Download 2. Material and Methods - Université de Montréal

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, email: [email protected].
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.
Phagocytosis (% of control)
140
140
120
120
100
100
80
80
60
60
40
40
20
20
0
100
Cell Mortality
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.
0
10-9 10-8 10-7 10-6 10-5 10-4 10-3
10-9 10-8 10-7 10-6 10-5 10-4 10-3
HgCl2 (M)
MeHgCl (M)
Mactromeris polynyma
Mytilus edulis
100
80
80
60
60
40
40
20
20
0
0
10-9 10-8 10-7 10-6 10-5 10-4 10-3
10-9 10-8 10-7 10-6 10-5 10-4 10-3
HgCl2 (M)
MeHgCl (M)
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.