Laura Canesi

Heavy metals and glutathione metabolism in mussel tissues

Abstract Data are presented on the tissue glutathione content and on the activity of two of the main enzymes involved in GSH metabolism (γ-glutamyl-cysteine synthetase (GCS), GSH transferase (GST)) in the gills and digestive gland of mussels (Mytilus galloprovincialis Lam.) exposed for 1, 4 and 7 days to sublethal concentrations of copper and of mercury, both in the inorganic form of HgCl2 and in the organic form of CH3HgCl. The results indicate that Cu2+ (0.6 μM) and CH3Hg+ (0.2 μM) can affect, although to a different extent, glutathione metabolism in mussel tissues; common traits were a decrease in tissue glutathione content, stimulation of GST activity and inhibition of GSH synthesis rate; on the other hand, inorganic Hg2+ (0.2 μM) did not significantly affect glutathione content and metabolism in mussel tissues. Exposure to copper (0.6 μM) resulted in a decrease in total glutathione content in both gills and the digestive gland that was maximal after the first day, followed by a tendency to recover at longer exposure times. Such a decrease seemed to be mainly related to a stimulation of GST activity, which was transient in gills and persistent in the digestive gland. The tissue glutathione content was also decreased in mussel exposed to methylmercury (0.2 μM); however, such a decrease seemed to be mainly due to inhibition of the GSH synthesis rate and, to a lesser extent, to an increased GST activity. On the contrary, inorganic Hg2+ (0.2 μM) did not significantly affect glutathione metabolism, although mussel exposure to the same concentration of organic and inorganic Hg2+ resulted in similar total metal tissue load in mussel tissues. The role of glutathione as a potential defense against heavy metals is discussed in relation to different routes of metal toxicity and homeostasis in mussel tissues.

Pro-oxidant processes and antioxidant defence systems in the tissues of the Antarctic scallop (Adamussium colbecki) compared with the Mediterranean scallop (Pecten jacobaeus)

Antioxidant defences (scavengers, enzymes), pro-oxidant processes (in vitro NAD(P)H-dependent iron/EDTA-mediated hydroxyl radical (·OH) production and susceptibility to lipid peroxidation) were investigated in the tissues of the antarctic scallop Adamussium colbecki (Smith, 1902) compared with a scallop species typical of temperate, mediterranean waters, Pecten jacobaeus L. Levels of free radical scavengers (total glutathione, vitamin E, total carotenoids) and activities of antioxidant enzymes (superoxide dismutase, EC 1.15.1.1.; catalane, EC 1.11.1.6.; glutathione peroxidase, EC 1.11.1.9.) were in the same range in both species and in both were higher in digestive gland than gills. The in vitro potential for -OH production was higher in the digestive gland than the gills for both species, consistent with the former tissues higher levels of antioxidant defences. Inhibition studies (A. colbecki) indicated the involvement of superoxide anion radical and hydrogen peroxide in ·OH formation. The digestive gland of A. colbecki showed a lower susceptibility to ‘in vitro’ stimulated lipid peroxidation than that of P. jacobaeus. The results are discussed in relation to the adaptation of A. coibecki to extreme Antarctic conditions, including low temperature, high oxygen tension and marked seasonality of food intake, all of which can influence reactive oxygen species formation.

Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAP kinases.

In this work the mechanisms of transduction triggered in Mytilus galloprovincialis hemocytes by bacterial challenge were investigated in an in vitro model of infection of hemocyte monolayers with Escherichia coli. Western blot analyses of hemocyte extracts with phospho-specific anti-MAPK (Mitogen Activated Protein Kinase) antibodies indicate that E. coli induced a time dependent activation of different classes of MAPKs, mainly of the stress-activated p38 MAPK. P38 activation was confirmed by the use of the selective kinase inhibitor SB203580. Moreover, hemocyte pretreatment with SB203580 significantly reduced bacterial killing, whereas PD98059, an inhibitor of extracellularly regulated kinase (ERK) activation, was ineffective. Interestingly, the PI3-kinase (phosphatidylinositol-3-OH-kinase) inhibitor, Wortmannin, reduced both p38 activation and bacterial killing, indicating a critical role also for this lipid kinase in the hemocyte immune response.

In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: the role of glutathione

Hexokinase (E.C. 2.7.1.1), the enzyme responsible for glucose phosphorylation to G-6P, is inactivated by SH reagents and oxyradicals, and its inhibition has been involved in heavy metal toxicity in mammalian systems. In this work, the possibility that hexokinase activity could be affected by both heavy metal binding and oxidative stress conditions also in mussel tissues (Mytilus galloprovincialis Lam.) was investigated. The results obtained in vitro demonstrate that heavy metals inhibited digestive gland hexokinase (with Cd2+ > Cu2+ > Hg2+ > Zn2+ > Pb2+) and suggest a role for GSH in the protection against the heavy metal effects. Hexokinase activity was also reduced by addition of iron/ascorbate, indicating a susceptibility of the enzyme to metal-mediated oxyradical production. The effects of Cu2+ treatment (3 days, 40 micrograms l-1 per animal) on hexokinase activity and on the GSH/GSSG status were then evaluated in mussels exposed to a cycle of air exposure/reimmersion. In Cu-exposed mussels, a significant decrease in hexokinase activity and a parallel reduction in tissue GSH levels were observed, suggesting that the two effects of metal treatment could be related; however, hexokinase activity progressively recovered during air exposure and reimmersion, whereas the level of GSH showed a further decrease during air exposure followed by recovery after reimmersion. The in vitro results therefore indicate that mussel digestive gland hexokinase is susceptible to inactivation by heavy metal binding and suggest a role for GSH in the protection against the effects of heavy metals. The effects of copper were confirmed by the results obtained in vivo. The possible relationship between hexokinase activity and the level of GSH in the digestive gland of control and Cu-exposed mussels during air exposure and reimmersion are discussed, taking into account the balance between pro-oxidant and antioxidant processes at different stages of exposure.

AGE-RELATED DIFFERENCES IN GLUTATHIONE METABOLISM IN MUSSEL TISSUES (MYTILUS EDULIS L.)

Previous data indicate that in the digestive gland of marine mussels (Mytilus edulis L.) aging process is accompanied by increased oxidative stress conditions and reduction in the antioxidant defense systems and, among these, in the tissue levels of glutathione (GSH). Data are presented here on the tissue glutathione content and on the activity of the main enzymes involved in GSH metabolism (γ-glutamyl-cysteine synthetase, γ-glutamyl transpeptidase, GSSG reductase, GSH transferase) in the gills and digestive gland of mussels of three different age groups. The results indicate that the glutathione content was progressively decreased in both tissues of aging mussels. Similarly, aging seems to affect the activity of GSH-related enzymes and, in particular, the activity of γ-glutamyl cysteine synthetase, the enzyme that represents the rate-limiting step in GSH biosynthesis. These data indicate that, in aging mussels, the increased susceptibility to oxidative stress is partly related to alteration of glutathione metabolism. The results are discussed in the light of the current hypotheses on the aging process.

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

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 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 Δfim; 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.

Bacterial killing by mytilus hemocyte monolayers as a model for investigating the signaling pathways involved in mussel immune defence

The signaling pathways involved in mussel immune defence were investigated utilizing a model of killing of Escherichia coli by Mytilus galloprovincialis hemocytes in a co-culture setting. In particular, the role played by different mitogen activated protein kinases (MAPKs) and by the production of eicosanoids were investigated utilising specific cell permeant, pharmacological enzyme inhibitors. Hemocyte pretreatment with the p38 MAPK inhibitor SB203580 significantly reduced bacterial killing, whereas PD98059 (an inhibitor of ERK--extracellularly regulated kinase--MAPK activation) had no significant effect. Wortmannin also inhibited bacterial killing, indicating a crucial role for PI3-kinase activation in the immune response. Killing of E. coli was also reduced by inhibitors of both PLA2 and cyclooxygenase activities, indicating that eicosanoid production is involved in mediating the response to bacterial challenge. The results demonstrate that bacterial killing by mussel hemocytes is particularly sensitive to inhibitors of the key steps involved in the transduction of bacterial signals into the host cell. Moreover, these data indicate that the hemocyte bactericidal activity can be suitably utilized not only for identifying the signaling pathways involved in the response to bacterial infection, but also as a potential investigative-toxicology model to test drugs and contaminants for their effect on the overall mussel immune defence.

Effects of tumour necrosis factor α (TNFα) on Mytilus haemocytes: role of stress‐activated mitogen‐activated protein kinases (MAPKs)

Background information. Many studies indicate that innate immunity in invertebrates can be modulated by a cytokine network like in vertebrates. In molluscs, the immune response is carried out by circulating haemocytes and soluble haemolymph factors. In the present study, the effects of heterologous TNFα (tumour necrosis factor α) on cell signalling and function in the haemocytes of the bivalve Mytilus galloprovincialis Lam. were investigated.

Tyrosine kinase-mediated cell signalling in the activation of Mytilus hemocytes: possible role of STAT-like proteins.

Abstract In bivalve molluscs, cell‐mediated immunity is carried out by circulating hemocytes, resembling the monocyte/macrophage lineage of vertebrates, that can kill the microbes through phagocytosis and various cytotoxic reactions. Previous data demonstrated that activation of MAPKs (Mitogen Activated Protein Kinases) is involved in the response of mussel hemocytes (Mytilus galloprovincialis Lam.) to bacterial challenge. In this work, the possibility that modulation of components of tyrosine kinase‐mediated cell signalling may participate in the activation of mussel hemocytes was investigated. Cell pre‐treatment with the macrophage activator IFNγ significantly increased the bactericidal activity of mussel hemocytes towards E. coli. Human recombinant IFNγ stimulated tyrosine phosphorylation of different members of STAT‐like proteins (Signal Transducers and Activators of Transcription), as evaluated by Western blotting of hemocyte protein extracts with specific anti‐phospho‐STAT antibodies. A similar increase in phosphorylation of immunoreactive STATs was observed in hemocytes incubated with E. coli, this indicating that tyrosine phosphorylation of STAT‐like members represents a physiological step in hemocyte activation. IFNγ lead to persistent phosphorylation of immunoreactive STAT1, a transcription factor that plays a critical role in innate immunity towards Gram negative bacteria in mammalian systems; moreover, hemocyte pretreatment with IFNγ significantly increased bacteria‐induced STAT1 phosphorylation, whereas IFNα did not. IFNγ also transiently affected the phosphorylation state of different MAPKs. The extent and time course of MAPK phosphorylation induced by IFNγ were distinct from those elicited by either IFNα or bacterial challenge. Overall, the results indicate that the hemocyte function can be modulated by heterologous cytokines and bacterial signals that act in concert through tyrosine kinase‐mediated transduction pathways converging on STAT‐ and MAPK‐like members.

Growth factor-mediated signal transduction and redox balance in isolated digestive gland cells from Mytilus galloprovincialis Lam.

In mammalian cells, a growing body of evidence indicates a relationship between cellular redox balance and tyrosine kinase-mediated cell signalling. The phosphorylative cascade activated by extracellular signals is inhibited by reducing conditions and stimulated by oxidative stress, in particular at the level of mitogen activated protein kinase (MAPK) activation. The mussel Mytilus typically shows variations in antioxidant defence systems and decreases in glutathione content in response to both natural and contaminant environmental stressors. In isolated mussel digestive gland cells, both epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) have been recently demonstrated to activate tyrosine kinase receptors leading to multiple responses; among these, stimulation of the key glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK). The present study investigates the possible relationship between the tyrosine kinase-mediated metabolic effects of growth factors and cellular redox balance in mussel cells. The results demonstrate that the effects of growth factors on glycolytic enzymes were abolished by cell pretreatment with the antioxidant N-acetyl-cysteine (NAC). On the other hand, in cells where the glutathione content and synthesis were lowered either in vitro (by cell pretreatment with buthionine sulfoximine (BSO)), or in vivo (by mussel exposure to Cu(2+)) the metabolic effects of growth factors were unaffected. Moreover, the results show that, in both control and glutathione-depleted cells, growth factors can also regulate the level of glutathione apparently by modulating, via phosphorylative mechanisms involving MAPK activation, the activity of gamma-glutamylcysteine synthetase (GCS), the rate limiting enzyme in GSH biosynthesis. Overall, this study extends the hypothesis that cell signalling is intimately related to redox balance in marine invertebrate cells.