Aquatic Toxicology 86 (2008) 197–204 Copper-induced oxidative stress in rainbow trout gill cells Stephanie K. Bopp 1 , Helge K. Abicht, Katja Knauer ∗ Department of Environmental Sciences, Programm Man Society Environment (MGU), University of Basel, Vesalgasse 1, CH-4051 Basel, Switzerland Received 8 August 2007; received in revised form 24 October 2007; accepted 28 October 2007 Abstract 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 2 DCF-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 M (corresponding to a free Cu 2+ of 0.11 M at pH 7) and higher, resulting in an EC 50 of Cu T = 29.2 M (Cu 2+ = 0.63 M, 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 M; Cu 2+ ≥ 2.2 M, 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 M 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+ . © 2007 Elsevier B.V. All rights reserved. Keywords: Reactive oxygen species (ROS); Cytotoxicity; DNA damage; Comet assay; Lipid peroxidation 1. Introduction The increasing worldwide contamination of freshwater systems with thousands of industrial and natural chemi- cal compounds is one of the key environmental problems (Schwarzenbach et al., 2006). Besides numerous organic com- pounds entering aquatic ecosystems, heavy metal input is still rising. Apart from cadmium, copper, a trace metal, is one of the most important pollution-causing metals. Despite the impor- ∗ Corresponding author. Current address: Programme Sustainability Research, University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland. Tel.: +41 661 2670438; fax: +41 61 2670775. E-mail address: katja.knauer@unibas.ch (K. Knauer). 1 Present address: European Commission – DG Joint Research Centre, Insti- tute for Environment and Sustainability, Ispra (VA), Italy. tance of copper as an essential trace element for all biota, aquatic organisms may suffer from exposure to concentrations, which might be 10–50 times higher than the required concentrations (Hall et al., 1998). Copper toxicity has been studied in several fish species. It is acutely highly toxic to rainbow trout (Oncorhynchus mykiss) with a LC 50 (96 h) of 210 g/L (De Boeck et al., 2004). Chronic copper exposure for 28 days to 1000 g/L had no effect on growth, condition factor or food consumption of juvenile rain- bow trout. Copper accumulated mainly in the liver, the gill, the gut, and least in the carcass (Kamunde et al., 2001; Handy et al., 2002). Copper toxicity is believed to be partly due to non- specific binding of the metal ion Cu 2+ to biologically important molecules. It binds to histidine-, cysteine- and methionine- residues in proteins with high affinity, which may result in their dysfunction (Camakaris et al., 1999). The influence of 0166-445X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2007.10.014