Cultured heart cells from oyster : an experimental approach for evaluation of the toxicity of the marine pollutant tributyltin

Abstract

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 (R)-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 P-31 nuclear magnetic resonance (NMR) spectroscopy and confirmed by the increase of the K-ATP 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.