desbruyères et al. 1982;
Tunnicliffe 1991). Blum and Fridovich (1984) suggested that several toxic compounds of the
fluid, in contact with the oxygen of deep-sea water, could produce reactive oxygen species
(ROS) such as superoxide anions (O2•−), hydrogen peroxide (H2O2) and hydroxyl radical
(OH•), a highly toxic component for organisms. ROS can oxidise most cellular constituents
such as proteins, lipids or DNA and markedly disturb vital cellular functions (Halliwell and
Gutteridge 1989).
To date, no data are known on the types and amounts of ROS around hydrothermal vents,
but there are many reasons to suspect the presence of ROS in this ecosystem (Blum and
Fridovich 1984; Dixon et al. 2002). Several abiotic parameters are indeed favourable to ROS
formation. The occurrence of high concentrations of metals, such as iron (II), reacting
spontaneously with H2O2 produces highly toxic OH• by Fenton’s reactions (Halliwell and
Gutteridge 1986; Fridovich 1998; Stohs and Bagchi 1995). Radionuclides, such as radon that
occur at high level in the hydrothermal habitats, could inflict oxidative damage (De Oliveira
et al. 2001). Spontaneous oxidation of sulphide in seawater produce ROS (Tapley et al. 1999)
and sulphide was also been detected in organism’s tissues (Edmond et al. 1982; CossonMannevy et al. 1988; Cherry et al. 1992; Cosson and Vivier 1997). High temperatures can as
well favour the formation of O2•- (Issels et al. 1986).
-3-
As in other aerobic organisms, another potential source of ROS is endogenously with the
activity of the mitochondrial respiratory chain which could continuously generate ROS at the
rate of 1-2% of the total oxygen consumed by animal tissues in resting conditions (Cadenas
and Davies 2000), even if this phenomenon is today highly debated (Staniek and Nohl 2000;
Keller et al. 2004). To cope with both endogenous and exogenous productions of ROS,
organisms possess a complex arsenal of antioxidative defence mechanisms such as vitamins,
glutathione, metallothioneins or specific enzymes. The most important of these enzymes are
superoxide dismutase (SOD) that converts O2•- into less toxic H2O2 via the reaction 2O2•- +
2H+→ H2O2 + O2, catalase (CAT) which decomposes H2O2 into H2O (2H2O2→ 2H2O + O2),
and glutathione peroxidase (GPX) which uses glutathione (GSH) as cofactor for reducing
peroxides according to 2GSH + ROOH→ GSSG + ROH + H2O. In many cases, the levels of
antioxidant enzymes are known to be adjusted to the threat posed by respiration-derived ROS.
In mammals, increasing the maximal aerobic capacity by exercise leads to increased levels of
various antioxidant enzymes (Vincent et al. 2000). In deep marine fishes SOD and GPX
activities are apparently adjusted to maximum aerobic metabolism capacity (Janssens et al.
2000), as measured by cytochrome c oxidase (COX) and citrate synthase (CS) activities
(Childress and Somero 1979; Thuesen and Childress 1993; Thuesen and Childress 1994).
The alvinellid Paralvinella grasslei Desbruyères and Laubier 1982 is a common endemic
polychaete from hydrothermal vents of the East Pacific Rise (Desbruyères and Laubier 1991;
Zal et al. 1995). Unlike other alvinellids, P. grasslei does not secrete a tube but is covered by
an abundant mucus. Alvinellids are highly aerobic organisms (Hand and Somero 1983;
Hourdez et al. 2000), although adapted for life in hypoxia through optimised oxygen
extraction in gills (Jouin and Gaill 1990) and a high oxygen carrying capacity (Toulmond et
al. 1990) . Indeed, this worm colonises environments, like black smoker chimneys where it is
exposed to low, highly fluctuating, oxygen concentrations (Johnson et al. 1988; Juniper and
-4-
Martineu 1995). Intertidal polychaetes, which also live in a fluctuating oxygen environment,
are well known to adapt their oxygen consumption and metabolism according to the oxygen
availability (Toulmond and Tchernigovtzeff 1984). Those worms down-regulate their
ventilatory activity under hyperoxic conditions in order to maintain their metabolic rates and
to possibly limit the toxic effects of oxygen occurring during an over-oxygenation (Fox and
Taylor 1955). Also, fluctuations of oxygen levels are known to favour the formation of ROS
in animal’s tissues, a phenomenon leading to the so-called ischemia–reperfusion injuries. (e.g.
Warner et al. 2004; Buja, 2005). Therefore, one could expect that the low and unstable
oxygen availability at vents could influence the aerobic metabolic rate as well as the
requirement for antioxidant enzymes.
For all the above reasons, one would first expect Paralvinella grasslei to possess a welldeveloped and specific antioxidant defence system aimed to reduce the toxic ROS occurring
in the hydrothermal fluid, and secondly, that its rate of oxidative metabolism enzymes and
antioxidant system could be linked and have some ability to respond to variations in oxygen
availability. To test these hypotheses, we have analysed the activities of antioxidant enzymes
in P. grasslei and the long-term effect of ambient oxygen concentration on antioxidants and
some oxidative metabolism enzymes, and looked for signs of oxidative damages in worms
maintained in hyperbaric aquaria.
-5-
Materials and methods
Animal collection
Collection of Paralvinella grasslei was performed at Elsa (N 12 48.1420 – W 103
56.3060) and Parigo (N 12 48.5960 – W 103 56.4270) vent sites (13°N on the East Pacific
Rise, ≈2600 meters of depth) during the PHARE cruise (May 2002), using the manipulated
claw or the «slurp gun» of the ROV Victor 6000. Specimens were collected on “Alvinella”
habitat where usually the temperature is comprised between 10°C and 30°C on the surface of
the colony. Animals were brought to the surface in watertight and temperature-insulated
containers. On board of the research ship N/O Atalante, they were quickly transferred to a
cold room (10°C) and animals of around 5 cm of length were selected and cleaned of their
mucus. For each in vivo experiments (cf. below), ten worms were dissected on ice and tissues
(i.e. gills, gut and body wall) were frozen immediately in liquid nitrogen and stored at - 80°C,
to serve as reference of physiological condition of the worm just after the decompression.
Those “Freshly Caught” animals were analysed and results compared to the worms of the
reconditioning experiments. Seven other individuals were immediately placed into perforated
50-ml Falcon tubes for reconditioning experiments, which were started within 3h of animal
collection on hydrothermal sites.
In vivo exposure to different partial pressures of oxygen
Animals just recovered on board, were immediately reconditioned in a high-pressure
chamber pressurised at 260 bars corresponding to habitat pressure at 2600 meters depth. The
reconditioning process was carried out in the pressure vessel system Ipocamp, thermostated to
15°C (Shillito et al. 2001). Oxygen levels were regulated upstream of Ipocamp using a
chemical-parameter controller system (Syrene, described in Chausson et al. 2004). Animals
were placed inside the chamber containing filtered seawater, re-pressurised (in 2 min), where
the desired oxygen level was reached after 4-8 hours of circulation at a flow rate of
-6-
approximately 5 l.h-1, and then the worm was held at that level for 12 hours. This
experimentation time was chosen to allow physiological recovery of animals as recently
demonstrated for the closely related species Alvinella pompejana (Shillito et al. 2004). The
seawater pH was maintained between 6.2 and 6.5 (i.e. corresponding to the pH found around
the worms in situ) with a proportional pH controller (Consort R305, Inc.) connected to two
metering pumps injecting either 1M NaOH or 1M HCl. The experiments at in situ pressure
were performed at three different oxygen concentrations on three set of different worms
(mean ± SD): (1) “hypoxia” (12.8 ± 6 µmol O2 l-1 or 1 ± 0.5 kPa or 7.5 ± 3.5 Torr); (2)
“normoxia” (78 ± 3 µmol O2 l-1 or 6.3 ± 0.2 kPa or 50 ± 2 Torr); (3) “hyperoxia” (203 ± 9
µmol O2 l-1 or 16.2 ± 0.8 kPa or 122 ± 5 Torr). The terminology used for the oxygen
conditions was chosen according to oxygen concentrations found in the habitat of this worm
(reviewed in Juniper and Martineu 1995, “hyperoxia” corresponding to air-saturated
seawater). Oxygen concentrations were measured at the outlet of the pressurised chamber
with an oxymeter (WTW oxi) throughout the experiments.
Sample preparation
Tissues (i.e. gill, body wall and gut) were dissected on ice, frozen in liquid nitrogen and
kept at – 80°C until analysed (i.e. around 6 months after collection). Tissues were weighed
and homogenised (w/v = 1:5) in phosphate buffered saline (PBS), pH 7.4, containing 1%
Triton X-100. An aliquot of the homogenate was used for the determination of the protein
content (Lowry et al. 1951), and the remaining was centrifuged at 15,000 g (15 min, 4°C).
The supernatant was used for enzyme activity assays as well as for the analysis of the
malondialdehyde contents. Enzymatic activity measurements were performed at 25°C in order
to allow comparisons with previous investigations, and because hydrothermal species are
predicted to have maximal sustained body temperature between 20-30°C (Dahlhoff and
Somero 1991).
-7-
Catalase assay (EC 1.11.1.6)
In a first tentative experiment, we attempted measuring catalase activity (CAT) using the
classical spectrophotometrical method reported by Blum and Fridovich 1984, but no activity
was detected by this assay. Consequently, CAT was measured by chemiluminescence
according to the method of Maral et al. (1977) modified for 96-well microplates by Janssens
et al. (2000). The consumption of H2O2 by CAT was followed at 25°C on a PC-controlled
microplate luminometer (Berthold LB96P). Fifty µl of 1 µmol l-1 H2O2 was added to 50 µl of
sample diluted in 100 µl of phosphate buffer 100 mmol l-1, pH = 7.8, containing 0.6 mmol l-1
EDTA. After a 30-min incubation at 25°C, the injection of 50 µl of 20 mmol l-1 luminol and
11.6 units ml-1 horseradish peroxidase produces an emission of light. The light intensity was
assumed to be proportional to the remaining quantity of H2O2. CAT activity in samples was
quantified by constructing standard curves using purified bovine liver CAT dissolved in PBSTriton buffer and expressed in international units (µmol H2O2 consumed.min-1).
Superoxide dismutase assay (EC 1.15.1)
The spectrophotometric method of Flohé and Ötting (1985) was adapted for microplate
measurements. The assay is based on the competition between superoxide dismutase (SOD)
and oxidised cytochrome c for O2•- generated by the reaction of hypoxanthine with xanthine
oxidase (EC 1.1.3.22). Reduction rate of cytochrome c (2 µmol l-1) was measured at 550 nm
(25°C), in 180 µl of phosphate buffer (50 mmol l-1, pH = 7.8) with 0.5 mmol l-1 EDTA, 5
µmol l-1 hypoxanthine and 10 µl diluted sample. The reaction was initiated by injecting 10 µl
xanthine oxidase (0.2 U ml-1). SOD activity in samples was estimated with Cu,Zn-SOD
purified from bovine erythrocytes and expressed in international units (1 unit corresponds to
the SOD amount inhibiting by 50% the rate of cytochrome c reduction).
-8-
Glutathione peroxidase assay (EC 1.11.1.9)
Glutathione peroxidase (GPX) was measured spectrophotometrically as reduction rate of
NADPH at 340 nm (25°C), using the protocol of Paglia and Valentine (1967) modified for
96-well microplates. Fifteen µl of diluted sample was added to 120 µl Tris-HCl buffer (50
mmol l-1, pH = 7.6) containing 0.1 mmol l-1 EDTA, 0.14 mmol l-1 NADPH, 1mmol l-1
glutathione and 1 unit glutathione reductase (EC 1.6.4.2). The reaction was initiated by the
addition of 15 µl t-butyl hydroperoxide 0.2 mmol l-1. GPX activity was estimated using the
molar extinction coefficient of NADPH (6.220 mol-1.l.cm-1) and expressed in international
units (µmol NADPH consumed.min-1).
Aerobic metabolism estimates
Aerobic metabolic capacity was estimated from citrate synthase (EC 4.1.3.7) and
cytochrome c oxidase activities (EC 1.9.3.1), two mitochondrial enzymes well correlated to
the oxygen consumption in vertebrates and invertebrate tissues (Childress and Somero 1979;
Thuesen and Childress 1993; Thuesen and Childress 1994).
Citrate synthase activity was measured according to the protocol of Sheperd and Garland
(1969) modified for multiwell plates. Twenty µl of diluted sample were added to 160 µl of
Tris-HCl buffer (100 mmol l-1, pH = 8.0) containing 15 µmol l-1 of 5,5’-dithio-bis(2nitrobenzoic acid) (DTNB) and 25 µmol l-1 acetyl-CoA. Consumption of acetyl-CoA was
followed at 420 nm after adding 20 µl oxaloacetate 50 µmol l-1 (25°C). CS activity was
estimated using the molar extinction coefficient of DTNB (13.600 mol.l-1.cm-1) and expressed
in international units (µmol DTNB consumed.min-1).
The spectrophometric assay method for cytochrome c oxidase (COX) of Hand and Somero
(1983) was adapted for microplates. The oxidation of 0.1 mol l-1 cytochrome c solubilized in
180 µl of Tris-HCl buffer (50 mmol l-1, pH = 7.6) was followed at 550 nm (25°C) after
addition of 20 µl of diluted sample. COX activity was estimated using the molar extinction
-9-
coefficient of cytochrome c (19.600 mol-1.l.cm-1) and expressed in international units (µmol
cytochrome c consumed.min-1).
Determination of malondialdehyde (MDA)
The occurrence of oxidative damage in tissues was investigated by assaying
malondialdehyde (MDA) that forms upon ROS attacks on unsaturated lipids (Storey 1996).
The concentration of MDA was estimated in gills and body wall of P. grasslei according to
Yagi’s method (1984) adapted for microplates. Thirty µl of sample were added to 48 µl of
thiobarbituric acid (0.67 %) in 0.3 mol l-1 NaOH and 24 µl of trichloroacetic acid at 15 %.
After 30-min incubation of microplates with a Teflon lid at 95°C and cooling to room
temperature, 100 µl butanol was added. Microplates were centrifuged for 5 min (800g, 4°C)
and the fluorescence was measured at 555 nm (excitation 515nm) with a fluorimeter Ascent
(Labsystems). MDA concentration was estimated from a standard curve made with
commercial MDA.
Reagents
All reagents were purchased from Sigma (St Louis, USA), except for Triton X-100, MDA,
GSH and glutathione reductase obtained from Boehringer (Mannheim, Germany).
Statistical analyses
The enzymatic activities and the concentrations were measured in triplicate and are
presented as means of a sample size of 7. Means are related to protein content and wet mass
of the tissue. Although the expression of enzyme activities as a function of the wet mass
seems to be more appropriate for comparing physiological capacities of the tissues, activities
expressed relatively to the protein content are often used in works published on different
species.
Mean comparisons were carried out using Statview 4.5 software. We used a nonparametric Kruskal-Wallis U-test and p < 0.05 was accepted to indicate a significant
- 10 -
difference. To evaluate correlation between enzyme activities in each tissue, linear regressions
were carried out on all experimental groups taken separately. Correlation tests were also
carried out for each tissue on all data combined in order to consider co-variation of enzyme
activities during experiments. Untransformed data were used to describe the relation between
COX and CS activities or on log-transformed data for antioxidant and metabolic enzyme
relationship, see Janssens et al. 2000. Furthermore, simple linear regression tests were carried
out using SAS software, considering the group effect as an external variable (i.e. “Freshly
Caught” (FC), hypoxia, normoxia, hyperoxia). Correlation was declared significant when P
value was less to 5%.
- 11 -
Results
Antioxidant and mitochondrial enzymes in P. grasslei
Activities of all enzymes (U g-1 wet weight) in tissues from “Freshly Caught” animals
(named “FC” in the following text) are shown in Table 1. CAT, SOD, GPX, COX and CS
activities were detected in gills, gut and body wall of P. grasslei.
The highest antioxidant activities for all enzymes were measured in the gut and in the body
wall, although some of these activities are not significantly different in comparison with the
gills (Table 1). The gills show the highest CS level, almost twice the value found for the gut
or the body wall. COX activity was similar in the gut and the gills, and was lower in body
wall. Surprisingly, CS/COX ratios were high: approximately 5 in gills, 3 in the body wall and
2 in the gut, for comparison purpose this ratio is comprise between 0.1 and 0.4 in fish tissues
(Lucassen et al. 2003).
Effects of conditioning at various oxygen concentrations on aerobic metabolism activities
Worms brought to the surface and reconditioned in pressure chambers were quite active
and exhibited parapodia movements after the experiments. The conditioning of P. grasslei at
different oxygen concentrations had important effects on COX and CS activities in the gills
and the body wall, but not in the gut (Table 2). However responses were different in gills and
body wall tissues. Fig. 1 shows the effect of oxygen concentration on COX and CS activities
(U g-1 wet weight) in gills. Both COX (Fig 1A) and CS (Fig. 1B) activities were lower of FC
values (p < 0.01) after conditioning in hypoxia (12 ± 6 µmol O2 l-1). In normoxia (78 ± 3 µmol
O2 l-1), COX and CS activities increased (p < 0.01) in comparison with hypoxia values. In
hyperoxia (203 ± 9 µmol O2 l-1) conditions, COX and CS activities decreased (p < 0.001 and
p < 0.05, respectively) below FC values. Fig. 2 reports the influence of oxygen concentration
on COX and CS activities in the body wall (U g-1 wet weight). While conditioning under
hypoxia and normoxia had no significant effect on COX activity in comparison with FC,
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COX activity slightly increased in hyperoxia conditions when compared to normoxia (p <
0.05). Similarly, CS activity also increased under hyperoxia conditions.
Fig. 3 shows the linear correlation between COX and CS activities (U g-1 wet weight) in
gills and body wall tissues.
When the different groups were considered separately, no
significant correlation between both enzymes was observed in gills. However, a highly
significant relationship between CS and COX was found when data of all experiments were
pooled together, emphasising that COX and CS activities in gills are similarly affected by
exposure to different oxygen concentrations. In body wall, significant correlations were
observed in FC, hypoxia and hyperoxia with quite similar slope coefficients; the relationship
observed in bulk data also indicates a correlation between COX and CS activities in this
tissue. These results show that, despite of the different responses to oxygen concentration in
gills and body wall tissues, COX and CS activities were linearly and positively correlated in
both tissues (R = 0.67, p < 0.001 and R = 0.43, p < 0.05, respectively, for all clump data). The
slopes characterising their relationship were similar in both tissues (CS = 1.92 COX + 2.99, in
gills; CS = 2.00 COX + 1.50, in body wall).
Antioxidant activities related to the oxygen concentration
Fig. 4 shows the effects of different oxygen concentrations on CAT and GPX activities in
gills and body wall during three acclimation experiments. In gills, a decrease of CAT activity
was observed (p < 0.01) after hypoxic conditioning (Fig. 4A) in comparison to FC group.
CAT activity was significantly higher in normoxia when compared to hypoxia (P < 0.01).
Differently, GPX activity in body wall tissues was affected by the oxygen concentration (Fig.
4B) as it increased more than twice after 12-h exposure to high oxygen (p < 0.001), while no
effect was observed at lower oxygen concentration. In another way, SOD activity seems not
to be affected by the different oxygen treatment and does not show any significant difference
in both tissues (data not shown).
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Relationships between antioxidant and aerobic metabolism activities
Fig 5A & B show the relationships between CAT and COX activities in gills and between
GPX and CS in body wall tissues (expressed on Log/Log axes), respectively. No significant
correlation was observed within the different groups taken separately . However, significant
relationships were found within experiments (bulk data, considering the effect of the group as
an external factor), underlining the link between CAT and COX activities in gills, and GPX
and CS activities in body wall. These relationships are best described as exponential increases
in antioxidant enzymes with increasing CS or COX activity (U g-1 wet weight) following the
equations CAT = 0.16 COX0.98 (p<0.001) and GPX = 2.84 CS0.27 (p < 0.01) (Fig. 5B).
Contrarily to what we observed for CAT and GPX, no relationship between SOD and COX
neither between SOD and CS (data not shown) could be found in any tissue whatever data
were expressed relatively to the protein content or the wet weight of the tissue, as revealed for
the gills (Fig 5C).
Induction of lipid peroxidation
Fig. 6 shows effects of ambient oxygen level in the aquarium on malondialdehyde levels in
gills and body wall tissues of P. grasslei. In FC specimens, MDA levels were higher in gills
(6.5 ± 0.8 nmol g-1 wet weight) than in body wall (3.8 ± 0.3 nmol g-1 wet weight). The
incubation of animals in hyperoxia increased (p < 0.01) the level of MDA in gills (12.6 ± 0.9
nmol g-1 wet weight), about two folds that measured in FC animals, testifying that some
oxidative damage may have occurred in this tissue. Quite remarkably, no variation of MDA
concentrations were observed in body wall tissues after any reconditioning experiments.
.
- 14 -
Discussion
Since free radicals are known to be produced continuously in all aerobic cells during the
electron transfer process in mitochondria, the positive correlation between GPX and CS in
body wall tissues (Fig. 5B) suggests that levels of GPX are adjusted to the peroxides
generated directly or indirectly by oxidative phosphorylation. Such correlations have been
demonstrated in other organisms, such as fish (e.g. Janssens et al., 1998; Morales et al., 2004)
and mammals (Leeuwenburgh et al., 1997). Furthermore, it seems that COX and CS are coregulated as their levels vary in parallel to each other in both gills and body wall tissues (Fig.
1, Fig. 2 and Fig. 3). This co-variation of those enzyme activities could suggest a regulation of
mitochondrial density, modifying the oxidative capacity of the tissues, as suggested for
Arenicola marina by Sommer and Pörtner 2002.
Blum and Fridovich (1984) reported the presence of SOD and GPX activities in the tubeworm Riftia pachyptila and the giant clam Calyptogena magnifica, but could not detect any
CAT activity in either species. Generally, CAT is a major antioxidant and in combination
with GPX detoxifies H2O2, formed by SOD and other exogenous sources. However, using a
more sensitive luminometric method operating at low H2O2 concentration (Maral et al. 1977
modified by Janssens et al. 2000), we were then able to detect very low levels CAT-like
activity in all tissues. CAT-like activity in body wall of P. grasslei is very low (0.21 ± 0.13 U
mg-1 protein) in comparison to Arenicola marina (8 ± 2 U mg-1 of protein) which possesses
the lowest CAT activity amongst non-hydrothermal vent annelids (Table 3). Such low
catalase levels suggest that this enzyme could be absent in P. grasslei tissues and that the
occurrence of this “catalase-like” activity may reflect the occurrence of H2O2-scavengers such
as antioxidant osmolytes or other enzymes. Interestingly, Yin et al. (2000) found high
concentration of potential antioxidant osmolytes like hypotaurine in hydrothermal vent
species. As another explanation, the consumption of H2O2 could be related to Fenton reactions
- 15 -
catalysed by metals likely to be present in tissue homogenates. In contrast, a high SOD
activity was measured: to date, P. grasslei possesses the highest SOD activity (30 ± 15 U mg-1
of protein in body wall) in comparison to hydrothermal vent (R. pachyptila 9.3 U mg-1 of
protein in muscle) or non-hydrothermal vent annelids (Heteromastus filiformis 17 ± 6 U mg-1
of protein in body wall). Thus, P. grasslei possesses a low CAT-like activity and a high SOD
activity in comparison to other annelids. Since Dixon et al. (2002) have shown that the
sensitivity of P. grasslei to exogenous H2O2 was similar to that recorded for shallow-water
polychaetes and that GPX levels are similar to those found in other annelids, it is possible that
the detoxification of SOD-derived H2O2 can be taken over other peroxidases. Another
interesting finding is that H2O2 could also be detoxified by chemical reaction with H2S
(Millero et al. 1989). Consequently, it would be judicious in future studies to measure H2O2
directly in the environment and inside the organisms and to look for other peroxydases in P.
grasslei.
Quite surprisingly, no correlation between SOD activity and oxidative metabolism
enzymes was detected. This absence of relationship seems to indicate that the high SOD
activity could be mainly aimed at superoxide anions generated through respirationindependent processes. The high metal content in tissues of P. grasslei (e.g. Cd, Fe, Hg and
As ; Cosson and Vivier 1997; Desbruyères et al. 1998), could be an important source of
superoxide anions. Highest SOD activities were associated with gut tissues in comparison to
body wall and gills tissues (Table 3). This gut tissue comprises the digestive tract with its
content, and the chloragog tissue. Buchner et al. (1996) have found SOD activity four-fold
higher in the chloragog than in body wall tissues of the intertidal polychaete A. marina. These
authors have proposed that chloragog was confronted to a large quantity of free radicals
resulting from hemoglobin autoxidation. In addition, comparing SOD levels in marine
invertebrates, Abele-Oeschger (1996), have observed that high SOD activities are often linked
- 16 -
to the presence of hemoglobin and have suggested that these respiratory pigments have a
tendency to form superoxide anions via autoxidation (Misra and Fridovich 1972; AbeleOeschger and Oeschger 1995, Hermes-Lima et al. 1998). It is important to note that P.
grasslei possesses a large amount of intra and extracellular hemoglobin (Toulmond et al.,
1990, Zal et al., 2000). Another interesting hypothesis suggested by Abele-Oeschger (1996) is
that SOD activity in benthic invertebrates could be related to in situ H2S concentrations like
those found in the environment of P. grasslei.
MDA concentration in P. grasslei was in range of those observed in other invertebrates
(Hermes-Lima et al. 1998), suggesting that no extensive oxidative damage to lipids occur in
FC specimens. However, MDA levels in gills seem to indicate that the antioxidant arsenal
could be insufficient in this issue but not in body wall high oxygen conditions. Indeed,
accumulation of MDA occurred in gills but not in body wall tissues when P. grasslei is
exposed to hyperoxic conditions (Fig.6). The higher susceptibility of gills might have to do
with the fact that these are directly exposed to ambient oxygen and act as a diffusion barrier
for oxygen into other tissues, which could be exposed to lower O2 levels. Another
consequence of this result is that it demonstrates the occurrence of oxidative stress in P.
grasslei submitted to air-saturated surface seawater. This observation is not highly surprising
since ecological data indicate that deep-sea hydrothermal vent organisms living on chimney
emitting anoxic fluids usually breath in poorly oxygenated (5 µmol l-1) seawater (Di MeoSavoie et al. 2004), and experience maximum oxygen concentrations of 120 µmol l-1 (Johnson
et al. 1988). This seems to confirm work by Dixon and co-workers (2002) who observed high
levels of DNA damage in P. grasslei conditioned 12 hours at 260 bar in air-saturated surface
seawater. Thus this further indicates that low oxygen concentration is necessary for keeping
these animals in physiological conditions.
- 17 -
Similarly, the intertidal polychaete Heteromastus filiformis, which was shown to never
encounter more than 150 µmol O2 l-1 in its natural biotope, is known to waste away under
well-aerated conditions (Abele et al. 1998). As a possible evidence that surface oxygen
concentrations could be toxic to hydrothermal organisms, the alvinellid Alvinella pompejana
that colonises an even more anoxic part of the hydrothermal ecosystem than Paralvinella
grasslei (Jollivet et al. 1995; Juniper and Martineu, 1995) had never been kept alive even for a
short period in hyperbaric aquaria circulated with air-saturated seawater. Only recently, some
A. pompejana were maintained in a pressure vessel (Ipocamp) alive and this coincided with
the reduction of oxygen pressure down to levels comparable to the hypoxic conditions of the
present study (Shillito et al. 2004). It is thus possible that the specific adaptations allowing
this animal to maintain normal aerobic metabolism at very low oxygen concentration (i.e. low
P50-hemoglobin, high branchial surface and presence of an internal oxygen reservoir, Hourdez
et al. 2000) could lead to a higher susceptibility to oxygen toxicity.
In conclusion, our results indicate that P. grasslei can regulate its enzymatic aerobic
metabolism capacity and antioxidant enzymes according to the ambient oxygen concentration.
During our experiments, when the oxygen concentration exceeded levels normally persisting
in the environment of the worms, GPX activity increased in body wall concomitantly to an
increase of aerobic enzyme activities, while CAT-like activity did not change compared to in
situ, on the other hand, aerobic enzyme activities declined in the gills. These modifications
were accompanied by signs of lipid peroxidation in gills, indicating that the upregulations of
GPX and CAT-like activities could not prevent oxidative stress to occur. The high SOD
activity in P. grasslei tissues and the absence of correlation between the level of this enzyme
and the aerobic metabolism indicators suggest that this enzyme could target high superoxide
anions levels related to non-respiratory mechanisms. On the other hand, the very low CAT-
- 18 -
like and the average GPX activities are suggestive of the presence of other mechanisms
dealing with H2O2 detoxification.
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Acknowledgements :
The authors would like to thank the captain and the crews of the N/O L'Atalante and ROV
Victor 6000. We are also very grateful to Nadine Le Bris and Françoise Gaill, the chief
scientists of the French research cruise PHARE’02. We thank Dominique Davoult for his
advice on statistical treatments. We would also like to thank Bruce Shillito for the
experiments realized in IPOCAMP and Cécile Marchand for assistance in the laboratory. We
thank the EAEM team from Roscoff for their advises and comments on this work. We are
also very grateful to the four anonymous referees for their useful comments and remarks,
which considerably improve the submitted form of this manuscript. These studies were
supported by the French Ministère des Affaires Etrangères under the Integrated Program
Action called Tournesol (N° 05365GT) and the Fonds de la Recherche Fondamentale
Collective (FRFC) convention 2.4595.06.
- 27 -
Figure legends
Figure 1 : Paralvinella grasslei. Aerobic metabolism activities (U g-1 wet weight) in gills
after incubations at various ambient oxygen concentrations. Animals were incubated at 260
bar, 12h in filtered sea-water maintained at 15°C once the oxygen conditions were stabilised,
in hypoxia (12 ± 6 µmol O2 l-1), normoxia (78 ± 3 µmol O2 l-1) and hyperoxia (203 ± 9 µmol
O2 l-1) (see materials and methods). A) COX activity in gills. B) CS activity in gills.

=
significantly different from the value at the inferior oxygen concentration point (P< 0.05,

P< 0.01,

P<0.001). Values are means ± SD (n = 7);
+
= significantly different from FC
value (+P< 0.05, ++P< 0.01, +++P<0.001). Kruskal-Wallis, non-parametric U-test.
Figure 2 : Paralvinella grasslei. Aerobic metabolism activities (U g-1 wet weight) in body
wall after incubations at various ambient oxygen concentrations like previously described. A)
COX activity in body wall. B) CS activity in body wall.

= significantly different from the
value at the inferior oxygen concentration point (P< 0.05). Values are means ± SD (n = 7); +
= significantly different from FC value (+P< 0.05,
++
P< 0.01,
+++
P<0.001). Kruskal-Wallis,
non-parametric U-test.
Figure 3 : Paralvinella grasslei. Linear relationships between COX (U g-1 wet weight) and
CS (U g-1 wet weight) in gills and body wall. In gills, no significant correlation was observed
within the different groups taken separately. The dotted line fits the equation of the covariation tendency of within experimentations (bulk data): CS activity= 1.92COX activity+
2.99, R = 0,67, P<0.001 (n = 26). In body wall, significant correlations were observed in FC,
hypoxia and hyperoxia (data not shown). Bulk data also shows similar relationship than in
gills (dotted line): CS activity= 2.00COX activity+1.50, R = 0.43, P<0.05 (n = 27).
- 28 -
Figure 4 : Paralvinella grasslei. Antioxidant activities (U g-1 wet weight) in gills (A) and
body wall (B) after incubations at various ambient oxygen concentrations. A) CAT activity.
B) GPX activity. Values are means ± SD (n = 7); = significantly different from the value at
the inferior oxygen concentration point (P< 0.01);
+
= significantly different from FC value
(+P< 0.05, ++P< 0.01, +++P<0.001). Kruskal-Wallis, non-parametric U-test.
Figure 5 : Paralvinella grasslei. Relationships between antioxidant activities (U g-1 wet
weight) and aerobic metabolism activities (U g-1 wet weight) in gills and body wall. A)
Exponential relationship between CAT and COX in gills. No significant correlation was
observed within the different groups taken separately. The dotted line fits the equation of the
co-variation tendency within experimentations (bulk data): COX activity=-0.80(CAT
activity)0.98, R = 0.65, P<0.001 (n = 26). B) Exponential relationship between GPX and CS in
body wall. No significant correlation into different group take separately was observed. The
dotted line fits the equation of the co-variation tendency of within experimentations (bulk
data): CS activity=0.44(GPX activity)0.29, R = 0.60, P<0.01 (n = 27). C) No significant
relationship was detected between SOD activity and aerobic metabolism activities, e.g. COX
activity both in gills and in body wall.
Figure 6 : Paralvinella grasslei. Concentration of malondialdehyde (nmol g-1 wet weight) in
gills and in body wall after incubations at various ambient oxygen concentrations. Animals
were incubated at 260 bar, 12h in filtered sea-water maintained at 15°C once the oxygen
conditions were stabilised, in hypoxia (12 ± 6 µmol O2 l-1), normoxia (78 ± 3 µmol O2l-1) or
hyperoxia (203 ± 9 µmol O2 l-1). Values are means ± SD (n = 3 individuals).  = significantly
- 29 -
different from the value at the inferior oxygen concentration point;
from FC value (P< 0.01) t-test.
- 30 -
+
= significantly different
CS activity (units
g-1 wet weight)
COX activity (units g-1
wet weight)
A
Fig. 1
+ 
++
B
++
+++ 

+
oxy
COX activity (units
g-1 wet weight)
1
A

0.5
0
FC
Hypoxia
Normoxia
B
++ 
4
CS activity (units g-1
wet weight)
Hyperoxia
++
+
3
2
1
0
FC
Hyperoxy
Fig. 2
Hypoxia
Normoxia
Hyperoxia
CS activity (units g-1
wet weight)
COX activity (units g-1 wet weight)
Fig. 3
GPX activity (units g-1 wet
weight)
CAT activity (units
g-1 wet weight)
A

++
B
+++ 
Fig. 4
10
A
COX activity (units
g-1 wet weight)
Gills
1
0.1
0.01
0.1
1
10
CAT activity (units g-1 wet weight)
10
B
CS activity (units g-1
wet weight)
Body wall
1
0.1
1
10
-1
GPX activity (units g wet weight)
COX activity (units g-1
wet weight)
10
Gills
Body wall
C
1
0.1
0.01
100
Fig.5
1000
SOD activity (units g-1 wet weight)
10000
16
Gills
++ 
MDA concentration
(nmol g-1 weit weight)
Body wall
12
8
4
0
FC
Fig. 6
Hypoxia
Normoxia
Hyperoxia
Table 1 Antioxidant and aerobic metabolism activities in different tissues
of Paralvinella grasslei « Freshly Caught » (U g–1 wet weight).
Paralvinella
grasslei
Gills
Gut
Body wall
Antioxidative enzymes
CAT
SOD
Aerobic metabolism enzymes
GPX
COX
CS
Units g-1 wet weight (Mean ± SE (n = 7))
aaa
5.9 ± 0.2
bb
21.2 ± 2.8
9.0 ± 2.4
920 ± 185
1377 ± 644
1367 ± 659
ab
0.54 ± 0.03
0.78 ± 0.11
0,81 ± 0.20
1.08 ± 0.32
0.98 ± 0.22
0.76 ± 0.18
aaabb
5.4 ± 0.8
1.9 ± 0.4
2.3 ± 0.2
All enzymatic activities were measured at 25°C
a
aaa
a = significantly different from the activity measured in gut ( P<0.05, P<0.0001). b = significantly different from the
b
activity measured in body wall ( P<0.05,
bb
P<0.001).
Table 2 : Antioxidant and aerobic metabolism activities of three tissues coming from the hydrothermal vent species
Paralvinella grasslei submitted at three oxygen partial pressures (U g-1 of wet weight)
Paralvinella
grasslei
Hypoxia
Gills
Gut
Body Wall
Normoxia Gills
Gut
Body Wall
Hyperoxia Gills
Gut
Body Wall
CAT
SOD
-1
GPX
COX
CS
Units g wet weight (Mean ± SE (n = 7))
1.28 ± 0.46
12.11 ± 7.45
7.98 ± 2.35
639.33 ± 371.55
1597.83 ± 454.44
2307.50 ± 566.50
0.64 ± 0.11
0.52 ± 0.20
0.98 ± 0.13
0.17 ± 0.11
0.31 ± 0.12
0.66 ± 0.10
2.65 ± 0.30
0.67 ± 0.50
3.07 ± 0.28
4.31 ± 1.30
17.13 ± 3.88
8.15 ± 0.48
1287.66 ± 464.00
1999.50 ± 1209.50
2245.50 ± 538. 60
0.66 ± 0.16
0,63 ± 0.13
0.85± 0.12
1.56 ± 0.14
0.87 ± 0.29
0.66 ± 0.06
6.12 ± 1.32
1.76 ± 0.61
2.94 ± 0.28
4.08 ± 1.65
9.53 ± 3.42
8.26 ± 3.88
1840.28 ± 743.75
2142.33 ± 698.88
2815.75 ± 648.43
0.84 ± 0.27
0.47 ± 0.01
2.26 ± 0.20
0.33 ± 0.10
0.15 ± 0.06
0.80 ± 0.09
3.78 ± 0.83
0.37 ± 0.20
3.63 ± 0.47
All enzymatic activities were measured at 25°C and values are the means and standard deviations of 7 different samples
Hypoxia = 12.8 ± 6 µmol O 2 l-1 or 1 ± 0.5 kPa or 7.5 ± 3.5 Torr
Normoxia = 78 ± 3 µmol O 2 l-1 or 6.3 ± 0.2 kPa or 50 ± 2 Torr
Hyperoxia = 203 ± 9 µmol O 2 l-1 or 16.2 ± 0.8 kPa or 122 ± 5 Torr
Table 3 Antioxidant and aerobic metabolism activities of hydrothermal and non-hydrothermal annelids (U mg–1 of protein).
Antioxidative enzymes
Species
CAT
SOD
Aerobic metabolism enzymes
GPX
COX
CS
-1
Source
Units mg of protein (Mean ± SE (number of specimens))
Hydrothermal Vent Annelids
Paralvinella grasslei
Riftia pachyptila
Alvinella pompejana
Gills
Gut
Body wall
Muscles
Gills
Body wall
0.16 ± 0.01 (7)
0.82 ± 0.11 (7)
0.20 ± 0.05 (7)
24 ± 5 (7)
53 ± 25 (7)
30 ± 15 (7)
0.014 ± 0.001 (7)
0.030 ± 0.004 (7)
0.018 ± 0.004 (7)
0.028 ± 0.009 (7) 0.142 ± 0.021 (7)
0.038 ± 0.008 (7) 0.073 ± 0.015 (7)
0.017 ± 0.004 (7) 0.051 ± 0.004 (7)
n.d. (1)
3.7 (1)
0.006 (1)
-
-
Blum & Fridovich
1984
-
-
-
0.08 (1)a
0.05 (1)a
-
Hand & Somero
1983
This study
Non Hydrothermal Vent Annelids
Arenicola marina
Body wall
8 ± 2 (5)
6 ± 3 (5)
-
-
-
Buchner et al. 1996
Nereis diversicolor
Body wall
57 ± 14 (10)
3 ± 1 (10)
-
-
-
Abele et al. 1994
Laeonereis acuta
Body wall
2.2 ± 0.4 (4)
3.5 ± 0.5 (4)
-
-
-
Heteromastus filiformis
Body wall
21 ± 6 (7)
17 ± 6 (6)
-
-
-
Geracitano et al.
2004
Abele et al. 1998
Phyllodoce mucosa
Body wall
-
7 ± 1 (8)
-
-
-
Abele-Oeschger
1996
Eisenia foetida
Body wall
91 ± 4 (3)
0.013 ± 0.004 (3)
Saint-Denis et al.
1998
All enzymatic activities were measured at 25°C and all protein concentrations were determined from homogenized tissue, except for (a ) which activities were
measured at 20°C and protein concentrations assayed from supernatant extracts. n.d. : not detectable, numbers in parentheses are the samples size.