Isolation and fractionation of gill cells from freshwater (Lasmigona costata) and seawater (Mesodesma mactroides) bivalves for use in toxicological studies with copper

Gills cells of the freshwater mussel Lasmigona costata and the seawater clam Mesodesma mactroides were isolated (mussel: chemical dissociation; clam: mechanical dissociation) and fractionated (Percoll gradient) into Fractions I and II. Mitochondrial dyes (DASPEI: mussel; MitoTracker(A (R)): clam) and Na+, K+-ATPase activity measurement were used to distinguish between cells of Fractions I and II. For mussel and clam, 80.5 +/- A 1.5 and 48.3 +/- A 3.2 % of cells were in Fraction II, respectively. For both species, cells of Fraction II had higher fluorescence emission and higher enzyme activity than those of Fraction I, being characterized as 'cells rich in mitochondria'. Cells of Fraction II were kept in saline solutions approximating the ionic composition of hemolymph either under control conditions (no Cu addition) or exposed (3 h) to copper (Cu: 5, 9 and 20 mu g Cu/L). Cell viability and Cu and Na+ content were measured. For both species, Cu content was higher and Na+ content was lower in cells exposed to 20 mu g Cu/L. Furthermore, a strong negative correlation was observed between cell Na+ and Cu content in the two bivalve species, indicating a possible competition between Cu and Na+ for ion-transporting mechanisms or binding sites at gill cells of Fraction II. Considering that Cu is an ionoregulatory toxicant in aquatic invertebrates, these preliminary toxicological data support the idea of using isolated gill cells rich in mitochondria to study the mechanisms underlying the acute toxicity of waterborne Cu in freshwater and marine bivalves.