Jean-Marc Lebel

EFFECTS OF DIFFERENT VERTEBRATE GROWTH FACTORS ON PRIMARY CULTURES OF HEMOCYTES FROM THE GASTROPOD MOLLUSC, HALIOTIS TUBERCULATA

A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [3H]-leucine and [3H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [3H]-leucine or for [3H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.

Effect of in vitro exposure to zinc on immunological parameters of haemocytes from the marine gastropod Haliotis tuberculata.

Environmental pollutants such as heavy metals exert immunotoxic effects on aquatic organisms. The immune defence of molluscs is comprised of cell-mediated and humoral mechanisms, in which haemocytes play a key role. In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of zinc in vitro. Cells were exposed for 24 h to ZnCl(2) concentrations of 0, 10, 100 or 1000 microM. The effects of zinc on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Immunotoxicity was reflected by a significant decrease in the number of viable haemocytes (LC(50)(24 h) = 314 microM). Moreover, the cell area was dramatically reduced, and the percentage of rounded cells increased with increasing zinc concentrations. Exposure to 1000 muM zinc induced a significant reduction in acid phosphatase activity, phagocytic activity and reactive oxygen species production in haemocytes. However, several haemocyte parameters increased significantly after 24 h of zinc exposure. In response to a 1000 microM exposure, the phenoloxidase level was 26-fold higher than that of the control, and non-specific esterase activity was increased by 69% above that of the control. These results suggest a relationship between zinc exposure and alterations in the functional responses of haemocytes from H. tuberculata.

Collagen study and regulation of the de novo synthesis by IGF-I in hemocytes from the gastropod mollusc, Haliotis tuberculata

To evidence a collagen synthesis and identify which type(s) of collagen is present in hemocytes from the mollusc Haliotis tuberculata, we have performed three separate approaches, namely, de novo synthesis by cultured cells, immunological approaches, and northern blot analysis. We demonstrated first that after 40-hr labeling, the de novo synthesis of collagen in the cell layer of cultured hemocytes represents 9.48 +/- 1.25% with respect to the total [(3)H]proline-labeled protein synthesis. In addition, IGF-I elicited a significant stimulation of collagen synthesis in cultured hemocytes in a dose-dependent manner from 10(-10) to 10(-8) M. The maximal stimulation (10(-9) M) induced an increase of 286 +/- 56% with respect to 100% control. By immunocytochemistry and immunoblotting, we showed that hemocytes present immunoreactive molecules to antibodies directed against the type I fibrillar collagen. In addition, using as a probe Hf 677 corresponding to a human pro alpha1(I) collagen cDNA and which encompasses the (Gly-X-Y) repeated sequence found in all Metazoa, four collagen transcripts of approximately 6.4, 5, 2.2, and 2 kb in length have been detected. These data suggest the presence of fibrillar type I collagen in hemocytes and are compatible with the concept that these cells are involved in the extracellular matrix deposition, a cardinal function in tissue repair as well as in developmental processes. Our model may appear as an excellent system to study the role of growth factors on the regulation of collagen synthesis by molluscan hemocytes. J. Exp. Zool. 287:275-284, 2000.

Acute toxicity of 8 antidepressants: What are their modes of action?

Currently, the hazard posed by pharmaceutical residues is a major concern of ecotoxicology. Most of the antidepressants belong to a family named the Cationic Amphipathic Drugs known to have specific interactions with cell membranes. The present study assessed the impact of eight antidepressants belonging to selective serotonin reuptake inhibitors or serotonin norepinephrine reuptake inhibitors by the combination of multi-approaches (in vivo, in vitro, in silico) and gives some insights on the mode of action for these molecules. Antidepressants were from the most to the least toxic compound for Daphnia magna: Sertraline (EC50=1.15 mg L(-1))>Clomipramine (2.74 mg L(-1))>Amitriptyline (4.82 mg L(-1))>Fluoxetine (5.91 mg L(-1))>Paroxetine (6.24 mg L(-1))>Mianserine (7.81 mg L(-1))>Citalopram (30.14 mg L(-1)) and Venlafaxine (141.28 mg L(-1)). These acute toxicities were found correlated to Log Kow coefficients (R=0.93, p<0.001) and to cytotoxicity assessed on abalone hemocytes through the neutral red uptake assay (R=0.96, p<0.001). If narcosis as mode of action is typically expected during acute ecotoxicity bioassays, we showed by molecular modeling that particular interactions can exist between antidepressants and phosphatidylcholine, a major component of cell membranes, leading to a more specific mode of action corresponding to a potential acidic hydrolysis of ester functions.

In Vitro Synthesis of Proteoglycans and Collagen in Primary Cultures of Mantle Cells from the Nacreous Mollusk, Haliotis tuberculata: A New Model for Study of Molluscan Extracellular Matrix

Abstract: In Mollusca, the mantle produces an organic matrix that mineralizes in time to make shell. Primary mantle cell cultures from the nacreous gastropod Haliotis tuberculata have been established as useful experimental model to investigate in vitro synthesis of both proteoglycans/glycosaminoglycans (PGs/GAGs) and collagen. First, we tested different enzymatic digestion procedures to find the method that gives the highest percentage of viable and adherent cultured cells. Enzymatic digestion with 0.1% pronase plus 0.1% collagenase was routinely used. Six days after the initiation of culture, about 80% of cells were viable, among which 20% were adherent as quantified by the MTT reduction assay. In addition, the protein synthesis estimated by [3H]leucine incorporation remained constant during this period. For the first time, we demonstrated a de novo synthesis of PGs/GAGs and collagen in primary cultures of mantle cells. After 48 hours of labeling, among the [3H]-d-glucosamine macromolecules synthesized, [3H]PGs/GAGs represented 43%, divided into 45% heparan sulfate, 37% chondroitin/dermatan sulfate, and 6% hyaluronic acid. Early elution on anion-exchange chromatography of these PGs/GAGs indicated that most of them appeared as undersulfated GAG molecules. De novo synthesis of collagen represents 4.52% ± 0.84% (SD) with respect to the total protein synthesis. Such a model will facilitate studies on the synthesis of PGs/GAGs and collagen as components of the extracellular matrix and its regulation in Mollusca. Both PGs/GAGs and collagen participate in molecular events that regulate cell adhesion, migration, and proliferation. Further studies with this type of in vitro model should provide knowledge about novel aspects of molluscan cell signaling, in relation to extracellular matrix components.

Effects of vertebrate growth factors on digestive gland cells from the mollusc Pecten maximus L.: an in vitro study

Abstract In relation with the digestive cycle, the digestive gland cells of bivalve molluscs undergo a sequence of cytological changes which is controlled by external and internal effectors such as putative gastrointestinal hormones and growth differentiation factors. A tissue dissociation method was developed to investigate the in vitro effect of the vertebrate growth and differentiation factors: insulin, insulin growth factor I (IGF-I), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on the digestive gland cells of the scallop Pecten maximus. All these vertebrate peptides induced a dose-dependent increased incorporation of 3H-leucine and 14C-uridine in whole digestive gland cell suspensions. However, after Percoll density gradient purification of the digestive cells, only stem and undifferentiated enriched cell fractions were responsive to the different peptides. In addition, insulin and IGF-I, but not EGF and bFGF, stimulated 3H-leucine incorporation in control dispersed mantle edge cells. These results suggest that insulin-related peptides could work as general growth promoting factors in molluscs. On the other hand, EGF and bFGF, or at least their molluscan counterparts, may be efficient growth differentiation factors in the regenerative processes occurring in the digestive gland of molluscs.

Responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata under 10-day exposure to cadmium chloride

Among metals, cadmium, a non-essential element, is an important pollutant that is released into aquatic environments. Due to its persistence and bioaccumulation, this metal has been shown to exert immunological effects on organisms. The objective of the present study was to investigate the in vitro effects of cadmium chloride using a haemocyte primary culture from the European abalone, Haliotis tuberculata. Most studies have maintained viable haemocytes in vitro for periods ranging from several hours to several days during acute exposures. Few investigations have reported the effects of metals using longer in vitro exposures, which are more realistic with regard to mimicking environmental conditions. In this study, we exposed abalone haemocytes to concentrations from 0.5 to 50,000 μgL(-1) of CdCl2 for 10 days. The effects of cadmium chloride were reflected in a significant decrease in the number of viable cells and morphological modifications in a concentration-dependent manner beginning at a concentration of 500 μgL(-1) as well as in some physiological processes, such as phagocytotic activity and the number of lysosome-positive cells. In contrast, phenoloxidase (PO) activity and reactive oxygen species (ROS) production were increased beginning at a concentration of 5 μgL(-1), which is consistent with environmental concentrations in polluted sites. For PO activity and ROS production, maximally 9-fold and 130% inductions, respectively, were recorded under the highest dose. These results thus indicate that cadmium chloride alters immune parameters of abalone haemocytes and that the long-term (10 days) primary culture system used here represents a suitable, sensitive in vitro model for assessing cytotoxic responses.

Assessment of cytotoxic and immunomodulatory properties of four antidepressants on primary cultures of abalone hemocytes (Haliotis tuberculata).

Pharmaceutical compounds like antidepressants found in surface waters raise concerns due to their potential toxicity on non-target aquatic organisms. This study aimed at investigating the in vitro cytotoxicity and immunomodulatory properties of four common antidepressants, namely Amitriptyline, Clomipramine, Citalopram and Paroxetine, on primary cultures of abalone hemocytes (Haliotis tuberculata), after 48 h-exposure. Effects on immunocompetence (phagocytosis, levels of reactive oxygen species, esterase activity and lysosomal membrane destabilization) were assessed. Results obtained by MTT assays revealed that acute toxicity is unlikely to occur in the environment since the LC50s of the four antidepressants are at the mg/L level. The different immunological endpoints displayed a biphasic response, with an increase at the lowest concentration (i.e. 1 μg/L) followed by a decrease at higher concentrations. Overall, Amitriptyline and Clomipramine, the two tricyclic antidepressants, had higher immunomodulatory capacities than the two selective serotonin reuptake inhibitors Citalopram and Paroxetine. Amitriptyline was the most potent and Citalopram the least potent drug in altering immune function in H. tuberculata.

Molecular cloning of a new member of the p53 family from the Pacific oyster Crassostrea gigas and seasonal pattern of its transcriptional expression level

Like other sessile filter-feeding molluscs, oysters may be exposed in the natural environment to a variety of contaminants. Long-term exposure to pollutants may be one factor affecting prevalence of cancerous-like disorders, such as neoplasia. Environmentally induced alterations in p53 protein expression, in relation to leukemia, have been reported in various mollusc species inhabiting polluted water, suggesting that p53 proteins can also be used as a marker for environmental research. This work reports the cloning and sequencing of a p53-like cDNA in the mollusc bivalve Crassostreagigas. The deduced amino acid sequences of p53 shared a high degree of homology with the homologues from other mollusc species, including typical eukaryotic p53 signature sequences. We examined the p53 transcription expression pattern during the annual cycle in oyster gills and whole soft tissues in four locations along the French coasts. Real-time PCR analysis suggested that strong variations at p53 mRNA level are probably synchronized with the seasonal cycle at the four locations investigated.

Cryopreservation of mantle dissociated cells from Haliotis tuberculata (Gastropoda) and postthawed primary cell cultures

Dissociated mantle cells from the gastropod mollusc Haliotis tuberculata were cultured after a freezing-thawing procedure using either 10% dimethyl sulfoxide (Me(2)SO) or 10% glycerol (Gly) as a cryoprotector. The survival rate of 2-day-old cultured cryopreserved cells after thawing, based on analysis of DNA and protein contents, was nearly 80% in comparison with 2-day-old cultured fresh cells. Cells thawed after cryopreservation exhibited the maintenance of all tested physiological activities. Metabolic activity (measured by the MTT test) and the activity of alkaline phosphatase (a plasma membrane-bound enzyme) were not decreased in comparison to those in cultured fresh cells. In addition, cryopreserved cultured cells maintained a physiological stimulation ability in response to treatment with growth factors. These results taken together represent one of the most convincing demonstrations of the survival and of the recovery of intact functional activities of molluscan cells after a freeze-thawing procedure. Our results suggest that in the future primary cultures of cryopreserved mantle cells will be able to be used for fundamental research, in toxicity tests, or in the field of biotechnology.