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 31 P 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.

Investigating the establishment of primary cultures of hemocytes from Mytilus edulis

“…All solutions were adjusted to a pH of 7 before use and were sterilized by autoclaving and filtering the solutions through a 0.22 lm filter. On the day of the extraction, mussels were sterilized in 1L of bleach solution (0.026% v/v) prepared in autoclaved ultra-pure water for 10 min as recommended in Droguet (2006). This step was done in order to sterilize the mussels and reduce the risk of contamination due to contaminants residing on the shell.…”

“…Mussels were then removed from ASW and the shells were sterilized with 70% ethanol and allowed to dry (Droguet et al 2012). A scalpel was then gently inserted into the ventral-anterior side of the animal to drain retained water that could potentially contaminate the cell culture.…”

Effects of Pollution on Marine Organisms

“…In addition, glutathione-S-transferase (GST) and lipid peroxidase were upregulated, gill histology changed, and the filtration rate and overall growth was reduced. A study by Droguet et al (2012) with oysters (Crassostrea gigas), both in vivo and in vitro with cultured oyster heart cells, found effects of TBT on ionic conductance, cytoplasmic Ca2+, ATP level, apoptosis, necrosis, heart beat rhythm, and mortality.…”

Modulation of the F1FO‐ATPase function by butyltin compounds

“…Since the F 1 F O ‐ATPase is a key entity in energy metabolism of the cell, in many cases it represents the molecular target of butyltins, and especially of TBT, responsible for the cell damage. A variety of biological effects of organotins such as alteration of the transmembrane ion gradient, inhibition of protein synthesis, immunotoxicity, hard tissue mineralization, decreased muscle contractility, neurotoxicity, reproductive efficiency and mode of cell death have been correlated with ATP depletion. Assuming that the catalytic activity is inhibited, the blocked step(s) and the mechanisms involved seem to tightly depend on the organotin compound and its concentration as well as on membrane properties.…”