Protein expression profiles in Bathymodiolus azoricus exposed to cadmium.

Proteomic 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.

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus.

The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.

A multi-biomarker approach in cross-transplanted mussels Mytilus galloprovincialis.

The present work integrates the active biomonitoring (ABM) concept in mussels Mytilus galloprovincialis from the South coast of Portugal transplanted during 28 days between two sites with different sources of contamination, and vice versa, in order to assess biological effects in these mussels. For that purpose a multibiomarker approach was used. The suit of biomarkers indicative of metal contamination were metallothioneins (MT) and the enzyme δ-aminolevulinic acid dehydratase (ALAD), for organic contamination mixed function oxidase system (MFO), glutathione-S-transferase (GST) and acetylcholinesterase (AChE), as oxidative stress biomarkers superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO). These biomarkers were used to determine an index to evaluate the stress levels in these two sites. Site A is strongly influenced by metallic contamination, with higher Cu, Cr and Pb in M. galloprovincialis, as well as higher MT levels, antioxidant enzymes activities and LPO concentrations, and lower ALAD activity. In site B organic compounds (PAHs) are prevalent and native mussels show higher activities of the MFO system components and GST. Transplanted mussels had significant alterations in some biomarkers that reflect the type of contaminants present in each site, which demonstrates the primary role of the environment in determining the physiological characteristics of resident mussels. Therefore the application of ABM using a battery of biomarkers turns out to be a useful approach in sites where usually complex mixtures of contaminants occurs. In this study the biomarkers that better differentiate the impact of different contaminants at each site were MT, CYP450, SOD and CAT.

Sub-lethal effects of cadmium on the antioxidant defence system of the hydrothermal vent mussel Bathymodiolus azoricus.

The 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.

A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality.

A multibiomarker approach was carried out for the first time in the South Portuguese Coast using Mytilus galloprovincialis, to assess environmental quality, establish if there are adverse biological responses associated to different sources of anthropogenic contamination and to determine spatial and seasonal trends. For this purpose the battery of biomarkers selected was: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx total and Se dependent), Cytochrome P450 component system, Glutathione-S-transferase (GST) and acetylcholinesterase (AChE), metallothionein (MT) and lead-delta-aminolevulinic acid dehydratase (ALAD), lipid peroxidation (LPO) and Condition Index (CI) along with the determination of PAHs and metals (Cd, Cu, Ni, Pb and Zn). Results show that despite the levels of both organic and metallic contaminants in these eight spots in the South Coast of Portugal not being particularly high compared with other contaminated/polluted sites worldwide, the selected battery of biomarkers responded efficiently to the environmental changes and allowed an environmental assessment between seasons and sites. Different spatial and seasonal responses were evident along the South Coast of Portugal, meaning that the contamination is not homogeneous. This does not only reflect different competition, origin and intensity of contamination, but also different environmental conditions (e.g. temperature, salinity). Along the South Portuguese Coast site 8 was the most contaminated, while site 2 was considered the least contaminated. Despite environmental factors possibly causing difficulties in the general interpretation of biomarker data, those that better responded to environmental contamination were CYP450, SOD-mit and T-GPx for the summation SigmaPAHs, MT (digestive gland) for metals (especially Cu), ALAD for Pb and LPO for both organic and metallic contamination. These biomarkers were also positively correlated with temperature in summer, revealing this as a more stressful/critical season. In future environmental contamination assessments there is no need to analyse the components b5, P418, NADH and NADPH of phase I MFO system, and MT in the gills, since their responses are not evident.

Detoxification mechanisms in shrimp: comparative approach between hydrothermal vent fields and estuarine environments.

Hydrothermal vents are extreme deep-sea habitats that, due to their singular features, still intrigue scientific communities. Swift growth rates and profuse biomass of biological communities can be observed, despite of their inherently unstable physical-chemical and toxic conditions, indicating that organisms inhabiting this environment must be well adapted to these inhospitable conditions. The caridean shrimp, Chorocaris chacei, Mirocaris fortunata and Rimicaris exoculata, together with bathymodiolid mussels, dominate the vent fauna along the Mid-Atlantic Ridge (MAR). Crustacean species are widely used as biological indicators of environmental alterations, since they play a key ecological role as planktivorous grazers, epibenthic scavengers or as prey species. The biological consequences of the hydrothermal metal-rich environment in shrimp species are still largely unknown. Therefore, the aim of this study was the determination of the metal levels (Ag, Cd, Cu, Fe, Mn and Zn), metallothioneins (MT) and lipid peroxidation (LPO) in shrimp species collected in Rainbow, Lucky Strike and Menez-Gwen vent sites, in order to evaluate their different adaptation strategies toward metals when compared with two common coastal shrimp species (Palaemon elegans and Palaemonetes varians) from a fairly unpolluted estuarine system in south Portugal (Ria Formosa). Results show significant differences in metal concentrations, MT levels and lipid peroxidation between vent and coastal shrimp and also between shrimp species from the same site. This indicates that biochemical responses in both vent and coastal shrimp are affected not only by the environmental characteristics but also by inter-specific differences. Nevertheless, these responses apparently grant a successful adaptation for the survival in a metal-extreme environment.

Antioxidant biochemical responses to long-term copper exposure in Bathymodiolus azoricus from Menez-Gwen hydrothermal vent.

Copper (Cu) is essential to various physiological processes in marine organisms. However, at high concentrations this redox-active transition metal may enhance the formation of reactive oxygen species (ROS) and subsequently initiate oxidative damage. High concentrations of Cu may increase oxidative damage to lipids, proteins and DNA. Bathymodiolus azoricus is a Mytilid bivalve very common in hydrothermal environments near the Azores Triple Junction continuously exposed to high metal concentrations, including Cu, emanating from the vent fluids. The knowledge of antioxidant defence system and other stress related biomarkers in these organisms is still scarce. The aim of this work was to study the effect of Cu (25 microg l(-1); 24 days exposure; 6 days depuration) on the antioxidant stress biomarkers in the gills and mantle of B. azoricus. The expression of stress related biomarkers was tissue-dependent and results suggest that other factors than metal exposure may influence stress biomarkers, since little variation in antioxidant enzymes activities, MT concentrations, LPO and total oxyradical scavenging capacity (TOSC) occurred in both control and Cu-exposed mussels. Moreover, there is a general tendency for these parameters to increase with time, in both control and Cu-exposed mussels, suggesting that reactive oxygen species (ROS) formation is not metal dependent, and may be related with poor physiological conditions of the animals after long periods in adverse conditions compared to those in hydrothermal environments.

Kinetic model of cadmium accumulation and elimination and metallothionein response in Ruditapes decussatus.

The aim of the present study was to determine the response of metallothionein (MT) during Cd accumulation and elimination in different tissues of the estuarine bivalve Ruditapes decussatus exposed to two nominal Cd concentrations (4 and 40 microg/L) for 40 d, followed by a depuration period of 50 d. Cadmium was accumulated in all tissues of R. decussatus at both exposure concentrations, and the accumulation was tissue dependent. Use of the kinetic model showed that in the gills and remaining tissues, Cd was assimilated faster at the beginning of the exposure and decreased with time, possibly limited by the diffusion rate of this metal within the cell. In the digestive gland, however, the Cd was continuously accumulated. This could reflect that the Cd uptake rate is considerably higher than the loss rate and, therefore, that this tissue has a higher capacity to accumulate Cd compared to the other two tissues. Moreover, the application of this kinetic model in the different subcellular fractions showed that the bioconcentration factor was significantly higher in the low-molecular-weight fraction (where MT is found), suggesting that this fraction binds Cd faster, with a high uptake rate (K(u) = 32/d), and eliminates this metal more slowly (K(1) = 0.005/d). During the depuration phase, MT decreased simultaneously with Cd elimination in all tissues, although with a shorter half-life. In conclusion, the MT response prevented Cd in the tissues of R. decussatus from interfering in the normal clam metabolism; therefore, MT acts as a detoxification mechanism of Cd.

Antioxidant enzyme activities, metallothioneins and lipid peroxidation as biomarkers in Ruditapes decussatus?

Antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPX)) were studied in the gills and digestive gland of clams Ruditapes decussatus from different sites of the Ria Formosa (south coast of Portugal) in order to validate these enzymes as biomarkers of metal exposure. Two other potential biomarkers, lipid peroxidation expressed as malondialdehyde (MDA), and metallothionein (MT) levels were also measured. This study demonstrates that mitochondrial and cytosolic SOD activity changed while CAT activity (cytosolic and mitochondrial) was higher in the gills of clams from the three sites affected by different sources of contamination. GPXs (total and Se-dependent) were inhibited in the gills of clams from sites A (directly influenced by sewage industrial wastes) and B (directly influenced by harbor facilities). MT and MDA levels were higher in the gills of clams from these two sites and in the digestive gland of clams from the third site (C, main navigation channel and exchange with Atlantic ocean). These results suggest that CAT and GPX activities and MDA levels especially in the gills of clams can be used as biomarkers for monitoring environmental pollution.

Response of antioxidant systems to copper in the gills of the clam Ruditapes decussatus.

Copper (Cu) is an essential element for biological systems, however, when present in excess, is toxic. Metallothioneins can play an important role in Cu homeostasis and detoxification. Moreover, Cu can catalyse the production of toxic hydroxyl radicals that cause lipid peroxidation but defence systems in the cells can limit the oxidative damage. The present study was performed to investigate the effect of three Cu concentrations (0.5, 2.5 and 25 microg l(-1)) on the response of antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), selenium-dependent glutathion peroxidase and total glutathion peroxidase), total proteins, metallothioneins (MT), malondialdehyde (MDA) concentrations in the gills of the clam, Ruditapes decussatus. The activity of antioxidant enzymes and total proteins, MT and MDA concentrations were measured in the gills of the clams after 1, 3, 7, 14, 21 and 28 days of Cu exposure. Results indicate that Cu only induces an imbalance in the oxygen metabolism during the first week of Cu exposure due to a decrease in mitochondrial SOD and CAT, selenium-dependent and total glutathion peroxidase activities. Cu also causes lipid peroxidation, measured by the MDA formation, that was Cu dependent. In the gills of clams exposed to 25 microg Cu/l, the excess of Cu triggers the induction of MT synthesis after 3 days of exposure.

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus.

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.