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- Biomarkers in nereis diversicolor (Polychaeta: Nereididae) as management tools for environmental assessment on the southwest Iberian coastPublication . Gomes, Tânia; Gonzalez-Rey, Maria; Rodriguez-Romero, Araceli; Trombini, Chiara; Riba, Immaculada; Blasco, Julian; Bebianno, Maria JoãoThe environmental quality of the southwest Iberian coast was assessed in different areas (Ria Formosa Lagoon, Guadiana Estuary and Cadiz Bay) focusing on metal contamination (Cd, Cu, Ni, Pb and Zn) in Nereis diversicolor whole tissues. In addition, associated toxicological effects were assessed using a multibiomarker approach combining several conventional biomarkers. The set of biomarkers selected included antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase), metallothionein and lipid peroxidation. N. diversicolor from the Ria Formosa Lagoon, Guadiana River and Cadiz Bay responded differently to metal contamination with different biomarker responses showing a clear site trend, suggesting different sources and/or magnitudes of contamination. Cadmium was a source of oxidative stress in polychaetes, mainly in Cadiz Bay, with a significant influence on antioxidant enzymes and enhancement of lipid peroxidation. The highest MT concentrations were in the Ria Formosa Lagoon and in the Guadiana River, where there was a direct relationship with high nickel concentrations. Biomarker responses of N. diversicolor are useful tools for environmental quality assessment on the southwest Iberian coast, and more specific metal biomarkers should be included in future assessments.
- 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.
- 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.
- Comparison of metal accumulation between ‘Artificial Mussel’ and natural mussels (Mytilus galloprovincialis) in marine environmentsPublication . Gonzalez-Rey, Maria; Lau, T. C.; Gomes, Tânia; Maria, Vera L.; Bebianno, Maria João; Wu, R.The passive sampler called 'Artificial Mussel' (AM) developed by Wu et al. (2007) can provide a time-integrated estimate of metal concentrations in the marine environment, and offers a potential device to assess and compare metal concentration in different marine environments worldwide. The aim of this study was to compare metal accumulation on AM and natural mussel Mytilus galloprovincialis at three sites with different metal loads along the Portuguese coast for four months. M. galloprovincialis were placed in cages alongside AMs at each site. Samples were collected monthly and Cd, Cr, Cu, Pb, and Zn concentrations in whole soft tissues and AMs compared. For both Cu and Cd, the results were similar between AMs and natural mussels. Higher concentrations of Zn were observed in natural mussels, whereas the inverse was shown for Pb (about 10-fold higher). Our results showed that AMs are promising tools for assessing metal concentrations in marine environments.
- Incidence of intersex in male clams Scrobicularia plana in the Guadiana Estuary (Portugal)Publication . Gomes, Tânia; Gonzalez-Rey, Maria; Bebianno, MariaEndocrine disrupting chemicals (EDCs) known to be present in the marine environment, have great potential to influence reproductive status in aquatic organisms. A widespread incidence of intersex linked with EDCs exposure has been reported in molluscs and fish species. The species Scrobicularia plana is inherently gonochoristic; nonetheless, the feminisation of male clams was described in estuaries from the United Kingdom. Thus, this study reports the presence of oocytes in the testis of male clams of S. plana collected in the Guadiana River (Portugal). Samples were collected between June and September (2007), and gonad tissue analysed by light microscopy. Intersex with several degrees of intensity was detected during the first three months of sampling, in which the percentage of male clams affected varied between 5.9, 58.3 and 71.4%. Little is known about the causes of intersex in S. plana, being the urban, industrial and agricultural discharges possible major sources of induction. In the Guadiana River, the presence of several EDCs may be directly linked to the incidence of the intersex condition detected in Scrobicularia plana.
- Effects of nanoparticles exposure in the mussel Mytilus GalloprovincialisPublication . Gomes, Tânia; Bebianno, Maria João da Anunciação FrancoNanotechnology is rapidly developing and attracting attention due to the exploitation of the novel materials at the nanoscale for application within biomedical, cosmetic, electronic, energy production and environmental sectors. Increased production and widespread use of these nanomaterials result in their release into the environment; nevertheless, the knowledge of their behaviour in aquatic systems is scarce. Accordingly, this thesis assessed the effects of two commercially available nanoparticles, copper oxide (CuO NPs) and silver nanoparticles (Ag NPs), using mussels Mytilus galloprovincialis as bioindicators. To understand the uptake, accumulation and effects of these NPs, mussels were exposed to a realistic environmental concentration of 10 !g.L-1 of CuO (31 ± 10 nm) and Ag NPs (<100 nm) for 15 days, comparative to their ionic counterparts. NPs were characterized and biomarkers of oxidative stress, metal exposure, genotoxicity and neurotoxicity evaluated in mussel tissues. To identify pathways of NP exposure and detect new biomarkers, a proteomic approach was undertaken. Oxidative stress is the major NP-induced toxicity, but with distinct modes of action. Gills are more susceptible to oxidative stress while the digestive gland is the preferential site for NPs accumulation. The oxidative (enzymatic activation/inhibition, metallothionein induction and lipid peroxidation), genotoxic (DNA strand breaks) and neurotoxic (acetylcholinesterase inhibition) changes suggest that NPs toxicity is associated with ROS that induced a cascade of pathways (via nucleus and mitochondria) that ultimately lead to apoptosis but by different mechanisms. New biomarkers candidates were identified: caspase 3/7-1, cathepsin-L and zinc-finger protein for CuO NPs and precollagen-P, major vault protein and ras partial for Ag NPs exposure. Overall, these results show that even though oxidative stress and apoptosis are similar outcomes for NP toxicity, particle composition, size, solubility, aggregation and chemistry are key elements for determining their mode of action. This study contributed to understand the CuO and Ag NPs behaviour, bioavailability and toxicity in aquatic systems and their uptake and effects in filter-feeding organisms.
- Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialisPublication . Gomes, Tânia; Araújo, Olinda; Pereira, Rita; Catarina Almeida, Ana; Cravo, Alexandra; Bebianno, Maria JoãoThough there is some information on cytotoxicity of copper nanoparticles and silver nanoparticles on human cell lines, there is no information on their genotoxic and cytotoxic behaviour in bivalve molluscs. The aim of this study was to investigate the genotoxic impact of copper oxide and silver nanoparticles using mussels Mytilus galloprovincialis. Mussels were exposed to 10 μg L⁻¹ of CuO nanoparticles and Cu²⁺ and Ag nanoparticles and Ag⁺ for 15 days to assess genotoxic effects in hemocytes using the comet assay. The results obtained indicated that copper and silver forms (nanoparticles and ionic) induced DNA damage in hemolymph cells and a time-response effect was evident when compared to unexposed mussels. Ionic forms presented higher genotoxicity than nanoparticles, suggesting different mechanisms of action that may be mediated through oxidative stress. DNA strand breaks proved to be a useful biomarker of exposure to genotoxic effects of CuO and Ag nanoparticles in marine molluscs.
- Proteomic changes in Corbicula fluminea exposed to wastewater from a psychiatric hospitalPublication . Bebianno, Maria João; Sroda, S.; Gomes, Tânia; Chan, P.; Bonnafe, E.; BUDZINSKI, Hélène; Geret, F.The increase use of pharmaceutical compounds in veterinary practice and human population results in the ubiquitous presence of these compounds in aquatic ecosystems. Because pharmaceuticals are highly bioactive, there is concern about their toxicological effects in aquatic organisms. Therefore, the aim of this study was to assess the effects of an effluent from a psychiatric hospital (containing a complex mixture of 25 pharmaceutical compounds from eleven therapeutic classes) on the freshwater clam Corbicula fluminea using a proteomic approach. The exposure of C. fluminea to this complex effluent containing anxiolytics, analgesics, lipid regulators, beta blockers, antidepressants, antiepileptics, antihistamines, antihypertensives, antiplatelets and antiarrhythmics induced protein changes after 1 day of exposure in clam gills and digestive gland more evident in the digestive gland. These changes included increase in the abundance of proteins associated with structural (actin and tubulin), cellular functions (calreticulin, proliferating cell nuclear antigen (PCNA), T complex protein 1 (TCP1)) and metabolism (aldehyde dehydrogenase (ALDH), alcohol dehydrogenase, 6 phosphogluconate dehydrogenase). Results from this study indicate that calreticulin, PCNA, ALDH and alcohol dehydrogenase in the digestive gland and T complex protein 1 (TCP1)) and 6 phosphogluconate dehydrogenase in the gills represent useful biomarkers for the ecotoxicological characterization of psychiatric hospital effluents in this species.
- A multibiomarker approach in Mytilus galloprovincialis to assess environmental qualityPublication . Cravo, Alexandra; Lopes, Belisandra; Serafim, M.A.; Company, Rui; Barreira, Luísa; Gomes, Tânia; Bebianno, Maria JoãoA 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.
- 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.
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