Antioxidative and oxidative changes in the digestive gland cells of freshwater mussels Unio tumidus caused by selected phenolic compounds in the presence of H2O2 or Cu2+ ions

Research on biomarkers as early bioindicators of perturbation in populations and individuals has received increasing interest during recent decades. These ecotoxicity studies allow us to measure the impact of environmental stressors and to monitor and evaluate the degradation or restoration of systems. In the present study we used bivalve molluscs (mussels), which are sensitive biomarkers of aquatic ecosystem pollution, to assess the effects of three polyphenols: tannic acid, ellagic acid and gallic acid. These compounds were used in the 1-60 mu M concentration range, alone and in the presence of H2O2 (40 and 100 mu M) or Cu2+ ions (50 mu M). The fluorescence probe dichlorofluorescein-diacetate (DCFH-DA) was used to measure reactive oxygen species (ROS). The oxidation of DCFH-DA to the fluorescent DCF (dichlorofluorescein) by the phenolic compounds was investigated spectrofluorimetrically. The results showed that the polyphenols tested can act as antioxidants against the ROS present in the digestive gland cells, but their activity is decreased after incubation with hydrogen peroxide or copper ions. SH-groups were determined spectrophotometrically using Ellman's reagent. The results showed that oxidative modification of proteins increased in a concentration-dependent manner in cells incubated with polyphenols (above 15 mu M) alone. Incubation of the cells with phenolic acids and H2O2 or Cu2+ ions revealed that the phenolic acids had prooxidant properties in all concentrations used except for 1 mu M tannic and ellagic acid and 40 mu M H2O2. DNA fragmentation was estimated by a fluorescence method using Hoechst 33258/propidium iodine binding. The data showed that the phenolic acids alone and in the presence of hydrogen peroxide or copper ions can induce apoptosis and necrosis. The methods used and results obtained indicate that the polyphenols selected can act not only as antioxidants but also as prooxidants in digestive gland cells of Unio tumidus. (c) 2006 Elsevier Ltd. All rights reserved.