Role of N-methyl-D-aspartate receptors in polychlorinated biphenyl mediated neurotoxicity

Abstract

Polychlorinated biphenyls (PCBs) are widespread persistent environmental pollutants. Chronic human and animal exposure to PCBs results in various harmful effects including neurotoxicity. This study investigates the effects of the PCB mixture Aroclor 1254 (A1254) and two PCB congeners (coplanar, non-ortho PCB 126, and non coplanar PCB 99) on the expression of N-methyl-D-aspartate receptors (NMDARs) and the subsequent toxic effects using a human SHS5-SY neuroblastoma cell line. NMDAR was measured using a radiolabeled phencyclidine receptor ligand [³H]-MK801, apoptosis was quantified using fluorogenic substrates specific for caspase-3 (DEVD-AFC) and cell death using lactate dehydrogenase (LDH) release. After treatment, a positive dose–response relationship of increasing NMDARS, increasing caspase-3 activity and cell death was observed in all PCB compounds. The non-coplanar PCB compounds were found to be significantly more toxic than the coplanar congener and the PCB mixture A1254. PCB-mediated cell death was attenuated with 10 μM NMDAR antagonists: 1-amino-3,5-dimethyladamantane hydrochloride (memantine) and (+)-5-methyl-10,11-dihydro-5H-debenzocyclhepten-5,10-imine maleate ((+)-MK-801), thus demonstrating the importance of NMDAR in PCB neurotoxicity. Intracellular calcium [Ca²⁺]_i chelator BAPTA-AM (1 μM) partially attenuated the neurotoxic effect of the PCBs suggesting a role of calcium homeostasis disruption in the neurotoxicity of PCBs. These results suggest that the neurotoxicity of PCBs can be mediated through activation of NMDARs.

Introduction

Polychlorinated biphenyls (PCBs) are widespread contaminants in the environment. Although their production and use have been strictly prohibited in developed countries for the last two decades, PCBs are still found accumulated in the lipid tissues of living organisms due to their lipophilic properties (Tanabe, 1988). Chronic exposure of humans and animals to PCBs occurs through the ingestion of contaminated foods and results in various harmful consequences, including neurotoxicity, by affecting nervous system development and function (Tilson et al., 1990, Mariussen et al., 2002, Canzoniero et al., 2006). Early exposure to levels of PCBs found in the environment has been associated with altered neurological function and impaired cognition in humans (Brouwer et al., 1999, Huisman et al., 1995, Jacobson and Jacobson, 1996) and altered behaviour in animal models (Schantz et al., 1995, Schantz et al., 1997, Bushnell and Rice, 1999).

PCBs are mixtures of 209 different possible PCB congeners differ with regard to the number and the location of the chlorine atoms on the two benzene rings (World Health Organization (WHO, 1993). The cellular and molecular mechanisms through which PCBs may alter neuronal development are not well understood, yet are the subject of intense in vitro and in vivo studies. Recent studies demonstrated PCBs to have effects on neurotransmitter systems like dopaminergic systems (Mariussen and Fonnum, 2001, Mariussen et al., 1999, Seegal et al., 2002, Caudle et al., 2006), muscarinic cholinergic systems (Coccini et al., 2006, Juarez de Ku et al., 1994), and glutaminergic systems (Altmann et al., 2001, Gafni et al., 2004). PCBs affect both ryanodine-sensitive calcium (Ca²⁺) channels (Wong et al., 1997, Gafni et al., 2004) and voltage-sensitive Ca²⁺ channels (VSCC) (Inglefield and Shafer, 2000). Both single congeners and PCB mixtures Aroclors have been shown to alter Ca²⁺ homeostasis in nerve cells (Kodavanti et al., 1993, Tilson and Kodavanti, 1998, Inglefield et al., 2001, Magi et al., 2001). PCBs can also induce oxidative stress through the formation of reactive oxygen species and the subsequent activation of several signaling cascades leading to cell death (Voie et al., 2000, Voie and Fonnum, 2000). Ca²⁺ influx through store- and voltage-operated Ca²⁺ influx channels have been shown to amplify Ca²⁺ signals mediated by N-methyl-D-aspartate receptors (NMDARs) (Netzeband et al., 1999). It is known that a NMDARs blockade leads to the impairment of neuronal plasticity (Collingridge and Bliss, 1995). The inappropriate or excessive stimulation of NMDARs can trigger a series of events leading to a cascade of biochemical and physiologic responses, which could induce neuronal cell death due to an overload of Ca²⁺ (Lipton and Rosenberg, 1994, Choi, 1992).

Boehning et al. (2003) established that a small amount of cytochrome c released from mitochondria can bind to and promote calcium conductance through inositol-1,4,5-trisphosphate (InsP3) receptors in the endoplasmic reticulum membrane. The released calcium then triggers a mass exodus of cytochrome c from all mitochondria in the cell, thus activating the caspase and nuclease enzymes that finalise the apoptotic process. Sánchez-Alonso et al. (2004) also demonstrated that PCB 77, PCB 153, as well as Aroclor 1248 and Aroclor 1260 can significantly induce apoptosis by via proteins of the Bcl-2 family and activating caspase-3 activities in primary cortical neuronal cultures.

NMDA receptor mediates excitatory neurotransmission in the CNS and plays a central role in the function of excitatory synapses, which are important in neuronal development, memory formation, and many forms of synaptic plasticity. The level of NMDA receptor function at the synapse critically regulates brain function and cell survival. At synapses, it is thought that the appropriate clustering of this receptor at the postsynaptic membrane is critical for efficient synaptic transmission and it is important to regulate a constant level by a dynamic balanced process of NMDA receptors at the surface level (Wenthold et al., 2003). Trafficking of NMDA receptor has emerged a key regulatory mechanism that underlies channel function. Experimental evidence supports that NMDA receptors are quite mobile within neurons, via endocytosis and lateral diffusion in the membrane (Rao and Craig, 1997). For example, chronic treatment with NMDA receptor antagonists leads to an increase in surface clusters of NMDA receptors and a shift to a more synaptic localization (Rao and Craig, 1997).

The role of NMDARs in the mechanism of PCBs- induced apoptosis and necrosis is not well understood, but a previous study stipulated that PCB may have significantly enhanced a NMDA and AMPA receptor-mediated calcium signal (Gafni et al., 2004). The objective of this study was to investigate the effects of two PCB congeners, including PCB 99 or 2,2′,3,4′,6′-pentachlorobiphenyl, a non-dioxin-like di-ortho-substituted PCB and PCB 126 or 3,3′,4,4′,5-pentachlorobiphenyl, which is a coplanar dioxin-like PCB with affinity for the aryl hydrocarbon receptor (AhR) and one of several commercial PCB mixture (A1254), on the concentration of NMDARs in the membrane of human SH-SY5Y neuroblastoma cells and the role of NMDARs in neuronal cell cytotoxicity. In order to clarify the roles of NMDARs in PCB induced human SH-SY5Y neuroblastoma cells toxicity, PCBs were co-incubated with a non-competitive antagonist of NMDAR, dizocilpine, also known as MK801 (Fix et al., 1993), and memantine, a low-affinity voltage-dependent uncompetitive antagonist at glutamatergic NMDARs (Robinson and Keating, 2006). Both MK801 and memantine bind inside the ion channel of the receptor and thus prevent the flow of ions such as Ca²⁺ through the channel. The possible role of Ca²⁺ in PCB-induced toxicity were further studied by incubating cells with PCBs in the presence or absence of the [Ca^2+]_i-chelator BAPTA-AM. BAPTA-AM is a membrane-permeable Ca²⁺ chelator (Watkins and Mathie, 1996). The presence of four AM groups in the molecule allows BAPTA-AM to cross-cellular membranes. Esterases located in the cytosol then cleave the AM groups, serving to trap the active chelator inside the cell where it binds to Ca²⁺ and effectively decreases the cytosolic free Ca²⁺ concentrations. The hypothesis is that PCBs bind to NMDARs and increase the number of NMDARs on the cell surface, triggering an influx of calcium, thereby inducing cascade of effects including increase in caspase-3 activities and leading to neuronal cell death by apoptosis and/or necrosis.