ToxSci Advance Access originally published online on January 30, 2006
Toxicological Sciences 2006 91(1):202-209; doi:10.1093/toxsci/kfj121
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Methylmercury Impairs Components of the Cholinergic System in Captive Mink (Mustela vison)
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* Department of Natural Resource Sciences, McGill University, Ste. Anne de Bellevue, Quebec, Canada, H9X 3V9; Center for Indigenous Peoples' Nutrition and Environment (CINE), McGill University, Ste. Anne de Bellevue, Quebec, Canada, H9X 3V9; Department of Plant and Animal Sciences and Canadian Centre for Fur Animal Research (CCFAR), Nova Scotia Agricultural College, Truro, Nova Scotia, Canada, B2N 5E3; Environmental and Resource Studies, Trent University, Peterborough, Ontario, Canada, K9J 7B8; ¶ School of Dietetics and Human Nutrition, McGill University, Ste. Anne de Bellevue, Quebec, Canada, H9X 3V9; and || National Wildlife Research Center, Canadian Wildlife Service, Environment Canada, Ottawa, Ontario, Canada, K1A 0H3
1 To whom correspondence should be addressed at Community Health Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada. Fax: (250) 960-5744. E-mail: lchan{at}unbc.ca.
Received December 21, 2005; accepted January 23, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of methylmercury (MeHg) on components of the cholinergic system were evaluated in captive mink (Mustela vison). Cholinergic parameters were measured in brain regions (occipital cortex, cerebellum, brain stem, basal ganglia) and blood (whole blood, plasma, serum) following an 89-day exposure to MeHg at dietary concentrations of 0, 0.1, 0.5, 1, and 2 ppm (n = 12 animals per treatment). There were no effects of MeHg on brain choline acetyltransferase, acetylcholine, and choline transporter. However, significantly higher densities of muscarinic cholinergic receptors, as assessed by 3H-quinuclidinyl benzilate binding, were measured in the occipital cortex (30.2 and 39.0% higher in the 1 and 2 ppm groups, respectively), basal ganglia (67.5 and 69.1% higher in the 0.5 and 1 ppm groups, respectively), and brain stem (64.4% higher in the 0.5 ppm group), compared to nonexposed controls. The calculated positive relationship between MeHg exposure and muscarinic cholinergic receptor levels in this dosing study were consistent with observations in wild mink. There were no MeHg-related effects on blood cholinesterase (ChE) activity, but ChE activity was significantly higher in the occipital cortex (17.0% in the 1 ppm group) and basal ganglia (34.1% in the 0.5 ppm group), compared to nonexposed controls. The parallel increases in muscarinic cholinergic receptor levels and ChE activity following MeHg exposure highlight the autoregulatory nature of cholinergic neurotransmission. In conclusion, these laboratory data support findings from wild mink and demonstrate that ecologically relevant exposures to MeHg (i.e., 0.5 ppm in diet) have the potential to alter the cholinergic system in specific brain regions.
Key Words: mink; methylmercury; muscarinic receptor; cholinesterase; brain; wildlife; neurotoxicology.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methylmercury (MeHg) is extremely neurotoxic, as it can readily cross the mammalian blood brain barrier and interact with protein thiols (ATSDR, 1999
; Clarkson, 1997). At sub- to low-micromolar concentrations MeHg can impede essential neurophysiological processes, including microtubule formation and calcium homeostasis (Castoldi et al., 2001). Although the neurotoxic effects of MeHg are mediated through multiple mechanisms, studies have shown that specific aspects of cholinergic neurotransmission are vulnerable to MeHg. In vitro, MeHg can inhibit the neuronal uptake of choline (Kobayashi et al., 1979), activity of choline acetyltransferase (ChAT) (Dwivedi et al., 1980; Kobayashi et al., 1979; Omata et al., 1982), and binding to the muscarinic acetylcholine (mACh) receptor (Abd-Elfattah and Shamoo, 1981; Basu et al., 2005c). In vivo, exposure to MeHg has been linked with decreased activity of ChAT (Dwivedi et al., 1980; Omata et al., 1982), increased levels of mACh receptors (Coccini et al., 2000), and reduced concentrations of acetylcholine (ACh) (Hrdina et al., 1976; Kobayashi et al., 1980). Furthermore, some of the clinical outcomes of cholinergic dysfunction (i.e., anorexia, salivation, tremors, reduced vision, seizures) (Kobayashi et al., 1980; Wess, 2004) have also been observed in Hg-poisoned individuals (ATSDR, 1999; Watanabe and Satoh, 1996), thus suggesting a possible role for this neurotransmission system in the progression of MeHg toxicosis.
Mercury (Hg) is a contaminant of global concern because elemental Hg (Hg0) can undergo long-range atmospheric transport and later be converted to MeHg, which biomagnifies through aquatic food webs (Chan et al., 2003; U.S. EPA, 1997; Wiener et al., 2003). Individuals at greatest risk of MeHg intoxication are obligate consumers of predatory fish. For example, ingestion of MeHg-contaminated fish by inhabitants of Minamata Bay and Niigata (Japan) circa 1950–1960 was implicated as the causative factor of Minamata disease (Watanabe and Satoh, 1996). Fish-eating wildlife are also susceptible to MeHg intoxication (Chan et al., 2003; Wiener et al., 2003), and it should be noted that symptoms resembling Minamata disease were observed in resident animals (e.g., dogs, cats, fish) nearly 4 years before the first documented human case (Watanabe and Satoh, 1996). Controlled dosing experiments have demonstrated that piscivorous wildlife, such as mink (Mustela vison; Aulerich et al., 1974; Wobeser et al., 1976; Wren et al., 1987), river otters (Lontra canadensis; O'Connor and Nielson, 1980), seals (Phoca sp.; Ronald et al., 1977), and loons (Gavia immer; Kenow et al., 2003), are sensitive to MeHg. Dietary levels as low as 1 ppm MeHg have been associated with a range of adverse outcomes at the tissue (e.g., neuronal lesions), whole-animal (e.g., effects on reproduction and neurobehavior), and possibly even population (e.g., decline in numbers) levels.
While MeHg has the potential to affect ecosystem health, nowadays fish-eating wildlife are seldom exposed to concentrations associated with overt toxic effects (i.e., >1 ppm MeHg in diet). Instead, animals are exposed to lower concentrations on a continual basis. The subtle biochemical and cellular perturbations associated with these exposures have gone largely unstudied. We recently documented that alterations in mACh receptor density can be associated with MeHg accumulation in the brains of wild mink (Basu et al., 2005a). Specifically, animals with higher MeHg accumulation in brain also had greater numbers of mACh receptors. Variations in neurochemical receptors (i.e., mACh and D2 receptors; Basu et al., 2005b) and enzymes (ChE and monoamine oxidase; Basu et al., under review) have also been linked with MeHg exposure in North American river otters. The existence of such neurochemical changes raises numerous questions regarding the ecophysiological consequences of MeHg on wildlife populations. As disruptions to neurochemistry are known to precede structural and functional damage to the nervous system (Manzo et al., 2001), alterations in brain chemistry may serve as an early warning for subsequent adverse neurological effects.
A major limitation in cross-sectional, epidemiological studies is the influence of multiple factors (e.g., co-contaminants, environmental stressors, and geographic isolation) on exposure-effect outcomes. As a result, before a causal link can be made between MeHg intake and neurochemical changes in fish-eating mammals, controlled dosing trials are required to characterize the underlying mechanisms and derive quantitative information. Wildlife are excellent models to validate the utility of neurochemical approaches, since exposure-response relationships can be assessed at multiple tiers of biological organization (i.e., laboratory experiments in vitro, whole-animal feeding trials, and field or ecosystem investigations), and brain tissue can be obtained for detailed analysis. Such multifaceted approaches are generally not permissible for rodents or humans. Accordingly, the present study was conducted to explore the effects of dietary MeHg on components of the cholinergic system in captive mink exposed to ecologically relevant concentrations of MeHg (i.e., 0 to 2 ppm) for 3 months.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Methyl Hg chloride (>95% purity) was obtained from Alfa Aesar (Ward Hill, MA). 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was purchased from Molecular Probes, Inc (Eugene, OR). 3H-Acetyl CoA (200 Ci/mmol), 3H-hemicholinium-3 (125 Ci/mmol), and 3H-quinuclidinyl benzilate (3H-QNB; 42 Ci/mmol) were obtained from NEN/Perkin Elmer (Boston, MA). All other laboratory reagents were purchased from Sigma-Aldrich (St. Louis, MO).
Animals.
Because mink are sensitive to many types of pollutants and can be studied both in captivity and in nature, they have been endorsed as excellent sentinels (U.S. EPA, 1997). Juvenile male mink (1763 ± 141 g), approximately 5 months of age at the initiation of study, were obtained from a commercial rancher and certified disease free. They were housed individually in raised wire mesh cages with nest boxes attached at the Canadian Centre for Fur Animal Research (Nova Scotia Agricultural College, Truro, NS, Canada). Mink were exposed to a natural photoperiod and temperature and had free access to water during the acclimation (
2 weeks) and exposure (13 weeks) periods. Each animal was checked twice daily, and all aspects of this study were approved by the Nova Scotia Agricultural College Animal Care and Use Committee and carried out in strict accordance to Canadian Council on Animal Care (CCAC) guidelines.
Experimental design.
Feed was prepared at the Nova Scotia Agricultural College and consisted of Atlantic herring (32%), beef tripe and liver (22%), cod (17%), barley (14%), herring oil (0.9%), and a preformulated vitamin–mineral mix. MeHg was incorporated into the diet at nominal concentrations of 0, 0.1, 0.5, 1, and 2 ppm, to reflect levels that would commonly be encountered in their natural environment (EPA, 1997). Animals were fed twice daily for a period of 89 days (August to November 2004). This length of exposure was chosen because it allows for the steady-state accumulation of Hg into tissues (Jernelov et al., 1976) and is approximately 10% of a wild mink's lifespan (Lariviere, 1999).
At the termination of the study, each animal was anesthetized with an im injection of xylazine (2 mg/kg bw) and ketamine hydrochloride (25 mg/kg bw). Blood was drawn via cardiac puncture, and the animals were sacrificed with an overdose of pentobarbital (105.6 mg/kg bw ic). Blood samples were kept on ice for approximately 4–6 h before they were separated into plasma and serum as described by Stamler et al. (2005), and then stored at –80°C. The entire brain was extracted from the skull, and specific regions (occipital cortex, cerebellum, brain stem, and basal ganglia) were dissected from the right hemisphere and stored at –80°C. These regions were studied because their structure and/or function have previously been shown to be affected by MeHg (ATSDR, 1999; Clarkson, 1997). Total Hg was measured in the feed and the tissues (brain and blood) according to Evans et al. (2000).
Neurochemical assays.
Tissues were prepared as described by Stamler et al. (2005) with minor modifications. All brain samples were homogenized for 30 s in cold Na/K buffer (50 mM NaH2PO4, 5 mM KCl, 120 mM NaCl, pH 7.4). For binding assays, cellular membranes were isolated by centrifuging the homogenate at 32,500 x g for 15 min at 4°C. The resulting pellet was washed twice under the same conditions, and the final pellet was resuspended in Na/K buffer. For ChAT, ACh, and ChE analyses, Triton-X (final concentration = 0.1% w/v) was added to the homogenate followed by a 20-s sonication. Protein concentration was determined using the Bradford protocol. Samples were stored at –80°C prior to analysis.
ChAT activity.
The activity of ChAT was determined by the method of Fonnum (1975) with modifications. The assay was carried out by incubating samples (10 µg protein) in 50 mM Na/K buffer containing 10 mM EDTA, 100 µM eserine, 100 mM choline chloride, and 0.2 µCi 3H-acetyl CoA for 30 min at 37°C. The reaction was terminated by adding an equal volume (200 µl) of 1.5% tetraphenyl boron, followed by vigorous shaking and centrifugation at 3750 x g (10 min, 25°C) to separate the phases. The activity of ChAT was determined by measuring ACh in the organic layer, and the results were expressed as fmol ACh formed/min/µg protein.
ACh concentration.
Concentrations of ACh were quantified using a commercially available kit (Molecular Probes Inc., Eugene, OR) with minor modifications. Samples (10 µg protein) were incubated in Na/K buffer including 100 µM Amplex Red, 200 mU horseradish peroxidase, 20 mU choline oxidase, and 5 U acetylcholinesterase (AChE). Formation of the assay end-product, resorufin, from ACh was determined following a three-step enzymatic reaction catalyzed by AChE, choline oxidase, and hydrogen peroxidase. Fluorescence of resorufin (
ex = 540, em = 590) was monitored in a microplate fluorometer (FLUOstar Optima, BMG Laboratories, Offenburg, Germany) following a 30 min incubation period. The concentration of ACh was determined from a standard curve (0–2 µM ACh chloride) and expressed as nM ACh per mg protein.
mACh receptor binding assay.
Binding to the mACh receptor was performed in a 96-well 1.0 µM GF/B glass filter system (Millipore, Boston, MA) as previously described (Stamler et al., 2005). Approximately 20 µg of membrane preparation in Na/K buffer was incubated with 1 nM 3H-QNB, a concentration that is indicative of mACh receptor density in mink (Stamler et al., 2005). Following a 60-min incubation period under gentle agitation, the binding assay was terminated by vacuum filtration, and the filters were washed three times with Na/K buffer and then allowed to soak overnight in scintillation cocktail. The radioactivity retained by the filters was quantified by a liquid scintillation counter (Beckman LS3801, Fullerton, CA) with approximately 60% counting efficiency. Specific binding was defined as the difference in 3H-QNB bound in the presence and absence of 100 µM atropine sulphate.
To compare the relationship between brain Hg and mACh receptor levels from the current study (i.e., data for four discrete brain regions) with a prior cross-sectional field experiment on wild mink (i.e., data for whole brains) (Basu et al., 2005a), receptor data from the current study were normalized to provide a relative measure of possible levels in the whole brain. This was achieved by taking into consideration that the average weight of the whole mink brain was 10.88 ± 0.81 g, and that approximate weights of the individual regions were: occipital cortex (1.0 g), cerebellum (1.2 g), brain stem (0.8 g), and basal ganglia (0.3 g). The weights of these regions were estimated by measuring the amount of tissue extracted during the necropsy, and the values compare favorably to rodents (Scheuhammer and Cherian, 1982). It should be noted that assumptions in this approach are that Hg levels are uniformly distributed throughout the mink brain, and that MeHg-related changes in mACh receptor levels are localized only to the specific brain regions we explored.
ChE activity.
The activity of ChE in brain and blood samples was determined according to protocols described by Stamler et al. (2005). The assay was carried out by incubating samples (0.1 µg of brain protein, or 1:5151 diluted whole blood, plasma, or serum) in Na/K buffer containing 100 µM Amplex Red, 200 mU horseradish peroxidase, 20 mU choline oxidase, and 100 µM ACh chloride. After a 30-min incubation period, the reaction end-product, resorufin, was detected as described in the section ACh concentration. The specific activity of ChE was expressed as nmol of resorufin formed per min per protein (µg) or volume (µl).
Choline transporter.
Binding to the high-affinity choline transporter was based on the method of Vickroy et al. (1984) and modified for a 96-well microplate filtration system. Approximately 20 µg of membrane preparation was preincubated for 30 min in Na/K buffer in filter plates (1.0 µM GF/B glass filters, Millipore, Boston, MA) that were presoaked with 0.1% polyethylenimine. Samples were then incubated with 2 nM 3H-hemicholinium-3 for 20 min at 25°C under gentle agitation. The binding assay was terminated, and radioactivity was quantified according to the methods described in the section mACh receptor binding assay. Specific binding was defined as the difference in 3H-hemicholinium-3 bound in the presence and absence of unlabelled hemicholinium-3 (10 µM).
Statistical analyses.
For quality control, method blanks and positive controls were included in all biochemical assays. An internal standard was created by pooling brain tissues from five untreated mink, and this sample was used to calculate inter- and intra-assay variation. All samples were assayed in triplicate.
A p value less than or equal to 0.05 was considered statistically significant in all analyses. All statistical analyses were performed using SPSS version 11.5 (Chicago, IL). Data are represented as means ± SD. The difference in the nominal and actual concentrations of Hg in the diet was assessed by a Mann-Whitney U test. Concentrations of MeHg in blood and brain were log-transformed to satisfy the assumptions of parametric statistics. One-way analysis of variance (ANOVAs) was used to determine the effects of dietary MeHg on the cholinergic parameters and Hg burden. When significant differences were found, post-hoc comparisons were performed with Tukey's HSD. Pearson correlations were used to determine the association between neurochemical parameters in components of blood and brain regions. The slopes of the regression plots relating brain Hg with mACh receptor levels from the laboratory and field study were compared using a general linear model.
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RESULTS |
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No mortalities or obvious changes to animal behavior were evident during the trial. The background level of Hg in the control diet (i.e., 0 ppm MeHg) was 22 ± 7 ng/g, and the measured concentrations of dietary MeHg in the treatments were not significantly different from the nominal values. Given that mean daily feed intake among all treatments was 313 ± 63 g feed per animal, dietary exposures ranged from 3.3 to 267.8 µg/kg b.w./day (F4,20 = 246.2, p < 0.001) (Table 1). A significant exposure-dependent increase in total Hg was measured in blood (F4,55 = 7.9, p < 0.001) and brain (F4,55 = 597.5, p < 0.001) (Table 1). There were no significant MeHg-related changes in feed intake, whole body, and brain weight.
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Cholinergic Measurements
The inter- and intra-assay variation in all assays was less than 15%, except for the measurement of choline transporter (intra-assay CV = 23.3%). There were no MeHg-related effects on ChAT activity, ACh concentrations, or choline transporter in different regions of the brain (data not shown). However, significant MeHg-dependent increases in mACh receptor levels were measured in all brain regions studied except for the cerebellum (Fig. 1). 3H-QNB binding in the occipital cortex was significantly (F4,55 = 3.8, p < 0.01) higher in the 1 and 2 ppm dietary groups (30.2 and 39.0%, respectively), compared to controls (Fig. 1A). In the basal ganglia, exposure to 0.5 and 1 ppm MeHg resulted in significantly (F4,55 = 2.8, p < 0.05) higher 3H-QNB binding (67.4 and 69.1%, respectively) compared to controls (Fig. 1C). In the brain stem, mean 3H-QNB binding was significantly (F4,55 = 2.9, p < 0.05) higher (64.4%) in the 0.5 ppm dietary group relative to controls (Fig. 1D).
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For ChE activity, MeHg-related increases were measured in the occipital cortex and basal ganglia (Fig. 2). The activity of ChE in the occipital cortex was significantly (F4,55 = 3.2, p < 0.05) higher in the 1 ppm MeHg group, compared to the 0 and 2 ppm dietary groups, by 17.0 and 18.4%, respectively (Fig. 2A). In the basal ganglia, ChE activity was significantly (F4,55 = 3.1, p < 0.05) higher by 34.1% in the 0.5 ppm group compared to nonexposed controls (Fig. 2C).
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In the components of blood tested (i.e., whole blood, plasma, serum) there were no effects of MeHg on the activity of ChE (data not shown). However, there were significant correlations between enzyme activity in plasma and brain stem (r = 0.516, p < 0.0001), plasma and occipital cortex (r = 0.250, p < 0.05), and whole blood and basal ganglia (r = –0.404, p < 0.001).
Comparison of mACh Receptor Levels between Lab and Field Studies
The relationship between brain Hg and mACh receptor levels were compared between the current laboratory study and a previous field experiment (Fig. 3). By normalizing the receptor data from the current dataset (i.e., studied in four discrete brain regions) to reflect values in the entire brain, the densities of mACh receptor (dependent variable) could be related to Hg (independent variable) in whole brains according to the following equation: y = 129.8Ln(x) + 1507.5 (r2 = 0.176, p < 0.001). This exposure-response relationship is similar to the field study (y = 118.8Ln(x) + 629.4; r2 = 0.299, p < 0.0001), as no statistically significant differences were obtained between the slopes of the two regression plots.
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) and wild (closed circles—•) mink. The data from the laboratory were normalized from receptor levels measured in four discrete brain regions (i.e., occipital cortex, cerebellum, basal ganglia, and brain stem). The data from the field were obtained from a previous publication (Basu et al., 2005a |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies in wild (Basu et al., 2005a
,b, under review) and laboratory (Coccini et al., 2000; Dwivedi et al., 1980; Hrdina et al., 1976; Kobayashi et al., 1980; Omata et al., 1982) animals have shown that MeHg can alter components of the cholinergic system. The effects on this system are nonspecific and are caused by the interactions of MeHg with sulfhydryl-containing proteins, which are ubiquitous in all cells. Thus, to properly evaluate the effect of MeHg on cholinergic neurotransmission, we have systematically investigated the key biochemical parameters in this pathway. While no changes to brain ChAT, ACh, or choline transporter were found, significant increases of mACh receptor levels (Fig. 1) and activity of ChE (Fig. 2) were related to dietary MeHg intake. The mechanisms underlying these observations can be attributed to the tightly regulated and homeostatic controlled cholinergic system.
Exposure of mammals to MeHg results in a net decrease of cholinergic signals through the central nervous system. In vitro, MeHg can inhibit the activity of ChAT (i.e., decreased synthesis of ACh; Dwivedi et al., 1980; Kobayashi et al., 1979; Omata et al., 1982), ligand binding to the mACh receptor (i.e., reduced signal transduction; Abd-Elfattah and Shamoo, 1981; Basu et al., 2005c), and reuptake of choline (i.e., reduced ACh turnover; Kobayashi et al., 1979). These results are supported in vivo, as MeHg can reduce ChAT activity (Dwivedi et al., 1980; Omata et al., 1982) and ACh content (Hrdina et al., 1976; Kobayashi et al., 1980). Despite these data, we did not measure any changes in ChAT activity, ACh concentration, or levels of choline transporter. Prior documentation of ChAT inhibition was obtained from acute, high-dose experiments on rats (i.e., 2–10 mg/kg bw/day for 1–4 weeks; Dwivedi et al., 1980; Omata et al., 1982). Conversely, our study was designed to mimic an ecologically relevant scenario (i.e., <0.27 mg/kg bw/day for 12.7 weeks). Perhaps no effects on ChAT were found because the burden of MeHg in the brain was not sufficiently high to affect enzyme activity, or the animals adapted to the continuous exposure. While MeHg can inhibit the reuptake of choline into the neuron in vitro (Kobayashi et al., 1979), to our knowledge, MeHg-induced changes in the levels of this transport protein have not been observed in vivo. Interestingly, the blockage of choline uptake in rats by MeHg or hemicholinium (a potent inhibitor of choline uptake) results in similar biochemical (i.e., reduced ACh content and turnover) and behavioral (e.g., tremors, staggered gait, and depression) outcomes (Kobayashi et al., 1980). It was not possible to postulate whether the neuronal content of ACh was affected by MeHg in our study. The accurate estimation of this neurotransmitter requires immediate decapitation or microwave irradiation, as ACh is rapidly hydrolyzed by ChE (turnover rate = 150 µsec; Goldberg and Hanin, 1976). Neither method was an option in the present study because of animal care protocols. Because chronic exposures to low levels of MeHg can be related to decreased ACh content in rat brains (Hrdina et al., 1976), carefully designed studies will be required to reconcile this outcome in wildlife.
The key finding of this study was that the variation in mACh receptor levels could be related to the intake of MeHg. The mACh receptor belongs to a highly conserved class of membrane-spanning proteins that transduce intracellular signals through a G-protein (Wess, 2004). MeHg can directly affect this receptor by inhibiting ligand binding (Abd-Elfattah and Shamoo, 1981). For example, the calculated IC50 in the cerebral cortex of mink (5.5 µM MeHg) was within four-fold of values in humans and rodents (Basu et al., 2005c). Because Hg can impair ligand binding, up-regulation of the mACh receptor is possibly a compensatory mechanism to ensure homeostasis in cholinergic transmission. Coccini et al. (2000) also found increased mACh receptors (20 to 44% over controls) in adult female Sprague-Dawley rats exposed to MeHg (0.5 mg/kg bw/day) for 16 days. However, these changes were measured 14 days following the termination of treatments and were localized to the hippocampus and cerebellum. No MeHg-related alterations in mACh receptor levels were found in the cerebral cortex. Whether there is a species difference in the susceptibility among different brain regions requires further study. A similar positive correlation was observed between concentrations of brain Hg and mACh receptor levels in wild mink collected from three study sites across Canada in a cross-sectional study (Basu et al., 2005a). More importantly, there was no significant difference in the slopes of the regression curves obtained from the current laboratory study and the previous field investigation (Fig. 3). Because consonant exposure-response relationships infer a common mode of action, these findings collectively suggest that exposure to ecologically relevant concentrations of MeHg can be related to higher levels of mACh receptors in populations of wild mink. The existence of this phenomenon in natural populations is further supported by considering that Hg-related changes to the cholinergic pathway have also been observed in feral river otters (Basu et al., 2005b, under review).
MeHg-related increases of ChE activity were measured in the occipital cortex and basal ganglia (Fig. 2). In vitro, MeHg does not inhibit ChE activity in mink (N. Basu, unpublished data) or rodents (Kobayashi et al., 1979, 1980). Therefore, changes in enzyme activity are likely secondary responses resulting from variations in mACh receptor levels. Several studies have shown that pharmacological agents and environmental pollutants can induce unidirectional changes to the mACh receptor and ChE activity. For example, decreases in both ChE activity and mACh receptors are common outcomes in animals poisoned by organophosphates (Costa et al., 1982). In nature, a positive correlation has been calculated between mACh receptor levels and ChE activity in wild birds exposed to pesticides (Burn and Leighton, 1996) and in wild river otters exposed to Hg (Basu et al., under review). These examples highlight the autoregulatory nature of the cholinergic system during periods of toxicant stress.
MeHg causes discrete lesions to the calcarine region of the occipital cortex and to the granule layer of the cerebellum in wildlife (Wobeser et al., 1976; O'Conner and Nielson, 1981), rodents, and humans (Watanabe and Satoh, 1996), and these homologous responses in different mammalian species suggest a common mode of action. While neurochemical changes would be expected in the two aforementioned brain regions, alterations in mACh receptors and ChE activity were measured only in the occipital cortex (Figs. 1A and 2A). The lack of observable response in the cerebellum (Figs. 1B and 2B) is likely due to the scarcity of cholinergic neurons in this brain region, although further research is required because prior studies have found MeHg-related effects on the mACh receptor in this region (Basu et al., 2005c; Coccini et al., 2000). Changes in cholinergic parameters were also measured in the basal ganglia and brain stem, and some clinical outcomes of MeHg poisoning have been linked to functional impairments in these brain structures. For example, MeHg-induced hand tremors (Fawer et al., 1983) and auditory-evoked potentials (Murata et al., 1999) can be related to damage to the basal ganglia and brain stem, respectively.
Monitoring neurochemistry is a novel approach to predict and/or detect early nervous system dysfunction, because alterations in cellular biochemistry are known to precede permanent tissue damage (Manzo et al., 2001). In the current study, a continuum of MeHg-related neurological effects was observed whereby exposure of mink to the lowest treatment (i.e., 0.1 ppm) resulted in significant uptakes of Hg into brain (Table 1), but no significant changes in neurochemistry. With increasing exposure to dietary MeHg, neurochemical effects became evident in animals exposed to 0.5 ppm MeHg (Figs. 1 and 2). Prior studies have shown the emergence of neuropathology in mink exposed to 1 ppm dietary MeHg (Wobeser et al., 1976; Wren et al., 1987). It is interesting to note that alterations in neurochemistry often subsided in the highest exposure groups (e.g., mACh receptor levels in the basal ganglia and brain stem, Figs. 1C and 1D, respectively), and this attenuation may be related to the animal's inability to maintain cellular homeostasis once cytotoxicity becomes imminent.
In summary, the present study demonstrated that MeHg can affect certain parameters of the cholinergic system in captive mink. Specifically, MeHg-related increases in mACh receptor levels (Fig. 1) and ChE activity (Fig. 2) were measured in discrete regions of the brain. Furthermore, the neurochemical changes occurred at a MeHg exposure level (i.e., 0.5 ppm) that is below dietary concentrations (i.e., 1 ppm; Wobeser et al., 1976; Wren et al., 1987) known to cause structural and functional damage. The results from this laboratory study corroborate ecological findings and suggest that high MeHg exposure is associated with increased mACh receptor density in mink (Fig. 3). Collectively, the emerging evidence from the laboratory and the field demonstrate that ecologically relevant concentrations of MeHg can affect cholinergic neurotransmission in fish-eating wildlife.
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ACKNOWLEDGMENTS |
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This study was funded by the Collaborative Mercury Research Network (COMERN) to A.S., R.D.E., and H.M.C., and a Discovery Grant from the Natural Science and Engineering Research Council of Canada (NSERC) to H.M.C. N.B. was a recipient of a NSERC Postgraduate Fellowship. We are thankful to the Canadian Centre for Fur Animal Research, especially Merridy Rankin, Rena Currie, Sarah Gatti-Yorke, Tanya Morse, Cindy Crossman, Jody Muise, and Margo White. Technical assistance from Donna Leggee, Chris Stamler, Sonja Ostertag, and Kimberly Bull is appreciated. No conflict of interest is declared.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ATSDR (1999). Toxicological Profile for Mercury. Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA.
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