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- Metallothioneins in the clam Ruditapes decussatus: an overviewPublication . Bebianno, Maria João; Serafim, MA; Simes, DinaThe clam Ruditapes decussatus is a suspension-feeding bivalve mollusc widely distributed in European waters and in the Mediterranean. Due to is economic importance it is heavily harvested in many countries, and particularly in Portugal. Its ability to accumulate high metal concentrations along with its economic importance was the main reason for its selection as a bioindicator. Metallothionein (MT) concentrations in the clams R. decussatus followed by gel filtration chromatography, differential pulse polarography and SDS-PAGE, after Cd exposure, revealed that MT is induced in different tissues (whole soft tissues, gills, digestive gland and remaining tissues) but the level of MT induction is tissue dependent. MT from the gills and the digestive gland give a more sensitive response to assess the effects of metal exposure directly from the water or from the food than in the whole soft tissues. MT levels were also measured in the gills, digestive gland and remaining tissues of R. decussatus collected in the Ria Formosa lagoon (Portugal) from areas of different metal load and during the period of sexual differentiation of the clam. Data revealed that there were significant differences of MT concentrations among sites and season but not among sex. Purification of MT from the digestive gland of R. decussatus revealed four MT isoforms. The molecular weight of one of these isoforms, determined by SDS-PAGE, was of the same order of magnitude as that of MT from other bivalve species. Similarly the amino acid sequence of the beta domain of the MT of the digestive gland of the clam also shows some degree of similarity with the similar MT sequence from mussels and oysters. It is, therefore suggested that there is some degree of similarity in the MT structure among these species.
- Sub-lethal effects of cadmium on the antioxidant defence system of the hydrothermal vent mussel Bathymodiolus azoricusPublication . Company, Rui; Serafim, M.A.; Cosson, Richard P.; Fiala-Médioni, Aline; Camus, Lionel; Serrão-Santos, Ricardo; Bebianno, Maria JoãoThe mussel Bathymodiolus azoricus is one of the most abundant species in the Mid-Atlantic Ridge hydrothermal vents and is continually exposed to the high-temperature venting fluids containing high metal concentrations and enriched in sulphides and methane, which constitute a potential toxic environment for marine species. The aim of this study was to assess the effects of a sub-lethal Cd concentration on the antioxidant defence system of this mussel. B. azoricus were collected at Menez Gwen vent site (37 degrees 51'N, 32 degrees 31'W) and exposed to Cd (50 microg l(-1)) during 24 days, followed by a depuration period of six days. A battery of stress related biomarkers including antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT; glutathione peroxidases-GPx), metallothioneins (MT), lipid peroxidation (LPO) and total oxyradical scavenging capacity (TOSC) were measured in the gills and mantle of B. azoricus. Cd was accumulated linearly during the exposure period in both tissues and no significant elimination occurred after the 6 days of depuration. Antioxidant enzymes activities were significantly higher in the gills. Cyt-SOD, T-GPx and Se-GPx were induced during the experiment but this was also observed in control organisms. Mit-SOD and CAT activities remained relatively unchanged. MT levels increased linearly in the gills of exposed mussels in the first 18 days of exposure. No significant differences were observed between LPO levels of control and exposed mussels. TOSC levels remained unchanged in control and exposed mussels. This suggests that although Cd is being accumulated in the tissues of exposed mussels, MT defence system is enough to detoxify the effect of Cd accumulated in the tissues. Furthermore, other factors besides the presence of Cd are influencing the antioxidant defence system in B. azoricus.
- Hepatic metallothionein concentrations in the golden grey mullet (Liza aurata): relationship with environmental metal concentrations in a metal-contaminated coastal system in PortugalPublication . Oliveira, M.; Ahmad, I.; Maria, Vera L.; Serafim, A.; Bebianno, Maria João; Pacheco, M.; Santos, M.A.This field survey was designed to assess the environmental metal contamination status of Ria de Aveiro (Portugal). To achieve that goal, the concentrations of Cd, Hg, Cu and Zn in the sediments and water were assessed and Liza aurata hepatic metallothionein (MT) determined. The relationships between MT and environmental metal concentrations and hydrological factors were examined. Results revealed a wide distribution of metals both in water and sediments throughout the lagoon, mainly at Rio Novo do Principe (RIO) and Laranjo (LAR), at concentrations that may affect biota. MT concentrations were higher at the sites with high metal content (RIO and LAR). A significant positive correlation was found between MT and Cd in the sediments as well as with MT and Hg and Cu in the water. Moreover, a negative correlation between MT and salinity was found. Thus, the current data support MT use as a biomarker of metal exposure emphasizing the importance of hydrological parameters in its concentrations. Results suggest the continued monitoring of this lagoon system.
- Comparison of metallothionein induction in response to cadmium in the gills of the bivalve molluscs Mytilus galloprovincialis and Ruditapes decussatusPublication . Bebianno, Maria João; Serafim, A.Metal binding characteristics and metallothionein induction differ markedly among the gills of the bivalve molluscs Mytilus galloprovincialis and Ruditapes decussatus exposed to sublethal cadmium concentrations (100 μg/l). The rates of cadmium uptake as well as the percentage of cadmium bound to the cytosol were significantly higher in the gills of M. galloprovincialis when compared with that of R. decussatus. Similarly metallothionein concentration detected in the gills of M. galloprovincialis increase fourfold while for the clam R. decussatus metallothionein concentrations increased only twofold.
- Effect of cadmium, copper and mercury on antioxidant enzyme activities and lipid peroxidation in the gills of the hydrothermal vent mussel Bathymodiolus azoricusPublication . Company, Rui; Serafim, Angela; Bebianno, Maria João; Cosson, R.; Shillito, B.; Fiala-Médioni, A.Metals are known to influence lipid peroxidation and oxidative status of marine organisms. Hydrothermal vent mussels Bathymodiolus azoricus live in deep-sea environments with anomalous conditions, including high metal concentrations. Although B. azoricus are aerobic organisms they possess abundant methano and thioautotrophic symbiotic bacteria in the gills. The enzymatic defences (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidase (Total GPx) and selenium-dependent glutathione peroxidase (Se-GPx)) and lipid peroxidation were determined in the gills of B. azoricus exposed to Cd (0.9 microM), Cu (0.4 microM) and Hg (0.1 microM) with different times of exposure. The experiments were performed in pressurized containers at 9+/-1 degrees C and 85 bars. Results show that vent mussels possess antioxidant enzymatic protection in the gills. Cd and Cu had an inhibitory effect in the enzymatic defence system, contrarily to Hg. These enzymatic systems are not completely understood in the B. azoricus, since reactive oxygen species might be produced through other processes than natural redox cycling, due to hydrogen sulphide and oxygen content present. Also the symbiotic bacteria may play an important contribution in the antioxidant protection of the gills.
- Variation of metal and metallothionein concentrations in a natural population of ruditapes decussatusPublication . Bebianno, Maria João; Serafim, A.The spatial and seasonal variation of total and subcellular distribution of Cd, Cu, and Zn was followed in different tissues (gills, digestive gland, and remaining tissues) of the clam Ruditapes decussatus collected along a metal contamination gradient in the Ria Formosa lagoon (southern Portugal) and compared with metallothionein (MT) concentrations.Total metal concentrations decreased according to the sequence digestive gland > gills > remaining tissues for Cd, digestive gland approximately gills > remaining tissues for Cu and gills > digestive gland > remaining tissues for Zn. MT concentrations in these tissues decreased according to the same sequence observed for Cd. In all the tissues, the highest subcellular concentration was in the cytosol for Cd and Cu and in the pellet for Zn. Among the three metals, Cd concentrations showed the most evident spatial variation. In all tissues, total and subcellular Cd concentrations decreased from the inner parts of the lagoon toward the ocean. However, no significant spatial or seasonal variation occurred in clam tissues for the other two metals, though marginal elevated Cu concentrations were observed in the inner parts of the lagoon. Therefore, Cu subcellular distribution in clam tissues was not significantly altered by Cu changes in the lagoon and are the baseline levels for normal metabolism of this clam population. The fact that total Zn concentrations remained unchanged both spatial and seasonal suggested that these clams regulate Zn in their tissues. In the three tissues, MT bind most significantly to Cd and Cu, while Zn, although binding to MT, is preferably bound to other ligands. MT concentrations showed the same spatial and seasonal variation of Cd and were significantly related with total and heat-treated cytosolic Cd in all tissues. For Cu a significant relationship between MT and total or cytosolic Cu was only observed in the remaining tissues. No relationship was observed between MT and total or cytosolic Zn concentrations. Metals and MT concentrations increased with the increase in the condition index for the gills and the digestive gland and decreased from the remaining tissues.Cd concentrations in the gills increased only in the heat-treated cytosolic fraction while Zn in this fraction decreased. Thus Cd concentrations in this tissue displaced Zn from the MT-fraction, leading to a modification of the soluble/insoluble Zn ratio once total Zn concentrations remained unchanged. This modification reflects a perturbation in the normal metabolism in this tissue due to the excess of Cd present. With the exception of the gills, Zn subcellular distribution in the other two tissues was similar among sites and season. The model that describes the relationship between MT, metals, and weight in the gills, digestive gland and remaining tissues also indicates that Cd was the only metal that influence MT synthesis significantly in all the tissues. The induced and/or existent MT was sufficient to bind free Cd ions present in the cells, preventing any damage to cellular metabolism in this clam population. Therefore, MT in the gills and digestive gland of R. decussatus can be used as an early warning signal for Cd exposure and are a useful biomarker to assess the toxicological status of this population in the Ria Formosa lagoon.
- Protein expression profiles in Bathymodiolus azoricus exposed to cadmiumPublication . Company, Rui; Antúnez, Oreto; Cosson, Richard P.; Serafim, M. A.; Shillito, Bruce; Cajaraville, Miren; Bebianno, Maria João; Torreblanca, AmparoProteomic changes in the "gill-bacteria complex" of the hydrothermal vent mussel B. azoricus exposed to cadmium in pressurized chambers ((Incubateurs Pressurises pour l'Observation en Culture d'Animaux Marins Profonds - IPOCAMP) were analyzed and compared with the non-exposed control group. 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) showed that less than 1.5% of the proteome of mussels and symbiotic bacteria were affected by a short-term (24 h) Cd exposure. Twelve proteins of the more abundant differentially expressed proteins of which six were up-regulated and six were down-regulated were excised, digested and identified by mass spectrometry. The identified proteins included structural proteins (actin/actin like proteins), metabolic proteins (calreticulin/calnexin, peptidyl-prolyl cis-trans isomerase, aminotransferase class-III, electron transfer flavoprotein, proteasome, alpha-subunit and carbonic anhydrase) and stress response proteins (chaperone protein htpG, selenium-binding protein and glutathione transferases). All differently expressed proteins are tightly connected to Cd exposure and are affected by oxidative stress. It was also demonstrated that B. azoricus was well adapted to Cd contamination therefore B. azoricus from hydrothermal vent areas may be considered a good bioindicator.
- Adaptation of the antioxidant defence system in hydrothermal-vent mussels (Bathymodiolus azoricus) transplanted between two Mid-Atlantic Ridge sitesPublication . Company, Rui; Serafim, Angela; Cosson, Richard; Fiala-Medioni, Aline; Dixon, David R.; Bebianno, Maria JoãoThe vent mussel Bathymodiolus azoricus is the dominant member of the Northern Mid-Atlantic Ridge (MAR) hydrothermal megafauna, and lives in an environment characterized by temporal and spatial variations in the levels of heavy metals, methane and hydrogen sulphide, substances which are known to increase reactive oxygen species levels in the tissues of exposed organisms. To evaluate the effects of two contrasting hydrothermal environments on the antioxidant defence system of this vent mussel species, a 2-week transplant experiment was carried out involving mussels collected from the relatively deep (2300 m), and chemical rich, Rainbow vent field. These were transplanted to the shallower (1700 m), and relatively less toxic, Lucky Strike vent field. To achieve this objective, levels of superoxide dismutase, catalase (CAT), total glutathione peroxidase (GPx), selenium-dependent glutathione peroxidase and lipid peroxidation (LPO) were measured in the gills and mantle tissues of resident and transplant mussels before and after the transplant experiment. With the exception of CAT, the gills of the transplanted mussels had significantly higher antioxidant enzyme activity compared with the basal levels in the donor (Rainbow) and recipient (Lucky Strike) populations; whereas the antioxidant enzyme levels in the mantle tissues of the transplants reflected the baseline levels of activity in the native Lucky Strike mussels after 2 weeks. In contrast, LPO levels were significantly higher in both tissue types in the transplants than in either the source or the recipient populations, which suggested a response to hydrostatic pressure change (note, the transplant animals were brought to the surface for transportation between the two vent fields). The fact that the Rainbow mussels survived the transplant experience indicates that B. azoricus has a very robust constitution, which enables it to cope behaviourally, physiologically and genetically with the extreme conditions found in its naturally contaminated deep-sea environment.
- Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmiumPublication . Serafim, Angela; Company, Rui; Bebianno, Maria João; Langston, W.JAlthough the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5+/-0.5 cm and large: 5.2+/-0.7 cm) were exposed to Cd (100 mug 1(-1)) at different water temperatures (5, 18 and 25 degreesC) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 degreesC) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 degreesC, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 degreesC there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.
- Effect of a polymetallic mixture on metal accumulation and metallothionein response in the clam Ruditapes decussatusPublication . Serafim, Angela; Bebianno, Maria JoãoThe toxic effects of metals are related to changes in natural physiological and biochemical processes in organisms, which are not easily detected. Marine invertebrates developed strategies to survive in the presence of toxic metal levels, by the removal of these compounds from the soluble fraction of the cell through different processes, including the synthesis of metallothionein (MT). To better understand the effect of Cd, Cu and Zn in MT response it is important to consider that in their natural environment these metals are normally mixed. The exposure to a metal mixture result in toxicological interactions that produce different biological responses when compared to the effects of a single metal exposure. The aim of this study was to assess metal accumulation and MT response in different tissues of the clam Ruditapes decussatus exposed to a mixture of sublethal Cd, Cu and Zn concentrations. Kinetic models were applied to determine metal uptake and loss to better predict and understand the detoxification mechanisms in this species. The kinetic model showed that the effect of this polymetallic exposure compared to a single exposure to these metals caused different metal uptake and loss rates in several tissues. Moreover, the exposure of R. decussatus to a polymetallic mixture caused an increase in MT induction compared to a single metal exposure. This is probably related to the interaction between the different metals and their different affinity to this protein. Metal interactions within aquatic organisms are very important in MT synthesis and the effect of polymetallic mixtures in the environment must be taken into account in field studies.