Harnessing the Bioactive Potential of Limonium spathulatum (Desf.) Kuntze: Insights into Enzyme Inhibition and Phytochemical Profile.

This study assessed the halophyte species Limonium spathulatum (Desf.) as a possible source of natural ingredients with the capacity to inhibit enzymes related to relevant human health disorders and food browning. Extracts using food-grade solvents such as water and ethanol were prepared by maceration from dried L. spathulatum leaves. They were evaluated for in vitro inhibition activity of enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), α-glucosidase, tyrosinase and lipase, related to Alzheimer's disease, type-2-diabetes mellitus, skin hyperpigmentation, and obesity, respectively. These extracts were also appraised for in vitro acute toxicity on tumoral and non-tumoral cell lines and their chemical composition by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). The extracts were more effective towards BChE than AChE. The best results were obtained with the hydroethanolic and water extracts, with IC50 values of 0.03 mg/mL and 0.06 mg/mL, respectively. The hydroethanolic extract had the highest capacity to inhibit α-glucosidase (IC50: 0.04 mg/mL), higher than the positive control used (acarbose, IC50 = 3.14 mg/mL). The ethanol extract displayed the best inhibitory activity against tyrosinase (IC50 = 0.34 mg/mL). The tested samples did not inhibit lipase and exhibited low to moderate cytotoxic activity against the tested cell lines. The hydroethanolic extract had a higher diversity of compounds, followed by the ethanol and water samples. Similar molecules were identified in all the extracts and were mainly hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids. Taken together, these results suggest that L. spathulatum should be further explored as a source of bioactive ingredients for the food, cosmetic, and pharmaceutical industries.

Nutritional anti-nutritional chemical composition and antioxidant activities of the leaves of the sea cliff dwelling species Limonium spathulatum (Desf.) Kuntze.

This work explored the nutritional and antioxidant properties of the leaves of the halophytic species Limonium spathulatum (Desf.) Kuntze from Tunisian sea cliffs. Furthermore, the analysis of the total phenolics and flavonoids contents and their individual compounds using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS) were also studied. L. spathulatum leaves had high levels of moisture, ash, neutral detergent fiber, and acid detergent fiber, but low concentrations of crude protein, crude fat and acid detergent lignin. It contained low carbohydrates levels, and low energetic values. The most abundant macroelements were Cl, Na and Ca while the microelements detected in the highest levels were Fe and Zn. No relevant α-amylase inhibition was observed, and no toxic metals (Pb and Cd) and phytic acid were detected. The ethanol and the hydroethanolic extracts had the highest capacity to scavenge free radicals, to chelate iron and copper and to inhibit lipid peroxidation. The same samples were also the most active towards oxidative haemolysis. These extracts contained high total phenolic and flavonoid contents. HPLC analysis, performed on ethanolic extracts identified 58 individual compounds known for their high antioxidant actvitiy including hydroxybenzoic acids (gallic, syringic acids), hydroxycinnamic acids (caffeic, coumaric, ferulic acids) and flavonoids (catechin, epigallocatechin gallate and naringin).In conclusion, the leaves of Tunisian accession of L. spathulatum were good source of minerals and fibers useful in the human diet for attaining nutritional sufficiency. The high in vitro and ex vitro antioxidant activities associated with high favonoids contents and compounds suggest the possibility to use the extracts of L. spathulatum in herbal products with the aim of improving general health and well-being, and/or as food additives for preventing lipid oxidation of lipid-rich foods.

The Medicinal Halophyte Frankenia laevis L. (Sea Heath) Has In Vitro Antioxidant Activity, α-Glucosidase Inhibition, and Cytotoxicity towards Hepatocarcinoma Cells.

This work explored the medicinal halophyte Frankenia laevis L. (sea heath) as a potential source of bioactive natural products. In this sense, methanol and dichloromethane extracts were prepared from aerial organs containing flowers, leaves and stems, and were profiled for their chemical composition using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). The extracts were evaluated for their in vitro antioxidant capacity using five complementary methods: enzyme inhibitory effects on enzymes related with neurodegeneration (acetyl (AChE) and butyrylcholinesterase (BuChE)), Type 2 diabetes (α-glucosidase and α-amylase), hyperpigmentation/food oxidation (tyrosinase), and cytotoxicity towards human hepatocarcinoma (HepG2) cells. Fifty-one molecules were identified in the extracts, including several derivatives of phenolic acids, lignans and flavonoids, monoterpenes, and hydroxylated derivatives of linoleic acid. The methanol extract was effective in DPPH and ABTS radical scavenging (EC50 = 0.25 and 0.65 mg/mL, respectively), copper chelation (EC50 = 0.78 mg/mL), and iron reduction (EC50 = 0.51 mg/mL) activities, whereas the dichloromethane extract had high iron chelating ability (EC50 = 0.76 mg/mL). Both extracts showed the capacity to inhibit α-glucosidase, especially the dichloromethane (EC50 = 0.52 mg/mL). This extract also exerted a significant selective cytotoxicity towards HepG2 cells (EC50 = 52.1 µg/mL, SI > 1.9). In conclusion, extracts from the aerial parts of sea heath were shown to be a promising source of natural products for pharmaceutical and/or food additive applications due to their high antioxidant, anti-diabetic, and cytotoxic properties.

Exploring the Biotechnological Value of Marine Invertebrates: A Closer Look at the Biochemical and Antioxidant Properties of Sabella spallanzanii and Microcosmus squamiger.

Sabella spallanzanii and Microcosmus squamiger were profiled for proximate composition, minerals, amino acids, fatty acids (FA), carotenoids, radical scavenging activity on the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC) and iron and copper chelating properties. Microcosmus squamiger had the highest level of moisture and crude protein, S. spallanzanii was enriched in crude fat and ash. Both species had similar levels of carbohydrates and energy. There was a prevalence of arginine and glycine in S. spallanzanii, and of taurine in M. squamiger. The most abundant minerals in both species were Na, Ca, and K. The methanol extract of S. spallanzanii had metal chelating properties towards copper and iron, while the methanol extract of M. squamiger was able to chelate copper. M. squamiger extracts had similar ORAC values. Fucoxanthinol and fucoxanthin were the major carotenoids in the M. squamiger dichloromethane extract. Saturated FA were more abundant than unsaturated ones in methanol extracts, and unsaturated FA prevailed in the dichloromethane extracts. Palmitic acid was the predominant FA in methanol extracts, whereas eicosapentaenoic (EPA) and dihomo-γ-linolenic acids were the major compounds in dichloromethane extracts. Low n-6/n-3 ratios were obtained. Our results suggests that both species could be explored as sources of bioactive ingredients with multiple applications.

Metabolomic Profile and Biological Properties of Sea Lavender (Limonium algarvense Erben) Plants Cultivated with Aquaculture Wastewaters: Implications for Its Use in Herbal Formulations and Food Additives.

Water extracts from sea lavender (Limonium algarvense Erben) plants cultivated in greenhouse conditions and irrigated with freshwater and saline aquaculture effluents were evaluated for metabolomics by liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS/MS), and functional properties by in vitro and ex vivo methods. In vitro antioxidant methods included radical scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric-reducing antioxidant power (FRAP), and copper and iron chelating assets. Flowers' extracts had the highest compounds' diversity (flavonoids and its derivatives) and strongest in vitro antioxidant activity. These extracts were further tested for ex vivo antioxidant properties by oxidative haemolysis inhibition (OxHLIA), lipid peroxidation inhibition by thiobarbituric acid reactive substances (TBARS) formation, and anti-melanogenic, anti-tyrosinase, anti-inflammation, and cytotoxicity. Extract from plants irrigated with 300 mM NaCl was the most active towards TBARS (IC50 = 81 µg/mL) and tyrosinase (IC50 = 873 µg/mL). In OxHLIA, the activity was similar for fresh- and saltwater-irrigated plants (300 mM NaCl; IC50 = 136 and 140 µg/mL, respectively). Samples had no anti-inflammatory and anti-melanogenic abilities and were not toxic. Our results suggest that sea lavender cultivated under saline conditions could provide a flavonoid-rich water extract with antioxidant and anti-tyrosinase properties with potential use as a food preservative or as a functional ingredient in herbal supplements.

A Review on Sarcocornia Species: Ethnopharmacology, Nutritional Properties, Phytochemistry, Biological Activities and Propagation.

Sarcocornia A. J. Scott is a halophytic edible succulent plant belonging to the Amaranthaceae family. To date, the genus includes 28 species distributed worldwide in saline environments, usually salt marshes. Sarcocornia (Scott) is similar to Salicornia (L.), which has a recognized commercial value in morphological and taxonomical traits. Species of both genera are commonly named samphire or glassworts in Europe, and their fleshy shoots are commercialized under their traditional names. Due to their nutritional, organoleptic and medicinal properties, Sarcocornia species have a high economic potential in various biotechnology sectors. Being highly tolerant to salt, they can be cultivated in saline conditions, and dissimilar to Salicornia, they are perennial, i.e., they can be harvested year-round. Therefore, Sarcocornia species are considered promising gourmet vegetables to be explored in the context of climate change, soil and water salinization and eco-sustainability. We hereby put together and reviewed the most relevant information on Sarcocornia taxonomy, morphology, nutritional and pharmacological properties, uses in ethnomedicine, potential applications in biotechnology, and propagation strategies.

In Vitro Anti-Trypanosoma cruzi Activity of Halophytes from Southern Portugal Reloaded: A Special Focus on Sea Fennel (Crithmum maritimum L.).

Marine halophytes are an outstanding reservoir of natural products and several species have anti-infectious traditional uses. However, reports about their potential use against neglected tropical ailments, such as Chagas disease, are scarce. This work evaluated for the first time the in vitro anti-Trypanosoma cruzi activity of extracts from the aromatic and medicinal species Helichrysum italicum subsp. picardii (Boiss. & Reut.) Franco (Asteraceae, everlasting) and Crithmum maritimum L. (Apiaceae, sea fennel). For that purpose, decoctions, tinctures, and essential oils from everlasting's flowers and sea fennel's stems, leaves, and flowers were tested against intracellular amastigotes of two T. cruzi strains. The extract from the sea fennel flower decoction displayed significant anti-trypanosomal activity and no toxicity towards the host cell (EC50 = 17.7 µg/mL, selectivity index > 5.65). Subsequent fractionation of this extract afforded 5 fractions that were re-tested in the same model of anti-parasitic activity. Fraction 1 was the most active and selective (EC50 = 0.47 µg/mL, selectivity index = 59.6) and was submitted to preparative thin-layer chromatography. One major compound was identified, falcarindiol, which was likely the one responsible for the observed anti-trypanosomal activity. This was confirmed using a commercially sourced molecule. Target-fishing studies showed falcarindiol as a ligand of T. cruzi spermidine synthase, pointing to a potential enzyme-inhibiting anti-trypanosomal mechanism of action. Overall, this work shows that sea fennel can provide effective anti-parasitic molecule(s) with potential pharmacological applications in the treatment of CD.

Greener Is Better: First Approach for the Use of Natural Deep Eutectic Solvents (NADES) to Extract Antioxidants from the Medicinal Halophyte Polygonum maritimum L.

In this study, natural deep eutectic solvents (NADES) formed by choline chloride (ChCl), sucrose, fructose, glucose, and xylose, were used to extract antioxidants from the halophyte Polygonum maritimum L. (sea knotgrass) and compared with conventional solvents (ethanol and acetone). NADES and conventional extracts were made by an ultrasound-assisted procedure and evaluated for in vitro antioxidant properties by the radical scavenging activity (RSA) on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, oxygen radical absorbance capacity (ORAC), and copper chelating activity (CCA). Samples were profiled by liquid chromatography (LC)-electrospray ionization (ESI)-QTOF-MS analysis. ChCl:fructose was more efficient in the DPPH assay, than the acetone extract. ChCl:sucrose and ChCl:fructose extracts had the highest ORAC when compared with the acetone extract. NADES extracts had higher CCA, than the acetone extract. The phenolic composition of the NADES extracts was less complex than the conventional extracts, but the proportions of major antioxidants, such as flavonols and flavan-3-ols, were similar in all the solvents. Myricitrin was the major flavonoid in all of the samples, while gallic acid was the main phenolic acid in the conventional extracts and present in a greater amount in ChCl:fructose. Results suggest that NADES containing ChCl and sucrose/fructose can replace conventional solvents, especially acetone, in the extraction of antioxidants from sea knotgrass.

In vitro enzyme inhibitory and anti-oxidant properties, cytotoxicity and chemical composition of the halophyte Malcolmia littorea (L.) R.Br. (Brassicaceae).

This work reports for the first time the in vitro anti-oxidant (towards DPPH, ABTS, copper and iron), enzymatic inhibitory (on AChE, BuChE, α-glucosidase, α-amylase and tyrosinase), cytotoxicity (towards HepG2 and HEK 293 cells), and metabolomics (by HPLC-MS) of extracts from organs of Malcolmia littorea (L.) R.Br. Extracts were constituted mainly by phenolic acids and flavonoids, and main compounds were salicylic acid and luteolin-7-O-glucoside. Samples showed reduced radical scavenging and metal chelating capacity, and only the methanol extracts reduced iron. The root's ethanol and methanol extracts, and the aerial organ's ethanol extract exhibited the highest AChE inhibition. The root's ethanol extract displayed dual anti-cholinesterase activity. Samples showed a low capacity to inhibit α-amylase, but a high α-glucosidase inhibition was obtained with the root's and flower's ethanol extracts, and flower's methanol extract. Overall, samples displayed a high inhibition against tyrosinase, reduced HepG2 cellular viability and were less toxic towards HEK 293 cells.

Phenolic Profile, Toxicity, Enzyme Inhibition, In Silico Studies, and Antioxidant Properties of Cakile maritima Scop. (Brassicaceae) from Southern Portugal.

Cakile maritima Scop. (sea rocket) is an edible halophyte plant with several ethnomedicinal uses. This work reports the chemical profile and bioactivities of food grade extracts from sea rocket organs. Toxicity was determined on mammalian cells, and phenolic profiling and the quantitation of the main metabolites were made by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Enzymatic inhibition was determined towards acetyl- and butyrylcholinesterase (AChE, BuChE), α-glucosidase, α-amylase, and tyrosinase. Docking studies were performed to tyrosinase, on the major metabolites, and samples were tested for antioxidant properties. Extracts were not toxic, were constituted mainly by flavonoids, and some compounds (roseoside and oleuropein) are here described for the first time in the species. The aerial organs' ethanol extract had relevant activity towards 2,2-diphenyl-1-picrylhydrazyl [DPPH, half maximal inhibitory concentration (IC50) = 0.59 mg/mL], and ferric-reducing activity power (FRAP, IC50 = 0.99 mg/mL). All samples were more active towards AChE than on BuChE. The ethanol fruits' extract inhibited α-glucosidase [2.19 mmol of equivalent of acarbose (ACAE)/g]. Samples were active against tyrosinase, especially the aerial organs' ethanol extracts [25.9 mg of equivalent of kojic acid (KAE)/g]. Quercetin and kaempferol glycosides fit well into the enzymatic pocket of tyrosinase. Our results suggest sea rocket as a candidate to be further explored as a source of bioactive products.

Proteomic response of mussels Mytilus galloprovincialis exposed to CuO NPs and Cu²âº: an exploratory biomarker discovery.

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.

Effects of silver nanoparticles exposure in the mussel Mytilus galloprovincialis.

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.

Genotoxicity in two bivalve species from a coastal lagoon in the south of Portugal.

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.

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

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.

Accumulation and toxicity of copper oxide nanoparticles in the digestive gland of Mytilus galloprovincialis.

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.

Effects of copper nanoparticles exposure in the mussel Mytilus galloprovincialis.

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.