Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes
Introduction
The role of reactive oxygen species (ROS) in cell death induced by metals has been amply documented over the past years (Stohs and Bagchi, 1995). In the case of copper (Cu), an essential trace element, its toxicity is believed to be mainly caused by participation in a Haber–Weiss reaction, whereby it catalyses the formation of ROS, and by peroxidation of membrane lipids (Britton, 1996, Chan et al., 1982). Recent detailed analyses by Pourahmad et al., 2001, Pourahmad et al., 2003 using rat hepatocytes indicated that the main sources of ROS generated in the presence of Cu were the lysosomes, whereas the mitochondria, a significant source of ROS under basal conditions (Kannan and Jain, 2000, Siraki et al., 2002), appeared to be not involved. In line with this, several studies have shown that during incubation with redox-reactive agents such as naphtazarin (Ollinger and Brunk, 1995) or H2O2 (Antunes et al., 2001, Persson et al., 2003), cell death is similarly related to lysosomal events. At the same time, however, mitochondria are clearly important targets of metal toxicity (Al-Nasser, 2000, Koizumi et al., 1994, Nieminen et al., 1990, Rauen et al., 2004, Xiang and Shao, 2003) and in many cases a close link between metal-induced radical stress and mitochondrial function has been established (Pourahmad and O'Brien, 2000, Zhao et al., 2003). In principle, mitochondria may be affected by radical stress in several ways, including (a) oxidation of membrane lipids causing direct damage to mitochondrial integrity (Kowaltowski et al., 1996); (b) elevation of matrix Ca2+ due to radical-induced disruption of cellular Ca2+ homeostasis (Orrenius et al., 2003); and (c) alteration of matrix redox status affecting the function of mitochondrial proteins (Kowaltowski et al., 2001). In addition, each of these processes may ultimately contribute to the induction of the mitochondrial permeability transition (MPT), a phenomenon of crucial importance in many models of necrotic and apoptotic cell death (Lemasters, 1999).
In recent studies on the acute toxicity of Cu on trout hepatocytes, we observed that, in these cells, cell death was also mainly related to Cu-induced radical formation (Manzl et al., 2003, Manzl et al., 2004). Besides enhancing ROS formation, Cu also elevated intracellular free Ca2+ (Ca2+i) and in previous studies it was shown that elevated Ca2+i per se may cause a depletion of the intracellular glutathione pool in trout hepatocytes (Krumschnabel et al., 1999), as does Cu in rat hepatocytes (Pourahmad and O'Brien, 2000). Overall, it thus appears that Cu may produce many of the effects described to affect the mitochondria. However, according to our knowledge, the impact of Cu on mitochondrial function has received comparatively little attention so far and, despite reports showing that Cu or Cu compounds may sensitize mitochondria to undergo MPT (Costantini et al., 1998, Garcia et al., 2000), it has not been directly investigated whether the MPT contributes to Cu-induced cell death in intact cells.
The aim of the present study was therefore to determine, in trout hepatocytes, the role of the mitochondria in Cu-induced cell death, their potential contribution to Cu-induced ROS production, and the possible involvement of the MPT in Cu toxicity. Furthermore, since the MPT may be involved in both necrotic and apoptotic cell death, we wanted to elucidate which mode of cell death is triggered by Cu and if inhibition of the MPT could confer protection against the detrimental effects of the metal.
Section snippets
Chemicals
Bisbenzimide H 33342 (Hoechst 33342), bovine serum albumin (BSA), carbonyl cyanide m-chlorophenyl hydrazine (CCCP), collagenase (Type VIII), cyanide (CN), deferoxamine mesylate (DFO), dimethyl sulfoxide (DMSO), 2′4-dinitrophenol (DNP), myxothiazol, poly-l-lysine (PLL), rotenone (ROT), thenoyltrifluoracetone (TTFA), and Trypan Blue were purchased from Sigma (Deisenhofen, Germany). Calcein-AM, dichlorofluorescein diacetate (DCF-DA), fura 2-AM, MitoTracker Orange,
Cell death and radical stress
Exposure of trout hepatocytes to 10 μM Cu for 2 h resulted in a significant increase in the number of both apoptotic and necrotic cells. The number of apoptotic hepatocytes, which were identified by typical apoptotic features such as chromatin condensation and nuclear fragmentation (Figs. 1B and C), more than doubled from 2.0% in controls to 4.7% in Cu-treated cells (Fig. 1A). A similar relative increase was seen in the percentage of necrotic cells, which after 2 h of incubation amounted to
Discussion
Our present data clearly demonstrate that Cu induces both necrotic and apoptotic cell death in trout hepatocytes. Furthermore, in line with previous observations, we found that Cu stimulated an increased production of ROS, and it appears that these oxygen radicals were the main toxic effect causing loss of cell viability. Thus, addition of the iron chelator DFO, which significantly reduced Cu-induced ROS formation, also protected against Cu-induced cell death. Several studies by Brunk and
Acknowledgments
This study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung in Österreich, project no. P16154-B06. The expert technical assistance of Veronika Lacher is gratefully acknowledged. We dedicate this work to the great comparative physiologist Prof. Wolfgang Wieser on the occasion of his 80th birthday.
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