ToxSci Advance Access originally published online on August 12, 2003
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Toxicological Sciences 76, 112-120 (2003)
Copyright © 2003 by the Society of Toxicology
NEUROTOXICOLOGY |
Neurobehavioral Derangements in Adult Mice Receiving Decabrominated Diphenyl Ether (PBDE 209) during a Defined Period of Neonatal Brain Development
* Department of Environmental Toxicology, Uppsala University, Norbyvägen, Uppsala, Sweden, and
Department of Environmental Chemistry, Stockholm University, Sweden
Received May 12, 2003; accepted July 28, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flame retardants are used to suppress or inhibit combustion processes in an effort to reduce the risk of fire. One class of flame retardants, polybrominated diphenyl ethers (PBDEs), has been found to be increasing in the environment and in human milk. Previous studies have shown that lower brominated PBDEs, tetra-, penta-, and hexabrominated diphenyl ethers, can cause developmental neurotoxic effects. The present study shows that the highly brominated PBDE 2,2',3,3',4,4',5,5',6,6'-decaBDE (PBDE 209) can be absorbed during neonatal life and induce developmental neurotoxic effects in adult mice, effects that also worsen with age. These effects seem to be inducible only during a defined critical period of neonatal life. Neonatal Naval Medical Research Institute (NMRI) male mice were exposed on day 3 to 2.22 or 20.1 mg PBDE 209/kg body weight, on day 10 to 1.34, 13.4, or 20.1 mg PBDE 209/kg body weight, or on day 19 to 2.22 or 20.1 mg PBDE 209/kg body weight, or to [U-14C]-2,2',3,3',4,4',5,5',6,6'-decaBDE. The oral neonatal administration of [U-14C]PBDE 209 on day 3, 10, or 19 showed that the compound distributes throughout the body and increases in the brain, from 24 h after administration to 7 days after administration, in 3-day-old and 10-day-old mice. The spontaneous behavior tests, observed in 2-, 4-, and 6-month-old mice, showed that the effect only occurred in mice exposed on day 3 and that this effect worsened with age. We conclude that more attention should be focused on the highly brominated PBDEs as possible developmental neurotoxic agents.
Key Words: decabrominated diphenyl ether; spontaneous behavior; neonatal; flame retardants; PBDEs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polybrominated diphenyl ethers (PBDEs) constitute a group of chemical substances used as additive flame retardants, meaning that PBDEs are mainly used to suppress or inhibit combustion processes in an effort to reduce the risk of fire, mainly in polymer products. The theoretical number of possible congeners is 209, and their chemical and physical characteristics are very similar to those of the polychlorinated biphenyls (PCBs; WHO, 1993
, 1994). Products that contain flame retardants are textiles, building materials, and electrical appliances such as computers and television sets (WHO, 1994). The most widely used congener of the PBDEs is 2,2',3,3',4,4',5,5',6,6'-decaBDE (PBDE 209; Hardy, 2000), with a worldwide use in 1999 of 54,800 metric tons. PBDEs are not fixed in the polymer product through chemical binding and can leak into the environment (Hutzinger and Thoma, 1987; Hutzinger et al., 1976).
Studies have shown that PBDEs are present in the global environment (de Boer et al., 1998; Johnson and Olson, 2001; Manchester-Neesvig et al., 2001; Strandberg et al., 2001) and that levels of PBDEs are increasing in the Swedish environment (Andersson and Blomkvist, 1981; Nylund et al., 1992; Sellström et al., 1993). Of the analyzed PBDEs, the most commonly found congeners in the environment are PBDE 47, PBDE 99, PBDE 100, and PBDE 153 (Darnerud et al., 2001). A recent report has shown the presence of PBDEs in Swedish human milk and also that the PBDEs have increased exponentially since 1972, whereas PCBs are steadily decreasing (Meironyté and Norén, 1999; Norén and Meironyté, 2000). Reports from Japan also indicate that PBDEs occur in human milk (Ohta et al., 2002). Analyses of various food sources (fish, vegetables, meat, and human milk) confirm the presence of PBDEs in them (Ohta et al., 2002). Recently, PBDE 209 has been found in chickens used for food (Huwe et al., 2002) and samples of human blood have also been shown to contain PBDEs and PBDE 209 (Klasson-Wehler et al., 1997; Sjodin et al., 2001). Occupational exposure to PBDEs has been demonstrated in workers in the electrical dismantling industry, who show increased levels of PBDEs in their blood (Sjodin et al., 1999). Furthermore, computer technicians show levels of PBDE 209 in their blood (Jakobsson et al., 2002).
During their development, mammals can be exposed to toxicants either while they are fetuses (via maternal intake of toxicants), during the neonatal period (via intake of human milk), or by direct ingestion or contact. We have earlier reported that exposure to low doses of persistent environmental agents (nondegradable), such as dichlorodiphenyltrichloroethane (DDT) and certain PCBs, and nonpersistent neurotoxic environmental agents, such as nicotine, pyrethroids, and organophosphates, during neonatal brain development in mouse can induce irreversible disruption in adult brain function observed as altered spontaneous behavior, learning and memory defects, and changes in cholinergic nicotinic and muscarinic receptors (Ankarberg et al., 2001; Eriksson, 1997, 1998; Eriksson et al., 1992, 2001a).
These disturbances in behavior and cholinergic transmitter systems have all been shown to be induced during the rapid growth period of the neonatal mouse brain. This period, called the brain growth spurt (BGS; Davison and Dobbing, 1968), is characterized by a series of rapid fundamental developmental changes, for example, maturation of dendritic and axonal outgrowth, the establishment of neural connections, and synaptogenesis and proliferation of glia cells with accompanying myelinization (Davison and Dobbing, 1968; Kolb and Whishaw, 1989). This is also when animals acquire many new motor and sensory abilities (Bolles and Woods, 1964) and when spontaneous motor behavior peaks (Campbell et al., 1969); additionally, the synthesis of brain lipids reaches its maximum at this time of development.
Studies have shown a pronounced retention of such lipophilic agents as DDT, PCBs, and PBDE 99 (Eriksson, 1984, 1998; Eriksson and Darnerud, 1985; Eriksson et al., 2002) in the neonatal mouse brain during the BGS. This stage of development is associated with numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult. The cholinergic transmitter system undergoes rapid development during BGS (Coyle and Yamamura, 1976; Fiedler et al., 1987), and many behavioral characteristics (Karzmar, 1975) and cognitive functions (Bartus et al., 1982; Drachman, 1977) are closely linked to the cholinergic transmitter system. In mammals, the period of BGS in terms of onset and duration varies from species to species. In rodents, rats and mice, the BGS is neonatal, spanning the first 3–4 weeks of life and reaching its peak around postnatal day 10. In the human, on the other hand, it begins during the third trimester of pregnancy and continues throughout the first 2 years of life.
In recent studies, we have shown that neonatal exposure to PBDE congeners 2,2',4,4',-tetraBDE (PBDE 47), 2,2',4,4',5-pentaBDE (PBDE 99), and 2,2',4,4',5,5'-hexaBDE (PBDE 153) (Eriksson et al., 2001b, 2002, 2002; Viberg et al., 2002, in press) can induce persistent dysfunction in adult mice, manifested as deranged spontaneous behavior, learning and memory defects, and derangements in the cholinergic system. These effects can also worsen with age (Eriksson et al., 2001b, 2002; Viberg et al., in press). We have also shown that the effects are inducible during a restricted period of neonatal life (Eriksson et al., 2002). Developmental neurotoxic effects by PBDEs has also been shown by Branchi et al. (2002).
With regard to our earlier observations of pronounced retention and developmental neurotoxic effects caused by DDT, PCBs, and PBDE 99, as well as the similarities among the PBDEs, DDT, and PCBs, the present study was undertaken to ascertain whether PBDE 209 could be absorbed during the neonatal brain development and induce persistent neurotoxic effects on the spontaneous motor behavior of the adult. To see whether the tissue distribution and/or a defined critical phase of brain development is the underlying cause of developmental effects, a study of the uptake and retention of 14C-labeled PBDE 209 at different stages of neonatal mouse brain development was conducted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals and Animals
Chemicals.
Both PBDE 209 and [U-14C]PBDE 209 [specific activity 17.5 mCi/mmol (650 MBq/mmol)] were dissolved in a mixture of egg lecithin (Merck, Darmstadt, Germany) and peanut oil (Oleum arachidis; 1:10 w/w) and then sonicated together with water to yield a 20% (w/w) fat emulsion vehicle containing, respectively, 0.134, 0.222, 1.34, or 2.01 mg PBDE 209/ml (0.14, 0.23, 1.4, or 2.1 µmol/ml) and 150 kBq [U-14C]PBDE/ml. The use of a 20% fat emulsion vehicle was to give a more physiologically appropriate absorption and, hence, distribution of the compound (Keller and Yeary, 1980
; Palin et al., 1982).
Animals and husbandry.
Pregnant Naval Medical Research Institute (NMRI) mice were obtained from B & K (Sollentuna, Sweden) and were housed individually in plastic cages in a room with an ambient temperature of 22°C and a 12/12 h cycle of light and dark. The animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. The pregnant NMRI mice were checked for births two times a day (at 0800 and 1800 h). The day of birth was assigned as day 0, and pups born during the night were considered to have day 0 the next day. The size of the litters was adjusted to 10–12 mice, within the first 48 h after birth, by the killing of excess pups. The litters contained pups of both sexes during the neonatal period. At the age of 4–5 weeks, the males were kept in their litters (in treatment groups) with their siblings and were placed and raised in groups of 4–7, in a room for male mice only, under the same conditions as detailed above.
Treatment of neonatal mice with PBDE 209 or [U-14C]PBDE 209.
In the behavioral study, 10-day-old mice were given 1.34, 13.4, or 20.1 mg PBDE 209/kg body weight (1.4, 14, or 21 µmol/kg body weight). These doses are the same, on a molar basis, as were used in studies of PBDE 99 and PBDE 153 previously (Eriksson et al., 2001b, 2002; Viberg et al., 2002, in press). Three-day-old and 19-day-old mice were given 2.22 or 20.1 mg PBDE 209/kg body weight (2.3 or 21 µmol/kg body weight). These doses were selected according to the dose used for [U-14C]PBDE 209 and the highest dose, on a molar basis, previously used in studies of PBDE 99 (Eriksson et al., 2001b, 2002; Viberg et al., 2002) and PBDE 153 (Viberg et al., in press). All doses were administered via a metal gastric tube, as a single oral dose, to 3-, 10-, and 19-day-old mice. Control mice, in each age category, were given 10 ml of the 20% fat emulsion vehicle/kg body weight. Each of the different treatment categories, in the different age categories, contained three to five litters.
In the retention study, 6–8 male offspring in each litter were given 1.5 MBq [U-14C]PBDE/kg body weight (40.5 µCi/kg body weight), as a single oral dose, via a metal gastric tube at the age of 3, 10, or 19 days. One male in each litter was used as a background control. Each age category contained two litters.
Spontaneous Behavior Tests
Spontaneous behavior was tested in the male mice at the ages of 2, 4, and 6 months, as in our previous studies regarding PBDEs (Eriksson et al., 2001b, 2002; Viberg et al., 2002, in press) and as described earlier (Ankarberg et al., 2001; Eriksson, 1997, 1998; Eriksson et al., 1992, 2001a). The animals were tested between 0800 and 1200 h under the same ambient light and temperature conditions as their housing conditions. A total of 10 mice were randomly picked from the three to five different litters in each treatment group. Motor activity was measured for a 60-min period, divided into three 20-min spells, in an automated device consisting of cages (40 x 25 x 15 cm) placed within two series of infrared beams (low and high level; Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden; Fredriksson, 1994).
Locomotion.
Counting took place when the mouse moved horizontally through the low-level grid of infrared beams.
Rearing.
Movement in the vertical plane was registered at a rate of 4 counts/s when a single high-level beam was interrupted, i.e., the number of counts obtained was proportional to time spent rearing.
Total activity.
All types of vibration within the cage, i.e., those caused by mouse movements, shaking (tremors), and grooming, were registered by a pick-up (mounted on a lever with a counterweight) connected to the test cage.
Radioactivity of [U-14C]PBDE Measured in the Neonatal Mouse Brain
Each of the two litters from the three different age categories was sacrificed by decapitation 24 h or 7 days, respectively, after administration. The skull was opened and the brain sectioned just behind the cerebellum. The brain samples were placed in preweighed vials, weighed, and individually solubilized in 1.5 ml Biolute tissue solubilizer (Zinsser Analytic GmbH, Frankfurt, Germany) at 48°C for 24 h (Eriksson, 1984). After solubilization, 18.5 ml scintillation liquid (Aquasafe 300+, Zinsser Analytic GmbH) was added to each vial. The heart samples were prepared in the same manner as the brain samples. To have the liver samples solubilized, the livers were divided into two samples; otherwise, the procedure was exactly the same as for the brain and heart samples. The samples were adapted overnight before being counted in a scintillation analyzer (Packard Tri-Carb 1900 CA). Counting efficiency was approximately 90% and quench correction was made by the external standard method. Counts/minute values not exceeding background + 3x background were excluded (Eriksson, 1984; Eriksson and Darnerud, 1985).
Statistical Analysis
Spontaneous behavior.
The data were subjected to a split-plot ANOVA (analysis of variance) and pairwise testing between treated groups; the control group was performed using a Tukey HSD (honestly significant difference) test (Kirk, 1968).
Habituation capability.
From the spontaneous behavior test, a ratio was calculated between the performance period 40–60 min and 0–20 min for the three different variables locomotion, rearing, and total activity in mice exposed neonatally to PBDE 209 on day 3. The following equations were used: 100 x (counts locomotion 40–60 min/counts locomotion 0–20 min), 100 x (counts rearing 40–60 min/counts rearing 0–20 min), and 100 x (counts total activity 40–60 min/counts total activity 0–20 min) (Eriksson et al., 2001b; Viberg et al., in press). Each ratio was used to analyze alteration in habituation between 2-month-old, 4-month-old, and 6-month-old mice. These data were subjected to a split-plot ANOVA.
Measurement of radioactivity of [U-14C]PBDE in neonatal brain.
The amount of radioactivity, as permillage (
) of total administered, found 24 h after administration was compared with that found 7 days after administration in the same age category, using Student’s t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There were no clinical signs of toxicity in the PBDE 209–treated mice at any given time during the experimental period, nor was there any significant difference in body weight gain or adult weight between the PBDE 209–treated and the vehicle-treated mice in the three different age categories.
The Effects on Spontaneous Behavior in Adult Mice
Adult mice exposed neonatally to the 20% fat emulsion vehicle.
Normal habituation, defined here as a decrease in the variables locomotion, rearing, and total activity, in response to the diminished novelty of the test chamber over a 60-min test period, divided into three 20-min periods, was seen in the control mice treated on neonatal day 3, 10, or 19 with the 20% fat emulsion vehicle. This normal habituation profile has been seen in a vast number of experiments conducted by the research group of Eriksson over the last 12 years (Eriksson, 1997, 1998; Eriksson et al., 1991, 2000, 2001b, 2002; Viberg et al., 2002, in press).
Adult mice exposed neonatally to PBDE 209 on postnatal day 3.
The results from the spontaneous behavioral variables locomotion, rearing, and total activity in 2-month-old, 4-month-old, and 6-month-old male mice, after exposure to a single oral dose of 2.22 mg or 20.1 mg PBDE 209/kg body weight (2.3 or 21 µmol/kg body weight) on postnatal day 3, are shown in Figures 1, 2, and 3, respectively.
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Two months after exposure to PBDE 209, on postnatal day 3, there were significant treatment x period interactions (F4, 54 = 13.72; F4, 54 = 13.39; F4, 54 = 15.00) for the locomotion, rearing, and total activity variables, respectively (Fig. 1
). Pairwise testing between PBDE 209 and control groups showed a significant dose-related change in all three test variables. In control mice, there was a distinct decrease in activity for all three behavioral variables over the 60-min period. Mice exposed, on neonatal day 3, to the highest dose of PBDE 209 (20.1 mg/kg body weight) displayed significantly less activity (p 
0.01), for all three behavioral variables, during the first 20-min period (0–20 min) compared with the controls, while during the third 20-min period (40–60 min) they were significantly more active than the control animals. These mice also showed significantly lower activity for rearing and total activity during the first 20 min period (0–20 min), and significantly higher activity, for all three variables, during the third period (40–60 min), compared with the mice receiving the lower dose of PBDE 209 (2.22 mg/kg body weight). Mice receiving the lower dose of PBDE 209 (2.22 mg/kg body weight) showed significantly lower activity (p 0.01) during the first 20-min period (0–20 min) for the total activity variable, compared with the controls.
Four months after exposure to PBDE 209, on postnatal day 3, there were significant treatment x period interactions (F4, 54 = 28.32; F4, 54 = 30.32; F4, 54 = 12.87) for the locomotion, rearing, and total activity variables, respectively (Fig. 2). Pairwise testing between PBDE 209 and control groups showed a similar significant dose-related change, as observed in 2-month-old mice, for all three test variables. However, mice receiving the lower dose of PBDE 209 (2.22 mg/kg body weight) did not show any significant differences in spontaneous behavior compared with the controls.
Six months after exposure to PBDE 209, on postnatal day 3, there were significant treatment x period interactions (F4, 54 = 40.29; F4, 54 = 35.69; F4, 54 = 20.68) for the locomotion, rearing, and total activity variables, respectively (Fig. 3). Pairwise testing between PBDE 209 and control groups showed a similar significant dose-related change, as observed in 2- and 4-month-old mice, for all three test variables. Additionally, during the middle period (20–40 min), mice treated with the highest dose of PBDE 209 showed significantly lower activity than the control animals for the rearing variable. During this period (20–40 min), mice treated with the highest dose of PBDE 209 showed significantly higher activity for the locomotion variable and lower activity for the rearing variable than the animals treated with the lower dose of PBDE 209 (2.22 mg/kg body weight). Mice receiving the lower dose of PBDE 209 (2.22 mg/kg body weight) showed significantly lower activity during the first 20-min period (0–20 min) for the rearing variable, compared with the controls.
Adult mice exposed neonatally to PBDE 209 on postnatal day 10.
In adult mice exposed neonatally to PBDE 209 on postnatal day 10, there were no significant treatment x period interactions for the locomotion, rearing, and total activity variables, respectively, after 2 months (F6, 72 = 0.61; F6, 72 = 0.34; F6, 72 = 1.24), 4 months (F6, 72 = 0.20; F6, 72 = 1.00; F6, 72 = 0.75), or 6 months (F6, 72 = 0.64; F6, 72 = 2.12; F6, 72 = 1.43); for locomotion, see Figure 4.
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Adult mice exposed neonatally to PBDE 209 on postnatal day 19.
In adult mice exposed neonatally to PBDE 209 on postnatal day 19, there were no significant treatment x period interactions for the locomotion, rearing, and total activity variables, respectively, after 2 months (F4, 54 = 0.30; F4, 54 = 0.15; F4, 54 = 0.07), 4 months (F4, 54 = 0.05; F4, 54 = 0.26; F4, 54 = 0.23), or 6 months (F4, 54 = 0.59; F4, 54 = 0.50; F4, 54 = 0.54); for locomotion, see Figure 4
.
The Effects on Habituation Capability in Adult Mice
By analyzing the habituation ratio between activity during the last and first (40–60 and 0–20 min) periods in the spontaneous behavior testing, information concerning the capability to habituate to a novel environment can be obtained. This information is used to analyze changes in habituation over time. The results of the habituation ratio, calculated from the spontaneous behavior variables (locomotion, rearing, and total activity) in 2-, 4-, and 6-month-old NMRI male mice, exposed on postnatal day 3 to PBDE 209, are presented in Figure 5. The habituation capability concerning the locomotion, rearing, and total activity variables was shown to significantly decrease (p 0.01) with age in mice exposed neonatally to 20.1 mg PBDE 209/kg body weight (21 µmol/kg body weight). Mice exposed on postnatal day 3 to 2.22 mg PBDE 209/kg body weight (2.3 µmol/kg body weight) did not show a significant decrease in habituation capability with age.
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For controls, only one variable, total activity, showed significant decrease in the ratio of activity at 40–60 min over activity at 0–20 min.
Retention of [U-14C]PBDE in Mouse Brain After 24 h and 7 Days Following an Oral Exposure on Day 3, 10, or 19
The amounts of radioactivity found in the brain, heart, and liver 24 h and 7 days after administration, given as permillage () of total administered, are shown in Figure 6.
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Brain.
In animals exposed to [U-14C]PBDE on postnatal day 3 or 10, the amounts of radioactivity in the brain were 4.8 or 4.0
, respectively, 24 h after administration; 7 days after administration, radioactivity was still present in the brain and had increased significantly (p 0.01) to 7.4 or 10.5, respectively. In contrast, in animals exposed to [U-14C]PBDE on postnatal day 19, the amount of radioactivity was 0.6 at 24 h after administration, and 7 days after administration no difference in the amount of radioactivity was seen in the brain.
Heart.
In animals exposed to [U-14C]PBDE on postnatal day 3 or 10, the amounts of radioactivity in the heart were 2.8 or 3.1, respectively, 24 h after administration; 7 days after administration, radioactivity was still present in the heart but no significant increase or decrease was seen (3.4. or 3.2 of total administered for 3-day-old or 10-day-old, respectively). In contrast, in animals exposed to [U-14C]PBDE on postnatal day 19, the amount of radioactivity was 2.2 at 24 h after administration, and 7 days after administration the amount of radioactivity had decreased significantly (p 0.01) to 0.8.
Liver.
In animals exposed to [U-14C]PBDE on postnatal day 3, 10, or 19, the amounts of radioactivity in the liver were 125.6, 94.1, or 57.8, respectively at 24 h after administration; 7 days after administration, radioactivity was still present in the liver but had decreased significantly (p 0.01) to 47.7, 46.3, and 3.12, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, the administration of PBDE 209 (20.1 mg/kg body weight) to mice on neonatal day 3, 10, or 19 only caused disturbances in the spontaneous motor behavior of the adult mice neonatally exposed on day 3. The effects on spontaneous motor behavior also seem to worsen with age when analyzing and comparing the habituation capability in 2-, 4-, and 6-month-old mice. Furthermore, neonatal exposure to [U-14C]PBDE in mice on day 3, 10, or 19 showed that the substance is absorbed and distributed in the body, and that the [U-14C]PBDE or its metabolites is/are present in the brain, heart, and liver 24 h after exposure. [U-14C]PBDE or its metabolites can still be detected in the body 7 days after administration in all three age categories. Of special concern is the fact that there is an increase over the 7-day period of radioactivity in the brain in animals exposed to [U-14C]PBDE 209 on postnatal day 3 or 10. The present results also indicate that there is a presence of [U-14C]PBDE 209 or its metabolites during a critical phase of neonatal brain development, which is responsible for the induction of neurobehavioral disturbances.
The spontaneous motor behavior data showed a disruption of habituation in adult mice neonatally exposed to the highest dose of PBDE 209 (20.1 mg/kg body weight) on day 3. This altered behavior profile has also appeared after neonatal exposure to lower brominated flame retardants such as PBDE 47, 99, and 153 (Eriksson et al., 2001b; Eriksson et al., 2002; Viberg et al., 2002, in press). In these studies, the lowest investigated dose to give an effect on spontaneous behavior was 10.5 mg PBDE 47/kg body weight (21 µmol/kg), 0.8 mg PBDE 99/kg body weight (1.4, µmol/kg), or 0.45 mg PBDE 153/kg body weight (0.7 µmol/kg).
The disrupted habituation observed after 2 months is also seen in mice at 4 and 6 months of age after neonatal exposure to the highest dose of PBDE 209 on neonatal day 3. This means that the neurotoxic effect of neonatal PBDE 209 exposure is persistent. In fact, when comparing the habituation capability between the spontaneous behavior testing at 2, 4, and 6 months of age, it is evident that the disruption of habituation worsens with age. This form of developmental neurotoxic effect has also been seen for PBDE 99, PBDE 153, and certain PCBs (Eriksson, 1998; Eriksson et al., 2001b; Viberg et al., in press).
In recent studies, we have seen that PBDE 99 and certain PCBs (Eriksson, 1998; Eriksson et al., 2002) elicit this kind of altered behavior when administered on postnatal day 10. It is also true that effects from these substances can be seen after exposure on postnatal day 3, but the effect is proposed to be due to the presence of the molecules in the brain during the peak of BGS, namely around day 10 (Eriksson, 1998; Eriksson et al., 1992, 2000).
The amount of toxic agent present in the brain at different neonatal ages may vary. Mice exposed to [U-14C]PBDE 209 on postnatal day 3 or 10 show approximately the same amount of radioactivity after 24 h: 4.8 and 4.0, respectively. This is about the same amount of radioactivity as has been seen earlier for the lower brominated PBDE, PBDE 99 (Eriksson et al., 2002). In our earlier studies, we have shown a pronounced retention of other lipophilic chlorinated hydrocarbons or their metabolites (DDT, PCB 52, PCB 153, and chlorinated paraffins) in the brain when administered on neonatal day 10 (Eriksson, 1984, 1998; Eriksson and Darnerud, 1985; Eriksson et al., 2002). During the course of time, the amount of radioactivity from these substances was the same or decreased over the 7-day period. In contrast, the radioactivity from [U-14C]PBDE 209 significantly increased, to 7.4 or 10.5, in animals exposed to [U-14C]PBDE 209 on postnatal day 3 or 10. For the other two organs analyzed, heart and liver, the radioactivity was the same or decreased during the 7-day period. This decrease has also been seen for DDT and certain PCBs (Eriksson, 1998; Eriksson and Darnerud, 1985).
There are only a few studies known to deal with the kinetics of PBDE 209. In fish, the dietary uptake of [U-14C]PBDE 209 is low. No measurements of fish brain tissue have been presented, but detectable amounts are found in liver, adipose tissue, and muscle (Kierkegaard et al., 1999). On the other hand, hexa- to nonabromodiphenyl ethers were overly represented, which was explained by possible debromination of the PBDE 209, to less brominated forms of PBDEs. The hypothesis concerning debromination of PBDE 209 has also been put forward in studies in mice (Sandholm, 2003). In another study, adult rats were fed with food containing [U-14C]PBDE 209. The uptake was low but still detectable (el Dareer et al., 1987). In that study, radioactivity was seen in a variety of organs and tissues, including the brain. In an early study by Norris and coworkers (Norris et al., 1975), [U-14C]PBDE 209 was taken up in adult rat, but most of it (about 91%) was found in the feces within 24 h and the rest within the next 24 h.
The two reference tissues we evaluated for radioactivity from [U-14C]PBDE 209, administered on postnatal day 3, 10, or 19, were liver and heart. The mean radioactivity from the liver 24 h after oral administration on day 3, 10, or 19 was 126, 94, and 58 of the total radioactivity administered, respectively. Over the 7-day period, there was a significant decrease of the radioactivity in the liver to 58, 46, and 3.1 for animals administered on day 3, 10, or 19, respectively. The mean radioactivity from the heart 24 h after oral administration on day 3, 10, or 19 was 2.8, 3.1, or 2.2 of the total radioactivity administered, respectively. Over the 7-day period, there was a significant decrease of radioactivity in the liver to 3.4, 3.2, and 0.8% for animals administered on day 3, 10, or 19, respectively. It is of special interest that even though an increase in the amount of radioactivity was seen in the brain during the 7-day period, the mean radioactivity from the liver and the heart tissues decreased or was unchanged after oral administration on day 3, 10, or 19. This and the fact that effects on spontaneous behavior were only seen in mice exposed neonatally on day 3 suggest that the effect is caused by one or more metabolites of the parent compound PBDE 209. For example, it has been shown for PCBs that the neonatal mouse is capable of metabolizing persistent organic compounds (Vodicnik et al., 1980). If the parent compound caused the neurotoxic effect, mice exposed to PBDE 209 on day 10 should have shown these neurotoxic effects because of the amount of radioactivity present 24 h after administration on day 10. For animals exposed to 20.1 mg PBDE 209/kg body weight on postnatal day 10, the calculated amount in the brain after 24 h would be 419 pmol, and for animals exposed to 20.1 mg PBDE 209/kg body weight on day 3, the calculated amount in the brain after 7 days would be 388 pmol, which is approximately the same amount. This is also the case when comparing these two groups according to radioactivity, as in Table 1. Therefore, the amount present around postnatal day 10, during the peak of BGS, is enough to induce persistent developmental neurotoxic effects.
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In conclusion, this study has shown that exposure in the mouse to PBDE 209 during a defined critical phase of neonatal brain development can give rise to irreversible changes in adult brain function. These changes are also seen to worsen with age, as seen previously for PBDE 99 and PBDE 153 (Eriksson et al., 2001b
; Viberg et al., in press). Additionally, it is shown that PBDE 209 can be taken up in the neonatal mouse brain and that the amount of radioactivity after neonatal exposure to [U-14C]PBDE actually increases in the brain during the first week after administration. The presence of radioactivity from the 14C-labeled PBDE 209 in the neonatal brain indicates that further studies are needed to evaluate whether it is the parent compound or debrominated metabolites, or other metabolites, that are present in the brain after neonatal exposure.
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ACKNOWLEDGMENTS |
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The authors would like to thank Ms. Anna Petterson for her excellent technical assistance. This work was supported by grants from the Foundation for Strategic Environmental Research.
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NOTES |
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1 To whom correspondence should be addressed at Department of Environmental Toxicology, Uppsala University, Norbyvägen 18A, S-752 36, Uppsala, Sweden. Fax: +46 18 518843. E-mail: henrik.viberg{at}ebc.uu.se.
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REFERENCES |
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