Copper-induced oxidative stress in rainbow trout gill cells

• Published in: Aquatic toxicology Vol. 86; no. 2; pp. 197 - 204
• Main Authors: Bopp, Stephanie K., Abicht, Helge K., Knauer, Katja
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 31.01.2008; Amsterdam; New York: Elsevier Science; Elsevier Science
• Subjects: Animal, plant and microbial ecology; Animals; Applied ecology; bioassays; Biological and medical sciences; Biological Assay - veterinary; Brackish; Cell Line; cells; Comet assay; Comet Assay - veterinary; copper; Copper Sulfate - toxicity; Cytotoxicity; DNA Damage; Ecotoxicology, biological effects of pollution; Freshwater; Fundamental and applied biological sciences. Psychology; General aspects; gills; Gills - cytology; Lipid peroxidation; Lipid Peroxidation - drug effects; Marine; Oncorhynchus mykiss; Oxidative Stress; reactive oxygen species; Reactive oxygen species (ROS); Reactive Oxygen Species - analysis; Survival Analysis; trout; viability; Water Pollutants, Chemical - toxicity; water pollution; Lipid peroxidation; Cytotoxicity; DNA damage; Comet assay; Reactive oxygen species (ROS); Oxidative stress; Reactive oxygen species; Gill; Ecotoxicology; Toxicity; Genotoxicity; Lipids; Oncorhynchus mykiss; Free radical; Aquatic environment; Vertebrata; Pisces; DNA; Copper; Damage; Peroxidation
• ISSN: 0166-445X; 1879-1514
• DOI: 10.1016/j.aquatox.2007.10.014

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 Cu 2+ concentrations in the medium were calculated using the programm ChemEQL 3.0. 2′,7′-Dichlorodihydrofluorescein-diacetate (H 2DCF-DA) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). 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 viability was significantly reduced at total copper (Cu T) concentrations of 5 μM (corresponding to a free Cu 2+ of 0.11 μM at pH 7) and higher, resulting in an EC 50 of Cu T = 29.2 μM (Cu 2+ = 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 Cu T concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell viability. 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 (Cu T ≥ 100 μM; Cu 2+ ≥ 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 Cu 2+ concentrations. A significant induction of DNA strand breaks was noted at Cu T of 1 and 2.5 μM with greater effects at pH 6 due to higher free Cu 2+ 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 Cu 2+.