Estrogenic compounds as exogenous modulators of physiological functions in molluscs: Signaling pathways and biological responses

Molluscs have been widely utilized to evaluate the effects of estrogenic compounds, one of the most widespread classes of Endocrine Disrupting Chemicals-EDCs. However, knowledge on steroid signaling and metabolism in molluscs has considerably increased in the last decade: from these studies, a considerable debate emerged on the role of 'natural' steroids in physiology, in particular in reproduction, of this invertebrate group. In this work, available information on the effects and mechanisms of action of estrogens in molluscs will be reviewed, with particular emphasis on bivalves that, widespread in aquatic ecosystems, are most likely affected by exposure to estrogenic EDCs. Recent advances in steroid uptake and metabolism, and estrogen receptors-ERs in molluscs, as well as in estrogen signaling in vertebrates, will be considered. The results so far obtained with 17\u3b2-estradiol and different estrogenic compounds in the model bivalve Mytilus spp., demonstrate specific effects on immune function, development and metabolism. Transcriptomic data reveal non genomic estrogen signaling pathways in mussel tissues that are supported by new observations at the cellular level. In vitro and in vivo data show, through independent lines of evidence, that estrogens act through non-genomic signaling pathways in bivalves. In this light, regardless of whether molluscs synthesize estrogens de novo or not, and despite their ERs are not directly activated by ligand binding, estrogens can interact with multiple signaling components, leading to modulation of different physiological functions. Increasing knowledge in endocrine physiology of molluscs will provide a framework for a better evaluation and interpretation of data on the impact of estrogenic EDCs in this invertebrate group

Physiological Roles of Serotonin in Bivalves: Possible Interference by Environmental Chemicals Resulting in Neuroendocrine Disruption

Contaminants of Emerging Concerns (CECs) are defined as chemicals not commonly monitored in aquatic ecosystems, but with the potential to cause adverse effects on biota. CECs include Endocrine Disrupting Chemicals (EDCs) and Neuro-Endocrine disruptors (NEDs) of vertebrates. However, most invertebrates only rely on neuroendocrine systems to maintain homeostatic processes. Although conserved neuroendocrine components have been characterized in ecologically relevant groups, limited knowledge on invertebrate neuroendocrinology makes it difficult to define EDCs and NEDs in most species. The monoamine serotonin (5-hydroxytryptamine, 5-HT) acts both as a neurotransmitter and as a peripheral hormone in mammals. In molluscs, 5-HT is involved in multiple physiological roles and molecular components of the serotonergic system have been identified. This review is focused on the effects of CECs on the serotonergic system of bivalve molluscs. Bivalves are widespread in all aquatic environments, estuarine and coastal areas in particular, where they are exposed to a variety of chemicals. In bivalves, 5-HT is involved in gametogenesis and spawning, oocyte maturation and sperm motility, regulates heart function, gill ciliary beating, mantle/siphon function, the ‘‘catch’’ state of smooth muscle and immune responses. Components of 5-HT transduction (receptors and signaling pathways) are being identified in several bivalve species. Different CECs have been shown to affect bivalve serotonergic system. This particularly applies to antidepressants, among the most commonly detected human pharmaceuticals in the aquatic environment. In particular, selective serotonin reuptake inhibitors (SSRIs) are frequently detected in seawater and in bivalve tissues. Information available on the effects and mechanisms of action of SSRIs on the serotonergic system of adult bivalves is summarized. Data are also reported on the effects of CECs on development of neuroendocrine pathways of early larval stages, in particular on the effects of model EDCs in the marine mussel Mytilus galloprovincialis. Overall, available data point at the serotonergic system as a sensitive target for neuroendocrine disruption in bivalves. The results contribute drawing Adverse Outcome Pathways (AOPs) for model EDCs and SSRIs in larvae and adults. However, basic research on neuroendocrine signaling is still needed to evaluate the potential impact of neuroendocrine disruptors in key invertebrate groups of aquatic ecosystems

Impact of bisphenol A (BPA) on early embryo development in the marine mussel Mytilus galloprovincialis: Effects on gene transcription.

Bisphenol A (BPA), a monomer used in plastic manufacturing, is weakly estrogenic and a potential endocrine disruptor in mammals. Although it degrades quickly, it is pseudo-persistent in the environment because of continual inputs, with reported concentrations in aquatic environments between 0.0005 and 12 \u3bcg/L. BPA represents a potential concern for aquatic ecosystems, as shown by its reproductive and developmental effects in aquatic vertebrates. In invertebrates, endocrine-related effects of BPA were observed in different species and experimental conditions, with often conflicting results, indicating that the sensitivity to this compound can vary considerably among related taxa. In the marine mussel Mytilus galloprovincialis BPA was recently shown to affect early development at environmental concentrations. In this work, the possible effects of BPA on mussel embryos were investigated at the molecular level by evaluating transcription of 13 genes, selected on the basis of their biological functions in adult mussels. Gene expression was first evaluated in trocophorae and D-veligers (24 and 48 h post fertilization) grown in physiological conditions, in comparison with unfertilized eggs. Basal expressions showed a general up-regulation during development, with distinct transcript levels in trocophorae and D-veligers. Exposure of fertilized eggs to BPA (10 \u3bcg/L) induced a general upregulation at 24 h pf, followed by down regulation at 48 h pf. Mytilus Estrogen Receptors, serotonin receptor and genes involved in biomineralization (Carbonic Anydrase and Extrapallial Protein) were the most affected by BPA exposure. At 48 h pf, changes in gene expression were associated with irregularities in shell formation, as shown by scanning electron microscopy (SEM), indicating that the formation of the first shelled embryo, a key step in mussel development, represents a sensitive target for BPA. Similar results were obtained with the natural estrogen 17\u3b2-estradiol. The results demonstrate that BPA and E2 can affect Mytilus early development through dysregulation of gene transcription

Photocatalytic Fe-doped n-TiO2: From synthesis to utilization of in vitro cell models for screening human and environmental nanosafety

The utilization of different types nanomaterials (NMs) in environmental remediation and wastewater treatment requires information on the potential harmful effects on human and environmental health. In this light, the utilization of human cell models together with cells from lower organisms, representative of different environmental compartments, could represent a valuable tool for the in vitro screening of the potential toxicity of different NMs used in nanoremediation. Among NMs, n-TiO2, because of its peculiar optical and chemical properties, is widely applied for photosensitized UV oxidation of organic pollutants. Moreover, development in design of metal- and non metal- doped TiO2 with extended photocatalytic activity in the visible region represents the subject of ongoing research. In this work, the cytotoxic effects of three different types of recently synthetized Fe-doped n-TiO2 were compared in two cell models widely utilized for screening cellular toxicity of NMs in humans and aquatic organisms, human vascular endothelial cells (HECV) and immune cells (hemocytes) of the marine invertebrate, the mussel Mytilus spp, respectively. Parallel studies were carried out using N-doped n-TiO2. The results indicate both distinct and common behavior (agglomeration state) in different media (human cell culture medium and mussel hemolymph serum) and biological effects (cytotoxicity, nitric oxide production) of different types of doped- n-TiO2 in different cell models. Although in vitro studies represent a first step in the toxicological assessment of NMs, studies comparing their effects on human and aquatic invertebrate cells that take into account the effects of different exposure media represent an useful tool for evaluating potential cytotoxicity of those NMs, like TiO2-based photocatalytic NMs, widely applied in environmental remediation, and whose potential risks are poorly understood

Responses of Mytilus galloprovincialis to challenge with the emerging marine pathogen Vibrio coralliilyticus

Vibrio coralliilyticus has emerged as a coral pathogen of concern throughout the Indo-Pacific reef. The interest towards understanding its ecology and pathogenic potential has increased since V. coralliilyticus was shown to be strongly virulent also for other species; in particular, it represents a serious threat for bivalve aquaculture, being one of the most important emerging pathogen responsible for oyster larval mortalities worldwide. V. coralliilyticus has a tightly regulated temperature-dependent virulence and it has been related to mass mortalities events of benthic invertebrates also in the temperate northwestern Mediterranean Sea. However, no data are available on the effects of V. coralliilyticus in the mussel Mytilus galloprovincialis, the most abundant aquacultured species in this area. In this work, responses of M. galloprovincialis to challenge with V. coralliilyticus (ATCC BAA-450) were investigated. In vitro, short term responses of mussel hemocytes were evaluated in terms of lysosomal membrane stability, bactericidal activity, lysozyme release, ROS and NO production, and ultrastructural changes, evaluated by TEM. In vivo, hemolymph parameters were measured in mussels challenged with V. coralliilyticus at 24h p.i. Moreover, the effects of V. coralliilyticus on mussel early embryo development (at 48 hpf) were evaluated. The results show that both in vitro and in vivo, mussels were unable to activate immune response towards V. coralliilyticus, and that challenge mainly induced lysosomal stress in the hemocytes. Moreover, V. coralliilyticus showed a strong and concentration-dependent embryotoxicity. Overall, the results indicate that, although M. galloprovincialis is considered a resistant species to vibrio infections, the emerging pathogen V. coralliilyticus can represent a potential threat to mussel aquaculture

De novo DNA methylation induced by circulating extracellular vesicles from acute coronary syndrome patients

DNA methylation is associated with gene silencing, but its clinical role in cardiovascular diseases (CVDs) remains to be elucidated. We hypothesized that extracellular vesicles (EVs) may carry epigenetic changes, showing themselves as a potentially valuable non-invasive diagnostic liquid biopsy. We isolated and characterized circulating EVs of acute coronary syndrome (ACS) patients and assessed their role on DNA methylation in epigenetic modifications

Interactions of cationic polystyrene nanoparticles with marine bivalve hemocytes in a physiological environment: Role of soluble hemolymph proteins

none12The bivalve Mytilus galloprovincialis has proven as a suitable model invertebrate for evaluating the potential impact of nanoparticles (NPs) in the marine environment. In particular, in mussels, the immune system represents a sensitive target for different types of NPs. In environmental conditions, both NP intrinsic properties and those of the receiving medium will affect particle behavior and consequent bioavailability/uptake/toxicity. However, the evaluation of the biological effects of NPs requires additional understanding of how, once within the organism, NPs interact at the molecular level with cells in a physiological environment. In mammalian systems, different NPs associate with serum soluble components, organized into a "protein corona", which affects particle interactions with target cells. However, no information is available so far on the interactions of NPs with biological fluids of aquatic organisms. In this work, the influence of hemolymph serum (HS) on the in vitro effects of amino modified polystyrene NPs (PS-NH2) on Mytilus hemocytes was investigated. Hemocytes were incubated with PS-NH2 suspensions in HS (1, 5 and 50µg/mL) and the results were compared with those obtained in ASW medium. Cell functional parameters (lysosomal membrane stability, oxyradical production, phagocytosis) were evaluated, and morphological changes were investigated by TEM. The activation state of the signalling components involved in Mytilus immune response (p38 MAPK and PKC) was determined. The results show that in the presence of HS, PS-NH2 increased cellular damage and ROS production with respect to ASW medium. The effects were apparently mediated by disregulation of p38 MAPK signalling. The formation of a PS-NH2-protein corona in HS was investigated by centrifugation, and 1D- gel electrophoresis and nano-HPLC-ESI-MS/MS. The results identified the Putative C1q domain containing protein (MgC1q6) as the only component of the PS-NH2 hard protein corona in Mytilus hemolymph. These data represent the first evidence for the formation of a NP bio-corona in aquatic organisms and underline the importance of the recognizable biological identity of NPs in physiological exposure medium when testing their potential impact environmental model organisms. Although the results obtained in vitro do not entirely reflect a realistic exposure scenario and the more complex formation of a bio-corona that is likely to occur in vivo, these data will contribute to a better understanding of the effects of NPs in marine invertebrates.openCanesi, Laura; Ciacci, Caterina; Fabbri, Rita; Balbi, Teresa; Salis, Annalisa; Damonte, Gianluca; Cortese, Katia; Caratto, Valentina; Monopoli, Marco P; Dawson, Kenneth; Bergami, Elisa; Corsi, IlariaCanesi, Laura; Ciacci, Caterina; Fabbri, Rita; Balbi, Teresa; Salis, Annalisa; Damonte, Gianluca; Cortese, Katia; Caratto, Valentina; Monopoli, Marco P; Dawson, Kenneth; Bergami, Elisa; Corsi, Ilari

Risk of latent tuberculosis infection among healthcare workers in Italy: a retrospective study with Quantiferon Test

OBJECTIVE. The latent tuberculosis infection (LTBI) is a state of persistent immune response to stimulation by Mycobacterium Tuberculosis antigens without clinical manifestation: the healthcare workers (HCWs) have a higher exposure risk so prevention is an important challenge for occupational medicine. The aim of our study is to evaluate the prevalence of LTBI among HCWs of the Foundation Policlinic “Tor Vergata”. METHODS. This is a retrospective study conducted by analyzing the clinical records of 825 HCWs of the PTV, from January 1st to December 31th 2016. To evaluate the TB infection we used the Quantiferon TB Gold interferon-gamma release assay. RESULTS. Our study underline the low prevalence of LTBI in the Italian healthcare workers. CONCLUSION. Although the LTBI status is not contagious, the diagnosis and the safety strategies require specific clinical and preventive considerations

Autophagic processes in Mytilus galloprovincialis hemocytes: Effects of Vibrio tapetis

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios