Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures

Abstract

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process.

Cobalt, in the ionized species Co²⁺, is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co²⁺ induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca²⁺ transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co²⁺ also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent ΔΨ drop, suggesting that Co²⁺ also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co²⁺ of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co²⁺ also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co²⁺ results in apoptosis induction with caspase activation and increased level of expression of HIF-1α.

All these observations allow us to state that, in the presence of calcium, Co²⁺ is an inducer of apoptosis triggered by mitochondrial oxidative stress.

Introduction

Cobalt is an oligoelement present in almost all the animal and vegetal organisms; its biological importance is due to its essential role in the formation of vitamin B₁₂ and other cobalamines. Vitamin B₁₂ is necessary for the organism, because it is involved in the formation of some proteins and in the normal functionality of the nervous system. Its lack can cause pernicious anaemia and peripheral nervous system diseases (Karovic et al., 2006).

Cobalt is potentially toxic in the ionic form, Co²⁺. Data in the literature indicate that cobalt is cytotoxic to many cell types, including neural cells (Wang et al., 2000) and can induce cell death by apoptosis and necrosis (Huk et al., 2004). It can cause DNA fragmentation (Zou et al., 2001), activation of caspases (Zou et al., 2002), increased production of reactive oxygen species (ROS) (Olivieri et al., 2001), augmented phosphorylation of mitogen-activated protein (MAP) kinases (Yang et al., 2004), and elevated levels of p53 (Chandel et al., 2000), as a consequence of the activation of hypoxia-inducible factor-1 (HIF-1) (Zou et al., 2001). In fact, in cultured cells, cobalt chloride mimics a hypoxic response. Like low oxygen tension, this metal is able to stabilize the α-subunit of HIF-1 (HIF-1α) by blocking its ubiquitination and proteasomal degradation (Epstein et al., 2001, Morwenna and Ratcliffe, 1997). Increased levels of HIF-1α stimulate overexpression of a set of genes encoding several proteins such as heat shock proteins, which promote a physiological response linked to the recovery of cell homeostasis. In the same way the transcription of many pro-apoptotic factors, such as NIP-3 and NIX, is achieved, with the effect of leading to cell death (Bruick, 2000).

Many experiments have been performed on alveolar macrophages and PC12 cells (Zou et al., 2001, Tomaro et al., 1991). The way by which Co²⁺ is able to induce apoptosis still has to be discovered, but there is some evidence that it activates both the extrinsic and the intrinsic pathway. Zou et al. used a caspase 3-like inhibitor, which is able to inhibit programmed cell death partially, suggesting the peculiar role of this protein in the cobalt-mediated process (Zou et al., 2002). In spite of these observations, the molecular mechanism by means of which cobalt leads to cell death still has to be understood.

There is some evidence that it acts by activating the intrinsic apoptotic pathway, because its effect is blocked by caspase 9-inhibitors (Araya et al., 2002). This suggests that production of ROS induced by the metal acts directly on mitochondria to provoke the release of cytochrome c (cyt c) from external mitochondrial membrane, which leads to the activation of caspase 9 and to apoptosis (Pulido and Parrish, 2003). Similar conclusions have also been reported by other authors studying the toxic effects of cobalt in primary cultures of mouse astrocytes (Karovic et al., 2006). The interaction of Co²⁺ with mitochondrial function has been preliminarily investigated at the level of ATP synthesis, with inhibition of this phenomenon, probably ascribable to the opening of the transition pore (Bragadin et al., 2007).

The aim of our work is to explain the mechanism of cobalt-induced cell death and which is the role of mitochondria in this phenomenon. Our studies were performed on hepatocyte primary cultures and isolated liver mitochondria, because the highest quantities of physiological Co²⁺ in the body is contained in the liver, as in kidney, heart and spleen, whereas low concentrations are detected in serum, brain and pancreas (Derelank and Hollinger, 2002).