Download Coexposure of Neonatal Mice to a Flame

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
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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
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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. PBDE 99
can interact with MeHg in a similar manner as earlier observed
between PCB 153 þ MeHg and PCB 52 þ PBDE 99. This is
important as the levels of PBDEs are increasing in mother’s
milk and in the environment.
FUNDING
Financial support was provided by the Swedish Research
Council for Environmental, Agricultural Sciences and Spatial
Planning, and the Foundation for Strategic Environment
Research.
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