Toxic effects of cobalt in primary cultures of mouse astrocytes

Abstract

Cobalt is suspected to cause memory deficit in humans and was reported to induce neurotoxicity in animal models. We have studied the effects of cobalt in primary cultures of mouse astrocytes. CoCl₂ (0.2–0.8 mM) caused dose-dependent ATP depletion, apoptosis (cell shrinkage, phosphatidylserine externalization and chromatin rearrangements) and secondary necrosis. The mitochondria appeared to be a main target of cobalt toxicity, as shown by the loss of mitochondrial membrane potential (ΔΨ_m) and release from the mitochondria of apoptogenic factors, e.g. apoptosis inducing factor (AIF). Pre-treatment with bongkrekic acid reduced ATP depletion, implicating the involvement of the mitochondrial permeability transition (MPT) pore. Cobalt increased the generation of oxygen radicals, but antioxidants did not prevent toxicity. There was also an impaired response to ATP stimulation, evaluated as a lower raise in intracellular calcium. Similarly to hypoxia and dymethyloxallyl glycine (DMOG), cobalt triggered stabilization of the α-subunit of hypoxia-inducible factor HIF-1 (HIF-1α). This early event was followed by an increased expression of HIF-1 regulated genes, e.g. stress protein HO-1, pro-apoptotic factor Nip3 and iNOS. Although all of the three stimuli activated the HIF-1α pathway and decreased ATP levels, the downstream effects were different. DMOG only inhibited cell proliferation, whereas the other two conditions caused cell death by apoptosis and necrosis. This points to cobalt and hypoxia not only inducing HIF-1α regulated genes but also affecting similarly other cellular functions, including metabolism.

Introduction

Cobalt is an essential element for humans since it is a necessary constituent for the formation of vitamin B12 (hydroxycobalamin). The human body contains about 1–2 mg of cobalt; most of it is found in the liver, kidney, heart and spleen, whereas low concentrations are detected in serum, brain and pancreas [1]. The minimum recommended daily intake of hydroxycobalamin of an adult is 3 μg, corresponding to 0.012 μg of cobalt. Dietary vitamin B12 deficiency is a cause of anemia and increases the risk of developmental abnormalities and growth failure in infants [2]. However, excessive levels of cobalt can be detrimental to the organism. The route of exposure is frequently dermal or via inhalation [3] and occurs mostly in industrial refining, in the production of alloys and in the tungsten carbide hard metal industry. In urban areas mean concentration of cobalt in air is small (about 1–2 ng Co/m³) but in heavily industrialised cities concentrations up to 10 ng/m³ have been reported with concentrations near industrial sources ranging up to 600 ng Co/m³[4]. In occupational setting levels can be as high as 1100 μg Co/m³[5]. Increased levels of cobalt were found in urine and blood of occupationally exposed workers. For example, Ichikawa et al. [6] have reported that cobalt concentration was 0.57–0.79 μg/dl in blood and 59–78 μg/dl in urine following exposure to 100 μg/m³.

Cobalt toxicity includes cardiomyopathy [7], adverse pulmonary effects [8] and carcinogenicity [9]. This heavy metal is also suspected to cause neurotoxic effects, as indicated by the report of memory deficit among workers exposed to hard metal both as dust powder and in mist form [10]. Uptake of cobalt in the nasal mucosa and transport to the olfactory bulbs may be an important route of brain exposure [11]. Experiments performed in the rat have shown that cobalt can cause decreased exploratory behavior [12] and depletion of neurotransmitters [13]. Besides inhibiting synaptic transmission by presynaptic blockade of calcium channels, cobalt can also block postsynaptic responses induced by neurotransmitters in vitro [14].

The data available in the literature indicate that cobalt is cytotoxic to many cell types, including neural cells [15], [16], [17] and can induce cell death by apoptosis and necrosis [18]. Cobalt can cause DNA fragmentation [19], [20], [21], activation of caspases [22], increased production of reactive oxygen species (ROS) [16], [19], [23], augmented phosphorylation of mitogen activated protein (MAP) kinases [17], [22] and elevated levels of p53 [23].

Cobalt has been used to induce ischemic pre-conditioning in vivo [24]. Some of the characteristic effects of cobalt are thought to be mediated by interaction with the cellular oxygen-sensing machinery. Like low oxygen tension, cobalt at normoxic conditions is able to stabilize the α-subunit of hypoxia-inducible factor HIF-1 by blocking its ubiquitination and proteasomal degradation [25]. Increased levels of HIF-1α result in higher transcription of a set of genes that encode several proteins, e.g. glycolytic enzymes, erythropoietin and heat shock proteins, important for the adaptation of cells to hypoxic stress [26], [27]. Many of the gene products regulated by HIF-1α are involved in a physiological response to promote cell survival and recovery after hypoxia, e.g. by maintenance of cellular energy supplies and stimulation of angiogenesis. However, recent work has also pointed to the increased transcription of pro-apoptotic factors, i.e. NIP3/BNIP3 and NIX [28], which can lead to cell death. Because of these effects cobalt ions may be used to elucidate the roles of HIF-1α in different types of cells.

Astrocytes play a crucial role in regulating functions and survival of neurons and controlling synapses [29], [30]. Although glia are generally considered more resistant to stress conditions as compared to neuronal cells, recent studies have indicated that astrocytes are also susceptible to a variety of neurotoxic stimuli, including hypoxic injury and heavy metals [31], [32], [33], [34], [35], [36], [37], [38]. It was reported that cobalt can activate extracellular signal-regulated protein kinase1/2 (ERK1/2) and cause cell death by apoptosis in C6 glioma cells [17], but otherwise little is known about cobalt effects in astrocytes. The purpose of the present study was twofold. First, we aimed to characterize cobalt cytotoxicity in primary astrocytes, by investigating the possible involvement of HIF-1 regulated genes, oxidative stress and calcium signaling, as well as by looking at mechanisms of cell death. On the other hand, we wanted to verify the hypothesis that cobalt is a valuable mimic of hypoxia in astrocytes. For this purpose, we have compared the effects of cobalt exposure to oxygen deprivation.