Isolation and fractionation of gill cells from freshwater (Lasmigona costata) and seawater (Mesodesma mactroides) bivalves for use in toxicological studies with copper
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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®: clam) and Na+, K+-ATPase activity measurement were used to distinguish between cells of Fractions I and II. For mussel and clam, 80.5 ± 1.5 and 48.3 ± 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 μg 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 μg 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.
KeywordsBioaccumulation Clam Copper Gills Isolated cells Mussel
We thank Rodney McInnis, Tina Hooey, Jim Bennett, Mark McMaster (Environment Canada) and Robert Boyle and Ana Cristina Kalb (Universidade Federal do Rio Grande, Brazil), and two anonymous reviewers for constructive comments. This research was supported by awards from the International Development Research Centre (IDRC, Ottawa, ON, Canada) to A. Bianchini and C.M. Wood, the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil), and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, DF, Brazil). A. Bianchini is a research fellow from the Brazilian CNPq (Proc. #304430/2009-9) and is supported by the International Research Chair Program from IDRC. C.M. Wood is supported by the Canada Research Chair Program. PL. Gillis is supported by Environment Canada.
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