title: Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes.
abstract: We have previously shown that copper is acutely toxic for trout hepatocytes, inducing enhanced influx of Ca(2+) and a loss of cell viability. The aim of the present study was to elucidate the pathways of Ca(2+) entry into the cells, the hypothetical role of reactive oxygen species (ROS) in copper toxicity, and the interaction of ROS formation and the disruption of Ca(2+) homeostasis. We found that, acutely, copper-induced cell death occurred independently from an increase of intracellular free Ca(2+) (Ca(2+)i), but could be prevented by addition of agents interfering with ROS production. Addition of the Ca(2+) channel blocker verapamil did not affect the Ca(2+)i increase evoked by copper, whereas in the presence of LaCl(3), an inhibitor of both Ca(2+) channels and Na(+)/Ca(2+)-exchange, this increase was significantly delayed. ROS formation, estimated by use of the fluorescence indicator 2',7'-dichlorofluorescin diacetate, was significantly enhanced by copper. Omission of extracellular Ca(2+) or addition of either verapamil or LaCl(3) did not diminish ROS formation induced by copper. In contrast, the hydroxyl radical scavenger dimethyl sulfoxide and the ferric ion chelator deferoxamine inhibited radical production. In addition, these agents either partially (dimethyl sulfoxide) or completely (deferoxamine) prevented an increase of Ca(2+)i. Altogether our results indicate that ROS formation is the crucial event leading to cell death during acute exposure to copper, whereas the increase of Ca(2+)i is a secondary, acutely less toxic, phenomenon. Furthermore, these findings suggest that Ca(2+) entry occurs via a LaCl(3)-sensitive pathway, presumably representing Na(+)/Ca(2+)-exchange, and non-specific membrane leaks induced by lipid peroxidation in the presence of copper.
authors.affiliation: Institut für Zoologie und Limnologie, Abteilung Okophysiologie, Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria.
authors.forename: Claudia
authors.initials: C
authors.lastname: Manzl
authors.forename: Julia
authors.initials: J
authors.lastname: Enrich
authors.forename: Hannes
authors.initials: H
authors.lastname: Ebner
authors.forename: Reinhard
authors.initials: R
authors.lastname: Dallinger
authors.forename: Gerhard
authors.initials: G
authors.lastname: Krumschnabel
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003300:Copper / Q000633:toxicity*; D022781:Hepatocytes / Q000187:drug effects*; D006706:Homeostasis / Q000187:drug effects*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000187:drug effects
chemical_list: D017382:Reactive Oxygen Species; D019831:Sodium-Calcium Exchanger; D003300:Copper; D002118:Calcium