Effects of Mercury on Neurochemical Receptors in ... - ACS Publications

Mar 22, 2005 - Peterborough, Ontario, Canada K9J 7B8, and Furbearers and. Upland Game, Nova Scotia Department of Natural Resources,. Kentville, Nova ...
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Environ. Sci. Technol. 2005, 39, 3585-3591

Effects of Mercury on Neurochemical Receptors in Wild River Otters (Lontra canadensis) N I L A D R I B A S U , †,‡ ANTON SCHEUHAMMER,§ NICOLE GROCHOWINA,| KATE KLENAVIC,| DOUGLAS EVANS,| MIKE O’BRIEN,⊥ AND H I N G M A N C H A N * ,†,‡,¶ Department of Natural Resource Sciences, Center for Indigenous Peoples’ Nutrition and Environment (CINE), and School of Dietetics and Human Nutrition, McGill University, Saint Anne de Bellevue, Quebec, Canada H9X 3V9, National Wildlife Research Center, Canadian Wildlife Service, Environment Canada, Ottawa, Ontario, Canada K1A 0H3, Environmental and Resource Studies, Trent University, Peterborough, Ontario, Canada K9J 7B8, and Furbearers and Upland Game, Nova Scotia Department of Natural Resources, Kentville, Nova Scotia, Canada B4N 4E5

Fish-eating wildlife, such as river otters (Lontra canadensis), accumulate mercury (Hg) at concentrations known to impair animal behavior, but few studies have explored the underlying biochemical changes that precede clinical neurotoxicity. The objective of this study was to determine if Hg exposure can be related to concentrations of neurochemical receptors in river otters. River otter carcasses (n ) 66) were collected in Ontario and Nova Scotia (Canada) by local trappers in 2002-2004. Concentrations of Hg (total and organic) were measured in the cerebral cortex and cerebellum. Saturation binding curves for the cholinergic muscarinic acetylcholine (mACh) receptor and dopamine-2 (D2) receptor were completed for each animal to calculate receptor density (Bmax) and ligand affinity (Kd). Negative correlations were found between concentrations of Hg and mACh receptor Bmax (rtotal Hg ) -0.458, rinorganic Hg ) -0.454, rorganic Hg ) -0.443) in the cerebral cortex. A negative correlation was also found between concentrations of total Hg and D2 receptor Bmax (r ) -0.292) in the cerebral cortex. These results suggest that neurochemical receptors may prove useful as novel biomarkers of Hg exposure and neurotoxic effects in wildlife. Given the importance of cholinergic and dopaminergic systems in animal physiology, the ecological implications of these changes need to be investigated.

Introduction Mercury (Hg) is a hazardous chemical whose concentrations continue to rise on a global scale due to anthropogenic * Corresponding author phone: (514) 398-7765; fax: (514) 3981020; e-mail: [email protected]. † Department of Natural Resource Sciences, McGill University. ‡ CINE, McGill University. § Environment Canada. | Trent University. ⊥ Nova Scotia Department of Natural Resources. ¶ School for Dietetics and Human Nutrition, McGill University. 10.1021/es0483746 CCC: $30.25 Published on Web 03/22/2005

 2005 American Chemical Society

industrial activities and long-range atmospheric transport (1, 2). Humans and wildlife are generally exposed to Hg through the consumption of predatory fish (1, 3-5). The main chemical form of Hg present in fish tissues is methylHg (6), which is of toxicological significance since MeHg is a potent neurotoxicant and is readily assimilated from the gut (>95% of ingested concentration) into the bloodstream. Substantial epidemiological evidence indicates that prolonged and excessive intake of MeHg-contaminated fish and seafood can result in adverse health outcomes in both humans (e.g., populations in Japan and Iraq; refs 1 and 4) and wildlife (e.g., wild mink, Mustela vison, and river otter, Lontra canadensis; refs 2 and 3). MeHg can enter the brain by conjugating with L-cysteine and exploiting the methionine-uptake pathway to traverse the protective blood-brain barrier (7). Pathological examinations of brains collected from Hg-poisoned mammals, including humans, demonstrate that neuronal lesions are generally localized to the occipital cortex and cerebellum (8, 9). Such focal damage is associated with neurobehavioral outcomes characteristic of Hg intoxication, including anorexia, paresthesia, ataxia, and visual impairment (8). The toxicological effects of Hg on brain structure and neurobehavioral function across a range of mammalian species (i.e., humans, rodents, river otter) are remarkably similar, supporting the use of interspecies extrapolations. There is increasing interest in utilizing molecular approaches to discover and characterize biomarkers of subclinical neurotoxicity in mammals (10, 11). The cholinergic and dopaminergic systems have a variety of important functions in animal physiology and behavior (12, 13), and rodent studies demonstrated that these neurochemical pathways are targets of Hg. For example, Hg can affect the synthesis (14, 15), release (16-18), and metabolism (15, 19) of neurotransmitters and receptors (20, 21) involved in the aforementioned systems. While the neurotoxicological effects of Hg have been characterized in laboratory rodents, ecological studies on fish and wildlife are limited. Binding of a radioligand specific for the muscarinic acetylcholine (mACh) receptor in the brains of river otter and mink was inhibited by HgCl2 and MeHg (20). Mummichogs (Fundulus heteroclitus) and spotted gar (Lepisosteus oculatus) inhabiting Hg-polluted waters had altered concentrations of neurotransmitters and impaired activities of associated enzymes (22, 23). Another field study demonstrated that changes in mACh and dopamine-2 (D2) receptor density (Bmax) and ligand affinity (Kd) can be correlated to concentrations of Hg in the brains of wild mink (24). Collectively, these studies demonstrated that chronic exposure of wildlife to concentrations of Hg found in the natural environment can impact physiologically important neurochemical systems. Fish-eating wildlife, such as mink and river otter, are ideal sentinel species to characterize the effects of contaminants on ecosystem health (2, 5, 25, 26). The measurement of Hgmediated responses (i.e., exposures and effects) in these wildlife offer an integrated weight of evidence approach that is necessary and relevant in human and ecological risk assessments. The purpose of the current study was to expand on previous findings from wild mink (24) and to determine if associations between brain Hg and neurochemical receptor binding characteristics (i.e., mACh and D2 receptor Bmax and Kd) could be observed in another mammalian piscivore, the North American river otter, and to quantify the responses in specific brain regions (i.e., cerebral cortex and cerebellum) as opposed to the entire brain. VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Experimental Section Sample Collection. River otter carcasses were obtained from licensed fur trappers during the winter collection periods between 2002 and 2004 from southern Ontario and southwestern Nova Scotia, Canada. All traps were checked on a daily basis, and upon collection, all carcasses were immediately stored at -20 °C until necropsy. While storage temperatures generally do not affect mACh and D2 receptor Bmax, multiple freeze-thaw cycles can increase Bmax and Kd values (27). To limit the potential effects of handling regimes on neurochemical parameters, we subjected each carcass to similar conditions (i.e., all were exposed to one or two freezethaw cycles and stored at -20 °C). Full brains were removed and dissected into the cerebral cortex and cerebellum at the Atlantic Veterinary College (Charlottetown, PEI, Canada). Gender was recorded and the lower jaw was removed to estimate the ages of these animals with cementum annuli readings (Matson’s Laboratory, Milltown, MT). Determination of Hg. Concentrations of total Hg and organic Hg in the brain regions were measured using either cold vapor atomic absorption spectrophotometry (CVAAS; Hitachi atomic absorption spectrophotometer model Z8200, Tokyo, Japan) or a total Hg analyzer (TMA; MA-2, Nippon Instruments Corp., Osaka, Japan). To quantify total Hg via CVAAS, approximately 0.3 g of freeze-dried brain tissue was digested in strong acids and analyzed with continuous flow CVAAS using standard methodologies previously described (28). To quantify total Hg with TMA, freeze-dried brain tissue was ground to a fine powder with a mortar and pestle, and approximately 50 mg was placed into a ceramic holder embedded in activated alumina powder. All Hg contained within the tissue was reduced to the elemental form following thermal decomposition of the sample at 800 °C for 6 min. The elemental Hg was selectively trapped by a gold amalgam and subsequently desorbed with heating. The concentration of Hg present in the sample was detected in an absorption cell at 253.7 nm. Organic Hg was isolated from the tissues by extracting the organic compound into toluene as a stabilized bromide salt, followed by back-extraction into a Na2S2O3 aqueous phase (28). For CVAAS detection, the aqueous sample was digested and stored in a mixture of strong acids. For TMA detection, 250 µL of the Na2S2O3 extract was embedded within carbonate powder, covered with activated alumina, and then placed into the TMA. Certified standard reference materials (DORM-2, dogfish muscle, Analytical Chemistry Unit, National Research Council, Ottawa, Canada) and method blanks were included in all batches for quality control purposes. Inorganic Hg was derived by subtracting the concentration of organic Hg from the concentration of total Hg. All data are expressed as dry weight (dw) concentrations, unless indicated. mACh Receptor Binding Assay. Cellular membranes were isolated from tissues, and receptor binding assays for the mACh receptor were completed as previously described (27). Briefly, 20 µg of membrane preparation was preincubated in NaK buffer (50 mM NaH2PO4, 5 mM KCl, 120 mM NaCl, pH 7.4) for 30 min at 25 °C in duplicate. Samples were mixed with 0.01-3.2 nM [3H]-quinuclidinyl benzilate ([3H]-QNB; 42 Ci/mmol; NEN/Perkin-Elmer, Boston, MA), a mACh receptor specific radioligand, for 60 min at 25 °C under constant agitation to develop saturation binding curves. The incubation was terminated by rapid vacuum filtration through 1.0 µM GF/B glass filters (Millipore Inc., Boston, MA). The filters were washed three times with 200 µL of NaK buffer and placed into glass scintillation vials. The filters were allowed to dissolve overnight in 5 mL of liquid scintillation cocktail (ICN Biomedicals, Aurora, OH). Radioactivity retained by the filters was quantified by a liquid 3586

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scintillation counter (LKB Wallac 1209 Rackbeta, Turku, Finland) with approximately 67% counting efficiency. Specific binding was defined as the difference in [3H]-QNB bound in the presence and absence of 100 µM atropine (Sigma-Aldrich, St. Louis, MO). D2 Receptor Binding Assay. D2 receptor binding assays were conducted as described previously (24). Briefly, 20 µg of membrane preparation were preincubated in Tris buffer (50 mM Tris, 5 mM KCl, 2 mM MgCl2, pH 7.4) for 30 min at 25 °C in triplicate. Samples were then mixed with 0.1-5.6 nM [3H]-spiperone (15.7 Ci/mmol; NEN/Perkin-Elmer), a D2specific radioligand, for 90 min at 25 °C under constant agitation to develop saturation binding curves. The incubation was terminated by rapid vacuum filtration through 1.0 µM GF/B glass filters (Millipore Inc., Boston, MA). Filters were washed three times with 200 µL Tris buffer and placed into glass scintillation vials. Radioactivity retained by the filters was determined as described earlier. Specific binding was defined as the difference in [3H]-spiperone bound in the presence and absence of 100 µM (+)-butaclamol (SigmaAldrich). To reduce nonspecific binding of the radioligand to the filters, the filters were soaked for 30 min in 0.5% (w/v) polyethylenimine prior to use, and 50 µM ketanserin (SigmaAldrich) was added to each well to prevent binding of [3H]spiperone to 5-HT2 receptors. Statistical Analysis. A p value less than, or equal to, 0.05 was considered statistically significant in all analyses (SPSS Version 11.5.0, SPSS Inc., San Rafael, CA), and data are represented as mean ( standard deviation. Data from all receptor binding studies were curve-fitted using GraphPad Prism (Version 3.02, GraphPad Software Inc., San Diego, CA) to calculate Bmax (fmol/mg) and Kd (nM). We have previously demonstrated that mACh receptor binding was best fit with a rectangular hyperbolic equation and D2 receptor binding was best fit with a three-parameter logistic equation (24). Hg (total, inorganic, and organic) data were log-transformed for statistical analysis (SPSS Version 11.5.0, SPSS Inc.). Pearson correlations were conducted to explore the relationship between brain Hg (total, inorganic, and organic) and neurochemical receptor binding characteristics (Bmax and Kd). Student’s T-tests were conducted to discern differences between the two study regions and brain regions with respect to concentrations of brain Hg or receptor binding characteristics. Analysis of variance (ANOVA) or Student’s T-tests were conducted to evaluate if any associations were related to age or gender.

Results Brain Hg Concentrations. For quality assurance, recovery of total Hg and organic Hg from DORM-2 standard reference material was within (10% of the certified value (93.6-100.8% for total Hg; 91.8-101.9% for organic Hg), regardless of the method of Hg analysis used. Concentrations of total Hg, inorganic Hg, and organic Hg in the otter brain ranged between 0.09 and 14.31, between 0.00 and 10.65, and between 0.08 and 8.54 µg/g, respectively (Table 1). There were no significant differences in Hg concentrations between the cortex and cerebellum from the same animal. Concentrations of brain Hg were significantly (p < 0.001) higher in Nova Scotia samples compared to ones collected from Ontario. Organic Hg accounted for 73.9 ( 26.4% of the total Hg, and this was consistent between the two collection areas and brain regions. Mean moisture content in the brain tissues was 75.5 ( 3.0%. Age and gender did not affect the concentrations of Hg. Receptor Binding Characteristics. In the cerebral cortex, mean mACh receptor Bmax (p < 0.01) and Kd (p < 0.01) were significantly higher in river otters collected from Ontario versus Nova Scotia (Table 2). No differences in mACh receptor binding characteristics between the two study regions were

TABLE 1. Mean ((Standard Deviation) Values of Hg Concentrations (Expressed as a Dry Weight Value) in the Cerebral Cortex and Cerebellum of North American River Otters Collected from Ontario and Nova Scotia during 2002-2004a

a

sample

n

total Hg (µg/g)

inorganic Hg (µg/g)

organic Hg (µg/g)

organic Hg (% of total Hg)

Nova Scotia cerebral cortex Nova Scotia cerebellum Ontario cerebral cortex Ontario cerebellum

40

4.78 ( 3.33a

1.95 ( 2.50a

2.78 ( 1.79a

70.92 ( 23.00a

40

4.05 ( 3.49a

1.73 ( 2.54a

2.38 ( 1.78a

68.40 ( 24.75a

23

1.23 ( 0.36b

0.29 ( 0.22b

0.94 ( 0.33b

76.57 ( 17.64a

26

1.05 ( 0.36b

0.28 ( 0.33b

0.78 ( 0.30b

78.28 ( 30.37a

Letter superscripts represent significant (p < 0.05) differences within a column.

noted in the cerebellum. Nonspecific binding, as determined by incubation of samples with atropine, was generally less than 5% of the total binding at 3.2 nM [3H]-QNB. Preliminary studies demonstrated that two saturable [3H]spiperone binding sites exist in the cerebral cortex and cerebellum of river otters (data not shown). These were similar to prior observations in mink (24), and as before, only the high-affinity D2 binding site was characterized. In the cerebral cortex, mean D2 receptor Bmax was higher (p < 0.001) in the Ontario river otters (Table 3). In the cerebellum, mean D2 receptor Kd was higher (p < 0.05) in the Nova Scotia river otters, while there was no difference in the D2 receptor Bmax. Nonspecific binding, as determined by incubation of samples with (+)-butaclamol, was between 45 and 55% of total binding at 5.6 nM [3H]-spiperone. mACh or D2 receptor binding characteristics were not affected by age or gender. Correlation of Hg with Receptor Binding Characteristics. In the cerebral cortex, there was a significant negative correlation between all forms of Hg and mACh receptor Bmax (Figure 1A-C) and Kd (Figure 2A-C) when the data from the two study regions were pooled together. When the data were separated according to study sites, a negative correlation between Hg and mACh receptor Bmax and Kd was still observed in the Nova Scotia river otters (mACh Bmax, rtotal Hg ) -0.413, rinorganic Hg ) -0.466, rorganic Hg ) -0.452; mACh Kd, rtotal Hg ) -0.322, rinorganic Hg ) -0.505, rorganic Hg ) -0.282), but not in those from Ontario. In the cerebellum, there were no correlations between Hg and mACh receptor Bmax when the data were pooled together or analyzed by individual regions, but significant negative correlations were calculated between concentrations of Hg (total Hg and inorganic Hg) and mACh receptor Kd on samples pooled from both regions (Table 4), or those collected from Nova Scotia (mACh Kd, rtotal Hg ) -0.375, rinorganic Hg ) -0.292).

For the D2 receptor, a significant negative correlation between total Hg and D2 receptor Bmax was calculated in the cerebral cortex when the samples were pooled from both study regions (Figure 3A), but such an association was not significant when the data were separated into the individual study sites. No significant correlations were calculated between any form of Hg and D2 receptor Kd in the cerebral cortex on samples pooled from both study regions (total Hg, r ) 0.004, p ) 0.980; inorganic Hg, r ) 0.004, p ) 0.978; organic Hg, r ) -0.029, p ) 0.856) or separated regions. In the cerebellum, there were no significant correlations between any form of Hg and D2 receptor Bmax and Kd (Table 4).

Discussion The major finding of the present study was that changes in neurochemical receptor binding characteristics in wild North American river otters were related to concentrations of brain Hg. Negative correlations were found in the cerebral cortex between Hg and mACh receptor Bmax, mACh receptor Kd, and D2 receptor Bmax on data pooled from Ontario and Nova Scotia (Figures 1-3). Correlative analysis on pooled samples provided a better indication regarding the association between brain Hg and neurochemical receptors as the range in Hg concentrations from these two regions was approximately 150-fold. These data agree with previous findings in wild mink (24) and suggest that environmentally relevant concentrations of Hg may exert subclinical neurotoxic effects on key species of fish-eating wildlife in the ecosystem. River otters bioaccumulate relatively high concentrations of Hg due to their strict piscivorous diet and high metabolism (29). Accordingly, studies on European (30, 31) and American (2, 26, 32) river otters have demonstrated that these popula-

TABLE 2. Mean ((Standard Deviation) Values of mACh Receptor Binding Characteristics in the Cerebral Cortex and Cerebellum of North American River Otters Collected from Ontario and Nova Scotia during 2002-2004a cerebral cortex

Nova Scotia Ontario a

cerebellum

Bmax (fmol/mg)

Kd (nM)

Bmax (fmol/mg)

Kd (nM)

1326.44 ( 765.31a 2018.17 ( 955.97b

0.12 ( 0.04a 0.15 ( 0.05b

296.70 ( 216.75a 338.46 ( 229.83a

0.12 ( 0.07a 0.13 ( 0.05a

Letter superscripts represent significant (p < 0.05) differences within a column.

TABLE 3. Mean ((Standard Deviation) Values of D2 Receptor Binding Characteristics in the Cerebral Cortex and Cerebellum of North American River Otters Collected from Ontario and Nova Scotia during 2002-2004a cerebral cortex

Nova Scotia Ontario a

cerebellum

Bmax (fmol/mg)

Kd (nM)

Bmax (fmol/mg)

Kd (nM)

70.67 ( 134.80 ( 76.21b

1.95 ( 1.98 ( 0.70a

131.71 ( 95.83 ( 38.78a

1.97 ( 0.42a 1.68 ( 0.67b

14.87a

0.26a

77.28a

Letter superscripts represent significant (p < 0.05) differences within a column.

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FIGURE 1. Relationship between cholinergic muscarinic acetylcholine (mACh) receptor density (Bmax) and concentrations of total Hg (A), inorganic Hg (B), and organic Hg (C) in the cerebral cortex of wild North American river otters (Lontra canadensis) collected from Nova Scotia (O) and Ontario (b) during 2002-2004. tions are sensitive to environmental pollutants. In the current study, concentrations of brain Hg from animals collected in Ontario were similar to previously published values (33, 34), suggesting no changes in Hg on a temporal scale. Also, the concentrations of brain Hg in river otters collected from Ontario (Table 1) were likely not high enough to induce any notable biochemical effects; thus, no correlations between Hg and neurochemical receptors were calculated in animals sampled from this region. However, significantly higher concentrations of brain Hg were measured in river otters collected from Nova Scotia (Table 1), and this provided an ideal situation to evaluate the neurochemical responses in a high Hg-exposed population. The spatial difference in Hg body burden between samples collected from Ontario and 3588

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FIGURE 2. Relationship between cholinergic muscarinic acetylcholine (mACh) receptor ligand affinity (Kd) and concentrations of total Hg (A), inorganic Hg (B), and organic Hg (C) in the cerebral cortex of wild North American river otters (Lontra canadensis) collected from Nova Scotia (O) and Ontario (b) during 2002-2004. Nova Scotia was not surprising as wildlife residing in Atlantic Canada generally have the highest levels of Hg in North America (2). For example, Hg in the blood of the common loon (Gavia immer), an avian piscivore, was at least 2-3 times higher in Kejimkujik National Park, Nova Scotia (35) than other locations across North America (36). Atmospheric Hg deposition, combined with lake characteristics that favor Hg methylation (i.e., low pH and high organic content), contribute to an enhanced bioaccumulation of Hg through the food chain in this region (35, 37). Chronic exposures to environmental Hg have the potential to alter wildlife behavior. The dopaminergic and cholinergic systems facilitate a range of physiologically important

TABLE 4. Pearson Correlations between Concentrations of Brain Hg and mACh and D2 Receptor Binding Characteristics in the Cerebellum of North American River Otters Collected from Nova Scotia and Ontario during 2002-2004 mACh receptor

Bmax (fmol/mg)

Kd (nM)

D2 receptor

Bmax (fmol/mg)

Kd (nM)

r p

-0.119 0.416

Total Hg -0.375 0.007

-0.023 0.874

0.069 0.639

r p

-0.056 0.727

Inorganic Hg -0.292 -0.084 0.004 0.565

-0.065 0.662

r p

-0.013 0.929

Organic Hg -0.238 0.128

-0.015 0.923

0.086 0.589

neurobehaviors, including learning and memory, motor function, thermoregulation, and cognition (12, 13). Rodent studies have demonstrated that components (i.e., synthesis, transport, and metabolism of neurotransmitters and receptors) of these neurological pathways are targets for Hg (15, 17-19, 38). This observation has been extended to wildlife by means of in vitro studies on cellular membranes isolated from mink and river otter brains that demonstrated Hg (IC50 ) 1.2-5.5 µM) can inhibit the binding of [3H]-QNB to the mACh receptor (20). Studies on wild mink demonstrated that reduced D2 receptor Bmax is associated with high Hg exposure (24). In the current study, similar findings were observed in the cerebral cortex of river otters (Figure 3). The mechanisms regarding this down-regulation in D2 receptor Bmax are not known, but evidence suggests that Hg can modulate dopaminergic neurons by causing the spontaneous release of dopamine (17, 18) and inhibition of monoamine oxidase activity (15, 19). As a result, Hg has the potential to enhance dopamine concentrations in the brain in exposed individuals, and a reduction in D2 receptor Bmax may represent an autoregulatory mechanism to maintain homeostasis of the dopaminergic system in both mink and river otters. In the cholinergic system, the association between Hg and mACh receptor Bmax and Kd was positive in the whole brains of mink (24) but negative in the cerebral cortex of river otters (Figures 1 and 2) and nonexistent in the cerebellum (Table 4). Conflicting results have also been reported in laboratory feeding trials with rodents. In one study there was increased mACh receptor Bmax in the cerebellum, but no change in the cerebral cortex, following a 16 day exposure to 0.5 mg of MeHg/kg of body weight/day (38), while in another study there was increased mACh receptor Bmax in the cerebral cortex and a decrease in the cerebellum following a 7 day exposure to 1 mg/kg of body weight/day (39). The mechanistic basis underlying these disparate results is unclear. However, the long latency period associated with Hg intoxication suggests that comparison of data from acute, laboratory dosing regimens against those obtained from wild animals that are chronically exposed to Hg via natural routes may not be easily translatable. While both in vitro and in vivo data are required in environmental risk assessments, steady-state toxicokinetics and physiological responses (e.g., long-term adaptation) will differ between animals exposed to Hg for acute and chronic durations, and in the laboratory versus the field. As such, Hg exposure and effects data acquired from mammalian wildlife piscivores may be more relevant to human populations than shortterm rodent bioassays. The high affinity of Hg for functional thiol groups renders numerous components of the cholinergic system as cellular targets for Hg. In vivo evidence from rodents (15, 40) and fish

FIGURE 3. Relationship between dopamine-2 (D2) receptor density (Bmax) and concentrations of total Hg (A), inorganic Hg (B), and organic Hg (C) in the cerebral cortex of wild North American river otters (Lontra canadensis) collected from Nova Scotia (O) and Ontario (b) during 2002-2004. (22, 41) suggests that acetylcholinesterase, the primary enzyme responsible for acetylcholine hydrolysis, is impaired by Hg. Most of our understanding regarding the biochemical and physiological significance of acetylcholinesterase inhibition has been obtained from studies on organophosphate pesticides. The toxicity of organophosphates is manifested by the phosphorylation of a hydroxyl group in the active site of acetylcholinesterase, thus inactivating the enzyme and allowing acetylcholine to accumulate in brain synapses and neuromuscular junctions (42). Organophosphate-mediated inhibition of acetylcholinesterase has also been associated with a reduction in mACh receptor Bmax, likely as a compensatory mechanism to prevent cholinergic overstimulation VOL. 39, NO. 10, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(43). Studies on knock-out mice corroborate these findings as concentrations of mACh receptor are significantly reduced when the genes for acetylcholinesterase were removed from animals (44). Therefore, down-regulation of mACh receptors in river otters may be an adaptive response to the buildup of acetylcholine in the brain as a result of chronic Hg exposure. However, further research is required to validate this proposition since Hg also has the potential to decrease the cellular pool of acetylcholine by suppression of choline acetyltransferase activity (15) and inhibition of voltage-gated acetylcholine uptake into synaptic endings (14). Even though mink and river otter inhabit common ecosystems and trophic levels, interspecies differences may help explain the variable neurochemical responses observed between these mustelids. River otters are obligate piscivores (i.e., 100% of diet is fish; ref 29) and bioaccumulate higher concentrations of Hg than mink, which are categorized as prey generalists that rely on fish for about half of their diet (45). However, in otter brains, 73.9% of total Hg was in the organic form, compared to approximately 90% for mink (24, 33), suggesting that river otters can better metabolize organic Hg into the inorganic form. River otters also accumulate selenium (Se), and a Se:Hg molar ratio of approximately 1:1 has been observed in the brains of river otters that have greater than 4 µg/g total Hg (46). Such a relationship was not observed in the brains of mink (46, 47). Given that Se is known to antagonize Hg toxicity by forming an inert compound (48), the coaccumulation of Hg and Se by river otters may render them less susceptible to Hg intoxication compared to mink. River otters are an appropriate sentinel species in human health (2, 26), especially for communities where elevated Hg ingestion via fish consumption is common (5). Risk assessments of Hg toxicity in human populations are generally based on characterizing recent exposure by quantifying the concentrations of Hg in the blood, hair, or nails of individuals or assessing subtle neurological effects by performing expensive, time-consuming, and complicated neurobehavioral tests (10, 27). As such, there is a lack of sensitive biomarkers to monitor the subclinical damages that precede adverse health outcomes associated with chronic exposure to low levels of Hg. The measurement of neurochemical parameters in blood samples from human populations has been proposed (10, 11) and tested (49), but numerous questions remain regarding their utility and relevance. The results of the current study suggest that monitoring neurochemical receptors in the brains of piscivorous wildlife may prove useful in assessing the negative impacts of Hg on the ecosystem. Further studies are required to determine the physiological consequences associated with prolonged Hgrelated changes in neurochemistry, as well as the implications this may have for human and ecological health.

Acknowledgments We gratefully acknowledge the support of trapping associations in Ontario and Nova Scotia for supplying carcasses. We would also like to thank Donna Leggee, Sonja Ostertag, and Chris J. Stamler for their assistance. This study was funded by a research grant from the Collaborative Mercury Research Network (COMERN) to H.M.C. and A.S. and a Discovery Grant from the Natural Science and Engineering Research Council (NSERC) of Canada to H.M.C. N.B. is a recipient of a NSERC graduate student fellowship.

Note Added after ASAP Publication This paper was released ASAP on March 22, 2005, with incorrect pagination for ref 24. The corrected version was posted on April 14, 2005. 3590

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Received for review October 18, 2004. Revised manuscript received February 2, 2005. Accepted February 10, 2005. ES0483746

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