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TOXICOLOGICAL SCIENCES 101(2), 275–285 (2008) doi:10.1093/toxsci/kfm271 Advance Access publication November 2, 2007 Coexposure of Neonatal Mice to a Flame Retardant PBDE 99 (2,2#,4,4#,5-Pentabromodiphenyl Ether) and Methyl Mercury Enhances Developmental Neurotoxic Defects Celia Fischer, Anders Fredriksson, and Per Eriksson1 Department of Environmental Toxicology, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden Received August 17, 2007; accepted October 7, 2007 Epidemiological studies indicate that exposure to environmental pollutants during early human development can have deleterious effects on cognitive development. The interaction between environmental pollutants is suggested as one reason for the observed defective neurological development in children from the Faeroe Islands as compared to children from the Seychelles. We have previously seen in mice that polychlorinated biphenyls (PCBs) can interact together with methyl mercury (MeHg), as well as PCB together with polybrominated diphenyl ether (PBDE 99) to exacerbate developmental neurotoxic effects when present during a critical period of neonatal brain development. PBDEs are a new class of global environmental contaminants. The present study shows that neonatal coexposure to PBDE 99 (0.8 mg/kg body weight) and MeHg (0.4 or 4.0 mg/kg body weight) can exacerbate developmental neurotoxic effects. These effects are manifested as disrupted spontaneous behavior, reduced habituation, and impaired learning/memory abilities. This is seen in the low dose range, where the sole compounds do no give rise to developmental neurotoxic effects. The effects seen are more than just additive. Furthermore, a significant effect of interaction was seen on the cholinergic nicotinic receptors in the cerebral cortex and hippocampus. This suggests that a mechanism for the observed cognitive defects is via the cholinergic system. Furthermore, PBDE can interact with MeHg causing developmental neurotoxic effects similar to those we previously have observed between PCB 153 1 MeHg and PCB 52 1 PBDE 99. This is of vital importance, as the levels of PBDEs are increasing in mother’s milk and in the environment generally. Key Words: PBDE; methyl mercury; behavior; cholinergic receptors; neonatal; neurotoxicity. Brominated flame retardants (BFRs) are a new class of chemicals where the polybrominated diphenyl ethers (PBDEs) appear to have an environmental dispersion similar to that of well-known persistent organic pollutants such as polychlorinated biphenyls (PCBs) and dichloro diphenyl trichloroethane 1 To whom correspondence should be addressed at Department of Environmental Toxicology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, S-752 36 Uppsala, Sweden. Fax: þ46-18-518843. E-mail: [email protected] (DDT) (Darnerud et al., 2001; de Boer et al., 1998; de Wit, 2002; Sellström et al., 1993). Methyl mercury (MeHg) is a well-known neurotoxic agent present in the environment. Environmental toxicants may interact and affect neurological development which can be one reason for the observed deficits in neurological development of children in the Faeroe Islands compared to children in the Seychelles. Children from both locations were exposed to MeHg, but children in the Faeroe Islands were also exposed to PCBs (Davidson et al., 2006; Grandjean et al., 2001; Myers and Davidson, 1998). Recently, we have observed that coexposure to PCB þ MeHg (Fischer et al., 2006), and PCB þ PBDE (Eriksson et al., 2006) can interact in newborn animals to enhance developmental neurotoxic effects. PBDEs are used in large quantities as flame-retardant additives in polymers for textiles, building materials, and in the manufacture of a wide variety of electrical and electronic appliances, including cases for television sets and computers (WHO, 1994). PBDEs are demonstrably present in the global environment (de Boer et al., 1998; de Wit, 2002). They have been found in samples taken from diverse sources, for example sediments (Sellström et al., 1993), fish (Asplund et al., 1999), and humans (Klasson-Wehler et al., 1997; Schecter et al., 2005; Sjodin et al., 2003). There have been several reports of PBDEs in human milk. The most commonly found congeners are PBDE 47 (2,2#,4,4#tetra-BDE), PBDE 99 (2,2#,4,4#,5-penta-BDE), PBDE 100, (2,2#,4,4#,6-penta-BDE), and PBDE 153 (2,2#,4,4#,5,5#-hexaBDE) (Darnerud et al., 2001; Fangstrom et al., 2005; Schecter et al., 2003). A breast-milk monitoring program in Sweden has shown that over the course of 20–30 years (1972–1997) the earliest organochlorine concentrations decreased by half, whereas PBDE levels have doubled every 5 years (Meironyte et al., 1999; Norén and Meironyté, 2000). A similar increase was observed in a time-trend study in Japan (1973–2000), in which the sum of PBDEs in human milk was of a magnitude similar to that in the Swedish study (Akutsu et al., 2003). Some samples of mother’s milk in the United States are reported to contain some of the highest levels of PBDEs worldwide, up to Ó The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected] 276 FISCHER, FREDRIKSSON, AND ERIKSSON 10–100 times that found in the Swedish and Japanese studies (Schecter et al., 2003, 2005). The body burden of PBDEs is also approaching that of the PCBs. There are an increasing number of studies indicating that PBDEs cause developmental neurotoxic effects (Branchi et al., 2002; Eriksson et al., 2001; Rice et al., 2007; Viberg et al., 2003, 2007). Neonatal exposure to PBDE 99 has been shown to disrupt spontaneous behavior, cause a loss of habituation, impair learning and memory abilities, alter response in the adult cholinergic system, or decrease the amount of cholinergic muscarinic receptors in the hippocampus (Branchi et al., 2002; Eriksson et al., 2001, 2002; Viberg et al., 2002, 2004b). A recent paper (Dingemans et al., 2007) indicates that PBDE 47 in neonatal mice affects longterm potential in the hippocampus, which is related to learning and memory processes. Methyl mercury (MeHg) is a well-known neurotoxic agent. Maternal exposure to high levels of methyl mercury (MeHg) can cause neurological damages in children as seen in Japan in the 1960#s through consumption of contaminated fish, in Iraq during the 1970#s after grain contaminated with a MeHg fungicide, and more recently in New Zealand (ATSDR, 1999; Shipp et al., 2000). The developmental neurological defects observed in children from Minamata in Japan included profound mental retardation. Notable from these studies was the observation that adults, including mothers of poisoned children, were less seriously affected than the children (see Grandjean and Landrigan, 2006). Developmental exposure to MeHg is known to cause neurobehavioral defects in animals (Day et al., 2005; Evans et al., 1975; Weiss et al., 2005). A recent study shows that neonatal exposure of mice to MeHg caused behavioral alterations, and these alterations were most pronounced when the exposure occurred after postnatal day 10 (PND 10) (Stringari et al., 2006). There are reports that show that MeHg can affect the cholinergic system, for example, reduce choline acetyltransferase activity (Kobayashi et al., 1979; Omata et al., 1982), acetylcholinesterase activity (Tsuzuki, 1981), and choline uptake (Bondy et al., 1979; Kobayashi et al., 1979). MeHg also has been shown to affect the muscarinic acetylcholine receptors in a variety of species, including humans (Basu et al., 2005, 2006). The cholinergic transmitter system is involved in many behavioral phenomena (Karczmar, 1975) and is closely related to cognitive functions (Drachman, 1977; Herlenius and Lagercrantz, 2004; Levin and Simon, 1998; Paterson and Nordberg, 2000; Perry et al., 1999). The brain growth spurt (BGS) (Davison and Dobbing, 1968) is the time when rapid developmental changes appear, transforming a feto/neonatal brain into a mature adult brain. During the BGS, the brain undergoes several fundamental phases, such as axonal and dendritic outgrowth, establishment of neural connections, acquisition of new motor and sensory faculties (Bolles and Woods, 1964; Davison and Dobbing, 1968; Kolb and Whishaw, 1989), and peak in spontaneous motor behavior (Campbell et al., 1969). The time for the BGS varies for different species (Davison and Dobbing, 1968). With humans this period begins during the third trimester of pregnancy and continues for the first 2 years of the child’s life. For mice and rats, this BGS period is neonatal and occurs during the first 3–4 weeks after birth. In rodents, the cholinergic transmitter system in the central nervous system undergoes rapid development during the first 3–4 weeks after birth (Coyle and Yamamura, 1976; Fiedler et al., 1987), when gradually increasing numbers of muscarinic and nicotinic receptors appear in the cerebral cortex and hippocampus (Falkeborn et al., 1983; Fiedler et al., 1987; Kuhar et al., 1980). In several studies we have shown that low-dose exposure to persistent environmental toxic agents such as PCBs, DDT, and BFRs during the BGS in neonatal mice can lead to persistent defects in adult behavior and to the cholinergic system (Eriksson, 1997, 2007; Viberg et al., 2003, 2004a,b). The disturbances are manifested as defective spontaneous behavior, lack of habituation, impaired learning and memory functions, and decrease in cholinergic muscarinic and nicotinic receptors. The aims of the present study were (1) to establish if coexposure to PBDE 99 and MeHg on PND 10 can interact to enhance developmental neurobehavioral effects, (2) to establish if neonatal coexposure to PBDE 99 and MeHg can interfere and affect learning and memory abilities, and (3) to investigate if coexposure to PBDE 99 and MeHg affects the cholinergic system by influencing the nicotinic cholinergic receptor density in the cerebral cortex and in the hippocampus. MATERIALS AND METHODS Animals and Chemicals For our neurotoxic recordings we used male NMRI mice to make this study comparable to our earlier studies on PBDEs (e.g., Eriksson et al., 2001, 2002; Viberg et al., 2004a,b) and MeHg (unpublished). Pregnant NMRI mice were purchased from B&K, Sollentuna, Sweden. Each litter, adjusted within 48 h of birth to eight to 12 mice by euthanasia of remaining pups, was kept together with its respective mother in a plastic cage housed in a room with an ambient temperature of 22°C and a 12-h light:12-h dark cycle. Each litter contained about an equal number of both male and female pups. At an age of 10 days, all pups were exposed to the vehicle or the test compounds. At the age of 4 weeks male mice were weaned, placed, and raised in groups of four to seven in a room for male mice only. The animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad labium. The PBDE 2,2#,4,4#,5-pentabromodiphenyl ether (PBDE 99) was a gift from Dr Åke Bergman at the Department of Environmental Chemistry, University of Stockholm, Sweden. The purity of the compound exceeded 98%. MeHg (methyl mercuric chloride, Merck, Darmstadt, Germany) was purchased from KEBO, Sweden. The PBDE was dissolved in a mixture of (1:10) egg lecithin (Merck) and peanut oil (Oleum arachidis) (Eriksson et al., 2001, 2006). Methyl mercury chloride was dissolved in water. The PBDE solution and the MeHg solution were mixed together and then sonicated to yield a 20% (wt:wt) fat emulsion vehicle containing various concentrations of the compounds. The substances were administered orally, at a volume of 10 ml/kg body weight, via a metal gastric-tube, as one single dose on PND 10. The amounts of the different compounds given are presented in Table 1. Control mice received 10 ml/kg body weight of 20% fat emulsion vehicle in the same manner as the treatment groups. Each treatment group comprised mice from three to four different litters. PBDE þ MeHg—NEURODEVELOPMENT DEFECTS TABLE 1 Treatment Table for Mice Exposed to a Single Oral Dose of PBDE 99, MeHg, PBDE 99 1 MeHg, or Vehicle on PND 10 Treatment Control PBDE 99 MeHg MeHg PBDE 99 þ MeHg PBDE 99 þ MeHg Dosages (mg/kg bw) 20% fat emulsion vehicle 0.8 0.4 4.0 0.8 þ 0.4 0.8 þ 4.0 This experimental design of neonatal exposure to xenobiotics has been used by our laboratory for several years and thereby generated historical controls as well as reproducible developmental neurotoxicological data on environmental toxicants (Eriksson, 1997, 2007; Eriksson et al., 2006). In this neonatal animal model, each of the different treatment groups comprise mice from three to four different litters. Randomly selecting animals from at least three different litters will have the same statistical effect and power compared to the use of litter based studies to evaluate developmental neurotoxicity in neonatal mice (Eriksson and Viberg, 2005; Eriksson et al., 2005). Behavioral Tests Spontaneous behavior. Spontaneous behavior previously described by Eriksson (Eriksson, 1997; Eriksson et al., 2006) was tested in male mice at the ages of 2, 4, and 6 months. A total of eight mice from each treatment group were randomly selected from three to four different litters and only tested once for each test occasion. The tests were performed between 8 and 12 A.M. under the same ambient light and temperature conditions. Motor activity was measured over 3 3 20 min in an automated device consisting of cages (40 3 25 3 15 cm) placed within two series of infrared beams (low level and high level) (RatO-Matic, ADEA Elektronik AB, Uppsala, Sweden) (Fredriksson, 1994). The cages were placed in individual soundproofed boxes with separate ventilation. Locomotion was registered when the mouse moved horizontally through the low-level grid of infrared beams. Rearing was the vertical movement registered at a rate of four counts per second, whenever the single high-level beam was interrupted, that is, the number of counts obtained was proportional to the time spent rearing up. For total activity, a pick-up (mounted on a lever with a counterweight) registered all types of vibrations within the test cage, that is, those caused by mouse movements, shaking (tremors), and grooming. Swim maze. The swim maze behavioral test was performed in male mice at the age of 4 months. A total of 13–22 male mice were selected from three to four litters form each treatment group. The swim maze behavioral test conducted was modeled after the Morris water maze type (Morris, 1981). The gray circular container 103 cm in diameter was filled with water to a depth of 15 cm from the brim at a water temperature of 23°C. In the middle of the northwest quadrant a metal mesh platform 12 cm in diameter was submerged 1 cm below the water surface. The relative positions of the observer and the Morris maze pool were the same throughout the course of the swim maze test. The behavioral test was performed for 5 consecutive days to test the mouse’s spatial learning ability to locate the platform for the first 4 days, trials 1–20. Each mouse was placed on the platform for 20 s and then released in the south position with its head pointed toward the wall of the container. The mice had 30 s to locate the submerged platform, and between each trial the mouse rested on the platform for 20 s. The time to reach the platform was measured by the observer; total search time for the five trials was set to 150 s. On the fifth day, the platform was relocated to the northeast quadrant and the mice were tested on their relearning abilities; otherwise the procedure was identical. Radial arm maze. Ten male mice were randomly selected from three to four litters and tested at 5 months of age for each treatment group. The radial 277 maze has eight arms (8 3 35 cm, surrounded by a 1.5 cm border) radiating from a circular platform (diameter 20 cm) (Eriksson and Fredriksson, 1996). The maze was raised 60 cm off the floor. Each arm was baited 3 cm from its outer most walls by placing a small food pellet (5 mg) behind a low barrier preventing the animal from seeing if a specific arm was baited or not. The animals were tested on 3 consecutive days, one trial per day. The tests were performed during the daytime between 9 A.M. and 3 P.M. The mice had free access to water but were deprived food 24 h before the initial trial. The first 2 days of the radial arm maze was used to accustom the mice to the test environment and to the maze itself. Only data from the final performance day were used for analyses. The start of each trial began with the mouse placed on the central platform always facing the same direction. The trial was terminated after 10 min or as soon as the mouse had eaten all eight food rewards. To perform well at this task, the mice had to store information continuously about which arm(s) had already been visited during a particular trial and which had not (working-memory, storing trial-specific information). The behavioral measures recorded were the time to find all eight pellets and the number of errors. Error is defined here as reentering an arm where the food pellet had already been devoured. Nicotinic receptor analysis. The male mice were euthanized by decapitation following the completion of the behavioral tests at 6 months of age. The brain was dissected into cerebral cortex and hippocampus on an ice-cold plate and immediately placed on dry ice and stored at 80°C until assayed. A crude synaptosomal P2 fraction was prepared from the cerebral cortex and hippocampus. The protein content was between 2.0 and 3.0 mg/ml for the cerebral cortex and hippocampus (measured according to Lowry et al., 1951). The nicotinic receptor assay was performed by measuring tritium labeled a-bungoratoxin ([3H] aBTX). The specific binding was carried out following the method by Falkeborn et al. (1983) as described by Viberg et al. (2003). Aliquots of P2 fractions (50 ll) were incubated with 20 ll [3H] aBTX (61.00 Ci/mmol, 20 nM in 0.1% bovine serum albumin) for 120 min at 25°C made up to a total of 200 ll with NaKPO4 buffer (pH 7.40). To measure nonspecific binding, parallel samples were incubated with 20 ll (5lM) of BTX. Each binding was determined in triplicate. Incubation was terminated by centrifugation at 20,000 3 g for 5 min. The pellet was washed with 200 ll of ice-cold NaKPO4 buffer and then transferred in miniscintillation vials and left overnight to dissolve the pellet in one milliliter of Aquasafe 300þ scintillation fluid (Zinsser Analytic, Ltd, UK). Four milliliters of Aquasafe 300þ scintillation fluid was added to each vial and radioactivity was determined in a liquid scintillation analyzer (Packard Tri-Carb, 1900 CA) after the samples had been kept in the dark for 8 h. Specific binding was determined by calculating the difference between the amounts of [3H] aBTX bound in the presence versus the absence of aBTX. Statistical Analysis The locomotion, rearing, and total activity data over three consecutive 20min periods (treatment, time, and treatment 3 time; between subjects, within subjects, and interaction factors, respectively), in the spontaneous behavior test, the time taken to find the submerged platform over 4 consecutive testing days (treatment, day, and treatment 3 day, between subjects, within subjects, and interaction factors, respectively), the time taken to find the submerged platform during the fifth day (treatment, trial, and treatment 3 trial, between subjects, within subjects, and interaction factors, respectively), in the swim maze test, were submitted to a split-plot ANOVA design (Kirk, 1968). The major advantages with a split-plot design compared with randomized block factorial design are that the estimates of the within-block effects are usually more accurate than estimates of the between-block estimates. Because the average experimental error over all treatments is the same for both designs, the increased precision on within-block effects is obtained by sacrificing precision on between block. The time required to obtain all eight pellets and the number of errors in the Radial Arm Maze test were after Barketts test for homogeneity subjected to Kruskal–Wallis test (Kirk, 1968). The data from [3H] aBTX binding were subjected to one-way ANOVA. Pairwise testing between the different treatment groups was performed with Duncan’s test. 278 FISCHER, FREDRIKSSON, AND ERIKSSON The results from the spontaneous behavior variables ‘‘locomotion,’’ ‘‘rearing,’’ and ‘‘total activity’’ in 2-, 4-, and 6-month-old NMRI male mice exposed to dosages given in Table 1 are presented in Figures 1–3. Two months after exposure, the significant group 3 period interactions are locomotion (F10,108 ¼ 13.73), rearing (F10,108 ¼ 44.89), and total activity (F10,108 ¼ 23.50). Pairwise testing between PBDE 99, MeHg, PBDE 99 þ MeHg and control groups showed a significant difference between the different treatment groups in all three-test variables. The activity level decreased for the control group for all variables throughout the 60-min period. This decrease in activity follows a normal spontaneous behavior profile (Eriksson, 1997; Fredriksson, 1994). The PBDE 99 group displayed normal behavioral pattern similar to those of the control group for both the locomotion and rearing variables. During the first 0–20 min, there was a decrease in activity compared with the control group ( p 0.01). The MeHg group (0.4 mg) also resembled the control group throughout the 60-min period for all variables. The activity in mice exposed to the highest dose of MeHg (4.0 mg) during the first 20-min period was decreased compared with the FIG. 1. Spontaneous behavior in 2-month-old mice exposed on PND 10 to a single oral dose of PBDE 99 (0.8 mg/kg bw), MeHg (0.4 or 4.0 mg/kg bw), PBDE 99 þ MeHg (0.8 þ 0.4 or 4.0 mg/kg bw), or the 20% fat emulsion vehicle. Statistical analysis, ANOVA with split-plot design and pairwise testing with Duncan’s test. The height of each bar represents the mean ± SD of eight animals. A ¼ p 0.01 versus vehicle; a ¼ p 0.05 versus vehicle; B ¼ p 0.01 versus PBDE 99; b ¼ p 0.05 versus PBDE 99; C ¼ p 0.01 versus MeHg (0.4 mg); c ¼ p 0.05 versus MeHg (0.4 mg); D ¼ p 0.01 versus MeHg (4.0 mg); d ¼ p 0.05 versus MeHg (4.0 mg); E ¼ p 0.01 versus PBDE 99 þ MeHg (0.4 mg); e ¼ p 0.05 versus PBDE 99 þ MeHg (0.4 mg). FIG. 2. Spontaneous behavior in 4-month-old mice exposed on PND 10 to a single oral dose of PBDE 99 (0.8 mg/kg bw), MeHg (0.4 or 4.0 mg/kg bw), PBDE 99 þ MeHg (0.8 þ 0.4 or 4.0 mg/kg bw), or the 20% fat emulsion vehicle. Statistical analysis, ANOVA with split-plot design and pairwise testing with Duncan’s test. The height of each bar represents the mean ± SD of eight animals. A ¼ p 0.01 versus vehicle; a ¼ p 0.05 versus vehicle; B ¼ p 0.01 versus PBDE 99; b ¼ p 0.05 versus PBDE 99; C ¼ p 0.01 versus MeHg (0.4 mg); c ¼ p 0.05 versus MeHg (0.4 mg); D ¼ p 0.01 versus MeHg (4.0 mg); d ¼ p 0.05 versus MeHg (4.0 mg); E ¼ p 0.01 versus PBDE 99 þ MeHg (0.4 mg); e ¼ p 0.05 versus PBDE 99 þ MeHg (0.4 mg). RESULTS There were no overt signs of clinical dysfunction in the treated mice throughout the experimental period. There were no significant deviations in body weight in the PBDE 99, MeHg, or PBDE 99 þ MeHg treated mice, compared with the vehicle treated mice. Spontaneous Behavior PBDE þ MeHg—NEURODEVELOPMENT DEFECTS FIG. 3. Spontaneous behavior in 6-month-old mice exposed on PND 10 to a single oral dose of PBDE 99 (0.8 mg/kg bw), MeHg (0.4 or 4.0 mg/kg bw), PBDE 99 þ MeHg (0.8 þ 0.4 or 4.0 mg/kg bw), or the 20% fat emulsion vehicle. Statistical analysis, ANOVA with split-plot design and pairwise testing with Duncan’s test. The height of each bar represents the mean ± SD of eight animals. A ¼ p 0.01 versus vehicle; a ¼ p 0.05 versus vehicle; B ¼ p 0.01 versus PBDE 99; b ¼ p 0.05 versus PBDE 99; C ¼ p 0.01 versus MeHg (0.4 mg); c ¼ p 0.05 versus MeHg (0.4 mg); D ¼ p 0.01 versus MeHg (4.0 mg); d ¼ p 0.05 versus MeHg (4.0 mg); E ¼ p 0.01 versus PBDE 99 þ MeHg (0.4 mg); e ¼ p 0.05 versus PBDE 99 þ MeHg (0.4 mg). control group ( p 0.01) for all variables. During the last 40- to 60-min period, an increase in activity was observed ( p 0.01) for locomotion and rearing variables. The coexposure group PBDE 99 þ MeHg (0.4 mg) displayed decreased activity compared with the control group and the PBDE 99 group, during the first 0–20 min. The final 40- to 60-min period for coexposure PBDE 99 þ MeHg (0.4 mg) displayed an increase in locomotion, rearing, and total activity compared with both the control group, PBDE 99 alone, and MeHg (0.4 mg) alone ( p 0.01). Mice given PBDE 99 þ MeHg (4.0 mg) also had a decrease ( p 0.01) in locomotion activity during the first 0–20 min compared with the control group and PBDE 99 group and an increase ( p 0.01) for all activity variables for the final 40–60 min compared with the control group, the PBDE 99 group, and the MeHg (0.4 mg) group. 279 Four months after the neonatal exposure to PBDE 99, MeHg, and PBDE 99 þ MeHg, the mice continued to display significant group 3 period interactions for locomotion (F10,108 ¼ 31.04), rearing (F10,108 ¼ 138.74), and total activity (F10,108 ¼ 30.22) (Fig. 2). Pairwise testing between PBDE 99, MeHg, PBDE 99 þ MeHg, and control groups showed a similar significant differences between these treatment groups in all three test variables as observed 2 months after exposure. The control mice continued to display normal spontaneous behavior with higher activity during the first 20 min with decreasing activity over time. The most pronounced additional changes were seen for the locomotion variable. The coexposure PBDE 99 þ MeHg (0.4 mg) group’s activity was lower ( p 0.01) during the first 20 min compared with the control group, PBDE 99 and MeHg (0.4 mg) group. During the last 40–60 min this group showed higher activity than the control group and the individual compound groups ( p 0.01). Six months after the neonatal exposure to PBDE 99, MeHg, and PBDE 99 þ MeHg the mice continued to display significant group 3 period interactions for locomotion (F10,108 ¼ 34.62), rearing (F10,108 ¼ 283.36), and total activity (F10,108 ¼ 30.20) (Fig. 3). Pairwise testing between PBDE 99, MeHg, PBDE 99 þ MeHg, and control groups showed a similar significant differences between these treatments groups for all three test variables as observed 4 months after the exposure. The control mice continued to display normal spontaneous behavior with higher activity during the first 20 min and decreasing activity over time. The most pronounced additional changes were seen in the locomotion variable. Mice coexposed to PBDE 99 þ MeHg (0.4 mg) showed an increase in activity during the last 40–60 min compared with all the single exposure groups and the control group ( p 0.01). The PBDE 99 þ MeHg (4.0 mg) group showed a higher activity during the final 40–60 min compared with all single exposure groups and the control groups ( p 0.01). Morris Swim Maze During the acquisition period of spatial learning abilities measured from day 1 to day 4, all mice improved their ability to locate the platform (F3,303 ¼ 143) (see Figure 4). The control group exhibits normal spatial learning abilities. Split-plot ANOVA revealed significant group 3 day interactions for the different treatment groups (F15,303 ¼ 3.11). Mice coexposed to PBDE 99 þ MeHg were significantly different ( p 0.01) from the control mice on day 3 and on day 4, as well as the highdose MeHg (4.0 mg). On day 4, the mice coexposed to low of doses PBDE 99 and MeHg (0.4 mg) were significantly different ( p 0.01) compared with the compounds by themselves as well as to the control group. Coexposure to PBDE 99 and MeHg (4.0 mg) differed significantly ( p 0.01) compared with the control group, PBDE 99 group, and the MeHg (0.4 mg) group but not to the MeHg (4.0 mg) group or the coexposure PBDE 99 þ MeHg (0.4 mg) group. PBDE 99 280 FISCHER, FREDRIKSSON, AND ERIKSSON Radial Arm Maze Kruskal–Wallis indicated a significant change for the times to acquire all eight pellets (H ¼ 27.17, p 0.01) and a significant change in number of errors made in acquiring all pellets (H ¼ 12.24, p 0.01) (Table 2). Pairwise testing using Duncan’s test showed that mice coexposed to PBDE 99 þ MeHg (0.4 mg) took significantly ( p 0.01) longer time in acquiring all eight pellets compared with the compounds by themselves as well as to control group. The mice given the highest dose of MeHg (4.0 mg) and the combination dose of PBDE 99 þ MeHg (4.0 mg) took significantly ( p 0.05) longer time in acquiring all pellets compared with the mice given the vehicle, PBDE 99, and MeHg (0.4 mg). All treatment groups had significantly ( p 0.05) more errors compared with the vehicle group, except the mice given MeHg (0.4 mg). FIG. 4. The Morris maze was preformed in 4-month-old NMRI male mice exposed to a single oral dose of PBDE 99, MeHg, combination dose of PBDE 99 and MeHg, or a vehicle (20% fat emulsion) on PND 10. Spontaneous behavior showed treatment 3 time effects (for statistical methods see exp. 1). The swim maze behavioral data, days 1–4, were submitted to an ANOVA using a split-plot design with Duncan’s test. Day 5 was analyzed by a one-way ANOVA and Duncan’s test. The data for days 1–4 showed a treatment 3 time effect, control < PBDE99 1.4, MeHg 0.4 < MeHg 4.0, PBDE 99 1.4 þ MeHg 0.4, PBDE 99 1.4 þ MeHg 4.0. Relearning on day 5 showed treatment 3 trail effect, control, PBDE 99 1.4, MeHg 0.4 < MeHg 4.0, PBDE 99 1.4 þ MeHg 0.4, PBDE 99 1.4 þ MeHg 4.0. had an effect on spatial learning on day 4, as did the MeHg (0.4 mg) ( p 0.05). MeHg (4.0 mg) affected the spatial learning abilities, which were significantly different ( p 0.01) from those for the control group, and the MeHg (0.4 mg) group. On day 5 the platform was relocated for relearning by reversal trials. In the initial trial on day 5, control mice displayed a longer time for locating the platform compared with the final trial on day 4. This is normal for relearning due to the relocation of the submerged platform because the mice initially begin their search near the previous platform location (Morris, 1981). The control mice quickly improved their ability to find the new location of the platform indicating normal relearning abilities. Split-plot ANOVA revealed significant group 3 trial interactions for the different treatment groups (F20,404 ¼ 2.82). Times for the mice given PBDE 99 and MeHg (0.4 mg) were similar to the control group. Mice given MeHg (4.0 mg) relearning abilities were significantly ( p 0.01) affected compared with the control group, and the MeHg (0.4 mg) group. The coexposure group PBDE 99 þ MeHg (0.4 mg) took significantly ( p 0.01) longer time to find the platform than the control, and the PBDE 99 and MeHg (0.4 mg) groups singly, thus indicating an interaction effect on relearning abilities. Coexposure to PBDE 99 þ MeHg (4.0 mg) caused significantly ( p 0.01) longer time to find the platform than the control group, PBDE 99 group, and the MeHg (0.4 mg) group. Nicotinic Receptors in the Cerebral Cortex and the Hippocampus The density of nicotinic receptors in the cerebral cortex and the hippocampus of 6-month-old mice exposed to PBDE 99, MeHg, PBDE 99 þ MeHg, or the vehicle on PND 10 are shown in Table 3. One-way ANOVA indicated a significant change in the densities of [3H] aBTX binding sites in the cerebral cortex (F5,72 ¼ 3.51, p 0.01) and in the hippocampus (F5,72 ¼ 2.83, p 0.01). In the cerebral cortex, all treatment groups significantly ( p 0.05) reduced the nicotinic receptor density from the vehicle group. In the hippocampus, there was a significant ( p 0.05) reduction in the density of [3H] aBTX binding sites in mice neonatally given PBDE 99 þ MeHg (0.4 mg), PBDE 99 þ MeHg (4.0 mg), and MeHg (4.0 mg) compared with the vehicle group. This indicates that coexposure to low doses of PBDE 99 and MeHg (0.4 mg) can interact and significantly promote an enhanced effect in the hippocampus. DISCUSSION This study shows that PBDE 99 and MeHg can interact during a critical period of rapid brain development in the neonatal mouse to exacerbate developmental neurobehavioral defects manifested as defective spontaneous behavior, lack of habituation, and impaired memory/learning. The behavioral defects were also sustained over time. This interaction was seen at low doses where the sole compounds singly did not cause an overall functional disorder. An interaction was also present in the cholinergic system where the combination of PBDE 99 and the low dose of MeHg caused a decrease in the density of nicotinic receptors in both the hippocampus and the cerebral cortex. This suggests that one of the mechanisms behind the behavioral disturbances is caused by changes in the cholinergic system. Neonatal mice coexposed to PBDE 99 þ MeHg (0.4 mg) on PND 10 displayed a significantly changed spontaneous PBDE þ MeHg—NEURODEVELOPMENT DEFECTS TABLE 2 Radial Arm Maze Performance in 6 Months Old Mice after Neonatal Exposure to PBDE 99 and MeHga Time to acquire all pellets (s) Treatment (mg/kg bw) (n) Median Vehicle PBDE 99 (0.8) MeHg (0.4) MeHg (4.0) PBDE 99 (0.8) þ MeHg (0.4) PBDE 99 (0.8) þ MeHg (4.0) 12 12 12 9 12 9 262 329 292 403 373 408 Min–Max 192–347 241–364 223–402 243–550ac 258–571abc 300–525abc Errors Median Min–Max 5 7.5 5 5 10 8 2–7 3–11ac 4–8 3–13ac 2–23ac 4–12ac a Male NMRI mice were exposed to a single oral dose of PBDE 99, MeHg, PBDE 99 þ MeHg, or 20% fat emulsion. The behavioral measures recorded were the time to acquire all eight pellets and the number of errors (errors made in acquiring all eight pellets). The statistical evaluation of times to acquire pellets was performed by using Kruskal–Wallis (H ¼ 27.17), two-tailed p 0.01, and pairwise testing using Duncan’s test. The statistical evaluation for errors was performed by using Kruskal–Wallis (H ¼ 12.24), two-tailed p 0.01, and pairwise testing using Duncan’s test. The letter a ¼ p 0.05 versus vehicle, b ¼ p 0.05 versus PBDE 99 0.8 mg/kg bw, and c ¼ p 0.05 versus MeHg 0.4 mg/kg bw. behavioral pattern compared with the control, PBDE 99 and MeHg (0.4 mg) exposed mice. This clearly shows an interaction between PBDE 99 and MeHg. This defective spontaneous behavior was present in 2-, 4-, and 6-month-old animals indicating a permanently modified habituation capability. Habituation is defined here as a decrease in locomotion, rearing, and total activity as a response to the diminishing novelty of the test chamber during the 60-min test period. Habituation was evident in the control animals, whereas mice exposed to PBDE 99 þ MeHg (0.4 mg) were obviously TABLE 3 Effects on Nicotinic Receptors in 6 Month Old Mice after Neonatal Exposure to PBDE 99 and MeHga [3H] aBTX binding (pmol/g protein) Treatment (mg/kg bw) (n) Vehicle PBDE 99 0.8 MeHg 0.4 MeHg 4.0 PBDE 99 0.8 þ MeHg 0.4 PBDE 99 0.8 þ MeHg 4.0 15 15 15 15 15 15 Hippocampus 106 92 87 81 74 80 ± ± ± ± ± ± 27 24 21 32a 22a 15a Cerebral cortex 66 51 48 46 47 50 ± ± ± ± ± ± 10 16a 16a 12a 18a 9a a Male NMRI mice were exposed to a single oral dose of PBDE 99, MeHg, PBDE 99 þ MeHg, or 20% fat emulsion. The animals were killed at 6 months of age and [3H] aBTX binding (mean ± SD) was assessed in the P2 fraction. The statistical evaluation was performed by using one-way ANOVA and pairwise testing using Duncan’s test. The letter a ¼ p 0.05 versus vehicle. 281 hypoactive early in the 60-min test period and became hyperactive toward the end. Spontaneous behavior and habituation were defective in neonatal mice exposed to the high doses of MeHg (4.0 mg). An important finding was that the developmental neurotoxic effects on spontaneous behavior after PBDE 99 þ MeHg (0.4 mg) were not different at doses 10 times higher of MeHg (4.0 mg). In mice receiving the higher dose of MeHg (4.0 mg) together with PBDE 99, there was no additional effect seen. This indicates that within the low-dose range the interaction effect on spontaneous behavior and habituation capability appears to be synergistic and the effect is sustained. The ability of adult mice to learn and memorize was studied using two different types of mazes, viz. a radial eight-arm maze and a swim maze of Morris water-maze type. Both mazes revealed an effect of interaction for mice coexposed to PBDE 99 þ MeHg (0.4 mg). Mice exposed to combination of compounds performed significantly worse than vehicle exposed animals and mice exposed to the sole compounds PBDE 99 and MeHg (0.4 mg). In the eight-arm maze, the mice coexposed to the combination PBDE 99 þ MeHg (0.4 mg) displayed significantly longer times to acquire the pellets and also made more errors, indicating impaired working memory in these animals. The swim-maze test revealed that adult mice coexposed to PBDE 99 þ MeHg (0.4 mg) performed significantly worse than vehicle exposed animals and mice exposed to the PBDE 99 and MeHg (0.4 mg) separately. During the 4-day acquisition period, all animals required less time to locate the submerged platform. During this acquisition period the animals coexposed to PBDE 99 þ MeHg (0.4 mg) spent more time locating the submerged platform compared with mice exposed to vehicle, PBDE 99, and MeHg (0.4 mg) alone. This deterioration during the acquisition period was also seen in mice given a 10 times higher dose of MeHg (4.0 mg) and in mice exposed to PBDE 99 þ MeHg (4.0 mg). These exposure groups did not differ from mice receiving PBDE 99 þ MeHg (0.4 mg). In the reversal trials on the fifth day there were similar changes between the groups as during the acquisition period. Mice exposed to PBDE 99 þ MeHg (0.4 mg) deviated from the control animals and mice exposed to just PBDE 99 or MeHg (0.4 mg) alone. This reduced ability to perform in a swim maze is similar to the impairments in spatial learning tasks seen in rodents with advancing age in the Morris water maze (Gage et al., 1984; Lamberty and Gower, 1989). Spatial learning is one form of memory in which humans also show significant impairments as they age (Barnes, 1988; Caplan and Lipman, 1995). This indicates that neonatal coexposure to both PBDE 99 and MeHg might interact and exacerbate this kind of aging process, and that this process can occur at low doses to these compounds. The cholinergic system plays an important role in many behavioral phenomena, for example, learning and memory, neurological syndromes, audition, vision, and aggression (Drachman, 1977; Karczmar, 1975; Perry et al., 1999). Several studies show that pharmacological manipulations of the 282 FISCHER, FREDRIKSSON, AND ERIKSSON cholinergic system are correlated to altered cognitive behavior (Decker et al., 1995; Murray and Fibiger, 1985). Spontaneous behavior is dependent on the integration of sensory input into motor output. As used here, spontaneous behavior measures habituation, which is essentially the integration of new information with previously attained information. Thus, spontaneous behavior is thereby a measurement of cognitive function. It is known that lesions to cholinergic nuclei, or cholinergic neurons projecting to the hippocampus or cortex, can cause learning and memory deficits (Berger-Sweeney et al., 1994; Nabeshima, 1993). The swim maze of Morris watermaze type with its submerged platform is designed to measure spatial learning, which has been suggested to be correlated with the cholinergic function (Lindner and Schallert, 1988; Whishaw, 1985). Behavioral performances of tasks requiring attention and rapid processing of information in humans and new/reversal learning and working memory in animals have been suggested to involve cholinergic transmission (Hodges et al., 1991). The cholinergic system is one of the major transmitter systems and is correlated to cognitive function. In the present study it was seen that neonatal coexposure to PBDE 99 þ MeHg (0.4 mg) significantly reduced the density of nicotinic cholinergic receptors of adult mice, in both the cerebral cortex and the hippocampus. The reduced density was observed as a decrease in the a-BTX binding sites that are connected to the nicotinic acetylcholine receptor (nACh) subunit a7 (Couturier et al., 1990; Orr-Urtreger et al., 1997). In earlier studies we have seen that neonatal exposure to PBDEs can affect the development of the cholinergic system (Viberg et al., 2002, 2003, 2004a, 2007). The changes have been observed as an increased susceptibility to the cholinergic acetylcholine agonist, nicotine, and as a decrease for nicotinic receptor densities in the hippocampus. The effects on nAChR were seen at a higher dose of PBDE 99 than the dose used in the present study together with MeHg. The a7 nAChRs are widely expressed throughout the mammalian brain and have been implicated in cognitive function and neuroprotection (Seo et al., 2001). Due to a7 nAChRs high Ca2þ permeability, it is proposed to be of special interest during this development (Ghosh and Greenberg, 1995; Wong and Ghosh, 2002). In vitro studies show that PBDEs can affect intracellular signaling processes and Ca2þ homeostasis in cerebellar granula cells (Kodavanti and Derr-Yellin, 2002; Kodavanti and Ward, 2005). In vitro experiments also show that apoptotic cell death of hippocampal progenitor cells can be induced by the induction of nicotinic receptors (Berger et al., 1998). However, the hippocampal cells were spared apoptotic cell death when these cells are differentiated. This apoptotic effect appears to be dependent on calbindin for the regulation of [Ca2þ] and activation of a7nAChRs. MeHg is also known to induce apoptotic cell death (Johansson et al., 2006; Tamm et al., 2006). Therefore, the changes in the cholinergic and apoptotic processes during a critical stage of the neonatal brain development are of special interest and require further studies. It is of special interest to compare the present study with a recent study in which we show that neonatal coexposure to low doses of PCB 153 and MeHg exacerbated developmental neurobehavioral deficits (Fischer et al., 2006). As found in the present study, the effects were seen at low doses where the compounds singly did not induce changes in spontaneous behavior or habituation plus these effects also were found to be sustained. Epidemiological studies have shown a discrepancy with regard to neuropsychological deficits during early development, where the deficits were seen in children from the Faeroe Islands but not in children from the Seychelles (Davidson et al., 2006; Grandjean et al., 2001; Myers and Davidson, 1998). Both populations have a high consumption of MeHg contaminated fish. The difference is that in the Faeroe Islands the children were exposed to PCBs via the mother’s dietary consumption of whale meat and blubber as well as to MeHg. The calculated doses of PCB 153 and MeHg indicated that the exacerbated developmental neurotoxic effects caused by coexposure to PCB 153 and MeHg can explain the observed differences between children in the Faeroe Islands and children in the Seychelles. A recent report indicates that the levels of PBDEs are approaching those for PCBs (Johnson-Restrepo et al., 2005). It is worth noting that the dose for PBDE 99 used in the present study has the same molar concentration as the dose for PCB 153 in combination with MeHg. In conclusion, this study shows that neonatal coexposure to PBDE 99 and MeHg can exacerbate developmental neurotoxic effects, manifested as disrupted spontaneous behavior, reduced habituation capability, and impaired learning/memory. The study also shows that PBDE 99 and MeHg can interact in the low dose range and the effect is more than just additive. Furthermore, a significant effect of interaction is seen on the cholinergic nicotinic receptors of the hippocampus. 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