A. Serafim

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.

Contamination assessment of a coastal lagoon (Ria de Aveiro, Portugal) using defence and damage biochemical indicators in gill of Liza aurata - an integrated biomarker approach.

Fish gill importance in toxicants uptake, bioconcentration and excretion allied to meagre knowledge on branchial damage/protection responses substantiate this study. Five critical sites in Ria de Aveiro (Portugal) were assessed in comparison with a reference site (Torreira), focusing on Liza aurata gill antioxidant defences versus damage (oxidative and genetic). Only in Barra fish displayed damage (lipid peroxidation) though no differences were found in antioxidants. In all other sites, except Rio, antioxidant alterations were found. Thus, fish from Gafanha, Laranjo and Vagos showed higher total glutathione, glutathione peroxidase and catalase. Higher glutathione reductase and glutathione S-transferase activity was also found in the first and the last sites, respectively. In Laranjo, metallothionein levels were higher though lower in Gafanha and Vagos. In general, damage was not accompanied by defences weakening confirming that predicting damage based on antioxidants depletion is not straightforward. The integrated biomarker response index ranked sites as: Gafanha>Barra>Laranjo>Vagos>Rio>Torreira.

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.

Multi-biomarker responses to estuarine habitat contamination in three fish species: Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps.

Several biomarker responses were determined in three fish species, Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps, from two estuaries of the Portuguese coast, Ria de Aveiro and Tejo. Both estuaries have significant anthropogenic influences from multiple sources (industrial, agricultural and shipping activities), which was evident from sediment chemical characterization concerning metal (copper, zinc, nickel, lead and chromium) and polycyclic aromatic hydrocarbon (PAH) concentrations. Spatial variability in fish responses was observed across species for most biomarkers of exposure [the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST), and metallothionein concentrations (MT)] and effect biomarkers [lipid peroxidation (LPO), RNA to DNA ratio (R:D), protein and lipid content]. In general, the interspecific differences in biomarker responses were greater than the spatial differences, due to differences in the behavior and habitat use of the species. Nevertheless, similarities were also observed considering both chemical load and biomarker responses. In highly polluted sites fish showed in general a significant antioxidant enzyme induction, associated with decreased R:D values, while fish from the least impacted site had little enzyme induction and better condition indices (high R:D and low LPO values). EROD activity was also higher for all species in the Tejo than Ria de Aveiro estuary, despite the generally higher total PAH measured in Ria de Aveiro, most likely due to a higher proportion of 4 and 6-ring PAHs, considered more toxic than low molecular weight PAHs, in the Tejo. In conclusion, this multi-biomarker approach considering multiple species provided improved understanding of the diverse responses and effects of exposure to contaminants and the effective risk it poses for different fish species.

A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal

The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence upon the surrounding areas. In this lagoon, the clam Ruditapes decussatus has a great economical importance and has been widely used as a biomonitor. A multibiomarker approach (δ-aminolevulinic acid dehydratase, metallothionein, lipid peroxidation, acetylcholinesterase, alkali-labile phosphates, DNA damage) was applied to assess the environmental quality of this ecosystem and the accumulation of contaminants and their potential adverse effects on clams. Clams were sampled in different shellfish beds in the period between July 2007 and December 2008 and abiotic parameters (temperature, salinity, pH and dissolved oxygen of seawater and organic matter in the sediment), condition index, metals (Cd, Cu, Zn, Ni, Pb), TBTs and PAHs concentrations were measured in clam tissues. Data was integrated using Principal Component Analyses and biomarker indices: IBR (Integrated Biomarker Response) and HSI (Health Status Index). This multibiomarker approach enabled discrimination of a time and space trend between sites with different degrees of anthropogenic contamination, identifying one of them (site 2) as the most stressful and summer months as the most critical period for clams due to an increase of environmental stress (anthropogenic pressure along with extreme environmental conditions, e.g. temperature, dissolved oxygen, organic matter in the sediments, etc). The selected biomarkers provided an integrated response to assess the environmental quality of the system, proving to be a useful approach when complex mixtures of contaminants occur.

Using biochemical and isotope geochemistry to understand the environmental and public health implications of lead pollution in the lower Guadiana River, Iberia: A freshwater bivalve study

Lead is a natural component of aquatic ecosystems with no known biological role and is highly toxic. Its toxicity stems from its ability to mimic biologically important metals and to produce membrane damage through lipid peroxidation (LPO). Most lead poisoning symptoms are thought to occur by interfering with an essential enzyme, delta-aminolevulinic acid dehydratase (ALAD), the activity of which is markedly inhibited by lead. The purpose of this work was to study the levels and effects of lead pollution (responses of ALAD and oxidative stress biomarker LPO) in the freshwater bivalve Corbicula fluminea along the lower Guadiana River (Portugal and Spain); a major river system impacted by historic mining pollution and more recent anthropogenic inputs. The results show that the enzymatic activity of ALAD is negatively correlated with the total Pb concentration of the whole tissue suggesting that ALAD has considerable potential as a biomarker of lead exposure in C. fluminea. To identify the sources of lead to which bivalves have been exposed, high precision (206)Pb/(204)Pb, (207)Pb/(204)Pb, (208)Pb/(204)/Pb ratios for C. fluminea confirm that historical mining activities in the Iberian Pyrite Belt are the dominant source of lead pollution in the lower Guadiana River. The isotope patterns however exhibit marked seasonal and geographic variation in response to rainfall and river water management. Locally, other anthropogenic sources of lead have been detected in C. fluminea close to population centres, thus adding to its versatility as a freshwater bio-indicator. Overall, the study highlights the value of natural ecosystems as monitors of water quality and their importance for public health assessment and surveillance.

European eel (Anguilla anguilla L.) metallothionein, endocrine, metabolic and genotoxic responses to copper exposure.

This study investigated Anguilla anguilla (European eel) physiological and genotoxic responses to copper (Cu) and their relation with metallothionein (MT) protection. Eels were exposed during 7 days to Cu 0.2 micromol/L. MT induction was assessed in gill and liver, revealing significant response only in liver. Endocrine responses displayed a plasma free triiodothyronine (T3) and cortisol significant decrease, though the thyroid-stimulating hormone (TSH) and free thyroxine (T4) concentrations were unaltered. A significant plasma glucose increase was observed whereas lactate was significantly decreased. Despite the absence of DNA integrity decrease in blood, gill, liver and kidney, erythrocytic nuclear abnormalities (ENA) frequency significantly increased in Cu exposed group. MT induction was insufficient to prevent endocrine and metabolic alterations as well as genotoxicity/clastogenicity in blood. However, MT protection was evident in liver by preventing DNA integrity loss. Globally, it was demonstrated that Cu environmentally realistic levels may pose a serious ecological risk to fish.

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.

Wild juvenile Dicentrarchus labrax L. liver antioxidant and damage responses at Aveiro Lagoon, Portugal

The Aveiro Lagoon, at the north-western coast of Portugal, has been under considerable anthropogenic pressure for the last 5 decades. In order to perform an adequate survey of the effects induced by the contaminants in presence, wild juveniles Dicentrarchus labrax (sea bass) were selected. Thus, sea bass was captured at five sites: Torreira (TOR, as reference site), Gafanha (GAF), Rio Novo Príncipe (RIO), Laranjo (LAR) and Vagos (VAG) in autumn 2005. Liver defence responses such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (GSHt), total non-protein thiols (NP-SH) and metallothioneins (MT) were measured. Liver damage was determined as lipid peroxidation (LPO) and DNA integrity loss. RIO, LAR and VAG presented lower CAT, GR, GST activities and NP-SH and GSHt depletion in comparison to TOR. VAG and LAR showed higher GPx activity when compared to TOR. The highest MT level was found at GAF and VAG. The NP-SH and DNA integrity decreased at GAF compared to TOR. This field study demonstrated that not only antioxidant induction but also inhibitory responses must be considered as a signal of contamination.