Establishment of primary cell culture of Ruditapes decussatus haemocytes for metal toxicity assessment.

In ecotoxicology, in vitro testing on cell cultures represents an ideal alternative to in vivo strategies for emerging contaminants. These tests have limited use particularly with marine invertebrates like the clams Ruditapes decussatus. In the present study, a primary culture of R. decussatus haemocytes was realized for the first time in order to determine the effect of metals (copper, zinc, and cobalt) on haemocyte parameters like viability and phagocytosis. Results showed that (i) among the studied medium, the modified Leibovitz (L-15) is the best for R. decussatus haemocytes primary culture. (ii) The primary culture system used here represents a suitable in vitro model for assessing cytotoxic responses, (iii) a decrease of cell viability and phagocytosis after 24 h exposure to 100 µg mL-1 CoSO4 and an increase of phagocytosis after 24 h exposure to 50 µg mL-1CuSO4.

Responses of primary cultured haemocytes derived from the marine gastropod Haliotis tuberculata to an industrial effluent exposure.

This study assessed the responses of primary cultured haemocytes from the marine gastropod Haliotis tuberculata exposed to the increasing concentrations of industrial effluent (0, 0.5, 1, 10, 15 and 20%) discharged into the Tunisian coastal area. Analyses showed the presence of metals such as cadmium (Cd), chromium (Cr), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni) and lead (Pb) in the effluent. The effects of this mixture of pollutants on abalone haemocyte parameters were reflected by a significant decrease of cell viability, phagocytotic activity and reactive oxygen species (ROS) production as well as morphological and lysosomal membrane alterations. Thus, these results indicated that our primary culture system represents a suitable in vitro model for monitoring of anthropogenic contaminants in aquatic environments.

The cell cultures and the use of haemocytes from marine molluscs for ecotoxicology assessment.

Among aquatic organisms suitable for biological monitoring, molluscs occupy a prominent place due to their wide geographic distribution, their abundance and accessibility in the field as well as in aquaculture. Molluscs reflect the degree of environmental contamination and are the most useful bioindicator tools. The study of modulation of immune system or immunomodulation in marine molluscs has become one of the privileged ways for evaluating the physiological effects of environmental factors. Physiological responses of molluscs to environmental stresses could be mediated by haemocytes. These cells are continually exposed to the external environment due to the open circulatory system of molluscs and are affected by pollutants. In fact, several studies showed the effects of different environmental contaminants on haemocyte functions (viability, phagocytosis, ROS production) as well as on proteins involved in cytoskeletal structure maintenance using the in vitro approaches. In ecotoxicology, in vitro approach is an alternative to animal testing due to the reduced use of experimental animals, low cost and rapid performance. Although several studies showed the importance of using in vitro cell models to determine the effects of different environmental contaminants on haemocyte parameters in marine molluscs, a few reviews highlight these effects. The main purpose of this paper is to summarize the recent data on the effect of some xenobiotics on haemocyte parameters in some mollusc species and then suggest future research prospects.

Does a short-term exposure to cadmium chloride affects haemocyte parameters of the marine gastropod Haliotis tuberculata?

In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of cadmium chloride in vitro. Cells were exposed for 24 h to CdCl2 concentrations of 0, 1 and 100 µg ml(-1). The effects of cadmium on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Results showed that cadmium has no significant effects on cell viability and phagocytotic activity under the tested conditions. However, haemocytes became more rounded after cadmium exposure, which could explain the significant decrease of cell area beginning at 1 µg ml(-1) of CdCl2.

Use of metallothioneins as biomarkers for environmental quality assessment in the Gulf of Gabès (Tunisia).

Detection and assessment of the impact of pollution on biological resources imply increasing research on early-warning markers such as metallothioneins (MTs) in metal exposure. In this paper, we have collated published information on the use of metallothioneins and metallothionein-like proteins (MTLPs) as biomarkers for environmental quality assessment in the Gulf of Gabès. In this area, some species of fish and bivalve were used as bioindicators of pollution. In these species, an induction of MTs/MTLPs by the essential metals such as Cu and Zn and the non-essential metals such as Cd was observed by different authors who suggest the potential use of these proteins as biomarkers. However, MT concentrations can be influenced by many biotic (sex, maturity stages, and tissues) and abiotic factors (temperature, salinity, and pH). This is essentially the case in field studies where many parameters can randomly affect MT levels, so the endogeneous regulation of MTs must be considered before using MTs as an indicator of heavy metal exposure. Moreover, the use of biomarker cannot be examined independently of the evaluation of techniques that enable its quantification. Therefore, the approach to the use of MTs/MTLP as biomarkers of exposure for an assessment of the physiological status of aquatic organisms is discussed in this paper.

Cloning and characterization of cDNA probes for the analysis of metallothionein gene expression in the Mediterranean bivalves: Ruditapes decussatus and Cerastoderma glaucum.

cDNA probes have been developed for subsequent use in monitoring the cadmium exposure of the clam Ruditapes decussatus and the cockle Cerastoderma glaucum using metallothionein (MT) gene expression in different tissues of these species. Two partial MT cDNAs were isolated from Ruditapes decussatus and Cerastoderma glaucum. The identification of the nucleotide sequences showed that the cDNAs consist of 480 bp coding 72 amino acid proteins containing 21 cysteine residues organized in Cys-X-Cys motifs as classically described for MTs. The induction of MT gene expression in CdCl(2) treated bivalves was confirmed by dot blot analysis and suggests a potential specific tissue expression rate.