Effect of methylmercury on glutamate metabolism in cerebellar astrocytes in culture
Introduction
Methylmercury (MeHg) is an organic form of mercury with toxic effects in multiple organs. In the brain, MeHg poisoning is characterized by damage to discrete anatomical areas and is the cause of the “so called” Minamata disease (for review, see Castoldi et al., 2001). The main route for human exposure involves the consumption of MeHg-adulterated fish from water polluted with organic mercury. Due to the high binding affinity of MeHg to sulfur, proteins and peptides bearing thiol groups are susceptible to structural and functional modifications by MeHg.
The mechanism of action for MeHg is not fully understood (Aschner, 2000). Several targets, such as blood–brain barrier, axonal transport, neurotransmisson, and synthesis of protein, DNA and RNA, have been proposed as sensitive sites for MeHg’s toxic effects (for review, see Castoldi et al., 2001). Numerous observations have established the role of astrocytes in mediating MeHg neurotoxicity. (a) MeHg selectively inhibits astrocytic uptake systems, such as cystine, and cysteine transport (Shanker et al., 2001, Shanker and Aschner, 2001, Allen et al., 2001b), compromising the redox potential and attenuating glutathione content. (b) Notably, astrocytes represent a preferential cellular site for MeHg accumulation (Aschner, 1996, Charleston et al., 1996, Garman et al., 1975). (c) MeHg inhibits astrocytic glutamate (and aspartate) uptake and stimulates its efflux (Mullaney et al., 1994, Allen et al., 2001c), increasing glutamate concentrations in the extracellular fluid and sensitizing neurons to excitotoxic injury (Coyle and Puttfarken, 1993, Rothstein et al., 1996). (d) MeHg-induced neuronal dysfunction is secondary to disturbances in astrocytes (Brookes, 1992), and the in vitro co-application of non-toxic concentrations of MeHg with glutamate results in the appearance of typical neuronal lesions associated with excitotoxic stimulation (Matyja and Albrecht, 1993).
Glutamate is the main excitatory neurotransmitter (Fonnum, 1984) and is released during neuronal excitation. The effect of glutamate is terminated by uptake into the surrounding cells, and astrocytes represent a major site for glutamate uptake (for review, see Gegelashvili and Schousboe, 1998). Intracellular glutamate is metabolized in astrocytes and its metabolites, such as glutamine, aspartate, and lactate, can be formed and released for uptake by neurons (for review, see Sonnewald et al., 1997). Such interaction is exquisitely important for normal neuronal function due to the drain of tricarboxylic acid (TCA) cycle intermediates for neurotransmitter synthesis.
MeHg has been shown to preferentially accumulate in astrocytes, where it induces cell swelling and specifically inhibits excitatory amino acid uptake (Aschner et al., 2000). In a previous study cortical astrocytes were exposed to MeHg and subsequently incubated with [U-]glutamate (Allen et al., 2001a), showing that MeHg affected mitochondria. Given these earlier observations, the present study was designed to examine the effects of MeHg on cerebellar astrocytes using [U-]glutamate and NMR spectroscopy.
Section snippets
Materials
Plastic tissue culture dishes were purchased from Nunc A/S (Roskilde, Denmark), fetal calf serum from Seralab Ltd. (Sussex, UK) and culture medium from GIBCO BRL, Life Technologies (Roskilde, Denmark). NMRI mice were purchased from Møllegaard Breeding Center (Copenhagen, Denmark). [U-]glutamate (98% enriched) and 99.9% D2O were from Cambridge Isotopes Laboratories (Woburn, MA, USA), and ethylene glycol from Merck (Darmstadt, Germany). All other chemicals were of the purest grade available
Results
Fig. 1 shows the transformation of [U-]glutamate in astrocytes. For detailed description of [U-]glutamate metabolism in cerebellar astrocytes see Qu et al. (2001). Briefly, [U-]glutamate enters the astrocytes and can be converted to glutamine and glutathione (GSH) directly. It can also enter the TCA cycle via 2-oxoglutarate for the synthesis of metabolites and energy production. [U-]aspartate and [U-]lactate can be formed from intermediates derived via the first turn of the TCA
Discussion
Several high-affinity glutamate transporters on the astrocytic membrane maintain optimal extracellular concentrations of glutamate (for review, see Gegelashvili and Schousboe, 1998). In the present study the amount of [U-]glutamate removed from the extracellular medium was unchanged when astrocytes were exposed to 25 μM MeHg for 2 h. The same was found in a study by Allen et al., 2001a using cortical astrocytes. However, the amount of [U-]glutamate removed from the media was decreased in
Acknowledgements
This research was supported by the Research Council of Norway, the Blix Foundation. The excellent technical assistance of Bente Urfjell is greatly appreciated.
References (33)
- et al.
Methylmercury has a selective effect on mitochondria in cultured astrocytes in the presence of [U-]glutamate
Brain Res.
(2001) - et al.
Methylmercury inhibits the in vitro uptake of the glutathione precursor, cystine, in astrocytes, but not in neurons
Brain Res.
(2001) - et al.
Methylmercury-mediated inhibition of -d-aspartate transporter in cultured astrocytes is reversed by the antioxidant catalase
Brain Res.
(2001) - et al.
Mercuric chloride, but not methylmercury, inhibits glutamine synthetase activity in primary cultures of cortical astrocytes
Brain Res.
(2001) - et al.
Methylmercury-induced alterations in excitatory amino acid transport in rat primary astrocyte cultures
Brain Res.
(1993) - et al.
Intracellular glutathione (GSH) levels modulate mercuric chloride (MC)- and methylmercuric chloride (MeHgCl)-induced amino acid release from neonatal rat primary astrocytes cultures
Brain Res.
(1994) - et al.
Methylmercury alters glutamate transport in astrocytes
Neurochem. Int.
(2000) In vitro evidence for the role of glutamate in the CNS toxicity of mercury
Toxicology
(1992)- et al.
Neurotoxicity and molecular effects of methylmercury
Brain Res. Bull.
(2001) - et al.
Cellular distribution and kinetic properties of high-affinity glutamate transporters
Brain Res. Bull.
(1998)
Ultrastructural evidence that mercuric chloride lowers the threshold for glutamate neurotoxicity in an organotypic culture of rat cerebellum
Neurosci. Lett.
The role of –SH groups in methylmercuric chloride induced d-aspartate and rubidium release from rat primary astrocyte cultures
Brain Res.
The effect of thiopental on [U-]glutamate metabolism in cultured cerebellar astrocytes
Neurosci. Lett.
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate
Neuron
Differential responses to methylmercury exposure and recorvery in neurblastoma and glioma cells and fibroblasts
Exp. Neurol.
Methylmercury inhibits cysteine uptake in cultured primary astrocytes, but not in neurons
Brain Res.
Cited by (18)
Contributions of the Scandinavian Countries to the Development of Non-Animal Alternatives in Toxicology
2018, The History of Alternative Test Methods in ToxicologyThe toxicology of mercury and its compounds
2014, Perspectives in MedicineThe toxicology of mercury and its compounds
2012, Journal of Trace Elements in Medicine and BiologyCitation Excerpt :Neuronal dysfunction has been proposed to be secondary to disturbances in astrocytes [164]. As referred to by Aschner and Syversen [165], astrocytes accumulate MeHg, where among other effects it potently inhibits astrocytic glutamate uptake and stimulates its efflux [166,167]. This causes an increase in glutamate levels in the extracellular fluid, which may cause exitotoxic injury towards neurons.
Comparison of alterations in amino acids content in cultured astrocytes or neurons exposed to methylmercury separately or in co-culture
2009, Neurochemistry International