EFEITOS DA EXPOSIÇÃO A NANOPARTÍCULAS NA ESPÉCIE MYTILUS GALLOPROVINCIALIS

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Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis

Publication . Gomes, Tânia; Araújo, Olinda; Pereira, Rita; Catarina Almeida, Ana; Cravo, Alexandra; Bebianno, Maria João

Though 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.

2013Journal article

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Effects of nanoparticles exposure in the mussel Mytilus Galloprovincialis

Publication . Gomes, Tânia; Bebianno, Maria João da Anunciação Franco

Nanotechnology 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.

2012Doctoral thesis

Open Access

Differential protein expression in mussels Mytilus galloprovincialis exposed to nano and ionic Ag

Publication . Gomes, Tânia; G. Pereira, Catarina; Cardoso, Cátia; Bebianno, Maria João

Ag 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.

2013Journal article

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Effects of silver nanoparticles exposure in the mussel Mytilus galloprovincialis

Publication . Gomes, Tânia; Pereira, Catarina; Cardoso, Cátia; Serrão Sousa, Vânia; Ribau Teixeira, Margarida; Pinheiro, José P.; Bebianno, Maria João

Silver nanoparticles (Ag NPs) have emerged as one of the most commonly used NPs in a wide range of industrial and commercial applications. This has caused increasing concern about their fate in the environment as well as uptake and potential toxicity towards aquatic organisms. Accordingly, mussels Mytilus galloprovincialis were exposed to 10 μg L(-1) of Ag NPs and ionic silver (Ag+) for 15 days, and biomarkers of oxidative stress and metal accumulation were determined. Accumulation results show that both Ag NPs and Ag+ accumulated in both gills and digestive glands. Antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were activated by Ag NPs and Ag+, showing different antioxidant patterns in both gills and digestive glands. Moreover, metallothionein was inducted in gills, directly related to Ag accumulation, while in the digestive glands only a small fraction of Ag seems to be associated with this protein. Lipid peroxidation was higher in gills exposed to Ag NPs, whereas in the digestive glands only Ag+ induced lipid peroxidation. Ag NPs and Ag+ cause oxidative stress with distinct modes of action and it's not clear if for Ag NPs the observed effects are attributed to free Ag+ ions associated with the nanoparticle effect.

2014Journal article

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