PHAGOCYTIC ACTIVITY OF EARTHWORM COELOMOCYTES USED
AS A SUBLETHAL BIOMARKER OF METAL TOXICITY
Sauvé S, Hendawi M., Brousseau P., Fournier M.
INRS-Institut Armand Frappier, 245 Hymus Boul., Pointe-Claire, Québec, Canada, H9R 1G6,
e-mail: sebastien.sauve@inrs-sante.uquebec.ca.
1. Introduction
Earthworms lead an essential ecological role in the cycling of nutrients through their effects on the
physicochemical soil properties and on their enhancement of microbial processes. Acute
toxicological tests on earthworms are only useful to establish grossly excessive contamination.
Various earthworm incubation assays show a significant metal accumulation in the invertebrate
tissues without apparent effects on their survival. Quantifying the metals levels leading to a reduced
efficiency of the invertebrate’s immune functions would permit to establish the threshold where the
fitness of the worm is impaired, before it is being killed outright.
2. Material and Methods
We extracted coelomocytes of Lumbricus terrestris, Eisenia fetida, Tubifex tubifex and an
indigenous species. Cells were extracted into a Lumbricus Balanced Salt Solution (LBSS – Fugère
et al. 1997). Total number of cells was determined microscopically. In parallel, viability was
defined as membrane permeability to propidium iodide determined by flow cytometry. To quantify
phagocytosis, yellow-green latex Fluoresbrite™ beads were added to cell suspensions and
incubated at room temperature. To establish the in vitro dose response curves, metals salts at
concentrations ranging from 10-9 to 10-4 M were added for CdCl2, CuCl2, HgCl2. MeHgCl, MoCl4,
PbNO3 and ZnCl2. 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). The
phagocytic activity was then evaluated using the mean fluorescence of the cells as determined by
flow cytometry. Further details on the data treatment can be obtained from Brousseau et al. (1999).
3. Results and Discussion
For all suspension considered, there was no significant difference in phagocytic activity for cells
cultured in the presence of any metals at concentrations ranging from 10-9 to 10-7 M. However,
metal-related immunotoxicity, expressed as decreased phagocytic activity, was noted for MeHgCl
and HgCl2 around 10-6 M. CdCl2 and CuCl2 became toxic at 10-5 M while MoCl4 and ZnCl2 became
toxic at 10-4 M. Comparison of the dose response of cell viability to metal exposure and the results
for phagocytosis activity clearly show that the coelemocytes immune function is impaired before
the onset of cell death. 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 hemocytes
from different worm species to the different metals varied, but still show surprisingly similar
patterns of response. 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 in vitro sensitivity for the worms can be ranked from most toxic to least as: CH3HgCl >
125
125
100
100
Viability (%)
Phagocytosis (%)
HgCl2 >> CuCl2 > CdCl2 >> MoCl4 > ZnCl2 >>PbNO3. These data indicate that, for all species
studied, mercury (in either forms) was the most potent inhibitor of the phagocytosis with IC50
values systematically lower than for the other metals. Compared to lethal or acute bioassays, the
measurement of phagocytosis by flow cytometry in worms represents a sensitive endpoint to
measure the adverse effects of heavy metals at sublethal concentrations.
75
50
25
0
-10
-9
-8 -7 -6 -5
MeHgCl (10 x M)
-4
-3
75
50
25
0
-10 -9
-8 -7 -6 -5
MeHgCl (10 x M)
-4
-3
E. fetida
Indigenous
T. tubifex
L. terrestris
Figure 1. Phagocytic response and immune cell viability as a function of Hg concentration.
4. Conclusions
We have shown that a simple in vitro assay of phagocytic activity can identify metals inducing
reduction of immune capability. The assay was only performed in vitro and further work is required
to link the levels observed in vitro to the actual worm tissue burden necessary to induce similar
immune function suppression and also the metal levels in soils that will induce bioaccumulation of
those metal levels.
Although the conditions of an 18 h in vitro assay are less realistic than in vivo soil exposure, it must
be considered that the short assay attempts to evaluate the organism’s response when faced with a
chronic sublethal exposure. This may help to elucidate the occurrence and absence of earthworms
in certain contaminated sites and possibly identify the physicochemical parameters conducive to
sublethal toxicological effects and the concomitant reduction in the worm fitness.
5. References
Brousseau, P., Y. Payette, H. Tryphonas, B. Blakley, H. Boermans, D. Flipo, and M. Fournier 1999.
Manual of immunological methods, CRS Press: Boca Raton, FL, USA.
Fugère, N., Brousseau, P., Krzystyniak, K., Coderre, D., Fournier, M. 1996. Heavy metal-specific
inhibition of phagocytosis and different in vitro sensitivity of heterogeneous coelomocytes
from Lumbricus terrestris (Oligochaeta). Toxicol. 109:157-166.