Phagocytic activity of freshwater and marine bivalves: in vitro exposure to metals (Ag, Cd, Hg, and Zn) Sauvé S. 1, Brousseau P. 1, Pellerin J. 2, Morin Y. 3, Senécal L. 1, Goudreau P. 3, Fournier M. 1,3

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PHAGOCYTIC ACTIVITY OF FRESHWATER AND MARINE BIVALVES: IN VITRO EXPOSURE TO METALS (Ag, Cd, Hg, and Zn)
Sauvé S.¹, Brousseau P.¹, Pellerin J.², Morin Y.³, Senécal L.¹, Goudreau P.³, Fournier M.^1,3
¹ INRS-Institut Armand Frappier, 245 Hymus Boul., Pointe-Claire, Québec, Canada, H9R 3G6, e-mail: sebastien.sauve@inrs-sante.uquebec.ca.

² Département d'Océanographie, Université du Québec à Rimouski, 300 Allée des Ursulines, C.P. 3300, Rimouski, Québec, Canada, G5L 3A1.

³ Marine Environmental Sciences Division, Maurice Lamontagne Institute, Fisheries and Oceans Canada, 850 de la Mer, Mont-Joli, Québec, Canada, G5H 3Z4.

1.Introduction

Sublethal toxic effects are discrete and difficult to observe. These cause a reduction in competitiveness of the affected species and it affects biological populations through higher disease, parasitism and predation. The highly regulated nature of the immune system renders it quite vulnerable to toxicants and there is a growing concern about the consequences of environmental contaminants on the immune system of aquatic species. Bivalve molluscs represent interesting specimens to perform immunotoxicological studies since they exist in direct contact with contaminated aquatic sediments and they are exposed to waterborne contaminants. Since many are filter-feeding organisms, bivalves have the potential to bioaccumulate contaminants. Immunotoxic effects of environmental exposure to chemical contaminants can be evaluated by monitoring cellular and functional parameters of the immune system of sentinel species (Wong et al. 1992).

2.Material and Methods

Hemolymph was extracted from the heart with a syringe. Cell concentration was determined using a drop of each cell suspension introduced into an improved Neubauer hemocytometer. Total number of cells was determined microscopically. In parallel, viability was defined as membrane permeability to propidium iodide (PI) determined by flow cytometry. To quantify phagocytosis, yellow-green latex FluoSpheres™ were added to cell suspensions and incubated at room temperature. After 18 h, an aliquot of each cell suspension was layered over a 3% bovine serum albumin gradient and centrifuged at 150 X g for 10 min, to remove free beads. The cell pellets were resuspended in 0.5 ml of hematall (Fisher Scientific, Ottawa, Ont, Canada) and the percent of phagocytic cells containing 3 beads and more was determined by flow cytometry. Further details on the data treatment can be obtained from Brousseau et al. (1999; 2000).

3.Results and Discussion

For all suspension considered, there was no significant difference in hemocyte viability for cells cultured in the presence of any metals at concentrations ranging from 10^-9 to 10^-5 M. However, metal-related cytotoxicity, expressed as decreased hemocyte viability, was noted for MeHgCl above 10^-4 M. HgCl₂ became cytotoxic at 10^-3 M while AgNO₃ became cytotoxic at ~10^-4 M. It is difficult to dissociate the onset of cell mortality from the co-occurring effects on phagocytosis.

As shown by these dose-response studies, the sensitivity of bivalve hemocytes to the different metals varied. This difference in sensitivity was evaluated by graphical determination of the concentration for each metal that induces a 50% suppression of the phagocytosis (IC₅₀). The average sensivity for bivalves can be ranked from most toxic to least as: CH₃HgCl >> AgNO₃ > HgCl₂> CdCl₂ >> ZnCl₂. These data indicate that, for all species studied, CH₃HgCl was the most potent inhibitor of the phagocytosis with IC₅₀ values systematically lower than for HgCl₂.Compared to lethal or acute bioassays, the measurement of phagocytosis by flow cytometry in bivalves represents a sensitive endpoint to measure the adverse effects of heavy metals at sublethal concentrations.

Figure 1. Phagocytic response and immune cell mortality as a function of Hg concentration.

4.Conclusions

The present study involves only short term in vitro exposure of hemocytes to heavy metals and demonstrates the potentially detrimental effects of sublethal concentrations of various metals to the immune system of bivalve molluscs. Therefore, consideration should be also given to elucidate the consequences under conditions of chronic exposure. These conditions, which represent reality for organisms living in contaminated sediments, could eventually render the bivalves much more vulnerable to infections, thus drastically affecting their survival and the equilibrium of aquatic ecosystems.

5.References

Brousseau, P., Y. Payette, H. Tryphonas, B. Blakley, H. Boernaus, D. Flipo, and M. Fournier 1999. Manual of immunological methods, CRS Press, Boca Raton, FL, USA.

Brousseau, P., J. Pellerin, Y. Morin, D. Cyr, B. Blakley, H. Boermans, and M. Fournier 2000. Flow cytometry as a tool to demonstrate the disturbance of phagocytosis in the clam Mya arenaria following in vitro exposure to heavy metals. Toxicol. 142: 145-146.

Wong, S., M. Fournier, D. Coderre, W. Banska, and K. Krzystyniak 1992. Environmental Immunotoxicology. In D. Peakall [eds.], “Animal biomarkers as pollution indicators.” Chapman and Hall.

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