Copper-induced Oxidative Stress in Rainbow Trout Gill Cells

Copper is known to pose a serious threat to aquatic organisms. However, the mechanisms of its toxicity still remain unclear. Cu is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS). The purpose of this work was therefore to link the exposure to copper at pH 6 and 7 to cellular formation of ROS and effects like cell viability and genotoxicity using the rainbow trout gill cell line RTgill-W1. To relate effects to bioavailable copper, free Cu2+ concentrations in the medium were calculated using the programm ChemEQL 3.0. H2DCF-DA (2¿,7¿-Dichlorodihydrofluorescein-Diacetate) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe CFDA-AM (5-carboxyfluorescein diacetate acetoxymethyl ester). DNA strand breaks were assessed using the comet assay, and lipid peroxidation was investigated using the thiobarbituric acid-reactive substances assay (TBARS). Copper treatment resulted in a dose-dependent elevation in cytotoxicity and formation of cellular ROS. Cell vitality was significantly reduced at total copper (CuT) concentrations of 5 µM (corresponding to a free Cu2+ of 0.11 µM at pH 7) and higher, resulting in an EC50 of CuT = 29.2 µM (Cu2+ = 0.63 µM, pH 7). Neither an impairment concerning the viability of control cells due to growth at pH 6 was observed, nor significant differences for cytotoxicity in cells exposed to the same nominal CuT concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell vitality. After normalizing ROS formation to cell viability, ROS induction up to 25-35 fold compared to the control was detected, but mainly for rather high concentrations (CuT = 100 µM; Cu2+ = 2.2 µM, pH 7). ROS formation rates were slightly higher when cells were exposed to Cu at pH 6 compared to pH 7, correlating with the higher free Cu2+ concentrations. A significant induction of DNA strand breakages was noted at CuT of 1 and 2.5 µM with greater effects at pH 6 due to higher free Cu2+ concentrations than at pH 7. No effects on lipid peroxidation were observed. These results lead to the hypothesis that copper-induced loss in viability and genotoxicity in trout gill cells are partially triggered by the generation of ROS and related to the free Cu2+.JRC.H.5-Rural, water and ecosystem resource