In vitro and whole animal evidence that methylmercury disrupts GABAergic systems in discrete brain regions in captive mink

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Abstract

The effects of mercury (Hg) on key components of the GABAergic system were evaluated in discrete brain regions of captive juvenile male American mink (Neovison vison) using in vitro and in vivo (whole animal) experimental approaches. In vitro studies on cortical brain tissues revealed that inorganic Hg (HgCl2; IC50 = 0.5 ± 0.2 µM) and methyl Hg (MeHgCl; IC50 = 1.6 ± 0.2 µM) inhibited glutamic acid decarboxylase (GAD; EC 4.1.1.15) activity. There were no Hg-related effects on [3H]-muscimol binding to GABA(A) receptors (IC50s > 100 µM). HgCl2 (IC50 = 0.8 ± 0.3 µM) but not MeHgCl (IC50 > 100 µM) inhibited GABA-transaminase (GABA-T; EC 2.6.1.19) activity. In a whole animal study, neurochemical indicators of GABAergic function were measured in brain regions (occipital cortex, cerebellum, brain stem, and basal ganglia) of captive mink fed relevant levels of MeHgCl (0 to 2 µg/g feed, ppm) daily for 89 d. No effects on GAD activity were measured. Concentration-dependent decreases in [3H]-muscimol binding to GABA(A) receptors and GABA-T activity were found in several brain regions, with reductions as great as 94% (for GABA(A) receptor levels) and 71% (for GABA-T activity) measured in the brain stem and basal ganglia. These results show that chronic exposure to environmentally relevant levels of MeHg disrupts GABAergic signaling. Given that GABA is the main inhibitory neurotransmitter in the mammalian nervous system, prolonged disruptions of its function may underlie the sub-clinical impacts of MeHg at relevant levels to animal health.

Introduction

Methylmercury (MeHg) is designated as a top priority pollutant of public health concern by several regulatory agencies (U.S. EPA, 1997, ATSDR, 1999, UNEP, 2003). Much of the initial concern over MeHg stemmed from tragic, accidental human poisoning events in which thousands of people died in Japan and Iraq between 1950 and 1970 following widespread ingestion of MeHg-containing foodstuffs. Currently, consumption of fish is the primary route by which humans are exposed to MeHg. It is estimated that 8–16% of U.S. newborns have cord blood Hg levels deemed unsafe (Trasande et al., 2005) and that this percentage is greater in groups that rely heavily on fish for sustenance such as indigenous peoples and Asians (Hightower et al., 2006).

Mercurial agents have a high affinity for protein thiols and can thus interact with and disrupt a range of neurological targets (Clarkson and Magos, 2006). While this makes it difficult to resolve precise mechanisms of neurotoxicity and develop MeHg-specific biomarkers, disruption of brain γ-aminobutyric acid (GABA) signaling is likely important. In vitro studies on rat brain showed that MeHg inhibited the catalytic activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of GABA from glutamate (Kung et al., 1987). In rat dorsal root ganglion neurons, both MeHg and inorganic Hg (Hg2+) were shown to interact with sulfhydryl groups in the extracellular domain of GABA(A) receptors and impair ligand binding (Huang and Narahashi, 1996). Electrophysiological studies showed that while HgCl2 augmented GABA-induced currents (Narahashi et al., 1994), MeHg antagonized this signal (Huang and Narahashi, 1996). Taken together, MeHg-induced disruption of GABAergic signaling can have profound consequences as GABA is the main inhibitory neurotransmitter in the mammalian brain, and may account for ∼ 50% of synapses in certain brain regions (Hendry et al., 1987). Many clinical outcomes associated with GABAergic disturbances (e.g., paraesthesia, loss of peripheral vision, sensorimotor processing, and motor coordination; Siegel et al., 2006) are also evident in MeHg-poisoned individuals (Clarkson and Magos, 2006). Moreover, many brain regions (e.g., cerebellar granule cells and occipito-parietal cortex) with known established sensitivity to MeHg are also rich in GABAergic neurons or receive GABAergic inputs (Hendry et al., 1987, Suñol et al., 2008).

Though several of the aforementioned studies have shown that MeHg impedes GABAergic neurotransmission, conclusions have primarily been drawn from experiments employing high doses, acute exposures, and/or in vitro methods. It is not well-resolved how chronic exposures to relevant levels of MeHg impact the function of key neurochemicals that underlie GABAergic signaling. Here, we studied brain regions (occipital cortex, cerebellum, brain stem, and basal ganglia) of captive American mink (Neovison vison, formerly known as Mustela vison) experimentally fed MeHg (0 to 2 μg/g or ppm feed as MeHgCl) on a daily basis for three months. Key indicators of GABAergic function (GAD and GABA-transaminase enzymes, GABA(A) receptor) were evaluated in these four brain regions. Furthermore, the results of our whole animal study were complemented by in vitro studies that aimed to determine if MeHgCl and HgCl2 could directly inhibit the function of the aforementioned GABAergic enzymes and receptors. The mink was used as a model since it is a fish-eating species with proven sensitivity to MeHg, it responds to MeHg in a manner analogous to humans (e.g., similar patterns of brain lesions, neurobehavioural and neurochemical changes), and it can be studied both in nature and in the laboratory enabling the derivation of linkages that are both causal and mechanistic (Basu et al., 2007a).

Section snippets

Whole animal study

The experimental details underlying the exposure of animals to MeHg are published elsewhere (Basu et al., 2006, Basu et al., 2007b). Briefly, juvenile male mink (n = 9 per treatment) were exposed to MeHg at the Nova Scotia Agricultural College (NSAC, Truro, Nova Scotia, Canada). Animals were exposed daily to dietary MeHgCl (> 95% pure, Alfa Aesar, Ward Hill, MA, USA) at nominal concentrations of 0, 0.1, 0.5, 1, and 2 ppm for three months (= 3.3, 17.5, 77.4, 162.5, and 267.8 µg MeHgCl per kg body mass

Results

In vitro, HgCl2 and MeHgCl significantly inhibited GAD activity in cortical samples (Fig. 1A; Table 1). Both inhibition curves were steep and parallel but HgCl2 was significantly more potent at inhibiting GAD activity than MeHgCl when IC50 values were compared (Table 1). Despite the inhibitory responses measured in vitro, there were no effects of MeHgCl on GAD activity in the whole animal, in vivo study (Fig. 2). Chronic dietary exposure to MeHgCl did not affect GAD activity in the occipital

Discussion

Here we show that MeHgCl under a probable and relevant exposure scenario impacts several components of GABAergic function in discrete brain regions of captive mink. In particular, significant decreases in GABA(A) receptor levels (upwards of 94%) and GABA-T enzyme activity (upwards of 71%) were measured in the brain stem and basal ganglia of mink chronically exposed to 0.1–2 ppm dietary MeHgCl. The concentrations of MeHg observed to cause these GABAergic effects are found in many fish species

Acknowledgements

This study was funded by a Collaborative Mercury Research Network (COMERN) grant to AS, RDE and HMC, Natural Science and Engineering Research Council of Canada (NSERC) grants to HMC and VLT, NSERC fellowship to NB, and University of Michigan School of Public Health start-up funding to NB. We are thankful to the Canadian Center for Fur Animal Research (especially Merridy Rankin, Rena Currie, Sarah Gatti-Yorke, Tanya Morse, Cindy Crossman, Jody Muise, and Margot White). The assistance of Della

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