ABSTRACT Estuaries are highly productive ecosystems that have been increasingly affected by anthr... more ABSTRACT Estuaries 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.
DNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decuss... more DNA 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.
Silver nanoparticles (Ag NPs) have emerged as one of the most commonly used NPs in a wide range o... more 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.
CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other c... more CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other commercial applications. Nevertheless, less is known about the mechanisms by which these NPs inflict toxicity in mussels and to what extent it differs from Cu(2+). The aim of this study was to investigate changes in protein expression profiles in mussels Mytilus galloprovincialis exposed for 15 days to CuO NPs and Cu(2+) (10 μg L(-1)) using a proteomic approach. Results demonstrate that CuO NPs and Cu(2+) induced major changes in protein expression in mussels' showing several tissue and metal-dependent responses. CuO NPs showed a higher tendency to up-regulate proteins in the gills and down-regulate in the digestive gland, while Cu(2+) showed the opposite tendency. Distinctive sets of differentially expressed proteins were found, either common or specific to each Cu form and tissue, reflecting different mechanisms involved in their toxicity. Fifteen of the differentially expressed proteins from both tissues were identified by MALDI-TOF-TOF. Identified proteins indicate common response mechanisms induced by CuO NPs and Cu(2+), namely in cytoskeleton and cell structure (actin, α-tubulin, paramyosin), stress response (heat shock cognate 71, putative C1q domain containing protein), transcription regulation (zinc-finger BED domain-containing protein 1, nuclear receptor subfamily 1G) and energy metabolism (ATP synthase F0 subunit 6). CuO NPs alone also had a marked effect on other biological processes, namely oxidative stress (GST), proteolysis (cathepsin L) and apoptosis (caspase 3/7-1). On the other hand, Cu(2+) affected a protein associated with adhesion and mobility, precollagen-D that is associated with the detoxification mechanism of Cu(2+). Protein identification clearly showed that the toxicity of CuO NPs is not solely due to Cu(2+) dissolution and can result in mitochondrial and nucleus stress-induced cell signalling cascades that can lead to apoptosis. While the absence of the mussel genome precluded the identification of other proteins relevant to clarify the effects of CuO NPs in mussels' tissues, proteomics analysis provided additional knowledge of their potential effects at the protein level that after confirmation and validation can be used as putative new biomarkers in nanotoxicology.
The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence u... more 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 (d-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.
CuO NPs are widely used in various industrial and commercial applications. However, little is kno... more CuO NPs are widely used in various industrial and commercial applications. However, little is known about their potential toxicity or fate in the environment. In this study the effects of copper nanoparticles were investigated in the gills of mussels Mytilus galloprovincialis, comparative to Cu(2+). Mussels were exposed to 10 μg Cu·L(-1) of CuO NPs and Cu(2+) for 15 days, and biomarkers of oxidative stress, metal exposure and neurotoxicity evaluated. Results show that mussels accumulated copper in gills and responded differently to CuO NPs and Cu(2+), suggesting distinct modes of action. CuO NPs induced oxidative stress in mussels by overwhelming gills antioxidant defense system, while for Cu(2+) enzymatic activities remained unchanged or increased. CuO NPs and Cu(2+) originated lipid peroxidation in mussels despite different antioxidant efficiency. Moreover, an induction of MT was detected throughout the exposure in mussels exposed to nano and ionic Cu, more evident in CuO NPs exposure. Neurotoxic effects reflected as AChE inhibition were only detected at the end of the exposure period for both forms of copper. In overall, these findings show that filter-feeding organisms are significant targets for nanoparticle exposure and need to be included when evaluating the overall toxicological impact of nanoparticles in the aquatic environment.
Given the wide use of CuO nanoparticles in various industrial and commercial applications they wi... more Given the wide use of CuO nanoparticles in various industrial and commercial applications they will inevitably end up in the aquatic environment. However, little information exists on their biological effects in bivalve species. Accordingly, mussels Mytilus galloprovincialis were exposed to 10 μg Cu L(-1) as CuO nanoparticles and Cu(2+) for 15 days, and biomarkers of oxidative stress (superoxide dismutase, catalase and glutathione peroxidase), damage (lipid peroxidation) and metal exposure (metallothionein) were determined along with Cu accumulation in the digestive glands of mussels. Cu was linearly accumulated with time of exposure in mussels exposed to CuO nanoparticles, while in those exposed to Cu(2+) elimination was significant by day 15. Both forms of Cu cause oxidative stress with distinct modes of action. Exposure to CuO nanoparticles induces lower SOD activity in digestive glands compared to those exposed to Cu(2+), while CAT was only activated after 7 days of exposure to nano and ionic Cu, with contradictory effects after 15 days of exposure and GPX activities were similar. Lipid peroxidation levels increased in both Cu forms despite different antioxidant efficiency. Moreover, a linear induction of metallothionein was detected with time in mussels exposed to CuO nanoparticles, directly related to Cu accumulation, whereas in those exposed to Cu(2+) metallothionein was only induced after 15 days of exposure. Since only a small fraction of soluble Cu fraction was released from CuO nanoparticles, the observed effects seem to be related to the nano form of Cu, with aggregation as a key factor. Overall, our results show that the digestive gland is susceptible to CuO nanoparticles related oxidative stress, and is also the main tissue for their accumulation.
In recent years, Cd-based quantum dots (QDs) have generated interest from the life sciences commu... more In recent years, Cd-based quantum dots (QDs) have generated interest from the life sciences community due to their potential applications in nanomedicine, biology and electronics. However, these engineered nanomaterials can be released into the marine environment, where their environmental health hazards remain unclear. This study investigated the tissue-specific responses related to alterations in the antioxidant defense system induced by CdTe QDs, in comparison with its dissolved counterpart, using the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and dissolved Cd for 14 days at 10μgCdL(-1) and biomarkers of oxidative stress [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (total, Se-independent and Se-dependent GPx) and glutathione-S-transferase (GST) activities] were analyzed along with Cd accumulation in the gills and digestive gland of mussels. Results show that both Cd forms changed mussels' antioxidant responses with distinct modes of action (MoA). There were tissue- and time-dependent differences in the biochemical responses to each Cd form, wherein QDs are more pro-oxidant when compared to dissolved Cd. The gills are the main tissue affected by QDs, with effects related to the increase of SOD, GST and GPx activities, while those of dissolved Cd was associated to the increase of CAT activity, Cd accumulation and exposure time. Digestive gland is a main tissue for accumulation of both Cd forms, but changes in antioxidant enzyme activities are smaller than in gills. A multivariate analysis revealed that the antioxidant patterns are tissue dependent, indicating nano-specific effects possibly associated to oxidative stress and changes in redox homeostasis.
ABSTRACT Estuaries are highly productive ecosystems that have been increasingly affected by anthr... more ABSTRACT Estuaries 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.
DNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decuss... more DNA 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.
Silver nanoparticles (Ag NPs) have emerged as one of the most commonly used NPs in a wide range o... more 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.
CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other c... more CuO NPs are one of the most used metal nanomaterials nowadays with several industrial and other commercial applications. Nevertheless, less is known about the mechanisms by which these NPs inflict toxicity in mussels and to what extent it differs from Cu(2+). The aim of this study was to investigate changes in protein expression profiles in mussels Mytilus galloprovincialis exposed for 15 days to CuO NPs and Cu(2+) (10 μg L(-1)) using a proteomic approach. Results demonstrate that CuO NPs and Cu(2+) induced major changes in protein expression in mussels' showing several tissue and metal-dependent responses. CuO NPs showed a higher tendency to up-regulate proteins in the gills and down-regulate in the digestive gland, while Cu(2+) showed the opposite tendency. Distinctive sets of differentially expressed proteins were found, either common or specific to each Cu form and tissue, reflecting different mechanisms involved in their toxicity. Fifteen of the differentially expressed proteins from both tissues were identified by MALDI-TOF-TOF. Identified proteins indicate common response mechanisms induced by CuO NPs and Cu(2+), namely in cytoskeleton and cell structure (actin, α-tubulin, paramyosin), stress response (heat shock cognate 71, putative C1q domain containing protein), transcription regulation (zinc-finger BED domain-containing protein 1, nuclear receptor subfamily 1G) and energy metabolism (ATP synthase F0 subunit 6). CuO NPs alone also had a marked effect on other biological processes, namely oxidative stress (GST), proteolysis (cathepsin L) and apoptosis (caspase 3/7-1). On the other hand, Cu(2+) affected a protein associated with adhesion and mobility, precollagen-D that is associated with the detoxification mechanism of Cu(2+). Protein identification clearly showed that the toxicity of CuO NPs is not solely due to Cu(2+) dissolution and can result in mitochondrial and nucleus stress-induced cell signalling cascades that can lead to apoptosis. While the absence of the mussel genome precluded the identification of other proteins relevant to clarify the effects of CuO NPs in mussels' tissues, proteomics analysis provided additional knowledge of their potential effects at the protein level that after confirmation and validation can be used as putative new biomarkers in nanotoxicology.
The Ria Formosa lagoon is an ecosystem whose water quality reflects the anthropogenic influence u... more 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 (d-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.
CuO NPs are widely used in various industrial and commercial applications. However, little is kno... more CuO NPs are widely used in various industrial and commercial applications. However, little is known about their potential toxicity or fate in the environment. In this study the effects of copper nanoparticles were investigated in the gills of mussels Mytilus galloprovincialis, comparative to Cu(2+). Mussels were exposed to 10 μg Cu·L(-1) of CuO NPs and Cu(2+) for 15 days, and biomarkers of oxidative stress, metal exposure and neurotoxicity evaluated. Results show that mussels accumulated copper in gills and responded differently to CuO NPs and Cu(2+), suggesting distinct modes of action. CuO NPs induced oxidative stress in mussels by overwhelming gills antioxidant defense system, while for Cu(2+) enzymatic activities remained unchanged or increased. CuO NPs and Cu(2+) originated lipid peroxidation in mussels despite different antioxidant efficiency. Moreover, an induction of MT was detected throughout the exposure in mussels exposed to nano and ionic Cu, more evident in CuO NPs exposure. Neurotoxic effects reflected as AChE inhibition were only detected at the end of the exposure period for both forms of copper. In overall, these findings show that filter-feeding organisms are significant targets for nanoparticle exposure and need to be included when evaluating the overall toxicological impact of nanoparticles in the aquatic environment.
Given the wide use of CuO nanoparticles in various industrial and commercial applications they wi... more Given the wide use of CuO nanoparticles in various industrial and commercial applications they will inevitably end up in the aquatic environment. However, little information exists on their biological effects in bivalve species. Accordingly, mussels Mytilus galloprovincialis were exposed to 10 μg Cu L(-1) as CuO nanoparticles and Cu(2+) for 15 days, and biomarkers of oxidative stress (superoxide dismutase, catalase and glutathione peroxidase), damage (lipid peroxidation) and metal exposure (metallothionein) were determined along with Cu accumulation in the digestive glands of mussels. Cu was linearly accumulated with time of exposure in mussels exposed to CuO nanoparticles, while in those exposed to Cu(2+) elimination was significant by day 15. Both forms of Cu cause oxidative stress with distinct modes of action. Exposure to CuO nanoparticles induces lower SOD activity in digestive glands compared to those exposed to Cu(2+), while CAT was only activated after 7 days of exposure to nano and ionic Cu, with contradictory effects after 15 days of exposure and GPX activities were similar. Lipid peroxidation levels increased in both Cu forms despite different antioxidant efficiency. Moreover, a linear induction of metallothionein was detected with time in mussels exposed to CuO nanoparticles, directly related to Cu accumulation, whereas in those exposed to Cu(2+) metallothionein was only induced after 15 days of exposure. Since only a small fraction of soluble Cu fraction was released from CuO nanoparticles, the observed effects seem to be related to the nano form of Cu, with aggregation as a key factor. Overall, our results show that the digestive gland is susceptible to CuO nanoparticles related oxidative stress, and is also the main tissue for their accumulation.
In recent years, Cd-based quantum dots (QDs) have generated interest from the life sciences commu... more In recent years, Cd-based quantum dots (QDs) have generated interest from the life sciences community due to their potential applications in nanomedicine, biology and electronics. However, these engineered nanomaterials can be released into the marine environment, where their environmental health hazards remain unclear. This study investigated the tissue-specific responses related to alterations in the antioxidant defense system induced by CdTe QDs, in comparison with its dissolved counterpart, using the marine mussel Mytilus galloprovincialis. Mussels were exposed to CdTe QDs and dissolved Cd for 14 days at 10μgCdL(-1) and biomarkers of oxidative stress [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (total, Se-independent and Se-dependent GPx) and glutathione-S-transferase (GST) activities] were analyzed along with Cd accumulation in the gills and digestive gland of mussels. Results show that both Cd forms changed mussels' antioxidant responses with distinct modes of action (MoA). There were tissue- and time-dependent differences in the biochemical responses to each Cd form, wherein QDs are more pro-oxidant when compared to dissolved Cd. The gills are the main tissue affected by QDs, with effects related to the increase of SOD, GST and GPx activities, while those of dissolved Cd was associated to the increase of CAT activity, Cd accumulation and exposure time. Digestive gland is a main tissue for accumulation of both Cd forms, but changes in antioxidant enzyme activities are smaller than in gills. A multivariate analysis revealed that the antioxidant patterns are tissue dependent, indicating nano-specific effects possibly associated to oxidative stress and changes in redox homeostasis.
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