ToxSci Advance Access originally published online on December 5, 2006
Toxicological Sciences 2007 96(1):115-122; doi:10.1093/toxsci/kfl179
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Dietary and In Utero Exposure to a Pentabrominated Diphenyl Ether Mixture Did Not Affect Cholinergic Parameters in the Cerebral Cortex of Ranch Mink (Mustela vison)
* Department of Natural Resource Sciences, McGill University, Macdonald Campus, Sainte-Anne-de-Bellevue, Quebec, Canada H9X 3V9
Wildlife Toxicology Division, National Wildlife Research Centre, Ottawa, Ontario, Canada K1A 0H3
Department of Public Health Sciences
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada T6G 2G3
¶ Department of Animal Science, Michigan State University, East Lansing, Michigan 48824
|| Canadian Wildlife Service, Environment Canada, Burlington, Ontario, Canada L7R 4A6
||| Community Health Program, University of Northern British Columbia, Prince George, British Columbia, Canada V2N 4Z9
1 To whom correspondence should be addressed at Community Health Program, University of Northern British Columbia, 3333 University Way, Prince George, British Columbia, Canada V2N 4Z9. Fax: +001 250 960-5744. E-mail: lchan{at}unbc.ca.
Received September 25, 2006; accepted November 28, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are recognized as global environmental contaminants and a potential health risk. They have been shown to elicit neurodevelopmental toxicity through disruption of the cholinergic neurotransmitter system in rodent models, but the effects of environmentally relevant exposures in wildlife species are unknown. The objective of this study was to assess the effects of the commercial pentabrominated diphenyl ether mixture DE-71 on cholinergic parameters in ranch mink (Mustela vison) following dietary exposure of adult females and in utero, lactational, and dietary exposure of their offspring. Adult females were fed diets containing 0, 0.1, 0.5, or 2.5 µg DE-71/g feed from four weeks prior to breeding through weaning of their kits at six weeks of age. A portion of the weaned kits were maintained on their respective diets through 27 weeks of age. Cholinergic parameters, including muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) binding, cholinesterase (ChE) activity, and acetylcholine (ACh) concentration, were assayed in the cerebral cortex, and ChE activity was measured in the plasma. In the cerebral cortex, results indicated a significant exposure-dependent increase in PBDE concentrations, but no significant effects of DE-71 on cholinergic parameters. There was a threefold increase in ChE activity in the plasma of adult females in the 2.5 µg DE-71/g feed group, but was likely due to effects on liver function. This study demonstrated that environmentally relevant exposures to DE-71 did not affect key parameters of the cholinergic neurotransmitter system in the brain of ranch mink.
Key Words: polybrominated diphenyl ether; cholinergic neurotransmitter system; congener concentration; congener profile; development; mink.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that are used in polymers (foam, plastics, and textiles) to increase fire safety (International Programme on Chemical Safety, 1994
). However, PBDEs are recognized as global environmental contaminants and a potential risk to human and wildlife health (European Chemicals Bureau, 2001). They are persistent, bioaccumulative, undergo long-range transport, and elicit adverse effects. There are three commercial PBDE mixtures, penta-BDE (DE-71), octa-BDE (DE-79), and deca-BDE (DE-83R). The predominant congeners in DE-71 (BDE-47, -99, -100, and -153) can readily be bioaccumulated and are thus the most abundant in biota and elicit the most potent adverse effects (Agency for Toxic Substances and Disease Registry, 2004). These congeners have been found to bioaccumulate in the liver of ranch mink following consumption of wild carp collected from the Saginaw River (Michigan, USA) (Bursian et al., 2006), as well as in the brain of wild otter collected from Nova Scotia (Basu et al., 2007b). They also have been shown to elicit adverse effects on the thyroid and immune systems in ranch mink following consumption of diets containing DE-71 (Martin et al., 2004, 2007). As the concentrations of PBDEs in freshwater fish in North America are high in certain species and locations and are increasing exponentially (Chernyak et al., 2005; Hale et al., 2001; Johnson and Olson, 2001; Rayne et al., 2003), the dietary exposure of humans and piscivorous wildlife species such as mink may pose a health risk.
Individual PBDE congeners have been shown to elicit neurodevelopmental toxicity through disruption of the cholinergic neurotransmitter system in rodent models (Dufault et al., 2005; Viberg et al., 2002, 2003, 2005). In addition to effects on spontaneous behavior, learning, and memory, neonatal exposure to BDE-99 alters the effects of adult exposure to nicotine (a nicotinic acetylcholine receptor, nAChR, agonist) on spontaneous behavior and decreases muscarinic acetylcholine receptor (mAChR) density in the hippocampus of adult rats (Viberg et al., 2002, 2005). Similarly, neonatal exposure to BDE-153 decreases nAChR density in the hippocampus of adult mice (Viberg et al., 2003). This evidence indicates that effects of PBDEs on neurobehavior may be mediated in part by changes in neurochemistry.
While the effects of PBDEs on the cholinergic neurotransmitter system have been studied in rodent models, the effects of environmentally relevant exposures on cholinergic parameters in wildlife species such as mink are unknown. The objective of this study was to assess the effects of the commercial penta-BDE mixture DE-71 on cholinergic parameters in the cerebral cortex and plasma of ranch mink (Mustela vison) following dietary exposure of adult females and in utero, lactational, and dietary exposure of their offspring. Concentrations of PBDE congeners in the in the cerebral cortex following DE-71 exposure were also analyzed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals
Acetone, hexane, and dimethyl sulfoxide were obtained from Fisher Scientific (Fair Lawn, NJ). The 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was purchased from Molecular Probes (Eugene, OR). Atropine and liquid scintillation cocktail were obtained from ICN Biomedicals (Aurora, OH). Bio-Beads S-X3 and Bio-Rad protein assay dye reagent concentrate were purchased from Bio-Rad Laboratories (Hercules, CA). [3H]-cytisine HCl ([3H]-CYT; 30.4 Ci/mmol) and [3H]-quinuclidinyl benzilate ([3H]-QNB; 42 Ci/mmol) were purchased from NEN/PerkinElmer (Boston, MA). Dichloromethane (DCM) was purchased from VWR Canlab (Mississauga, ON). Heparin was obtained from USB Corporation (Cleveland, OH). Pentabromodiphenyl ether (DE-71) was a gift from Great Lakes Chemical Corporation (West Lafayette, IN). PBDE congener standards (BDE-28, -47, -66, -100, -99, -85, -154, and -153), the internal standard ([13C]-BDE-138), the instrument performance internal standard polychlorinated biphenyl (PCB-199), and the certified reference material (CRM, WMF-01, fish) were obtained from Wellington Laboratories (Guelph, ON). Florisil, nonane, Ottawa sand, sodium sulphate (Na2SO4), acetylcholine (ACh), acetylcholinesterase (AChE), 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentan-3-one dibromide (BW284c51), bovine serum albumin, choline oxidase, horseradish peroxidase, hydrogen peroxide, (-)-nicotine hydrogen tartrate, resorufin, tetraisopropyl pyrophosphoramide (iso-OMPA), Triton X-100, and all other reagents were purchased from Sigma-Aldrich (St Louis, MO) and were of analytical grade or higher.
Animals and Treatment
Mink were housed and necropsied at the Michigan State University (MSU) Experimental Fur Farm (East Lansing, MI) as previously described (Bursian et al., 2006). The MSU Experimental Fur Farm ranch diet was used as the base of the four treatment diets, which contained 0, 0.1, 0.5, or 2.5 µg DE-71/g feed. Samples of each treatment diet were stored at – 80°C for subsequent analysis of PBDE congener concentrations.
In an earlier 70-day DE-71 mink feeding trial, there was a decrease in feed consumption and body weight at dietary concentrations of 5 µg DE-71/g feed and 10 µg DE-71/g feed (Martin et al., 2007). As a result, a maximum dietary concentration of 2.5 µg DE-71/g feed was selected for the current 1-generation DE-71 mink feeding trial.
Forty 1-year-old, virgin, natural dark, female mink from the MSU Experimental Fur Farm herd were randomly assigned to the four treatment groups (10 mink/group). Animals were started on their respective treatment diets beginning 2 February 2004. Feed and water were available ad libitum. Dietary intake of DE-71 was estimated from feed consumption and body weight of females measured weekly from weeks 1 to 4 of the study (Bursian et al., in preparation).
The females were mated to untreated males between weeks 5 and 7 of the study. Whelping occurred from weeks 12 to 14 of the study. Nest boxes were checked daily, and live and stillborn kits were counted at birth and their gender determined. Reproductive parameters were measured, including breeding success, whelping success, litter size, and survivability of kits from birth to six weeks of age (Bursian et al., in preparation).
Kits were completely weaned by six weeks of age. All adult females and six kits from each treatment group at, approximately, six week of age were necropsied on week 19 of the study. Ten kits from each treatment group were maintained on their respective treatment diets. At, approximately, 27 weeks of age, all juveniles from each treatment group were necropsied on week 45 of the study. Samples of brain tissue were collected, immediately frozen in liquid nitrogen, and stored at – 80°C until further analysis. Whole blood samples were collected in syringes containing heparin, transferred to heparinized Vacutainer tubes (BD, Franklin Lakes, NJ), and gently mixed for at least 2 min. Plasma was isolated by centrifugation of whole blood at 900 x g for 10 min at room temperature and stored at 4°C.
Quantitative PBDE Congener Analysis
Dissected cerebral cortex tissues were ground, loaded (1–3 g) into accelerated solvent extraction (ASE 200, Dionex, Sunnyvale, CA) cells (22 ml) with prewashed Ottawa sand, and spiked with the internal standard ([13C]-labeled BDE-138). The ASE extraction was carried out using DCM/acetone (1:1) at 100°C and 1500 psi with a 5-min heat-up period and two static cycles. The extracts were dried by passing them through columns (7.5 cm x 1.5 cm internal diameter, glass) packed with 8 g of anhydrous Na2SO4, eluted with 5 ml of hexane and reduced to dryness under a gentle stream of nitrogen. The residues were weighed to calculate the lipid content, then dissolved in 3 ml of DCM/hexane (1:1) and fractionated by size exclusion chromatography using columns (55 cm x 27 mm internal diameter, glass) packed with 60 g of Bio-Beads S-X3 (200–400 mesh) and eluted with DCM/hexane (1:1). The first 140-ml fraction, containing the lipids, was discarded, and the second 220-ml fraction, containing the analytes, was collected and reduced to, approximately, 1 ml by rotary evaporation. The analyte fractions were further cleaned by passing them through columns (30 cm x 10 mm internal diameter, glass) packed with 8 g of florisil (60–100 mesh, 1.2% deactivated), eluted with 100 ml of hexane, reduced to dryness, and solvent exchanged into 500-µl nonane. The instrument performance internal standard (PCB-199) was then added.
PBDE concentrations were analyzed by gas chromatography/mass spectrometry (GC/MS) using a HP6890 gas chromatograph (Agilent, Palo Alto, CA) with a DB-5MS capillary column (30 m x 0.25 mm internal diameter x 0.25 µm film thickness, Agilent J&W Scientific, Palo Alto, CA) and helium as the carrier gas (2 ml/min), coupled to a HP5973n MSD mass spectrometer (Agilent) operated in electron ionization mode. The injections (1 µl) were made in splitless mode at 230°C. The GC oven temperature was initially 110°C, ramped to 250°C at 32.5°C/min, then to 275°C at 10°C/min, and to 325°C at 20°C/min and held for 5 min. The transfer line temperature of the GC/MS interface and the ion source temperature were held at 300°C and 150°C, respectively, and the electron energy was 69.6 eV. Congeners BDE-28, -47, -66, -100, -99, -85, -154, and -153 were quantified based on relative response factors, and the recovery was generally between 70% and 100%. The method was verified using the CRM (WMF-01, fish), of which all analytes were quantified within the range of certified reference values. For statistical analysis, concentrations below the method detection limit (MDL) were assigned a value of half the MDL (ng) divided by the sample weight (g).
Neurochemical Analysis
Sample preparation.
Brain tissues were prepared as described by Stamler et al. (2005) with minor modifications. Dissected cerebral cortex or whole brains were homogenized (Tissue Tearor, Model 398, BioSpec Products, Bartlesville, OK) for 30 s in ice-cold Na/K buffer (50mM NaH2PO4, 5mM KCl, 120mM NaCl, pH 7.4). Membrane fractions were prepared by centrifugation of the homogenate at 32,000 x g for 15 min at 4°C. The resulting pellet was washed twice under the same conditions and resuspended in ice-cold Na/K buffer. Enzyme fractions were prepared by sonication (Sonic Dismembrator, Model 60, Fisher Scientific, Pittsburgh, PA) of the homogenate for 10 s in ice-cold Na/K buffer and 0.1% Triton X-100, followed by centrifugation at 12,000 x g for 10 min at room temperature to obtain the supernatant. The membrane and enzyme preparations were immediately frozen in liquid nitrogen and stored at – 80°C. The concentration of protein was determined using the Bradford assay (Bradford, 1976) with bovine serum albumin as the standard.
mAChR binding assay.
mAChR binding was measured as described by Stamler et al. (2005) with minor modifications. Cerebral cortex membrane fractions (20 µg protein) were added in triplicate to a 96-well, 0.22-µm GF/B glass filter system (Millipore, Boston, MA). Samples were preincubated with Na/K buffer (total binding) or 100µM atropine (a mAChR antagonist) (nonspecific binding) for 30 min at room temperature and then incubated with 1nM [3H]-QNB for 60 min at room temperature with gentle agitation. It has been shown that specific binding at 1nM [3H]-QNB is indicative of mAChR density in mink brain tissues (Stamler et al., 2005). The incubation was terminated by rapid vacuum filtration and the filters were washed twice with ice-cold Na/K buffer. Filters were extracted and allowed to soak overnight in liquid scintillation cocktail. Radioactivity retained by the filters was quantified by a liquid scintillation counter (LS 3801, Beckman Instruments, Irvine, CA) with, approximately, 60% counting efficiency. Nonspecific binding was, approximately, 3% of total binding. Intra- and interassay variation were generally less than 10% and 3% relative standard deviation (RSD), respectively.
nAChR binding assay.
nAChR binding was measured as previously described (Trauth et al., 1999) with the following modifications. Cerebral cortex membrane fractions (25 µg protein) were added in triplicate to a 96-well, 0.22-µm GF/B glass filter system (Millipore). Samples were preincubated with Tris buffer (50mM Tris, 5mM KCl, 2mM CaCl2, 2mM MgCl2, pH 7.4) (total binding) or 100µM (-)-nicotine hydrogen tartrate (a nAChR agonist) (nonspecific binding) for 30 min at room temperature and then incubated with 1nM [3H]-CYT for 1–2 min at room temperature with gentle agitation followed by 75 min at 4°C without agitation. The incubation was terminated by rapid vacuum filtration and the filters were washed three times with ice-cold Tris buffer. Radioactivity retained by the filters was quantified as described above. Nonspecific binding was, approximately, 35% of total binding. Intra- and interassay variation were generally less than 10% and 6% RSD, respectively.
ChE activity assay.
ChE activity was measured as described by Stamler et al. (2005) with minor modifications. Cerebral cortex enzyme fractions (0.5 µg protein) or plasma (20 nl plasma) were added to a 96-well microplate and incubated with Na/K reaction buffer (20µM Amplex Red, 1 U/ml horseradish peroxidase, 0.1 U/ml choline oxidase, and 50µM ACh). Resorufin standard solutions (0–2.5µM resorufin) were also added to the microplate. ChE hydrolyzes ACh to form the coproduct choline. The oxidation of choline by choline oxidase to form the coproduct H2O2 is coupled to the oxidation of Amplex Red by horseradish peroxidase to form the fluorescent product resorufin. Fluorescence was measured every 5 min between 30 and 90 min at 540/590 nm (excitation/emission) by a microplate fluorometer (Wallac Victor2, PerkinElmer) at room temperature. Intra- and interassay variation were generally less than 5% and 10% RSD, respectively.
ACh concentration assay.
ACh concentration was measured as described by Basu et al. (2006) with minor modifications. Cerebral cortex enzyme fractions (20 µg protein) and ACh standard solutions (0–2.5µM ACh) were added to a 96-well microplate and incubated with Na/K reaction buffer (50µM Amplex Red, 1 U/ml horseradish peroxidase, 0.1 U/ml choline oxidase, and 0.5 U/ml AChE). Fluorescence was measured every 5 min between 45 and 60 min as described above. Intra- and interassay variation were generally less than 5% and 10% RSD, respectively.
Statistical Analysis
Statistical analyses were performed using SAS Release 8.02 (SAS Institute, Cary, NC). Although the number of adult females and kits used for PBDE concentration analysis was not large enough to model an ANOVA or perform t-tests, descriptive statistical analyses were performed using SAS PROC REG and SAS PROC CORR. In all cases only one kit from each litter and only paired adult females and kits were included in the analysis, with the exception of the 2.5 µg DE-71/g feed group in which the adult females did not whelp any kits. SAS PROC GLM was used to model a one-way ANOVA for the effect of treatment on the cholinergic parameters assayed for adult females, kits, and juveniles. Either one kit or one juvenile from each litter was used as a statistical unit. In most cases only one female and/or one male offspring from each litter were present. In the cases where more than one offspring from a given litter were present, the average of the offspring was taken to avoid overinflation of statistical power. No gender effect was found on the cholinergic parameters assayed in the offspring as tested and gender was excluded from the analyses. A p value of p < 0.05 was considered statistically significant in all analyses. When a significant difference was detected, the Tukey multiple comparison test was performed.
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RESULTS |
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Dietary Intake of DE-71
The dietary intake of DE-71 in adult females prior to breeding was estimated to be, approximately, 0, 0.01, 0.05, and 0.25 mg/kg bw/d for the 0, 0.1, 0.5, and 2.5 µg DE-71/g feed groups, respectively (Bursian et al., in preparation).
Treatment Groups
There were no significant differences between litter sizes of adult females in the 0, 0.1 and 0.5 µg DE-71/g feed groups. Adult females in the 2.5 µg DE-71/g feed group did not whelp. Average litter sizes (± standard error of the mean) of adult females fed 0, 0.1, and 0.5 µg DE-71/g feed were 3.9 ± 1.1, 4.9 ± 0.95, and 5.6 ± 0.95 kits, respectively. The ratios of male to female kits in the 0, 0.1 and 0.5 µg DE-71/g feed groups were 1:1.3, 1.7:1, and 1.2:1, respectively (Bursian et al., in preparation).
Quantitative PBDE Congener Analysis
The cerebral cortex of two adult females and two 6-week-old kits in the 0, 0.1, and 0.5 µg DE-71/g feed groups and three adult females (no kits were whelped) in the 2.5 µg DE-71/g feed group were analyzed for PBDE concentrations (Table 1). Congeners BDE-47, -99, -100, and -153 were detected in the cerebral cortex of adult females in the control group (0 µg DE-71/g feed), but the total concentrations (
BDEs) were below 3 ng/g wet weight (ww). There was a significant linear increase in BDEs in the cerebral cortex with increasing dietary concentrations for both adult females and 6-week-old kits (p < 0.0001 and p = 0.0022, respectively). There was also a significant positive correlation between BDEs in the cerebral cortex of paired adult females and 6-week-old kits (r = 0.98, p = 0.0009), and the concentrations were twofold to threefold higher in the adult females than in the 6-week-old kits.
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Congener profiles for BDE-47, -99, -100, -153, and -154 in the cerebral cortex of adult females and 6-week-old kits in the 0.5 µg DE-71/g feed group, as well as in the feed, are shown in Figure 1. Although the sample size was not large enough to perform statistical analysis, there were noticeable differences in the congener profiles between the diet and the cerebral cortex. While the predominant congener in the feed was BDE-99, the predominant congeners in the cerebral cortex were BDE-47, -99, and -153. The contributions of BDE-47 and -153 to
BDEs were higher in the cerebral cortex than in the feed for both adult females (by 23% and 19%, respectively) and 6-week-old kits (by 17% and 7.9%, respectively). Conversely, the contributions of BDE-99, -100, and -154 to BDEs were lower in the cerebral cortex than in the feed for both adult females (by 32%, 5.3%, and 5.0%, respectively) and 6-week-old kits (by 20%, 2.9%, and 3.9%, respectively).
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Neurochemical Analysis
There were no significant effects of exposure to DE-71 on mAChR binding, nAChR binding, ChE activity, or ACh concentration in the cerebral cortex of adult females, 6-week-old kits, and 27-week-old juveniles (Table 2).
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ChE activity in the plasma of adult females in the highest treatment group (2.5 µg DE-71/g feed) was threefold higher than that in all other treatment groups (p < 0.0001) (approximately, 300 and 100 nmol resorufin/min/mg protein, respectively) (Fig. 2). There was no significant effect of DE-71 on ChE activity in the plasma of 6-week-old kits or 27-week-old juveniles (Fig. 2), although no offspring were whelped in the 2.5 µg DE-71/g feed group. There were no correlations between ChE activity in the plasma and cerebral cortex, but there were significant positive correlations between ChE activity in the plasma and both liver weight (r = 0.46, p = 0.0029) and liver-to-body weight ratio (r = 0.62, p < 0.0001) in adult females.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Quantitative PBDE Congener Analysis
The concentrations of total BDEs (
BDEs) in the cerebral cortex of adult females and 6-week-old kits ranged from 2.1 to 110 ng/g ww (0–2.5 µg DE-71/g feed groups) and 2.4 to 6.8 ng/g ww (0–0.5 µg DE-71/g feed groups), respectively. The BDEs were twofold to threefold higher in the adult females than in the 6-week-old kits (0.1 and 0.5 µg DE-71/g feed groups). While there is little data available for comparison to the current study, the BDEs measured in the brain of wild otter collected from Nova Scotia was 0.33 ng/g ww (Basu et al., 2007b), an order of magnitude lower than those measured in the cerebral cortex of adult females and 6-week-old kits in the control group in the current study. The concentrations of BDE-47, the predominant congener, in the cerebral cortex of adult females and 6-week-old kits ranged from 0.37 to 42 ng/g ww (0–2.5 µg DE-71/g feed groups) and 1.0 to 2.2 ng/g ww (0–0.5 µg DE-71/g feed groups), respectively. In mice, the concentrations of BDE-47 in the brain of adult females and pups (postnatal day 10) peaked at 104 ng/g ww and 77 ng/g ww, respectively, 8 h following a single oral dose of 1 mg BDE-47/kg body weight (bw), and were higher in adult females than in pups at early time points (Staskal et al., 2005, 2006a). The concentrations of BDE-47 in the brain of adult females were over twofold higher following repeated exposure than single exposure (18.07 ng/g ww, 5 days following repeated oral doses of 1 mg/kg bw/d for 10 days; 7.17 ng/g ww, 5 days following a single oral dose of 1 mg/kg bw) (Staskal et al., 2006b). While there are many differences between the current study and those of Staskal et al. (2005, 2006a,b), the concentrations of BDE-47 in the cerebral cortex of mink are comparable to those in the brain of mice, and the concentrations in adult females are higher than those in kits or pups.
The congener profiles of BDE-47, -99, -100, -153, and -154 in the cerebral cortex of adult females and 6-week-old kits in the 0.5 µg DE-71/g feed group were different from those in the feed. The percentages of BDE-47 and -153 were higher in the cerebral cortex than in the feed, while the percentages of BDE-99, -100, and -154 were lower in the cerebral cortex than in the feed. This may indicate selective uptake, retention, metabolism, or excretion of these congeners in mink, and a study of the relative importance of these processes in this species is ongoing. In a mass balance study of DE-71 in rats (Hakk et al., 2001), the percentages of BDE-47, -99, -100, -153, and -154 were very similar in the feed and the tissue (liver and carcass) following exposure to 32 ng DE-71/rat/day for 21 days, but were not measured in the brain.
Neurochemical Analysis
There were no significant effects of exposure to DE-71 on mAChR binding, nAChR binding, ChE activity, or ACh concentration in the cerebral cortex of adult females, 6-week-old kits, and 27-week-old juveniles. The mAChR binding, ChE activity, and ACh concentration measured in the cerebral cortex and ChE activity measured in the plasma were similar to those measured in ranch mink in a previous study (Basu et al., 2006). However, the results of the current study do not corroborate those of Viberg et al. (2003, 2005), in which neonatal exposure to BDE-99 decreased mAChR density in the hippocampus of adult rats and neonatal exposure to BDE-153 decreased nAChR density in the hippocampus of adult mice. Neonatal exposure to BDE-99 and -153 also disrupted spontaneous behavior (locomotion, rearing, and total activity) and impaired learning and memory in adult mice and rats (Eriksson et al., 1998, 2002; Viberg et al., 2002, 2003, 2004, 2005), while neonatal exposure to BDE-99 altered the effects of adult exposure to nicotine (a nAChR agonist) on spontaneous behavior (Viberg et al., 2002). Neurochemical effects were only observed in the highest treatment groups (administered single oral doses of 16 mg BDE-99/kg bw and 9 mg BDE-153/kg bw). There are several important differences between the current study and those of Viberg et al. (2003, 2005) (Table 3), which may account for the differences in the results.
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DE-71 contains several predominant congeners, including BDE-99 and -153 (used in the studies of Viberg et al. 2003
, 2005), and other environmentally relevant congeners such as BDE-47 and -100 (Wellington Laboratories Inc., 2005). Commercial mixtures may also contain contaminants not present in individual congeners. While differences in the toxicokinetics and toxicodynamics of PBDEs between mink and rodents have not yet been determined, as a wildlife species the mink is a relevant animal model for environmental risk assessment (Basu et al., 2007a). Moreover, differences in the distribution of the mAChR and nAChR subtypes in the cerebral cortex and hippocampus between mink and rodents have not yet been determined.
In the current study, mink were administered continual, low-concentration doses of DE-71 (approximately 0.01–0.25 mg/kg bw/d in adult females prior to breeding; Bursian et al., in preparation) that mimicked environmental exposure. Mink consume 140–200 g/kg bw/d of food, and their diet consists of approximately 50% fish (Aulerich et al., 1999; Sample and Glenn, 1999). The concentrations of PBDEs in freshwater fish in North America are in the order of 0.01–1 µg/g ww (Hale et al., 2001; Johnson and Olson, 2001) and directly comparable to the range of dietary concentrations used in the current study (0.1–2.5 µg DE-71/g feed). Therefore, exposure estimates in mink based on dietary intake of fish are in the order of 0.7–100 µg/kg bw/d, also comparable to the range of dietary intake in the current study (approximately 10–250 µg/kg bw/d; Bursian et al., in preparation).
The effects of exposure to BDE-99 and -153 on the cholinergic neurotransmitter system observed by Viberg et al. (2002, 2003, 2005) in rodents may not be direct but rather mediated in part by other mechanisms such as thyroid hormone disruption. Thyroid hormones regulate neurodevelopment, including the cholinergic neurotransmitter system in the cerebral cortex and hippocampus (Porterfield, 2000). PBDEs have been reported to affect thyroid function. For example, a significant decrease in T4 was observed in the fetuses and offspring of rat dams exposed to 1 mg DE-71/kg bw/d (Zhou et al., 2002), in rat dams exposed to 30 mg DE-71/kg bw/d (Zhou et al., 2001, 2002), and in ranch mink exposed to 5 µg DE-71/g feed for 70 days (Martin et al., 2004). The role of thyroid hormone disruption in the neurodevelopmental toxicity of PBDE warrants further investigation.
There were no significant effects of exposure to DE-71 on ChE activity in the plasma of 6-week-old kits and 27-week-old juveniles. However, there was a threefold increase in ChE activity in the plasma of adult females in the highest treatment group (2.5 µg DE-71/g feed) from that in all other treatment groups. As ChE in the plasma is synthesized in the liver and secreted via very low density lipoproteins (Kutty and Payne, 1994), the increase in ChE activity in the plasma may indicate effects of DE-71 on liver function rather than on neurochemistry. This is supported by significant positive correlations between ChE activity in the plasma and both liver weight and liver-to-body weight ratio in adult females, as well as a lack of correlations between ChE activity in the plasma and the cerebral cortex. The results of the current study corroborate those of Zhou et al. (2001, 2002), in which an increase in liver weight and liver-to-body weight ratio was also observed in rats exposed to DE-71.
In conclusion, this study demonstrated that environmentally relevant exposures to DE-71 did not affect key parameters of the cholinergic neurotransmitter system in the cerebral cortex of ranch mink. The lack of effects of DE-71 on cholinergic parameters in mink did not corroborate the effects of BDE-99 and -153 on cholinergic receptors observed by Viberg et al. (2003, 2005) in rodents, although there are several important differences between these studies. Further studies are needed to determine the differences across species and brain regions in the effects of PBDEs on the cholinergic neurotransmitter system and the mechanism of neurodevelopmental toxicity following environmentally relevant exposures.
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ACKNOWLEDGMENTS |
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This study was funded by the Natural Sciences and Engineering Research Council of Canada (K.B., N.B., J.W.M., and H.M.C.), Alberta Health and Wellness (J.W.M.), the MSU Department of Animal Science (S.B.), the Northern Contaminants Program (H.M.C.), and ArcticNet (L.H.M.C.). The application to use animals in research (05/03-069-00) was approved by the MSU Institutional Animal Care and Use Committee. No conflict of interest is noted. This paper has been presented orally in whole at the Eighth Annual Workshop on Brominated Flame Retardants in the Environment (Toronto, Ontario, Canada; 27–29 June 2006).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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