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- Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic AgPublication . Gomes, Tânia; G. Pereira, Catarina; Cardoso, Cátia; Bebianno, Maria JoãoAg NPs are one of the most commonly used NPs in nanotechnology whose environmental impacts are to date unknown and the information about bioavailability, mechanisms of biological uptake and toxic implications in organisms is scarce. So, the main objective of this study was to investigate differences in protein expression profiles in gills and digestive gland of mussels Mytilus galloprovincialis exposed to Ag NPs and Ag(+) (10 μg L(-1)) for a period of 15 days. Protein expression profiles of exposed gills and digestive glands were compared to those of control mussels using two-dimensional electrophoresis to discriminate differentially expressed proteins. Different patterns of protein expression were obtained for exposed mussels, dependent not only on the different redox requirements of each tissue but also to the Ag form used. Unique sets of differentially expressed proteins were affected by each silver form in addition to proteins that were affected by both Ag NPs and Ag(+). Fifteen of these proteins were subsequently identified by MALDI-TOF-TOF and database search. Ag NPs affected similar cellular pathways as Ag(+), with common response mechanisms in cytoskeleton and cell structure (catchin, myosin heavy chain), stress response (heat shock protein 70), oxidative stress (glutathione s-transferase), transcriptional regulation (nuclear receptor subfamily 1G), adhesion and mobility (precollagen-P) and energy metabolism (ATP synthase F0 subunit 6 and NADH dehydrogenase subunit 2). Exposure to Ag NPs altered the expression of two proteins associated with stress response (major vault protein and ras partial) and one protein involved in cytoskeleton and cell structure (paramyosin), while exposure to Ag(+) had a strong influence in one protein related to stress response (putative c1q domain containing protein) and two proteins involved in cytoskeleton and cell structure (actin and α-tubulin). Protein identification showed that Ag NPs toxicity is mediated by oxidative stress-induced cell signalling cascades (including mitochondria and nucleus) that can lead to cell death. This toxicity represents the cumulative effect of Ag(+) released from the particles and other properties as particle size and surface reactivity. This study helped to unravel the molecular mechanisms that can be associated with Ag NPs toxicity; nevertheless, some additional studies are required to investigate the exact interaction between these NPs and cellular components.
- Do microplastic contaminated seafood consumption pose a potential risk to human health?Publication . Vital, S. A.; Cardoso, Cátia; Avio, C.; Pittura, L.; Regoli, F.; Bebianno, MariaMicroplastics are present in all parts of the ocean and can have deleterious effects on marine resources. The aim of this work was to map the presence of microplastics in commercial marine species such as bivalves (mussels Mytilus galloprovincialis and clams Scrobicularia plana), crabs (Carcinus maenas) as well as fish (Mullus surmuletus) to relate microplastics levels to pollution sources, assess possible impact on marine food chains and on human health. These species were collected from several sites of the Ria Formosa lagoon and along the south coast of Portugal. A quantitative assessment (number, size and color) and typology of microplastics were made in these species. Only one green fragment of polypropylene was detected in the gills of the crabs, while a blue polyethylene fragment was detected in the hepatopancreas of the mullets. Moreover, no microplastics were present in S. plana nor in the crabs whole soft tissues. Among mussels, 86% of microplastics were present from all sites and the number, size and color were site specific. Mussels from the west side of the coast (Sites 1–3) had the highest levels of MPs per mussel and per weight compared to the other sites, probably related to the impact of touristic activity, fishing gears, fresh water and sewage effluents along with the hydrodynamics of the area.
- Changes in metallothionein transcription levels in the mussel Mytilus galloprovincialis exposed to CdTe quantum dotsPublication . Lopes Rocha, Thiago; Bilbao, Eider; Cardoso, Cátia; Soto, Manu; Bebianno, Maria JoãoQuantum dots (QDs) are a class of engineered nanoparticles (ENPs) with several biomedical, industrial and commercial applications. However, their metabolism and detoxification process in aquatic invertebrates and environmental health hazards remain unclear. This study investigate the transcriptional changes of metallothioneins (MTs) isoforms (mt10IIIa and mt20IV) induced by CdTe QDs, in comparison with its dissolved counterpart, in the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and to the same Cd concentration (10 µg Cd L-1) of dissolved Cd for 14 days and mt transcription levels were measured by real time quantitative PCR (qPCR). Tissue specific mt transcription patterns were observed in mussels exposed to both Cd forms, wherein the gills were a more sensitive organ compared to the digestive gland. No significant changes were observed in mt10IIIa transcription levels in mussels exposed to both Cd forms. In contrast, transcription of mt20IV was tissue and exposure time dependent, with higher mt20IV mRNA levels in mussels exposed to QDs and dissolved Cd when compared to unexposed mussels. Multivariate analysis indicates particle-specific effects after 14 days of exposure and a dual role of MTs in the QD metabolism and in the protection against oxidative stress in mussels exposed to Cd-based ENPs.
- A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of PortugalPublication . Cravo, Alexandra; Pereira, C.; Gomes, Tânia; Cardoso, Cátia; Serafim, M.A.; Almeida, Cheila; Rocha, T.; Lopes, Belisandra; Company, Rui; Medeiros, A.; Norberto, R.; Pereira, R.; Araújo, O.; Bebianno, Maria JoãoThe 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 (d-aminolevulinic acid dehy- dratase, 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.
- Changes in protein expression in mussels Mytilus galloprovincialis dietarily exposed to PVP/PEI coated silver nanoparticles at different seasonsPublication . Duroudier, Nerea; Cardoso, Cátia; Mehennaoui, Kahina; Mikolaczyk, Mathilde; Schäfer, Jörg; Gutleb, Arno C.; Giamberini, Laure; Bebianno, Maria João; Bilbao, Eider; Cajaraville, Miren P.Potential toxic effects of Ag NPs ingested through the food web and depending on the season have not been addressed in marine bivalves. This work aimed to assess differences in protein expression in the digestive gland of female mussels after dietary exposure to Ag NPs in autumn and spring. Mussels were fed daily with microalgae previously exposed for 24 hours to 10 µg/L of PVP/PEI coated 5 nm Ag NPs. After 21 days, mussels significantly accumulated Ag in both seasons and Ag NPs were found within digestive gland cells and gills. Two-dimensional electrophoresis distinguished 104 differentially expressed protein spots in autumn and 142 in spring. Among them, chitinase like protein-3, partial and glyceraldehyde-3-phosphate dehydrogenase, that are involved in amino sugar and nucleotide sugar metabolism, carbon metabolism, glycolysis/gluconeogenesis and the biosynthesis of amino acids KEGG pathways, were overexpressed in autumn but underexpressed in spring. In autumn, pyruvate metabolism, citrate cycle, cysteine and methionine metabolism and glyoxylate and dicarboxylate metabolism were altered, while in spring, proteins related to the formation of phagosomes and hydrogen peroxide metabolism were differentially expressed. Overall, protein expression signatures depended on season and Ag NPs exposure, suggesting that season significantly influences responses of mussels to NP exposure.
- Immunocytotoxicity, cytogenotoxicity and genotoxicity of cadmium-based quantum dots in the marine mussel Mytilus galloprovincialisPublication . Lopes Rocha, Thiago; Gomes, Tânia; Cardoso, Cátia; Letendre, Julie; Pinheiro, José Paulo; Serrão Sousa, Vânia; Ribau Teixeira, Margarida; Bebianno, MariaThere is an increased use of Quantum Dot (QDs) in biological and biomedical applications, but little is known about their marine ecotoxicology. So, the aim of this study was to investigate the possible immunocytotoxic, cytogenotoxic and genotoxic effects of cadmium telluride QDs (CdTe QDs) on the marine mussel Mytilus galloprovincialis. Mussels were exposed to 10 μg L(-1) of CdTe QDs or to soluble Cd [Cd(NO3)2] for 14 days and Cd accumulation, immunocytotoxicity [hemocyte density, cell viability, lysosomal membrane stability (LMS), differential cell counts (DCC)], cytogenotoxicity (micronucleus test and nuclear abnormalities assay) and genotoxicity (comet assay) were analyzed. Results show that in vivo exposure to QDs, Cd is accumulated in mussel soft tissues and hemolymph and induce immunotoxic effects mediated by a decrease in LMS, changes in DCC, as well as genotoxicity (DNA damage). However, QDs do not induce significant changes in hemocytes density, cell viability and cytogenetic parameters in opposition to Cd(2+). Soluble Cd is the most cytotoxic and cytogenotoxic form on Mytilus hemocytes due to a higher accumulation of Cd in tissues. Results indicate that immunotoxicity and genotoxicity of CdTe QDs and Cd(2+) are mediated by different modes of action and show that Mytilus hemocytes are important targets for in vivo QDs toxicity.
- Lipid composition and some bioactivities of 3 newly isolated microalgae (Tetraselmis sp. IMP3, Tetraselmis sp. CTP4, and Skeletonema sp.)Publication . Cardoso, Cátia; Pereira, H.; Franca, J.; Matos, J.; Monteiro, I.; Pousão-Ferreira, P.; Gomes, A.; Barreira, Luísa; Varela, Varela J.; Neng, N.; Nogueira, J. M.; Afonso, C.; Bandarra, N. M.Recently isolated microalgae Tetraselmis sp. IMP3, Tetraselmis sp. CTP4, and Skeletonema sp. were studied. The three novel strains contained relatively high levels of polyunsaturated fatty acids (PUFA) and n3 PUFA. However, highly unsaturated n3 FA contents were relatively low (5.7-13.0% of the total FA). In general, eicosapentaenoic acid (EPA, 20:5 n3) contents were low (< 6.4% of the total FA). However, in Skeletonema biomass, EPA levels were higher than 10% of the total FA. alpha-Linolenic acid (ALA, 18:3 n3) and 16:3 n4 were the main PUFA in Tetraselmis strains and Skeletonema, respectively. High contents of myristic (14:0) and palmitoleic (16:1 n7) acids were found in Skeletonema (exceeding 20% of the total FA in both cases), whereas the Tetraselmis strains were rich in palmitic (16:0), 15-27% of the total FA, and oleic (18:1 n9) acids, 12-19% of the total FA. Linoleic acid (18:2 n6) content was low in Skeletonema (< 1% of the total FA). This microalga had the highest total polyphenol content, reaching 300-400 mg/100 g dw. Gentisic acid was the main phenolic compound in the aqueous and ethanolic extracts of this microalga. The highest antioxidant activity was displayed by Skeletonema. The ferric ion reducing antioxidant power (FRAP) and 2,2 '-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods showed higher antioxidant power for Skeletonema sp. extracts, reaching an ABTS reduction of more than 80%. Concerning anti-inflammatory activity, ethanolic extracts of Skeletonema sp. exhibited the highest inhibitory capacity of cyclooxygenase-2 (COX-2), 82 +/- 2%, which compares to 36 +/- 9% in Tetraselmis sp. CTP4 and 45 +/- 5% in Tetraselmis sp. IMP3. Aqueous extracts had always a lower anti-inflammatory capacity, 6-30%. Therefore, these microalgae have potential for multiple applications, ranging from bioactive feedstocks to aquaculture and nutraceutical uses.
- Genotoxicity in two bivalve species from a coastal lagoon in the south of PortugalPublication . Catarina Almeida, Ana; G. Pereira, Catarina; Gomes, Tânia; Cardoso, Cátia; Bebianno, Maria João; Cravo, AlexandraDNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decussatus and Mytilus galloprovincialis from the Ria Formosa lagoon (south Coast of Portugal). Clams and mussels were sampled from sites close to each other to determine interspecific responses to similar environmental conditions, considering also seasonal and gender differences. Coupled with genotoxic effect, another damage biomarker (lipid peroxidation) was analysed to verify if the conditions that instigate DNA damage can be related with injury to cell membranes. For both species, DNA damage was low, reflecting the low levels of genotoxic contaminants in the lagoon, and no interspecific differences were found. However, seasonal differences were established for both bivalve species, reflecting higher environmental stress in summer. Regarding gender susceptibility, only clams showed differences in percentage of Tail DNA, with females more sensitive to DNA damage than males. Additionally, results for clams point out that factors responsible for LPO may not be the same as those causing genotoxicity. This study demonstrated that DNA damage is a sensitive biomarker to discriminate spatial, temporal and gender differences, being an appropriate biomarker for genotoxicity evaluation even in places of low contamination, such as the Ria Formosa lagoon.
- Interspecific variability of endocrine disruption and oxidative stress in two bivalve species from the Ria Formosa Lagoon (south coast of Portugal)Publication . Pereira, Catarina; Gomes, Tânia; Cardoso, Catia; Almeida, Ana Catarina; Araújo, Olinda; Bebianno, Maria João; Cravo, AlexandraEstuaries are highly productive ecosystems that have been increasingly affected by anthropogenic activities. In this study, the presence of endocrine disrupting compounds (EDCs) and their relation to oxidative stress were assessed in two bivalve species (mussels Mytilus galloprovincialis and clams Ruditapes decussatus) from the Ria Formosa Lagoon (south Portugal). We applied the alkali-labile phosphate (ALP) assay to determine vitellogenin-like protein levels, and the lipid peroxidation (LPO) method to assess oxidative damage. Mussels and clams exhibited the same pattern of ALP concentrations, with no differences between genders and higher levels in winter. R. decussatus showed higher ALP values than M. galloprovincialis, possibly associated with a stronger responsiveness of clams to EDCs or due to the influence of the reproductive cycle. Mussels exhibited a more evident seasonal effect for LPO, indicating that these organisms are more influenced by seasonal fluctuations in the water column compared to the sediment-associated clams. Both species appear to be under the influence of EDC exposure (which could induce Vg-like protein synthesis) and oxidative stressors (leading to lipid peroxidation) in the Ria Formosa Lagoon, particularly in winter, reflecting the presence of different contaminants in Ria Formosa and environmental changes, as well as complex interactions between them.
- Are shallow-water shrimps proxies for hydrothermal-vent shrimps to assess the impact of deep-sea mining?Publication . Mestre, Nélia; Auguste, M.; de Sá, L.C.; FONSECA, TAINÁ; Cardoso, Cátia; Brown, A.; Barthelemy, D.; Charlemagne, N.; Hauton, Chris; Machon, J.; Ravaux, J.; Shillito, B.; Thatje, S.; Bebianno, Maria JoãoPolymetallic seafloor massive sulphide deposits are potential targets for deep-sea mining, but high concentrations of metals (including copper - Cu) may be released during exploitation activities, potentially inducing harmful impact. To determine whether shallow-water shrimp are suitable ecotoxicological proxies for deep-sea hydrothermal vent shrimp the effects of waterborne Cu exposure (3 and 10 days at 0.4 and 4 μM concentrations) in Palaemon elegans, Palaemon serratus, and Palaemon varians were compared with Mirocaris fortunata. Accumulation of Cu and a set of biomarkers were analysed. Results show different responses among congeneric species indicating that it is not appropriate to use shallow-water shrimps as ecotoxicological proxies for deep-water shrimps. During the evolutionary history of these species they were likely subject to different chemical environments which may have induced different molecular/biochemical adaptations/tolerances. Results highlight the importance of analysing effects of deep-sea mining in situ and in local species to adequately assess ecotoxicological effects under natural environmental conditions.