Metal bioaccumulation and physiological condition of the Pacific oyster (Crassostrea gigas) reared in two shellfish basins and a marina in Normandy (northwest France).

A 5-month experiment combining a geochemical survey of metals with a bioaccumulation study in batches of Crassostrea gigas was conducted in two shellfish farming areas and a marina in Normandy (France). Various endpoints at different levels of biological organization were studied. ROCCH data showed differences in biota contamination between the two shellfish areas but the present study revealed only slight differences in metallic contamination and biomarkers. By contrast, significantly different values were recorded in the marina in comparison with the two other sites. Indeed, higher levels of Cd, Cu and Zn were measured in the oysters from the marina, and these oysters also showed a poorer physiological condition (e.g., condition index, histopathological alterations and neutral lipid content). For coastal monitoring, the multi-biomarker approach coupled with an assessment of metallic contamination in biota appeared to be suitable for discriminating spatial differences in environmental quality after only a few months of exposure.

Toxicity of five antidepressant drugs on embryo-larval development and metamorphosis success in the Pacific oyster, Crassostrea gigas.

Unlike conventional pollutants, pharmaceutical residues are continuously discharged at low levels (low to mid ng l(-1) concentrations), which results in the chronic contamination of non-target organisms, but little is known about the effects of these residues. The purpose of this study was to provide the first assessment of the ecotoxicity of five antidepressants (selective serotonin reuptake inhibitors [SSRIs] fluoxetine and sertraline, tricyclic antidepressants [TCAs] clomipramine and amitriptyline, and serotonin norepinephrine reuptake inhibitor [SNRI] duloxetine) at a wide range of concentrations from 0.1 to 100,000 µg l(-1) on two early life stages in the Pacific oyster. The toxicity was quantified in D-shaped larvae after 36 h of exposure, and in 21-day-old pediveliger larvae after 24 h of exposure using the percentage of normal larval development and the metamorphosis rate as endpoints, respectively. The embryotoxicity assays reported that the EC50 values were within the same range of concentrations (67 to 192 µg l(-1)) for all of the tested molecules. The metamorphosis tests revealed that the antidepressants can be ranked along an increasing severity gradient: clomipramine < amitriptyline < duloxetine ~ fluoxetine. Sertraline appeared to be the less toxic molecule on this endpoint; however, a different concentration range was used. The embryotoxicity test was more sensitive than the metamorphosis bioassay for three of the five molecules tested, but the latter test showed more practical benefits. Overall, the obtained toxicity values were at least 10,000-fold higher than the reported environmental concentrations.

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.

Shell repair process in the green ormer Haliotis tuberculata: a histological and microstructural study.

In the present paper, juvenile and adult shells of the green ormer Haliotis tuberculata ('Oreille de Saint-Pierre') were perforated in a zone close to the shell edge and the shell repair process was followed at two levels: (1) by observing the histology of the calcifying mantle in the repair zone and (2) by analyzing with SEM the microstructure of the shell repair zone. Histological data clearly show the presence of calcium carbonate granules into the connective tissues, but not in the epithelial cells. This suggests that calcium carbonate granules are synthesized by sub-epithelial cells and actively transported through the epithelium to the repair zone, via a process which may be similar to that described by Mount et al. [Mount, A.S., Wheeler, A.P., Paradkar, R.P., Snider, D., 2004. Hemocyte-mediated shell mineralization in the eastern oyster. Science 304, 297-300]. Furthermore, SEM observations show that the repair zone exhibits different stratified microstructures (spherulitic, thin prismatic, blocklike, sub-nacreous, nacreous, foliated-like), some of which are not continuous (i.e. lenticular) along the repair zone. This suggests a complex secreting regime of the calcifying mantle and an elaborate geometry of the epithelium involved in shell repair.

Preliminary study of the effects of commercial lactobacilli preparations on digestive metabolism of juvenile sea bass (Dicentrarchus labrax).

Two forms of the same commercial product (SORBIAL, Allonnes, France), one with live bacteria (PSA) and the other with heat-inactivated bacteria (PSI), containing a mixture of 2 strains of lactobacilli and their growth medium were tested as a diet complement for juvenile sea bass (Dicentrarchus labrax) during a 103-day experiment. In addition to zootechnical parameters (survival, growth, conformation), some effects on digestive metabolism were studied, including enzymatic, ultrastructural and microbial aspects. Microbial preparations improved survival rate. The ventral, dorsal and operculum malformations which usually occur in juveniles did not appear in those receiving PSA and PSI. Furthermore, they stimulated, but not constantly, trypsin and acid phosphatase activities. Intestinal ultrastructure showed an increase in the number of endocytosis vesicles at the apical pole of enterocytes in fishes receiving enrichments. Bacterial flora was not modified in terms of quantity, especially the lactic acid bacteria counts, which were not changed in fishes receiving live lactobacilli (PSA). The mode of action of these multiple beneficial effects appears complex and could be caused by different molecules inside the bacterial cell or excreted into their medium.

Influence of cryoprotective agent and cooling rate on frozen and thawed hemocytes from the Mollusk Haliotis tuberculata.

Cultures of circulating cells from abalone (Haliotis tuberculata) may be used in fundamental research or in biotechnology. This paper describes attempts to develop a cryoconservation method for these hemocytes in order to constitute a standardized cell stock. Among a panel of five distinct cryoprotective solutions, 10% v/v glycerol ('G solution') was the most effective and better post-thaw recovery was achieved after cooling at 1 degrees C/min than after more rapid cooling (3 degrees C or 9 degrees C/min). In 2-day-old cultures, cell viability, assessed by DNA or protein content, was 83 and 78%, respectively, and metabolic activity, measured by the MTT reduction assay, reached 96%. Viability rates were only slightly reduced after 6 days of culture, suggesting a low proportion of damaged cells among the surviving hemocytes. This study identified a cryoprotective solution and a freezing protocol that allow thawed hemocytes to recover a large part of their viability.

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.

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.

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

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.

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.

Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells.

To determine the functions of the alpha 1 and beta 1 thyroid hormone receptors (TRs) in neural differentiation, we have established stable transfected neuronal cell lines (Neuro-2a) that overexpress either TR alpha 1 or TR beta 1. 3,5,3'-Triiodothyronine (T3) treatment of cells that overexpress TR beta 1 blocks proliferation by an arrest of cells in G0/G1 and induces morphological and functional differentiation of Neuro-2a cells as indicated by the marked increase in the number of perisomatal filopodia-like neurites and in acetylcholinesterase (AChE) activity. The effect on AChE activity was dose-dependent, and the time-course analysis reveals that this effect occurs after 24 hr of T3 treatment, with a maximal increase occurring after 48 hr of treatment. The increase of AChE activity is paralleled by an increase of AChE mRNAs. Last, we present evidence that shows that the effects of T3 on differentiation are independent of its effect on proliferation. T3 had no effect on the differentiation of Neuro-2a cells that overexpressed TR alpha 1. Our results indicate that TR beta 1 may play a key role in the effects of T3 in neuroblastoma cell differentiation.

Thyroid hormones and putative nuclear T3 receptors in tissues of the ascidian, Phallusia mammillata cuvier.

Thyroid hormones were measured by radioimmunoassay in blood plasma and in extracts (butanol/chloroform/ammonia) of pharynx, alimentary canal, and tunic of Phallusia mammillata. Other animals were injected with [125I]T3 and its distribution in the same tissues was determined from 6 to 48 hr after injection. Last, the saturable binding of [125I]T3 to salt-extracted nuclear proteins in the pharynx and alimentary canal was studied in vitro. T4 was found in all tissues examined and in the same order of magnitude (2.7 to 8.4 ng/g) whereas plasma concentration was low (0.2 ng/ml). Tissue T3 concentrations were always much lower than T4 tissue concentrations, being highest in alimentary canal (0.8-1.1 ng/g) and very low in the tunic as well as in plasma, in which T3 was generally below 0.02 ng/ml. The tissue distribution of [125I]T3 was correlated with T3 concentrations. Tissue/plasma ratios were approximately 10 in the alimentary canal, 5 in the pharynx, and 0.18 in the tunic. Saturable binding of T3 to nuclear proteins in the alimentary canal and pharynx was demonstrated. The affinity (Kd) was similar to that found in tissues from other chordates but the maximal binding capacity was much lower. The very low levels of plasma T3 and low T3/T4 ratios may indicate that the endostyle releases primarily T4 into the body fluid. On the other hand, the high levels of T3 and the high T3/T4 ratios in the alimentary canal suggest that this metabolically active target tissue is the main site of the process of deiodination of T4 into T3, a process which has been previously shown in P. mammillata in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid hormone up-regulates thyroid hormone receptor beta gene expression in rat cerebral hemisphere astrocyte cultures.

Oligonucleotide probes complementary to specific regions of three thyroid receptor cDNAs were used to study the effects of thyroid hormone on the expression of the mRNAs encoding two alpha (alpha 1 and alpha 2) and one beta-thyroid (beta 1) receptors isoforms in rat cerebral hemisphere astrocyte cultures. Both genes are expressed by type 1 astrocytes. The levels of the alpha 1-, alpha 2-, and beta 1-mRNAs did not significantly change between day 8 and day 22, in cultures grown in the absence of thyroid hormone. L-triiodothyronine (L-T3) treatment of the cultures increased the levels of beta 1-mRNAs by fivefold without changing either the levels of the alpha 1- and alpha 2-mRNAs or L-T3 binding capacity. The effect of L-T3 on beta 1-mRNAs was observed after 4 h of treatment and was independent of protein synthesis, suggesting that this effect is likely to be a direct one. Treatment of the cultures by cytosine arabinosine, a drug that kills dividing cells, specifically decreased level of the alpha 1- and alpha 2-mRNAs by 60% and 38%, respectively. Finally, by immunocytochemistry, we showed that the beta 1 receptor-immunoreactivity was either located in the perinuclear region and the cytoplasm or in the nuclei of astrocytes. Taken together with previous data obtained in neuronal cultures where no effect of L-T3 was observed on the levels of the beta 1-mRNAs, our findings indicate that the beta 1 gene is differentially regulated in neurons and astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine is necessary for the action of growth hormone in acclimation to seawater of brown (Salmo trutta) and rainbow trout (Oncorhynchus mykiss).

Brown (BT) and rainbow trout (RT) in freshwater (FW) were treated with ovine growth hormone (GH), GH + iopanoic acid (IOP), and GH + IOP plus triiodothyronine (T3) for RT only. After 1 week of treatment, trout were transferred to 30 o/oo SW and treatment continued. In FW, GH treatment increased significantly plasma T3 level (BT) and T3/T4 ratio (BT and RT) by stimulating T4 to T3 deiodination. In the GH + IOP group, the plasma T3 levels and T3/T4 ratio fell significantly as T4 to T3 deiodination was inhibited. In GH + IOP + T3-treated RT, plasma T3 and T3/T4 ratios increased significantly relative to other groups. No mortality occurred and plasma osmolarity (PO) was not altered by any treatment in FW. After transfer to SW, all IOP + GH trout died within 2 (BT) or 3 days (RT). All GH-treated or control BT survived to the end of the experiment (6 days). RT survival rates tended to be improved in GH and GH + IOP + T3 groups relative to controls. Correlatively on day 1 the PO increase was significantly higher in IOP + GH groups (BT and RT) than in the other groups and significantly lower in GH and GH + IOP + T3 treated RT than in controls from days 1 to 6. These data confirm the requirement of T3 and deiodination of T4 to T3 for the development of hypoosmoregulatory mechanisms in SW as previously shown (Lebel and Leloup 1992). Furthermore, the suppression of the hypoosmoregulatory effect of GH, when conversion of T4 to T3 was inhibited by IOP and the reversal when T3 was added to IOP + GH treatment suggests that GH osmoregulatory action in SW acts via the simulation of T4-5' monodeiodination which increases T3 production.

[Triiodothyronine is needed for the acclimatization of brown trout (Salmo trutta) or rainbow trout (Oncorhynchus mykiss) to seawater]. / La triiodothyronine est nécessaire à l'acclimatation à l'eau de mer de la truite fario (Salmo trutta) ou arc en ciel (Oncorhynchus mykiss).

Brown and rainbow trout, held in freshwater at 13 +/- 1 degrees, were injected, every 3 days, with iopanoic acid (IOP: 5 mg/100 g body wt), an inhibitor of deiodination of thyroxine (T4) to triiodothyronine (T3). One group of IOP-treated rainbow trout was immersed in T3 (20 micrograms/l water). In IOP trout, plasma T3 fell to very low levels by day 7, while changes in T4 levels were less marked. In IOP + T3 trout plasma T3 increased fivefold, plasma T4 being unchanged. No mortality occurred and plasma osmolarity (OP) was not altered by any treatment. After direct transfer to seawater (30/1000), IOP trout were unable to acclimate to salinity: all died within 2 or 3 days, while the survival at day 3 was 100% in control brown trout and 45 and 74% in control and IOP + T3 rainbow trout respectively. OP increased more in IOP and less in IOP + T3 than in controls. There was a significant inverse correlation between T3, but not T4, plasma level, at the time of transfer and the OP 1 day later. In conclusion, although T3 does not play a significant role in osmoregulation in freshwater, T3 and therefore the deiodination of T4 into T3, were required for the development of hypo-osmoregulatory capacity involved in acclimation of trout to seawater.

Triiodothyronine binding to putative solubilized nuclear thyroid hormone receptor in liver and gill of the brown trout (Salmo trutta) and the European eel (Anguilla anguilla).

The binding of 3,5,3'-triiodothyronine (T3) to salt-extracted nuclear protein of liver and gill from trout and eel was studied in vitro. [125I]T3 binding depended on the temperature and protein concentration. Binding equilibria were achieved in the two tissues of each species between 15 and 24 hr at 4 degrees. The binding was reversible in the presence of excess unlabeled T3. Scatchard analysis showed a single class of high affinity and low capacity T3 binding species considered, 2.71 x 10(-10) M for eel liver and gill. Association (k + 1) and dissociation (k-1) rate constants, calculated from the kinetics of hormone association, were respectively 8.3 x 10(8) M.hr-1 and 0.216 hr-1 for trout liver and 8.3 x 10(8) M.hr-1 and 0.257 hr-1 for trout gill. Their ratio, the equilibrium dissociation constant for liver (2.60 x 10(-10)M) and for gill 3.10 x 10(-10) M), was in good agreement with the apparent Kd from the Scatchard plot. Half-times (t1/2) of dissociation of T3 calculated from association curves, liver (3.2 hr), and gill (2.7 hr) were in reasonable agreement with corresponding values determined directly, liver (7.7 hr) and gill (11.5 hr). These data are consistent with a reversible bimolecular process to describe the binding of T3 to nuclear extracts. The maximal binding capacity (MBC) was lower in gill than in liver. MBC values were similar in liver of trout, 163, and eel, 234, but were different in gill, 82 for trout and 29 fmol/mg protein for eel.(ABSTRACT TRUNCATED AT 250 WORDS)