Department of Environmental Toxicology, Uppsala University, Norbyvägen 18A, SE-75236 Uppsala, Sweden
Received August 22, 2001; accepted January 8, 2002
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ABSTRACT |
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Key Words: behavior; cholinergic system; developmental neurotoxicity; flame retardant (PBDE); neonatal.
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INTRODUCTION |
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In recent studies, we have shown that neonatal exposure to certain PBDEs and PCBs during the period of rapid brain development (Davison and Dobbing, 1968) can cause similar persistent disturbances in spontaneous motor behavior and dysfunctions in learning and memory in adult animals (Eriksson, 1998
; Eriksson and Fredriksson, 1996a
,b
; Eriksson et al., 2001
). Earlier studies have shown that neonatal exposure of mice to PCBs affects the cholinergic transmitter system. Effects seen were alterations in nicotinic receptors and also an adult response to the cholinergic agent nicotine that was the opposite of animals neonatally exposed to the vehicle (Eriksson and Fredriksson, 1996b
). In view of these findings, the objective of this study was to determine whether recently reported changes in spontaneous behavior in adult mice neonatally exposed to PBDE 99 would include effects on the cholinergic system, and thereby would alter the response in the adult animal to the cholinergic agent nicotine.
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MATERIALS AND METHODS |
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Sixteen pregnant NMRI mice were obtained from B&K, Sollentuna, Sweden; they were housed individually in plastic cages in a room with an ambient temperature of 22°C and a 12/12-h light/dark cycle. The animals were supplied with standardized pellet food (Lactamin, Sweden) and tap water ad libitum. The pregnant NMRI mice were checked for births twice a day (0800 and 1800 h). The day of birth was assigned day 0, and pups born during the night were considered to have day 0 as the day they were found. The sizes of the litters were adjusted to 1012 mice within the first 48 h after birth, and excess pups were killed. The litters contained pups of both sexes during the neonatal period and no separation with regard to sex was made in the preweanling mice. At the age of 45 weeks, all females were sacrificed; males were kept in litters (in treatment groups) with their siblings and were placed and raised in groups of 47 in a room for male mice only, under the same conditions as detailed above. Only male mice were used.
Ten-day-old mice received, as a single oral dose, 8 mg 2,2`,4,4`,5-pentaBDE/kg body weight (bw) (14 µmol 2,2`,4,4`,5-pentaBDE/kg bw) via a metal gastric tube, and the mice serving as controls received vehicle in the same manner: 10 ml/kg bw of 20% fat emulsion only. Both the treatment group and the control group contained 34 litters, respectively.
The animals were weighed twice during the course of the experiment: first on the day of administration of PBDE 99 (day 10) and then at the time of spontaneous and nicotine-induced behavior testing (age 2 months). Additionally, during the course of the experiment, the animals were checked by visual examination once a day for clinical signs of toxic effects.
At the adult age of 2 months, the mice were subjected to spontaneous behavior testing. The animals were tested between 0800 and 1200 h under the same ambient light and temperature conditions as in their cages. Twelve mice were randomly picked from 34 different litters in the different treatment groups. Motor activity was measured for a 60-min period, divided into three 20-min periods, in an automated device consisting of 12 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) (Eriksson et al., 2001; Fredriksson, 1994
). Three variables were measured:
Directly after the spontaneous behavior test, nicotine-induced behavior was studied. The mice were picked up from the test cage and were directly given a single sc injection of 80 µg nicotine base/kg bw [nicotine-bi-(+)-tartrate (Sigma, St. Louis, MO)] or 10 ml 0.9% NaCl/kg bw, sc. This amount of nicotine is known to cause an increased activity in normal adult NMRI mice (Eriksson et al., 2000). Directly after the nicotine injection, the mice were replaced in the test chamber. The nicotine-induced behavior was measured during another 60-min period (60120 min, in the same way as was the spontaneous behavior) in which motor activity was recorded with regard to the three variables: locomotion, rearing, and total activity. The 60-min period was divided into three 20-min periods.
The statistical data concerning body weight were evaluated by a Student's t-test. The statistical data obtained from the behavioral tests were evaluated by ANOVA (analysis of variance) using a split-plot design. Pairwise testing between treated and control groups was performed using a Tukey HSD (honestly significant differences) test (p = 0.01) (Kirk, 1968).
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RESULTS |
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Shown in Figure 1 are the results from the spontaneous behavior variables, locomotion, rearing, and total activity in 2-month-old mice exposed to a single oral dose of 8.0 mg PBDE 99/kg bw and from controls receiving 10 ml/kg bw of the 20% fat emulsion vehicle, during the first 60-min period. There were significant group x period interactions [F(6,56) = 14.33; F(6,56) = 16.29; F(6,56) = 15.91] for the variables locomotion, rearing, and total activity, respectively, and pairwise testing between PBDE 99-treated and control animals showed a significant change in all three test variables. In the vehicle-treated mice, a decrease in activity over time was observed in response to the diminished novelty of the test chamber, but PBDE 99-treated animals displayed significantly less activity during the first 20-min period (020 min), compared with the controls, while during the last 20-min period (4060 min) the PBDE 99-treated animals had significantly increased activity compared to the controls.
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DISCUSSION |
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The mice treated neonatally with PBDE 99 showed the same nonhabituating behavior at the age of 2 months and 4 months as earlier reported (Eriksson et al., 2001). Habituation, here defined as a decrease in the three behavioral variables, locomotion, rearing, and total activity, in response to the diminished novelty of the test chamber over the 60-min period, was observed in the control animals. The PBDE 99-treated animals, on the other hand, were significantly hypoactive during the first 20 min of the 60-min period (020 min), but during the last 20-min period (4060 min) a hyperactive behavior was evident.
In the nicotine-induced behavior test, the response to nicotine was drastically changed and the PBDE 99-treated animals showed a totally opposite response compared to the vehicle-treated mice. In the control animals, hyperactivity was seen after injection of 80 µg nicotine base, while the neonatally treated mice showed hypoactivity. This response to nicotine, after neonatal exposure to PBDE 99, is the same as earlier seen for animals neonatally exposed to PCB 52 (Eriksson and Fredriksson, 1996b) or nicotine (Eriksson et al., 2000
; Nordberg et al., 1991
). These animals showed hypoactivity after adult exposure to nicotine, in contrast to animals neonatally exposed to 20% fat emulsion vehicle or saline that showed hyperactivity. In these animals it was also shown that the cholinergic nicotinic receptors were affected. The neonatal exposure on postnatal day 10 coincides with the rapid development of the cholinergic system (Falkeborn et al., 1983
; Fiedler et al., 1987
) and this indicates that PBDE 99 has an effect on the cholinergic system. Furthermore, it is of special interest to note that the hyperactive condition in PBDE 99-treated mice is again observed at the end of the test period (100120 min.), indicating the persistence of the adverse spontaneous motor behavior defect.
In conclusion, the present study shows that neonatal exposure to PBDE 99 affects the cholinergic system, seen as changes in the adult response to nicotine exposure. It also reproduces the effects on spontaneous behavior in the adult mouse after neonatal exposure to PBDE 99. These findings show similarities to observations made from neonatal exposure to PCBs and nicotine, compounds shown to affect cholinergic nicotinic receptors. This indicates that PBDE 99 can affect the cholinergic system and might thereby interact with other environmental toxicants.
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ACKNOWLEDGMENTS |
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NOTES |
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