Teresa Balbi

In vivo effects of n-TiO2 on digestive gland and immune function of the marine bivalve mytilus galloprovincialis

Due to the increasing production of nanoparticles (NPs) and their potential release in the aquatic environment, evaluation of their biological impact on aquatic organisms represents a major concern. Suspension feeding invertebrates, in particular bivalve mollusks, may play a role in NP biotransformation and transfer through food webs and may represent a significant target for NP toxicity. In this work, the in vivo effects of titanium dioxide (n-TiO2), one of the most widespread NPs in use, were investigated in the bivalve Mytilus galloprovincialis, largely utilised as a sentinel for marine contamination. Mussels were exposed for 96h to different concentrations of n-TiO2 suspensions (1, 10 and 100μgL(-1)) and multiple responses were evaluated in the digestive gland and immune cells, the haemocytes. In the digestive gland, n-TiO2 affected lysosomal and oxidative stress biomarkers and decreased transcription of antioxidant and immune-related genes. In the haemocytes, n-TiO2 decreased lysosomal membrane stability-LMS and phagocytosis, increased oxyradical production and transcription of antimicrobial peptides; moreover, pre-apoptotic processes were observed. The effects of n-TiO2 on digestive gland and haemocytes were distinct, also depending on the endpoint and on nominal NP concentrations, with many significant responses elicited by the lowest concentrations tested. The results show that n-TiO2, at concentrations close to predicted environmental levels, significantly affected different functional and molecular parameters of mussel digestive gland and immune cells. In particular, the observed changes in immune parameters that represent significant biomarkers of exposure at the organism level suggest that exposure to n-TiO2 may pose a serious risk to mussel health.

Interactive effects of n-TiO2 and 2,3,7,8-TCDD on the marine bivalve Mytilus galloprovincialis

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. The bivalve Mytilus sp, largely utilized as a sentinel for marine contamination, has been shown to represent a significant target for different types of NP, including n-TiO2, one of the most widespread in use. In this work, the possible interactive effects of n-TiO2 and 2,3,7,8-TCDD, chosen as models of NP and organic contaminant, respectively, were investigated in Mytilus galloprovincialis. In vitro experiments with n-TiO2 and TCDD, alone and in combination, were carried out in different conditions (concentrations and times of exposure), depending on the target (hemocytes, gill cells and biopsies) and the endpoint measured. Mussels were also exposed in vivo to n-TiO2 (100 μg L(-1)) or to TCDD (0.25 μg L(-1)), alone and in combination, for 96 h. A wide range of biomarkers, from molecular to tissue level, were measured: lysosomal membrane stability and phagocytosis in hemocytes, ATP-binding cassette efflux transporters in gills (gene transcription and efflux activity), several biomarkers of genotoxicity in gill and digestive cells (DNA damage, random amplified polymorphic DNA-RAPD changes), lysosomal biomarkers and transcription of selected genes in the digestive gland. The results demonstrate that n-TiO2 and TCDD can exert synergistic or antagonistic effects, depending on experimental condition, cell/tissue and type of measured response. Some of these interactions may result from a significant increase in TCDD accumulation in whole mussel organisms in the presence of n-TiO2, indicating a Trojan horse effect. The results represent the most extensive data obtained so far on the sub-lethal effects of NPs and organic contaminants in aquatic organisms. Moreover, these data extend the knowledge on the molecular and cellular targets of NPs in bivalves.

Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis

We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media.

Co-exposure to n-TiO2 and Cd2 + results in interactive effects on biomarker responses but not in increased toxicity in the marine bivalve M. galloprovincialis

The increasing production of nanoparticles (NPs) will lead to their release into the aquatic environment, where they could modify the bioavailability/bioconcentration and consequent biological impact of other contaminants. Interactive effects of n-TiO2, one of the most widespread NP type, and Cd(2+), a common heavy metal pollutant, have been described in freshwater species, whereas no information is available in marine organisms. In this work, the effects of co-exposure to n-TiO2 and Cd(2+) were investigated in the marine bivalve Mytilus galloprovincialis. Experimental conditions (100 μg/L, 96 h), were chosen in order to induce early but measurable stress responses (biomarkers) without toxicity. Several biomarkers, from molecular to tissue level, were measured in hemolymph and digestive gland; the effects on embryo development were also evaluated. In hemolymph, Cd(2+) abolished the increase in immune parameters induced by n-TiO2 (NO production and lysozyme activity). In the digestive gland, distinct interactive effects of n-TiO2 and Cd(2+) were observed on different lysosomal biomarkers (lysosomal membrane stability, lipid accumulation and lysosome/cytoplasm volume ratio) and transcription of the immune genes lysozyme and toll-like receptor (TLR). However, n-TiO2 did not affect specific metal-induced responses (metallothionein induction) and tissue metal accumulation. Cd(2+) alone, but not in combination with n-TiO2, affected embryo development. The interactive effects observed on different biomarkers were not apparently due to differences in bioavailability/bioaccumulation of Cd(2+) in the presence of n-TiO2 agglomerates; these effects may result from interactions of either contaminant with both common and distinct targets/mechanisms of action at different levels of biological organization. Overall, the results indicate that co-exposure to n-TiO2 and Cd(2+) did not result in increased adverse effects in M. galloprovincialis. These data underline the need for further knowledge on the potential interactions of NPs with existing contaminants in marine organisms.

Interactive effects of nanoparticles with other contaminants in aquatic organisms: Friend or foe?

The increasing production and use of nanoparticles (NPs) will lead to their release into the aquatic environment, posing a potential threat to the health of aquatic organisms. Both in the water phase and in the sediments NPs could mix and interact with other pollutants, such as organic xenobiotics and heavy metals, leading to possible changes in their bioavailability/bioconcentration/toxicity. However, whether these interactive effects may lead to increased harmful effects in marine organisms is largely unknown. In this work, available data mainly obtained on carbon based NPs and n-TiO2, as examples of widespread NPs, in aquatic organisms are reviewed. Moreover, data are summarized on the interactive effects of n-TiO2 with 2,3,7,8-TCDD and Cd(2+), chosen as examples of common and persistent organic and inorganic contaminants, respectively, in the model marine bivalve Mytilus. The results reveal complex and often unexpected interactive responses of NPs with other pollutants, depending on type of contaminant and the endpoint measured, as well as differences in bioaccumulation. The results are discussed in relation with data obtained in freshwater organisms. Overall, information available so far indicate that interactive effects of NPs with other contaminants do not necessarily lead to increased toxicity or harmful effects in aquatic organisms.

Interactions between Mytilus galloprovincialis hemocytes and the bivalve pathogens Vibrio aestuarianus 01/032 and Vibrio splendidus LGP32.

Marine bivalves can accumulate large numbers of bacteria, in particular Vibrio species, whose persistence in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating hemocytes and hemolymph soluble factors. The interactions between vibrios and hemolymph have been investigated, in particular in bivalve species susceptible to infection by certain Vibrio spp. and strains. In this work, the effects of two bivalve pathogens, Vibrio splendidus LGP32 (V.s.) and Vibrio aestuarianus 01/032 (V.a.), isolated from oyster mortality outbreaks, on the hemocytes of Mytilus galloprovincialis were investigated. In vitro, V.s., but not V.a., induced a dramatic decrease in lysosomal membrane stability-LMS in the hemocytes; both vibrios induced a moderate lysozyme release, with V.s. > V.a.. The V.s.-induced decrease in LMS was mediated by activation of PI-3Kinase, as shown by use of different kinase inhibitors. TEM analysis showed rapid internalization of both vibrios; however, V.s. lead to cellular and lysosomal damage and was able to survive within the hemocytes, whereas significant killing of V.a. was observed. In vivo, in mussels challenged with either vibrio and sampled at 6, 24 and 96 h post-injection, transient decreases in hemocyte LMS and progressive increases in serum lysozyme activity were observed, with V.s. > V.a.. Moreover, whereas V.a. was efficiently cleared from hemolymph, V.s. showed significant growth, that was maximal at 24 h p.i. when lowest LMS values were recorded in the hemocytes. Both vibrios also induced significant decreases in LMS in the digestive gland, again with V.s. > V.a.. The results indicate distinct interactions between mussel hemocytes and the two vibrio strains tested. The effects of V.s. may be due to the capacity of this strain to interfere with the signaling pathways involved in hemocyte function, thus escaping the bactericidal activity of the host cell, as observed for certain mammalian pathogens. Although V.s. is considered not pathogenic to Mytilus, this vibrio strain can affect the lysosomal function at the cellular and tissue level, thus leading to stressful conditions.

Seasonal variability of different biomarkers in mussels (Mytilus galloprovincialis) farmed at different sites of the Gulf of La Spezia, Ligurian sea, Italy.

Mussels (Mytilus spp.) are worldwide utilized in marine biomonitoring by a multi-biomarker approach. However, for a correct interpretation of different biomarker responses, information is needed on their natural seasonal variability due to environmental/physiological factors. In this work, the seasonal variations of different biomarkers were investigated in M. galloprovincialis from 4 different sites from the gulf of La Spezia (Ligurian sea, Italy), an intensive rearing area in the north-western Mediterranean near La Spezia harbor, an important commercial and touristic port. Lysosomal membrane stability-LMS, stress on stress-SoS, phagocytosis, tissue metallothionein-MT content, oxidative stress related enzyme activities (GST, catalase), and nitric oxide (NO) production were evaluated. The results underline the importance of LMS and SoS as core descriptors of the mussel health status in relation to seasonal variations in temperature and reproduction. These data represent the baseline information for ongoing biomonitoring studies related to dredging activities in this area.

Utilization of Mytilus digestive gland cells for the in vitro screening of potential metabolic disruptors in aquatic invertebrates

In vertebrate systems, many endocrine disruptors (EDs) can also interfere with energy and lipid metabolism, thus acting as metabolic disruptors. At the cellular level, these effects are mainly mediated by interactions with nuclear receptors/transcription factors, leading to the modulation of genes involved in lipid homeostasis, as well as by rapid, receptor-independent pathways. Several potential metabolic disruptors are found in aquatic environments. In fish, different EDs have been shown to affect hepatic lipid homeostasis both in vivo and in vitro. However, little information is available in aquatic invertebrates due to our poor knowledge of the regulatory pathways of lipid metabolism. In this work, primary cell cultures from the digestive gland of the bivalve Mytilus galloprovincialis were utilized to investigate the effects of model EDs (bisphenol A (BPA) and perfluorooctane sulphonate (PFOS)) on lipid homeostasis. Both compounds (at 24 and 3h of exposure) increased intracellular lipid and tryglyceride-TAG content, with strongest effects of PFOS at 10-7M. Acyl-CoA oxidase activity was unaffected, whereas some changes in the activity of glycolytic, antioxidant/biotransformation enzymes were observed; however, no clear relationship was found with lipid accumulation. Evaluation of mitochondrial membrane potential Δψm and determination of extracellular TAG content indicate that PFOS interferes with mitochondrial function and lipid secretion, whereas BPA mainly affects lipid secretion. Experiments with specific inhibitors showed that activation of PI-3 kinase and extracellularly regulated mitogen-activated protein kinase (ERK MAPK) plays a key role in mediating lipid accumulation. Mussel digestive gland cells represent a simple in vitro model for screening the metabolic effects of EDs in marine invertebrates.

Diclofenac affects early embryo development in the marine bivalve Mytilus galloprovincialis

Diclofenac-DCF, one of the most widely prescribed non-steroidal anti-inflammatory drug, is globally detected in environmental compartments. Due to its occurrence in freshwater and potential impact on aquatic organisms, it has been added to the watch list of chemicals in the EU Water Directive; consequently, research on the impact of DCF in model aquatic organisms has great regulatory implications towards ecosystem health. DCF is also detected in coastal waters at concentrations from ng/L to 1 μg/L, as well as in marine organisms, such as the mussel Mytilus. Increasing evidence indicates that environmental concentrations of DCF have multiple impacts in adult mussels. Moreover, in M. galloprovincialis, DCF has been shown to affect early embryo development. The developmental effects of DCF in mussels were further investigated. DFC (1 and 10 μg/L) was added at different times post-fertilization (30 min and 24 hpf) and the effects were compared in the 48 hpf embryotoxicity assay. Shell mineralization and morphology were investigated by polarized light microscopy, X-Ray Spectrometry-XRD and Scanning Electron Microscopy-SEM. Transcriptional profiles of 12 selected genes physiologically regulated across early embryo development were assessed at 24 and 48 hpf. DCF induced shell malformations, irrespectively of concentration and time of exposure. DCF phenotypes were characterized by convex hinges, undulated edges, fractured shells. However, no changes in biomineralization were observed. DCF affected gene transcription at both times pf, in particular at 1 μg/L. The most affected genes were those involved in early shell formation (CS, CA, EP) and biotransformation (ABCB, GST). The results confirm that Mytilus early development represents a significant target for environmental concentrations of DCF. These data underline how the standard embryotoxicity assay, in combination with a structural and transcriptomic approach, represents a powerful tool for evaluating the early impact of pharmaceuticals on mussel embryos, and identification of the possible underlying mechanisms of action.

Cytotoxicity of CeO 2 nanoparticles using in vitro assay with Mytilus galloprovincialis hemocytes: Relevance of zeta potential, shape and biocorona formation

Over the last decades, the growth in nanotechnology has provoked an increase in the number of its applications and consumer products that incorporate nanomaterials in their formulation. Metal nanoparticles are released to the marine environment and they can interact with cells by colloids forces establish a nano-bio interface. This interface can be compatible or generate bioadverse effects to cells. The daily use of CeO2 nanoparticles (CeO2 NPs) in industrial catalysis, sunscreen, fuel cells, fuel additives and biomedicine and their potential release into aquatic environments has turned them into a new emerging pollutant of concern. It is necessary to assess of effects of CeO2 NPs in aquatic organisms and understand the potential mechanisms of action of CeO2 NP toxicity to improve our knowledge about the intrinsic and extrinsic characteristic of CeO2 NPs and the interaction of CeO2 NPs with biomolecules in different environment and biological fluids. The conserved innate immune system of bivalves represents a useful tool for studying immunoregulatory responses when cells are exposed to NPs. In this context, the effects of two different CeO2 NPs with different physico-chemical characteristics (size, shape, zeta potential and Ce+3/Ce+4 ratio) and different behavior with biomolecules in plasma fluid were studied in a series of in vitro assays using primary hemocytes from Mytilus galloprovincialis. Different cellular responses such as lysosome membrane stability, phagocytosis capacity and extracellular reactive oxygen species (ROS) production were evaluated. Our results indicate that the agglomeration state of CeO2 NPs in the exposure media did not appear to have a substantial role in particle effects, while differences in shape, zeta potential and biocorona formation in NPs appear to be important in provoking negative impacts on hemocytes. The negative charge and the rounded shape of CeO2 NPs, which formed Cu, Zn-SOD biocorona in hemolymph serum (HS), triggered higher changes in the biomarker of stress (LMS) and immunological parameters (ROS and phagocytosis capacity). On the other hand, the almost neutral surface charge and well-faceted shape of CeO2 NPs did not show either biocorona formation in HS under tested conditions or significant responses. According to the results, the most relevant conclusion of this work is that not only the physicochemical characterization of CeO2 NPs plays an important role in NPs toxicity but also the study of the interaction of NPs with biological fluids is essential to know it behavior and toxicity at cellular level.