Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes

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Abstract

We have previously shown that, in trout hepatocytes, exposure to a high dose of copper (Cu) leads to disruption of Ca2+ homeostasis and elevated formation of reactive oxygen species (ROS), with the latter ultimately causing cell death. In the present study, we aimed at identifying, using a lower Cu concentration, the role of mitochondria in this scenario, the potential involvement of the mitochondrial permeability transition (MPT), and the mode of cell death induced by the metal. Incubation with 10 μM Cu resulted in a strong stimulation of ROS formation, and after 2 h of exposure a significant increase of both apoptotic and necrotic cells was seen. Co-incubation of Cu-treated hepatocytes with the iron-chelator deferoxamine significantly inhibited ROS production and completely prevented cell death. The origin of the radicals generated was at least partly mitochondrial, as visualized by confocal laser scanning microscopy. Furthermore, ROS production was diminished by inhibition of mitochondrial respiration, but since this also aggravated the elevation of intracellular Ca2+ induced by Cu, it did not preserve cell viability. In a sub-population of cells, Cu induced a decrease of mitochondrial membrane potential and occurrence of the MPT. Cyclosporin A, which did not inhibit ROS formation, prevented the onset of the MPT and inhibited apoptotic, but not necrotic, cell death. Cu-induced apoptosis therefore appears to be dependent on induction of the MPT, but the prominent contribution of mitochondria to ROS generation also suggests an important role of mitochondria in necrotic cell death.

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.

References (58)

  • G. Krumschnabel et al.

    Metabolic responses to epinephrine stimulation in goldfish hepatocytes: evidence for the presence of alpha-adrenoceptors

    Gen. Comp. Endocrinol.

    (2001)
  • M. Lam et al.

    Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization

    J. Biol. Chem.

    (2001)
  • J.J. Lemasters et al.

    The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy

    Biochim. Biophys. Acta

    (1998)
  • C. Manzl et al.

    Copper, but not cadmium, is acutely toxic for trout hepatocytes: short-term effects on energetics and ion homeostasis

    Toxicol. Appl. Pharmacol.

    (2003)
  • C. Manzl et al.

    Copper-induced formation of reactive oxygen species causes cell death and disruption of calcium homeostasis in trout hepatocytes

    Toxicology

    (2004)
  • A.L. Nieminen et al.

    Toxic injury from mercuric chloride in rat hepatocytes

    J. Biol. Chem.

    (1990)
  • K. Ollinger et al.

    Cellular injury induced by oxidative stress is mediated through lysosomal damage

    Free Radical Biol. Med.

    (1995)
  • H.L. Persson et al.

    Prevention of oxidant-induced cell death by lysosomotropic iron chelators

    Free Radical Biol. Med.

    (2003)
  • J. Pourahmad et al.

    A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+

    Toxicology

    (2000)
  • J. Pourahmad et al.

    Lysosomal involvement in hepatocyte cytotoxicity induced by Cu(2+) but not Cd(2+)

    Free Radical Biol. Med.

    (2001)
  • J. Pourahmad et al.

    Carcinogenic metal induced sites of reactive oxygen species formation in hepatocytes

    Toxicol. In Vitro

    (2003)
  • T. Qian et al.

    The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187

    Toxicol. Appl. Pharmacol.

    (1999)
  • B. Qu et al.

    Mechanism of clofibrate hepatotoxicity: mitochondrial damage and oxidative stress in hepatocytes

    Free Radical Biol. Med.

    (2001)
  • U. Rauen et al.

    Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron

    Free Radical Biol. Med.

    (2003)
  • U. Rauen et al.

    Iron-induced mitochondrial permeability transition in cultured hepatocytes

    J. Hepatol.

    (2004)
  • F. Ruiz et al.

    Cyclosporin A targets involved in protection against glutamate excitotoxicity

    Eur. J. Pharmacol.

    (2000)
  • A.G. Siraki et al.

    Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes

    Free Radical Biol. Med.

    (2002)
  • S.J. Stohs et al.

    Oxidative mechanisms in the toxicity of metal ions

    Free Radical Biol. Med.

    (1995)
  • N. Zamzami et al.

    Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis

    FEBS Lett.

    (1996)
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