Toxicology 249 (2008) 251–255 Contents lists available at ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Methylmercury increases N-methyl-d-aspartate receptors on human SH-SY 5Y neuroblastoma cells leading to neurotoxicity Leopold Tchapda Ndountse, Hing Man Chan ∗ Community Health Sciences Program, University of Northern British Columbia, Prince George, BC, Canada article info Article history: Received 25 January 2008 Received in revised form 29 April 2008 Accepted 16 May 2008 Available online 28 May 2008 Keywords: Methylmercury NMDA receptors SH-SY 5Y neuroblastoma cells Neurotoxicity abstract Methylmercury (MeHg) is a known neurotoxin, yet the mechanism for low dose chronic toxicity is still not clear. While N-methyl-d-aspartate receptors (NMDARs) were found to be induced after exposure to MeHg in a mink model, its role on neurotoxicity is not known. The aims of this study were to inves- tigate the expression and the functional roles of NMDARs on the induction of cell death in the human SH-SY 5Y neuroblastoma cell line after exposure to MeHg. NMDARs were measured using a radiolabeled phencyclidine receptor ligand [ 3 H] (MK801) and cell death was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and lactate dehydrogenase (LDH) release. We found a significant increase in NMDARs followed by increased caspase-3 activity after 4 h of exposure to MeHg (0.25–1 M). Necrotic cell death was found after 4 and 24 h of exposure to MeHg (0.25–5 M). The NMDAR antagonists dizocilpine ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate [(+)-MK801]) and Memantine (1-amino-3,5-dimethyl-adamantane) (10 M) completely attenuated MeHg-mediated cell death by blocking NMDARs, thus demonstrating the importance of NMDARs in mercury neurotoxic- ity. Intracellular calcium chelator BAPTA-AM (1 M) partially attenuated the neurotoxicity effect of 1 M MeHg. These results suggest that MeHg toxicity can be mediated through the binding and increase of NMDARs. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Methylmercury (MeHg) is one of the most ubiquitous envi- ronmental toxins found in high levels in human brains (Clarkson et al., 2003) and fish-eating wildlife (Wiener et al., 2006). MeHg alters the normal structure and function of the central nervous system (CNS), particularly in prenatal exposure (Clarkson, 1997). The underlying mechanism through which it inhibits the devel- opment of the nervous system is the subject of intensive in vivo and in vitro research investigations. Current efforts to elucidate the mechanisms of mercury toxicity include evaluating their effects on mitochondrial function (Carranza-Rosales et al., 2005), calcium homeostasis perturbation (Limke et al., 2003; Sirois and Atchison, Abbreviations: (+)-MK801, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-iminemaleate]; BAPTA/AM, bis-(o-aminophenoxy)-ethane- N,N,N ′ ,N ′ -tetraacetic acid/acetomethyl ester; Ca 2+ , calcium; DMSO, dimethyl sulfoxide; DTNB, 5,5 ′ -dithiobis-(2-nitrobenzoic acid); EDTA, ethylene diamine tetracetic acid; FBS, fetal bovine serum; GSH, reduced glutathione; HEPES, 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid; LDH, lactate dehydrogenase; MeHg, methylmercury; Memantine, 1-amino-3,5-dimethyl-adamantane; NMDARs, N-methyl-d-aspartate receptors; PBS, phosphate buffered saline. ∗ Corresponding author. Tel.: +1 250 960 5237; fax: +1 250 960 5892. E-mail address: lchan@unbc.ca (H.M. Chan). 2000), glutathione balance (Ou et al., 1999; Olivieri et al., 2001; Kaur et al., 2006), the increase of reactive oxygen species forma- tion (Usuki et al., 2001; Dar ´ e et al., 2000), proteasomal perturbation and apoptosis (Shenker et al., 2002), and on the reduction of monoamine oxidase-B (Chakrabarti et al., 1998; Stamler et al., 2006; Beyrouty et al., 2006). Several studies demonstrated the high affinity of MeHg for the thiol groups of proteins (Castoldi et al., 2001). Thiol conjugates of MeHg (especially a cysteine S-conjugate) mimic amino acids and are transported into target cells by amino acid transporters. This characteristic makes neuron cell components particularly vulnera- ble to MeHg toxic action. One of the main effects of MeHg is its direct action on specific neurotransmitters such as N-methyl-d-aspartate (NMDA) (Basu et al., 2007; Rajanna et al., 1997) and glutamate (Glu); the main excitatory neurotransmitter (Aschner et al., 2000; Meldrum, 2000; Fonfr´ ıa et al., 2005). Glutamatergic function plays a crucial role in MeHg neurotoxicity, since the inhibition of glutamate uptake by MeHg leads to the overexpression of NMDARs, which can trigger an excitotoxic cascade (McDonald and Johnston, 1990; Coyle and Puttfarcken, 1993). Inappropriate or excessive stimula- tion of NMDARs can trigger a series of events leading to a cascade of biochemical and physiologic responses, which could induce neu- ronal cell death (Lipton and Rosenberg, 1994). Our previous results show a concentration-dependent decrease in NMDAR levels in the 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.05.011