European Community regulations on chemicals promote alternative methods to test
substances presenting potential risks for the environment. In the present work, cultured
atrial cells isolated from oyster (Crassostrea gigas) were used as an
experimental model to investigate the toxicity of tributyltin (TBT) after
short-time exposure at concentrations representative of those that can be measured in
seawater, marine sediments and/or bivalves bioaccumulating this pollutant. In
vitro and in vivo assays produce values of the same order of
magnitude for both animal/cell survival and heart/cardiomyocyte beating rate. The survival
rate of whole animals decreased from 10-6 M TBT after 3 days.
For cultured cells, the viability, evaluated using 3-(4, 5-dimethylthiazolyl-2)-2,
5-diphenyltetrazolium bromide (MTT) assay, significantly decreased after two days of
treatment with 10-6 M TBT, and after six days with
10-10 M TBT. The percentage of apoptotic cells, quantified
by flow cytometry and YO-PRO®-1 iodide, a nucleic acid stain that only
permeates cells that are beginning to undergo apoptosis, increased significantly in these
cases. Moreover, intracellular concentration of Ca++ had increased after 10 min
of exposition to 10-6 M, and could be associated with apoptotic processes. As
patch clamp experiments showed that Ca++ conductance was decreased,
intracellular calcium increase could mainly be due to a release from internal stores. The
decreases in beating rhythm could be explained by the decrease in adenosine triphosphate
(ATP) production revealed by 31 P nuclear magnetic resonance (NMR) spectroscopy
and confirmed by the increase of the KATP channel conductance. The related
hyperpolarization and the disturbances of the energetic metabolism were clearly related to
the loss of the atrial cell contractility and viability.