Manzl Claudia, Enrich Julia, Ebner Hannes, Dallinger Reinhard, Krumschnabel Gerhard
Institut für Zoologie und Limnologie, Abteilung Okophysiologie, Universität Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria.
Toxicology. 2004 Mar 1;196(1-2):57-64. doi: 10.1016/j.tox.2003.11.001.
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.
我们之前已经表明，铜对鳟鱼肝细胞具有急性毒性，会导致钙离子（Ca(2+)）内流增加以及细胞活力丧失。本研究的目的是阐明Ca(2+)进入细胞的途径、活性氧（ROS）在铜毒性中的假设作用，以及ROS形成与Ca(2+)稳态破坏之间的相互作用。我们发现，急性情况下，铜诱导的细胞死亡独立于细胞内游离Ca(2+)（Ca(2+)i）的增加，但可通过添加干扰ROS产生的试剂来预防。添加Ca(2+)通道阻滞剂维拉帕米并不影响铜诱导的Ca(2+)i增加，而在同时抑制Ca(2+)通道和Na(+)/Ca(2+)交换的LaCl(3)存在时，这种增加会显著延迟。使用荧光指示剂二氯荧光素二乙酸酯评估的ROS形成，被铜显著增强。去除细胞外Ca(2+)或添加维拉帕米或LaCl(3)均不会减少铜诱导的ROS形成。相反，羟基自由基清除剂二甲基亚砜和铁离子螯合剂去铁胺抑制自由基产生。此外，这些试剂要么部分（二甲基亚砜）要么完全（去铁胺）阻止了Ca(2+)i的增加。总之，我们的结果表明，ROS形成是急性暴露于铜期间导致细胞死亡的关键事件，而Ca(2+)i的增加是一种次要的、急性毒性较小的现象。此外，这些发现表明Ca(2+)通过一条对LaCl(3)敏感的途径进入细胞，这条途径可能代表Na(+)/Ca(2+)交换，以及在铜存在下由脂质过氧化诱导的非特异性膜渗漏。
