Lucia Cecilia Lorusso

Publications of Lucia Cecilia Lorusso

• Short-term effects of environmentally relevant concentrations of EDC mixtures on Mytilus galloprovincialis digestive gland.

  Authors: Laura Canesi, Cristina Borghi, Caterina Ciacci, Rita Fabbri, Lucia Cecilia Lorusso, Laura Vergani, Antonio Marcomini, Giulio Poiana

  Aquatic toxicology (Amsterdam, Netherlands). 06/2008; 87(4):272-9.

  Endocrine disrupting compounds (EDCs), including both natural estrogens and estrogenic chemicals, are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus galloprovincialisEndocrine disrupting compounds (EDCs), including both natural estrogens and estrogenic chemicals, are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus galloprovincialis different estrogenic compounds, both individually and in mixtures, were shown to affect the immune function both in vitro and in vivo. Moreover, individual estrogens, the natural estrogen 17beta-estradiol (E(2)) and the xenoestrogen bisphenol A (BPA), have been recently demonstrated to alter functional parameters and gene expression in mussel digestive gland, a tissue that plays a central role in metabolism and in nutrient distribution to the gonad during gamete maturation, with possible consequences on gametogenesis. In this work, the possible effects of a synthetic mixture of EDCs on the digestive gland were evaluated. The mixture contained seven estrogenic chemicals (17beta-estradiol, 17alpha-ethynyl estradiol, mestranol (MES), nonylphenol, nonylphenol monoethoxylate carboxylate (NP1EC), BPA, benzophenone (BP)), in proportions similar to those previously found in water samples of a coastal lagoon. Mussels were injected with different concentrations of the mixture (approximate nominal concentrations of total EDCs: 0.0177, 0.177, 1.77 and 177 ng/g dw) and tissues sampled 24 h post-injection. The mixture induced significant changes in lysosomal biomarkers (lysosomal membrane stability (LMS), neutral lipid (NL) and lipofuscin (LF) accumulation) as well as in the activities of catalase, glutathione transferase (GST), and of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). Moreover, downregulation of the gene transcription for the Mytilus estrogen receptor MeER1 isoform and for catalase, as evaluated by quantitative RT-PCR, were observed. Significant changes in lysosomal biomarkers, enzyme activities and gene transcription were also recorded at 72 h post-injection. The results demonstrate that short-term exposure to environmentally relevant concentrations of EDC mixtures can interfere with the lysosomal function, redox-related enzyme activities and gene transcription of mussel digestive gland.

• Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms.

  Authors: Laura Canesi, Caterina Ciacci, Lucia Cecilia Lorusso, Michele Betti, Gabriella Gallo, Giulio Pojana, Antonio Marcomini

  Comparative biochemistry and physiology. Toxicology & pharmacology : CBP. 05/2007; 145(3):464-72.

  Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action.Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action. Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) (TCS), is a common antibacterial agent that is found in significant amounts in the aquatic environment. In this work, the possible effects and modes of action of TCS were investigated in the marine bivalve Mytilus galloprovincialis Lam. In mussel immune cells, the hemocytes, in vitro short-term exposure to TCS in the low microM range reduced lysosomal membrane stability (LMS) and induced extracellular release of lysosomal hydrolytic enzymes. The effects on LMS were mediated by activation of ERK MAPKs (Extracellularly Regulated Mitogen Activated Protein Kinases) and PKC (protein kinase C) alpha and betaII isoforms, as demonstrated by both specific kinase inhibitors and Western blotting with specific anti-phospho-antibodies. The effects of TCS were confirmed in vivo, in the hemocytes of mussels injected with different concentrations of TCS (corresponding to 0.29, 2.9 and 29 ng/g dry weight) and sampled at 24 h post-injection. The possible in vivo effects of TCS were also evaluated on the activity of different enzymes in the digestive gland, the tissue mainly involved in accumulation and metabolism of organic contaminants in mussels. Significant increases were observed in the activity of the glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), as well as of GST (GSH transferase) and GSR (GSSG reductase), whereas a decrease in catalase activity was observed. The results demonstrate that in mussels TCS can act on kinase-mediated cell signalling, lysosomal membranes and redox balance in different systems/organs. Although further studies are needed in order to evaluate possible consequences of environmental exposure to TCS on mussel health, the results represent the first data on the possible modes of action of this widespread antibacterial in aquatic invertebrates.

• Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies.

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Marco Rocchi, Giulio Pojana, Antonio Marcomini

  Aquatic toxicology (Amsterdam, Netherlands). 03/2007; 81(1):36-44.

  Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recentlyEndocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17beta-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17alpha-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.

• Immunomodulation by 17beta-estradiol in bivalve hemocytes.

  Authors: Laura Canesi, Caterina Ciacci, Lucia Cecilia Lorusso, Michele Betti, Tiziana Guarnieri, Simona Tavolari, Gabriella Gallo

  American journal of physiology. Regulatory, integrative and comparative physiology. 10/2006; 291(3):R664-73.

  In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, theIn mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17beta-estradiol (E(2)) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E(2) in Mytilus hemocytes, the cells responsible for the innate immune response. E(2) at 5-25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E(2) inhibited phagocytosis. E(2)-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine and superoxide dismutase, indicating involvement of NO and O(2)(-); NO production was confirmed by nitrite accumulation. The effects of E(2) were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E(2) induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-alpha- and -beta-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E(2) on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E(2)-injected mussels. E(2) significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E(2) resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E(2) in immunomodulation is conserved from invertebrates to mammals.

• Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C.

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Gabriella Gallo

  Aquatic toxicology (Amsterdam, Netherlands). 12/2005; 75(3):277-87.

  Brominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individualBrominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individual BFR used in industry. Although TBBPA and its derivatives can be found in environmental samples, data are very limited on the presence of this compound in biota. Research on mammals indicates that TBBPA has low toxicity in vivo; however, in vitro TBBPA can act as a cytotoxicant, neurotoxicant, immunotoxicant, thyroid hormone agonist and has a weak estrogenic activity; in particular, the effects of TBBPA have been recently ascribed to its interactions with cellular signaling pathways, in particular with mitogen activated protein kinases (MAPKs). TBBPA has high acute toxicity to aquatic organisms, such as algae, molluscs, crustaceans and fish; however, little is known on the mechanisms of action of this compound in the cells of aquatic species. In this work, we investigated the possible effects and mechanisms of action of TBBPA on the immune cells, the hemocytes, of the marine mussel Mytilus galloprovincialis. The results demonstrate that TBBPA in the low micromolar range induces hemocyte lysosomal membrane destabilization. The effect was reduced or prevented by hemocyte pre-treatment by specific inhibitors of MAPKs and of protein kinase C (PKC). TBBPA stimulated phosphorylation of MAPK members and PKC, as evaluated by electrophoresis and Western blotting with anti-phospho-antibodies, although to a different extent and with distinct time-courses. A rapid (from 5 min) and transient increase in phosphoryation of the stress-activated JNK MAPKs and of PKC was observed, followed by a later increase (at 30-60 min) in phosphorylation of extracellularly regulated MAPKs (ERK2 MAPK) and of the stress-activated p38 MAPK. TBBPA significantly stimulated the hemocyte microbicidal activity towards E. coli, lysosomal enzyme release, phagocytic activity and extracellular superoxide (O2-) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

• Interactions between Mytilus haemocytes and different strains of Escherichia coli and Vibrio cholerae O1 El Tor: role of kinase-mediated signalling.

  Authors: Laura Canesi, Michele Betti, Caterina Ciacci, Lucia Cecilia Lorusso, Gabriella Gallo, Carla Pruzzo

  Cellular microbiology. 06/2005; 7(5):667-74.

  Marine bivalves accumulate large amounts of bacteria from the environment (mainly Vibrionaceae and coliforms). Although persistence of different bacteria in bivalve tissues largely depends on theirMarine bivalves accumulate large amounts of bacteria from the environment (mainly Vibrionaceae and coliforms). Although persistence of different bacteria in bivalve tissues largely depends on their sensitivity to the bactericidal activity of circulating haemocytes and haemolymph soluble factors, the mechanisms involved in bacteria-host cell interactions in these invertebrates are largely unknown. In the mussel Mytilus, differences in interactions between haemocytes and different Escherichia coli and Vibrio cholerae strains [E. coli MG155, a wild-type strain carrying type 1 fimbriae, and its unfimbriated derivative, AAEC072 Deltafim; V. cholerae O1 El Tor biotype strain N16961, carrying the mannose-sensitive haemagglutinin (MSHA), and its MSHA mutant] lead to differences in bactericidal activity in the presence of serum. Here we show that different bacteria induced distinct patterns of phosphorylation of mitogen-activated protein kinases (MAPKs), in particular of the stress-activated MAPKs involved in the immune response. Differences in phosphorylation of PKC-like proteins were also observed. The results support the hypothesis that, like in mammalian host cells, different bacteria can modulate the signalling pathways of mussel haemocytes. The lower anti-bacterial activity towards the mutant E. coli strain and wild-type V. cholerae compared with wild E. coli may result from a reduced capacity of activating MAPKs. Moreover, the mutant V. cholerae strain that was the most resistant to the haemocyte bactericidal activity induced downregulation of cell signalling and showed the strongest effect on lysosomal membrane stability, evaluated as a marker of bivalve cell stress. These data suggest that certain bacteria could evade the bactericidal activity of mussel haemocytes through disruption of the host signalling pathways.

• 'In vivo' effects of Bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways.

  Authors: Laura Canesi, Michele Betti, Lucia Cecilia Lorusso, Caterina Ciacci, Gabriella Gallo

  Aquatic toxicology (Amsterdam, Netherlands). 02/2005; 71(1):73-84.

  Endocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the musselEndocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the mussel Mytilus galloprovincialis Lam. both natural and environmental estrogens in vitro can rapidly affect the phosphorylation state of components of tyrosine kinase-mediated cell signalling, in particular of mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STAT), that are involved in mediating the hemocyte immune response. These effects were consistent with the hypothesis that 'alternative' modes of estrogen action involving kinase-mediated pathways similar to those described in mammalian systems are also present in invertebrate cells. This possibility was investigated in vivo with Bisphenol A (BPA): mussels were injected with BPA and hemocytes sampled at 6, 12, and 24 h post-injection. The results show that BPA (25 nM nominal concentration in the hemolymph) lead to a significant lysosomal membrane destabilisation at all times post-injection, indicating BPA-induced stress conditions in the hemocytes, whereas lower concentrations were ineffective. BPA induced significant changes in the phosphorylation state of MAPK and STAT members, as evaluated by SDS-PAGE and WB of hemocyte protein extracts with specific antibodies, although to a different degree at different exposure times. In particular, BPA induced a dramatic decrease in phosphorylation of the stress-activated p38 MAPK, whose activation is crucial in mediating the bactericidal activity. Moreover, BPA decreased the phosphorylation of a CREB-like transcription factor (cAMP-responsive element binding protein). The results demonstrate that BPA can affect kinase-mediated cell signalling in mussel hemocytes also in vivo, and suggest that EDCs may affect gene expression in mussel cells through modulation of the activity of transcription factors secondary to cytosolic kinase cascades. Overall, these data address the importance of investigating full range responses to EDCs in ecologically relevant marine invertebrate species.

• Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways.

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Massimiliano Zampini, Gabriella Gallo

  General and comparative endocrinology. 09/2004; 138(1):58-69.

  Estrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine molluscEstrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine mollusc Mytilus the natural estrogen 17beta-estradiol (E(2)) can affect the immune function through rapid tyrosine kinase-mediated signalling pathways converging on phosphorylation of both mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs), whose activation plays a key role in the immune response. In this work the effects of synthetic estrogens (such as DES), estrogenic chemicals (such as Bisphenol A, Nonylphenol), and plant estrogens (genistein) on mussel hemocytes were evaluated. The results demonstrate that all the EDCs tested exert in vitro effects similar to those of E(2) on lysosomal membrane stability, although at concentrations 1000 times higher than those of the natural estrogen. When the effects of DES, BPA, and NP on tyrosine kinase-mediated cell signalling were investigated, estrogenic compounds showed distinct effects on the phosphorylation state of MAPK and STAT members. In particular, only DES, like E(2), induced p38 MAPK phosphorylation, whereas BPA and NP seem to have opposite effects. Moreover, different EDCs significantly decreased the tyrosine phosphorylation state of STAT3 and STAT5, showing a distinct effect with respect to E(2). Experiments with specific kinase inhibitors showed that activation of p38 MAPK, but also of ERK MAPK and PI3-kinase, plays a key role in mediating the effect of DES. On the other hand, the effects of NP were partly mediated by ERK MAPK activation. BPA-induced lysosomal membrane destabilisation was unaffected by either MAPK or PI3-K inhibitors. However, hemocyte pre-treatment with the PKC inhibitor GF109203X prevented the effects of both BPA and NP, this indicating that kinase pathways other than those involving MAPKs are also responsible for mediating the effects of certain EDCs. Overall, the results support the hypothesis that EDCs may rapidly modulate the function of mussel hemocytes through activation of transduction pathways involving different kinase-mediated cascades. Moreover, the effects of EDCs on the phosphorylation state of transcription factor STATs suggest that these compounds may lead to changes in gene expression secondary to modulation of kinase/phosphatases. Our data address to the importance of investigating full range responses to estrogenic chemicals and may help understanding their basic mechanisms of action in ecologically relevant invertebrate species.

• Rapid effects of 17beta-estradiol on cell signaling and function of Mytilus hemocytes.

  Authors: Laura Canesi, Caterina Ciacci, Michele Betti, Lucia Cecilia Lorusso, Barbara Marchi, Sabrina Burattini, Elisabetta Falcieri, Gabriella Gallo

  General and comparative endocrinology. 04/2004; 136(1):58-71.

  Estrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17beta-estradiol (E2) has both dose- and cell-type specific effectsEstrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17beta-estradiol (E2) has both dose- and cell-type specific effects and the responses to E2 seem to be mediated by rapid, non-genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects, such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, including mitogen activated protein kinases (MAPKs). In this work, the short-term effects of E(2) and the possible mechanisms of estrogen-mediated cell signaling were investigated in the hemocytes, the immune cells of the bivalve mollusc, the mussel Mytilus galloprovincialis Lam. The results show that E2 (25nM) caused a rapid and significant increase in hemocyte cytosolic [Ca2+]; lower concentrations (5 nM) showed a smaller, not significant effect. Both E2 concentrations affected the phosphorylation state of the components of tyrosine kinase-mediated signal transduction MAPK- and STAT- (signal transducers and activators of transcription) like proteins within 5-15 min from E2 addition. A greater effect and clearer time course were observed with 25 nM E2: in particular, E2 induced a transient increase in p-ERK2 MAPK and a persistent increase in p-p38 MAPK. Moreover, both STAT3 and STAT5 were tyrosine phosphorylated in response to E2. E2 (5 nM) induced both morphological (as evaluated by SEM) and functional changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, and stimulation of the bactericidal activity) within 10-30 min from addition. Lysosomal membrane destabilisation induced by both E2 concentrations was abolished by hemocyte preincubation with the p38 MAPK inhibitor SB203580, and significantly reduced by PD98059 and Wortmannin (inhibitors of ERK MAPK and PI3-K, respectively), this suggesting that rapid activation of kinase cascades is involved in mediating the effects of E2 in mussel hemocytes. The antiestrogen Tamoxifen prevented or strongly reduced most, but not all, the effects of E2. Western blotting with heterologous anti-ERalpha-anti-ERbeta-antibodies revealed the presence of immunoreactive ERalpha- and ERbeta-like proteins in hemocyte protein extracts. Overall, our data support the hypothesis that the rapid effects and mechanisms of action of 17beta-estradiol are extremely conserved and that they may play a crucial role in endocrine-immune interactions in invertebrates.

• Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Massimiliano Zampini, Gabriella Gallo

  General and Comparative Endocrinology.

  Estrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine molluscEstrogens and estrogenic chemicals can affect several vertebrate non-reproductive functions, the immune response in particular. We have previously shown that in the hemocytes of the marine mollusc Mytilus the natural estrogen 17β-estradiol (E2) can affect the immune function through rapid tyrosine kinase-mediated signalling pathways converging on phosphorylation of both mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs), whose activation plays a key role in the immune response. In this work the effects of synthetic estrogens (such as DES), estrogenic chemicals (such as Bisphenol A, Nonylphenol), and plant estrogens (genistein) on mussel hemocytes were evaluated. The results demonstrate that all the EDCs tested exert in vitro effects similar to those of E2 on lysosomal membrane stability, although at concentrations 1000 times higher than those of the natural estrogen. When the effects of DES, BPA, and NP on tyrosine kinase-mediated cell signalling were investigated, estrogenic compounds showed distinct effects on the phosphorylation state of MAPK and STAT members. In particular, only DES, like E2, induced p38 MAPK phosphorylation, whereas BPA and NP seem to have opposite effects. Moreover, different EDCs significantly decreased the tyrosine phosphorylation state of STAT3 and STAT5, showing a distinct effect with respect to E2. Experiments with specific kinase inhibitors showed that activation of p38 MAPK, but also of ERK MAPK and PI3-kinase, plays a key role in mediating the effect of DES. On the other hand, the effects of NP were partly mediated by ERK MAPK activation. BPA-induced lysosomal membrane destabilisation was unaffected by either MAPK or PI3-K inhibitors. However, hemocyte pre-treatment with the PKC inhibitor GF109203X prevented the effects of both BPA and NP, this indicating that kinase pathways other than those involving MAPKs are also responsible for mediating the effects of certain EDCs. Overall, the results support the hypothesis that EDCs may rapidly modulate the function of mussel hemocytes through activation of transduction pathways involving different kinase-mediated cascades. Moreover, the effects of EDCs on the phosphorylation state of transcription factor STATs suggest that these compounds may lead to changes in gene expression secondary to modulation of kinase/phosphatases. Our data address to the importance of investigating full range responses to estrogenic chemicals and may help understanding their basic mechanisms of action in ecologically relevant invertebrate species.

• Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes

  Authors: Laura Canesi, Caterina Ciacci, Michele Betti, Lucia Cecilia Lorusso, Barbara Marchi, Sabrina Burattini, Elisabetta Falcieri, Gabriella Gallo

  General and Comparative Endocrinology.

  Estrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17β-estradiol (E2) has both dose- and cell-type specific effects andEstrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17β-estradiol (E2) has both dose- and cell-type specific effects and the responses to E2 seem to be mediated by rapid, non-genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects, such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, including mitogen activated protein kinases (MAPKs). In this work, the short-term effects of E2 and the possible mechanisms of estrogen-mediated cell signaling were investigated in the hemocytes, the immune cells of the bivalve mollusc, the mussel Mytilus galloprovincialis Lam. The results show that E2 (25 nM) caused a rapid and significant increase in hemocyte cytosolic [Ca2+]; lower concentrations (5 nM) showed a smaller, not significant effect. Both E2 concentrations affected the phosphorylation state of the components of tyrosine kinase-mediated signal transduction MAPK- and STAT- (signal transducers and activators of transcription) like proteins within 5–15 min from E2 addition. A greater effect and clearer time course were observed with 25 nM E2: in particular, E2 induced a transient increase in p-ERK2 MAPK and a persistent increase in p-p38 MAPK. Moreover, both STAT3 and STAT5 were tyrosine phosphorylated in response to E2. E2 (5 nM) induced both morphological (as evaluated by SEM) and functional changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, and stimulation of the bactericidal activity) within 10–30 min from addition. Lysosomal membrane destabilisation induced by both E2 concentrations was abolished by hemocyte preincubation with the p38 MAPK inhibitor SB203580, and significantly reduced by PD98059 and Wortmannin (inhibitors of ERK MAPK and PI3-K, respectively), this suggesting that rapid activation of kinase cascades is involved in mediating the effects of E2 in mussel hemocytes. The antiestrogen Tamoxifen prevented or strongly reduced most, but not all, the effects of E2. Western blotting with heterologous anti-ERα–anti-ERβ-antibodies revealed the presence of immunoreactive ERα- and ERβ-like proteins in hemocyte protein extracts. Overall, our data support the hypothesis that the rapid effects and mechanisms of action of 17β-estradiol are extremely conserved and that they may play a crucial role in endocrine–immune interactions in invertebrates.

• Effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: Involvement of MAP kinases and protein kinase C

  Authors: Laura Canesi, Lucia Cecilia Lorusso, Caterina Ciacci, Michele Betti, Gabriella Gallo

  Aquatic Toxicology.

  Brominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individualBrominated flame retardants (BFRs) are a large group of compounds added to or applied as a treatment to polymeric materials to prevent fires. Tetrabisphenol A (TBBPA) is the most important individual BFR used in industry. Although TBBPA and its derivatives can be found in environmental samples, data are very limited on the presence of this compound in biota. Research on mammals indicates that TBBPA has low toxicity in vivo; however, in vitro TBBPA can act as a cytotoxicant, neurotoxicant, immunotoxicant, thyroid hormone agonist and has a weak estrogenic activity; in particular, the effects of TBBPA have been recently ascribed to its interactions with cellular signaling pathways, in particular with mitogen activated protein kinases (MAPKs).TBBPA has high acute toxicity to aquatic organisms, such as algae, molluscs, crustaceans and fish; however, little is known on the mechanisms of action of this compound in the cells of aquatic species. In this work, we investigated the possible effects and mechanisms of action of TBBPA on the immune cells, the hemocytes, of the marine mussel Mytilus galloprovincialis. The results demonstrate that TBBPA in the low micromolar range induces hemocyte lysosomal membrane destabilization. The effect was reduced or prevented by hemocyte pre-treatment by specific inhibitors of MAPKs and of protein kinase C (PKC). TBBPA stimulated phosphorylation of MAPK members and PKC, as evaluated by electrophoresis and Western blotting with anti-phospho-antibodies, although to a different extent and with distinct time-courses. A rapid (from 5 min) and transient increase in phosphoryation of the stress-activated JNK MAPKs and of PKC was observed, followed by a later increase (at 30–60 min) in phosphorylation of extracellularly regulated MAPKs (ERK2 MAPK) and of the stress-activated p38 MAPK. TBBPA significantly stimulated the hemocyte microbicidal activity towards E. coli, lysosomal enzyme release, phagocytic activity and extracellular superoxide (O2−) production. The results demonstrate that TBBPA in vitro activates the immune function of mussel hemocytes through kinase-mediated cell signaling and that common transduction pathways are involved in mediating the effects of this BFR in mammalian and aquatic invertebrate cells.

• ‘In vivo’ effects of Bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways

  Authors: Laura Canesi, Michele Betti, Lucia Cecilia Lorusso, Caterina Ciacci, Gabriella Gallo

  Aquatic Toxicology.

  Endocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the musselEndocrine disrupting chemicals (EDCs) include a variety of natural and synthetic estrogens, as well as estrogen-mimicking chemicals. We have previously shown that in the hemocytes of the mussel Mytilus galloprovincialis Lam. both natural and environmental estrogens in vitro can rapidly affect the phosphorylation state of components of tyrosine kinase-mediated cell signalling, in particular of mitogen activated protein kinases (MAPKs) and signal transducers and activators of transcription (STAT), that are involved in mediating the hemocyte immune response. These effects were consistent with the hypothesis that ‘alternative’ modes of estrogen action involving kinase-mediated pathways similar to those described in mammalian systems are also present in invertebrate cells.This possibility was investigated in vivo with Bisphenol A (BPA): mussels were injected with BPA and hemocytes sampled at 6, 12, and 24 h post-injection. The results show that BPA (25 nM nominal concentration in the hemolymph) lead to a significant lysosomal membrane destabilisation at all times post-injection, indicating BPA-induced stress conditions in the hemocytes, whereas lower concentrations were ineffective. BPA induced significant changes in the phosphorylation state of MAPK and STAT members, as evaluated by SDS-PAGE and WB of hemocyte protein extracts with specific antibodies, although to a different degree at different exposure times. In particular, BPA induced a dramatic decrease in phosphorylation of the stress-activated p38 MAPK, whose activation is crucial in mediating the bactericidal activity. Moreover, BPA decreased the phosphorylation of a CREB-like transcription factor (cAMP-responsive element binding protein). The results demonstrate that BPA can affect kinase-mediated cell signalling in mussel hemocytes also in vivo, and suggest that EDCs may affect gene expression in mussel cells through modulation of the activity of transcription factors secondary to cytosolic kinase cascades. Overall, these data address the importance of investigating full range responses to EDCs in ecologically relevant marine invertebrate species.

• Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: Possible modes of action on non target organisms

  Authors: Laura Canesi, Caterina Ciacci, Lucia Cecilia Lorusso, Michele Betti, Gabriella Gallo, Giulio Pojana, Antonio Marcomini

  Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology.

  Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action.Pharmaceuticals and Personal Care Products (PPCPs) are a class of emerging environmental pollutants with the potential of affecting various aquatic organisms through unexpected modes of action. Triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether) (TCS), is a common antibacterial agent that is found in significant amounts in the aquatic environment. In this work, the possible effects and modes of action of TCS were investigated in the marine bivalve Mytilus galloprovincialis Lam. In mussel immune cells, the hemocytes, in vitro short-term exposure to TCS in the low μM range reduced lysosomal membrane stability (LMS) and induced extracellular release of lysosomal hydrolytic enzymes. The effects on LMS were mediated by activation of ERK MAPKs (Extracellularly Regulated Mitogen Activated Protein Kinases) and PKC (protein kinase C) α and βII isoforms, as demonstrated by both specific kinase inhibitors and Western blotting with specific anti-phospho-antibodies. The effects of TCS were confirmed in vivo, in the hemocytes of mussels injected with different concentrations of TCS (corresponding to 0.29, 2.9 and 29 ng/g dry weight) and sampled at 24 h post-injection. The possible in vivo effects of TCS were also evaluated on the activity of different enzymes in the digestive gland, the tissue mainly involved in accumulation and metabolism of organic contaminants in mussels. Significant increases were observed in the activity of the glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), as well as of GST (GSH transferase) and GSR (GSSG reductase), whereas a decrease in catalase activity was observed. The results demonstrate that in mussels TCS can act on kinase-mediated cell signalling, lysosomal membranes and redox balance in different systems/organs. Although further studies are needed in order to evaluate possible consequences of environmental exposure to TCS on mussel health, the results represent the first data on the possible modes of action of this widespread antibacterial in aquatic invertebrates.

• Effects of 17beta-estradiol on mussel digestive gland.

  Authors: Laura Canesi, Cristina Borghi, Rita Fabbri, Caterina Ciacci, Lucia Cecilia Lorusso, Gabriella Gallo, Laura Vergani

  General and comparative endocrinology. 153(1-3):40-6.

  In bivalve molluscs the digestive gland (hepatopancreas) plays a central role in metabolism. In this work, the effects of 17beta-estradiol (E(2)) on digestive gland were evaluated in MytilusIn bivalve molluscs the digestive gland (hepatopancreas) plays a central role in metabolism. In this work, the effects of 17beta-estradiol (E(2)) on digestive gland were evaluated in Mytilus galloprovincialis. Mussels were injected into the adductor muscle sinus with different amounts of the hormone (5, 25 and 100pmol) and tissues were sampled 24h post-injection. Functional parameters (lysosomal membrane stability-LMS, lysosomal accumulation of neutral lipids-NL and of lipofuscin-LF), as well as the activity of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase), and of the antioxidant enzyme catalase were evaluated. Selected genes, whose expression can be modulated by estrogens in mammalian systems and whose sequences have been identified in Mytilus, were investigated as possible targets for the action of E(2). E(2) induced a concentration-dependent decrease in LMS; such an effect was accompanied by an increase in NL accumulation, whereas the level of lipofuscin showed a slight, although not significant decrease. E(2) exposure also led to a significant increase in the activity of PFK and catalase but not of PK. Moreover, E(2) induced significant changes in the pattern of gene expression at the lower concentrations tested (5 and 25pmol) as evaluated by quantitative RT-PCR. In particular, increased transcription of catalase, as well as of the metallothionein 20 (MT20) isoform were observed; on the other hand, a decreased transcription of the p53 gene was detected. The results demonstrate that in Mytilus the digestive gland represents a target for the action of E(2), and that the hormone can modulate the lysosomal function, as well as lipid and glucose metabolism. Moreover, these data suggest that E(2) may also alter oxidative stress conditions in this tissue, as indicated by the increased transcription of genes (metallothionein and catalase) that play a role in antioxidant defences. Overall, the results indicate that E(2) can modulate both functional parameters and gene expression in mussel hepatopancreas and underline the importance of investigating also non-reproductive effects of estrogenic compounds in bivalve molluscs.