Variation of cholinergic biomarkers in brain regions and blood components of captive mink.

Studies are increasingly using cholinergic parameters as biomarkers of early neurotoxicity, but few have characterized this system in ecologically relevant model organisms. In the present study, key neurochemicals in the cholinergic pathway were measured and analyzed from discrete parts of brain and blood from captive mink (Mustela vison). Similar to other mammals, the regional distribution of cholinergic parameters in the brain could be ranked from highest to lowest as: basal ganglia > occipital cortex > brain stem > cerebellum (F (3,192) = 172.1, p < 0.001). Higher variation in cholinergic parameters was found in the cerebellum (coefficient of variation = 34.9%), and the least variation was measured in the brain stem (19.7%). Variation was also assessed by calculating the difference between the lowest and highest measures among individual animals: choline acetyltransferase (1.6x fold difference), cholinesterase (2.0x), muscarinic receptor levels (2.4x), acetylcholine (3.7x), nicotinic receptor levels (3.9x), and choline transporter (5.0x). In blood samples, activity and inter-individual variation of cholinesterase was highest in whole blood and lowest in plasma and serum. By using captive mink of a common genetic source, age, gender, and rearing conditions, these data help establish normal levels, ranges, and variations of cholinergic biomarkers among brain regions, blood components, and individual animals. Such information may better enable the utility of cholinergic biomarkers in environmental assessments.

The effects of mercury on muscarinic cholinergic receptor subtypes (M1 and M2) in captive mink.

A combination of in vitro (competitive binding assays) and in vivo (tissues from animals exposed to dietary methyl mercury, MeHg) experimental procedures was employed to assess the effects of mercury (MeHg, HgCl(2)) on the two-key muscarinic cholinergic (mACh) receptor subtypes (M1, M2) in two brain regions (occipital cortex, brain stem) of captive mink (Mustela vison). In vitro, HgCl(2) and MeHg were equipotent in inhibiting [(3)H]-pirenzipine binding to the M1 receptor in the occipital cortex, but in the brain stem, MeHg was about 65x more potent than HgCl(2). For the M2 receptor, both HgCl(2) and MeHg were more potent at inhibiting [(3)H]-AFDX-384 binding in the occipital cortex than in the brain stem. Within each brain region, HgCl(2) was more potent at inhibiting [(3)H]-AFDX-384 binding than MeHg. In vivo exposure of captive mink to MeHg (0.5, 1, and 2ppm MeHg in the diet for 89 days) resulted in greater binding of radioligands to the M1 and M2 receptor in the occipital cortex, but not in the brain stem, when compared to control animals. Based on the in vitro results, we could not conclude which mACh receptor subtype or brain region was most sensitive to Hg, but the in vivo findings suggest that Hg preferentially affects mACh receptor subtype (M1 and M2) levels in the occipital cortex. By studying distinct mACh receptors, these results extend upon previous studies in laboratory rodents and wildlife that showed Hg to affect the global population of mACh receptors.

Decreased N-methyl-D-aspartic acid (NMDA) receptor levels are associated with mercury exposure in wild and captive mink.

Mercury (Hg) impairs glutamate homeostasis but little is known about its effects on the N-methyl-d-aspartic acid (NMDA) receptor. Here, we investigated NMDA receptor levels, as determined by [(3)H]-MK801 binding, in both wild and captive mink (Mustela vison) that experienced different levels of methylmercury (MeHg) exposure. Competitive in vitro binding experiments showed that inorganic Hg (HgCl(2); IC(50)=1.5-20.7 microM), but not MeHg (MeHgCl; IC(50)>320 microM), inhibited binding to the NMDA receptor in several brain regions of mink. In a survey of trapped wild mink, NMDA receptor levels in the brain were negatively correlated (p<0.005) with concentrations of total Hg (R=-0.618) and MeHg (R=-0.714). These findings were supported by a laboratory feeding study in which captive mink were exposed to dietary MeHg (0-2 ppm) for 89 days. Concentration-dependent decreases in NMDA receptor levels were found in the basal ganglia, cerebellum, brain stem and occipital cortex. These findings are of physiological and ecological concern because they demonstrate that Hg, at dietary concentrations as low as 0.1 ppm, can significantly reduce NMDA receptor levels.

Methylmercury impairs components of the cholinergic system in captive mink (Mustela vison).

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.

Effects of mercury on neurochemical receptors in wild river otters (Lontra canadensis).

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 (r(total) Hg = -0.458, r(inorganic) Hg = -0.454, r(organic) 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.