Glutamate cysteine ligase (GCL) in the freshwater bivalve Unio tumidus: impact of storage conditions and seasons on activity and identification of partial coding sequence of the catalytic subunit.

Glutamate cysteine ligase (GCL; EC 6.3.2.2) is the first enzyme involved in the synthesis of glutathione. A HPLC method with fluorimetric detection was used to measure GCL activity in the gills and the digestive gland of the freshwater bivalve, Unio tumidus. Storage conditions were optimized in order to prevent decrease of GCL activity and consisted in freezing the cytosolic fraction in the presence of protease (1 mM phenylmethylsulfonic fluoric acid) and gamma-glutamyltranspeptidase (1 mM L-serine borate mixture and 0.5 mM acivicin) inhibitors. Seasonal variations of activity in the digestive gland and to a lesser extent in the gills were found with activity increasing in spring compared to winter. No sex differences were revealed. The GCL coding sequence was identified using degenerated primers designed in the highly conserved regions of the catalytic subunit of GCL. The partial sequence identified encoded for 121 amino acids. The comparison of the identified partial coding sequence of U. tumidus with those available from vertebrates and invertebrates indicated that GCL sequence was highly conserved.

Response of the bivalve Unio tumidus and freshwater communities in artificial streams for hazard assessment of methyl methacrylate.

In artificial streams (pilot rivers) supplied with the river water of Gave de Pau (France), we studied the effects of methyl methacrylate (MMA) on the autochthonous bivalve Unio tumidus transferred into the streams and on natural freshwater communities colonizing the channels. Unio tumidus and freshwater communities were exposed to MMA for 15 and 30 d, respectively, at measured concentrations ranging from 0.6 to 122 mg/L. Biomarkers studied in the digestive gland and gills of U. tumidus comprised detoxication systems (namely, antioxidant enzyme activities and glutathione status) and lipid peroxidation as a marker of cytotoxicity. Biocoenotic indicators were used to evaluate effects on benthic invertebrates and diatoms. In bivalves, a decrease in antioxidant levels was found at the lowest concentrations tested (0.6 and 6 mg/L), whereas an increase in lipid peroxidation and mortality was registered at 30 mg/L after 15 d of exposure. Disturbances in freshwater communities occurred after 30 d at MMA concentrations of greater than 30 mg/L. Antioxidant responses in bivalves were recorded at the lowest MMA concentration tested, which was close to the predicted no-effect concentration (0.74 mg/L), and cytotoxicity was registered at a concentration corresponding to the 21-d no-observed-effect concentration for Daphnia magna. On the basis of the criteria studied, antioxidant biomarkers of bivalves appeared to be more sensitive than biocoenotic indicators to MMA.