Toxicology, 74 (1992) 151-160 Elsevier Scientific Publishers Ireland Ltd.
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Behavioural effects of neonatal metallic mercury exposure in rats A. F r e d r i k s s o n a, L. D a h l g r e n b, B. D a n i e l s s o n a, P. Eriksson c, L. D e n c k e r a a n d T. Archer b aDepartment of Toxicology, Uppsala University, Box 594, S-75124 Uppsala, bDepartment of Psychology, University of Gothenburg, Box 14158, S-400 20 Gothenburg and CDepartment of Zoophysiology, Uppsala University, Box 560, S-75122 Uppsala (Sweden) (Received December 1 lth, 1991; accepted June 9th, 1992)
Summary The effect of neonatal exposure of rats to mercury vapour (Hg°), at the concentration 0.05 mg/m 3, 1 h (low dose) or 4 h (high dose), on the behaviour in adulthood were studied. Exposure occurred on days 11-17 (the period of rapid brain growth). Tests for spontaneous motor activity were performed at the ages of 2 and 4 months. Rats exposed to the high dose Hg ° showed a marked increase in variables locomotion and total activity but a decrease for rearing when tested at 2 months of age. At 4 months of age these rats showed a marked hypoactivity with respect to all three variables. Rats exposed to the low dose showed no significant differences at 2 months compared to controls. However, at the age of 4 months the same pattern (increase in variables locomotion and total activity but a decrease for rearing) already noticed in the high dose group at 2 months was observed. In the spatial learning tasks applied, the radial arm maze and circular swim maze, neonatally exposed pups showed a retarded acquisition to the former, while there was no difference compared to controls in the latter. These data indicate that neonatal exposure to mercury vapour results in similar behaviour changes as reported from offspring prenatally exposed to mercury vapour or methylmercury. Furthermore, exposure for 1 week to concentrations around Swedish threshold values (TLV) for 1 or 4 h resulted in dose and age-related behavioural changes.
Key words: Mercury; Neuro behaviour; Neuro development; Behavioural teratology
Introduction Exposure to inorganic mercury may originate from occupational work [1-3], setting, removing and polishing amalgam restorations [4,5] or else in the environment. While inorganic mercury in the ionic form does not enter the brain in high concentrations [6,7], the more lipid soluble metallic mercury passes the blood brain barrier rapidly. Metallic mercury is oxidized by catalase to the inorganic form followed by its binding to intracellular proteins [8-11]. Metallic mercury, like Correspondence to. M.K. Anders Fredriksson, Department of Toxicology, Uppsala University, Box 594, S-75124 Uppsala, Sweden. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
152 methylmercury is an established neurotoxicant. The sensitivity of the developing central nervous system to metallic mercury has not been studied in detail and is thus largely unknown. In recently reported experiments [12-15] we have observed that neonatal exposure to environmental toxic agents such as DDT, PCBs and pyrethroids, potently induces permanent aberrations in spontaneous behaviour and cholinergic system in CNS of the adult animal. The changes were induced even with low doses to neonatal mice provided that the toxic agent was given during the period of rapid development of the brain [16], 'the brain growth spurt' [17]. This species-related period of development (around partus in humans and 10 days after birth in rats) is characterized by the maturation of axonal and dendritic outgrowth and establishment of neural connections, synaptogenesis. This stage of development is also associated with a large number of biochemical changes that will transform the feto-neonatal brain into that of the mature adult. The development of the dopaminergic system in the striatum [18] belongs to these biochemical changes. During this period an animal acquires new motor and sensory qualities [19] and the spontaneous motor behaviour peaks [20]. The present study was undertaken to investigate whether neonatal rats exposed to low levels of mercury (Hg °) vapour during 'the brain growth spurt' would alter physical development or spontaneous behaviour and learning capacity of the adult. Method
Animals Thirty pregnant Sprague-Dawley rats, - 3 months old, were purchased from ALAB, Sollentuna, Sweden. They were housed in plastic cages in a room with a 12-h light-dark cycle, thermostatically maintained at 22 ± I°C. Rats were allowed free access to food (R3, Ewos, S6dertfilje, Sweden) and tap water. The day of birth was designated day 0.
Study design Three treatment groups, 8 litters per group normalized to 8 individuals on Day 3, were exposed to metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) or air (2 h in a chamber without Hg vapour in control group). Daily exposure occurred in the chambers from Day 11 to Day 17 of age. Body weights and general appearance were monitored during the pre-weaning period. On Day 23 the pups were separated from their mothers and placed in plastic cages (6 males in each cage) according to treatment group. On Day 25, 6 pups from each group were sacrificed for quantitative analysis of Hg in brain, liver and kidney. All treatment groups were tested for spontaneous motor activity, learning in the radial arm maze and in a circular swim maze.
Test procedures and apparatus Hg exposure and analysis Hg ° vapour was generated into the exposure chamber by blowing a stream of
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nitrogen over a surface of metallic Hg. Air was aspirated through the chamber with a vacuum pump, resulting in a pressure slightly below atmospheric. Air samples were taken every 10 rain with a gas-tight syringe and bubbled into an acidified KMnO4 solution for Hg analysis. Flameless atomic absorption spectrophotometry was used to quantify the content of Hg in the samples (detection limit 0.1 ng Hg/sample). To permit analysis organic samples were digested under pressure in metallic bombs (MeAna - - Konsult, S-740 22 B/ilinge, Sweden) under the influence of acid and heat (2 ml conc. HNO3, 1 h at 175°C).
Spontaneous motor activity An automated device, consisting of cages (40 x 25 x 15 cm) placed within two series of IR beams (high and low level), was used to measure spontaneous motor behaviour (Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden). Each rat was alone in its cage. In each group 10 animals were tested. The following variables were measured: Locomotion. Registered by the low level grid of invisible IR beams. Counts occurred only when the rat moved horizontally, showing predominantly locomotion behaviour. Rearing. Registered by the high level IR beams when the rat raised their front legs and/or rested on their haunches. Counts (at a rate of 4 per s) occurred as long as a single high level beam remained interrupted, i.e., the number of rearing counts was proportional to the amount of time the animal reared and interrupted the beam. Total activity. Registered by a pick-up, mounted on a lever with a counter-weight, resting on the wall of the cage. The pick-up registered all types of vibrations within the test cage, e.g., particular rat movements (tremors, headshakes or grooming behavior) as well as rearing and locomotion.
Radial arm maze The radial arm maze test, a procedure sensitive to deficits in learning performance [21,22], was adapted both to evaluate spontaneous motor activity and cognitive function [231. The maze had 8 arms (each 54 cm long, 10 cm wide and 25 cm high) which extended radially from a central hub, 25 × 25 cm. At the extremity of each arm an extra, removable, back wall concealed food cups. For the activity testing the arms were marked into 3 units each, giving a total of 25 units. Ambulation and rearing were measured for 10 min by direct observation. Ambulation is defined as the passage of a rat's body from one unit to another and rearing was scored each time a rat raised itself onto its hindlegs. The maze was carefully cleaned after each test. After the activity testing, rats were placed on total food deprivation for 48 h. For the learning tests, the back walls were removed, exposing the 8 food cups (one at the distal end of each arm) into which a food pellet was placed. At the start of each test the rat was placed in the central hub and then monitored for its performance, i.e., the latency until all pellets were collected and the number of arms visited during the session. Immediately after the second test the rats were given free access to food. Testing was performed when the offspring were about 6 months old.
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Swim maze The swim maze was a circular bath (diameter 140 cm) thermostatically controlled to 25°C. The bath included a platform, placed at a constant position, 1 cm below the water level. Before testing, all rats were given swim training, for which the platform was removed and the animals allowed to swim for 60 s. After the platform had been placed at a set position in the pool, each animal had one test session on each of 3 consecutive days. On each test session, five trials were presented to the rat. For all trials, the rat was placed at the same point in the pool and allowed to swim around to find the submerged platform and escape from the water. On reaching the platform the rat was allowed to remain upon it for 30 s before being placed in the water again for the next trial. If a rat failed to locate the platform within 65 s it was placed on the platform for its 30-s orientation period. Statistical analysis Locomotion, rearing and total activity measures of spontaneous motor activity over three consecutive 20-rain periods at 2 and 4 months of age were submitted to a split-plot A N O V A [24]. The variables from the radial arm maze (latency and error data) were all subjected to one-way A N O V A analyses based on a completely randomized design. The latency data in the water maze was also subjected to split-plot ANOVA. Pairwise testing was performed using the Tukey HSD test. The results from the analysis of mercury were subjected to one-way A N O V A analyses based on a completely randomized design.
Results Chamber concentrations o f Hg ° To evaluate the conditions during the exposure samples were drawn every l0 rain and the concentration of Hg ° was estimated. Figure 1 shows the mean concentration of Hg ° + SD during the exposure for the low Hg ° dose group.
7503 tO
45 E 30.
o 0
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0 0
10
20
30
40
50
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155 TABLE I THE AMOUNT OF Hg IN THE BRAIN, LIVER AND KIDNEY OF 25-DAY-OLD RATS EXPOSED TO Hg ° BETWEEN DAYS 11 AND 17a OF AGE
Control Hg-low Hg-high
Brain
Liver
Kidney
0.002 0.017 b 0.063 b
0.002 0.084 b 0.219 b
0.002 1.247b 6.734 b
aThe rats were subjected to daily exposure of metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days l I to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg vapour. The amount of Hg is expressed as mg/kg organ. blndicates significant differences between means (P < 0.01) versus control and the other dose group. One-way ANOVA based on a completely randomized design [24] was significant for all three organs (F(2,15) > 90.30). Pairwise testing using Tukey HSD tests [24] showed a dose-dependent increase of the amount of Hg °.
Analysis of Hg All three g r o u p s were a n a l y z e d for contents o f H g in animals s a c r i f i c e d on D a y 25. Thus, o n e - w a y A N O V A b a s e d on a c o m p l e t e l y r a n d o m i z e d design was significant for all three o r g a n s (F(2,15) >_ 90.30, for brain, liver a n d kidney). Pairwise testing using T u k e y H S D tests showed a d o s e - d e p e n d e n t increase o f the a m o u n t o f H g (Table I).
Clinical observations of the litters N o changes in b o d y weight o f the pups were seen as a result o f the H g exposure, n o r were any clinical signs o f d y s f u n c t i o n o b s e r v e d in any o f the groups.
Spontaneous motor activity H g ° - t r e a t e d rats s h o w e d m a r k e d changes in the different variables o f m o t o r activity that were to some extent d e p e n d e n t u p o n the age at which they were rested (Fig. 2). Two-month-old rats. R a t s e x p o s e d to the high H g dose showed increases in the l o c o m o t i o n a n d t o t a l activity variables but a decrease in rearing b e h a v i o u r at the age o f 2 m o n t h s . There were significant G r o u p s x Test Periods interaction (F(4,42) = 8.49, F(4,42) = 3.79, F(4,42) = 5.90) for l o c o m o t i o n , rearing and total activity, respectively. These rats h a d significantly ( P _ 0.01) m o r e counts at the 20-, 40- a n d 60-rain p e r i o d s for l o c o m o t i o n a n d total activity, whereas rearing counts were significantly ( P _ 0.05) decreased at the 20- a n d 40-rain periods, c o m p a r e d with controls. A t the age o f 2 m o n t h s no significant changes in m o t o r activity were observed in rats e x p o s e d to the low H g dose. Four-month-old rats. R a t s exposed to the high H g dose showed decreases in all three variables o f activity at the age o f 4 m o n t h s , whereas rats exposed to the low H g dose showed increases in the l o c o m o t i o n a n d total activity variables a n d
156
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20-40 TIME (rain)
40-60
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20 - 40
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Fig. 2. Spontaneous motor activity in 2- and 4-month-old rats exposed to Hg ° as neonates. The rats were subjected to daily exposure of Hg ° vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Day 11-17 of age. Control rats were exposed to air for 2 h in a chamber without Hg ° vapour. Each treatment group comprised of 10 rats from at least 3 different litters. Regarding measurement of motor activity, see methods section. Statistical analysis of behavioural data was performed using ANOVA with a split-plot design [24]. There were significant Groups x Periods interaction (F(4,42)= 11.0, F(4,42) = 16.96, F(4,42) = 20.73) for the locomotion, rearing and total activity variables, respectively. Pairwise testing between Hg-low, Hg-high and control groups was performed with the Tukey HSD test [24]. The statistical difference from the control is indicated by *P -< 0.05; **P _< 0.01. rl, Control; k~ Hglow; I , Hg-high.
decreases in the r e a r i n g v a r i a b l e . T h e r e were s i g n i f i c a n t G r o u p s x Test P e r i o d s i n t e r a c t i o n (F(4,42) = l l . 0 , F(4,42) = 16.96, F(4,42) --- 20.73) for l o c o m o t i o n , reari n g a n d total activity, respectively. R a t s c o n s t i t u t i n g the high H g dose g r o u p h a d significantly ( P _< 0.05, P _< 0.01) fewer c o u n t s at the first 20-rain p e r i o d for the v a r i a b l e s l o c o m o t i o n , r e a r i n g a n d total activity a n d also at the 40-rain p e r i o d for the r e a r i n g a n d total activity v a r i a b l e , c o m p a r e d with c o n t r o l s . R a t s c o n s t i t u t i n g the low H g dose g r o u p s h o w e d s i g n i f i c a n t l y ( P _< 0.05, P _< 0.01) m o r e c o u n t s for the
157
600
450
NEONATAL, Hg ° ** xx xx
124 -18 m ,m
>-
300-
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-12 XX
150.
-9 NN XN
Fig. 3. Radial arm maze performance in 6-month-old rats exposed to Hg ° as neonates. The rats were subjected to daily exposure of Hg ° vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days 11 to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg ° vapour. Each treatment group comprised of 8 rats from at least 3 different litters. Regarding measurement of radial arm maze performance, see methods section. Statistical analysis of behavioural data was performed using one-way ANOVA based on a completely randomized design [24]. There were significant changes for both latency to acquire all 8 pellets (F(2,24)= 7.13) and number of errors made in acquiring all 8 pellets (F(2,24) = 3.68). Pairwise testing between Hg-low, Hg-high and control groups was performed with the Tukey HSD test [24]. The statistical difference from the control is indicated by *P-< 0.05; **P _< 0.01. n, Control; ~1, Hg-low; •, Hg-high.
locomotion and total activity variables and fewer counts for rearing variable at the 20- and 40-min intervals, compared with controls. Radial arm maze
Rats treated neonatally with Hg ° showed at 6 months of age dose-related impairment in radial arm maze learning. Thus, one-way ANOVA based on a completely TABLE II SWIM MAZE L E A R N I N G IN 5-MONTH-OLD RATS EXPOSED TO Hg ° AS NEONATES a Day
Control Hg-low Hg-high
Latency to find platform (s) 1
2
3
44.2 45.7 39.4
26.6 25.9 23.5
20.2 18.8 24.6
aThe rats were subjected to daily exposure of metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days 11 to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg vapour. The result is expressed in seconds in latency to find platform. Split-plot ANOVA [24] showed neither a Groups nor a Groups x Days interaction effect. Significant days effect (F(2,52) = 32.3) was observed indicating that all three groups learned the task of locating the platform within the circular pool from day to day.
158 randomized design was significant for both latency to acquire all 8 pellets (F(2,24) = 7.13, P < 0.01) and number of errors made in acquiring all 8 pellets (F(2,24) = 3.68, P _ 0.05). Pairwise testing using Tukey HSD tests indicated that the high Hg dose group showed significantly longer latencies (q = 5.33, P _ 0.01) and more errors (q = 4.28, P _< 0.05) than the control group. Figure 3 shows the performance of control and Hg-exposed groups in the radial arm maze. The low Hg dose group showed higher latencies and more errors than the control group but did not reach significantly different values. Swim maze learning
Control and Hg-exposed rats were tested for swim maze learning performance at 5 months of age. No difference among the three groups was obtained. Thus, splitplot ANOVA showed neither a Groups (F(4,52) < 0.06) nor a Groups x Days interaction effect (F(4,52) ___ 0.89), but only a Days effect (F(2,52) = 32.3) indicating that all three groups learned the task of locating the platform within the circular pool. Table II presents the latencies to reach the platform by the control and Hgexposed groups. Discussion Neonatal exposure of rat pups to metallic mercury, during Day 11-17 of age, 'the period of brain growth spurt' caused behavioural changes in these animals when they were tested at adult ages. The results may be summarized as follows: (i) There was a dose-dependent concentration of mercury in the organs of animals sacrificed 1 week after exposure to Hg °. For example, the brain concentration of Hg in the high dose group was about 4 times higher than in the low dose group; 63/~g/kg and 17 #g/kg, respectively. (ii) Rats exposed to the high dose Hg ° showed a marked increase in locomotion and total activity but a decrease for rearing when tested at 2 months of age. At 4 months of age these rats showed a marked hypoactivity for all three variables. Rats exposed to the low dose showed no difference at 2 months compared to controls. However, at the age of 4 months the same pattern (increase in variables locomotion and total activity but a decrease for rearing) already noticed in the high dose group at 2 months was observed. (iii) Spatial learning ability was measured in the radial arm maze and a dose-related retardation of the Hg°-exposed offspring was observed when tested at 6 months of age. (iv) Spatial navigation was studied in a Morris-type circular swim maze at 5 months of age. No significant difference was obtained. The exposure concentration used (0.05 mg/m 3) corresponds to levels of mercury in the air found in chloralkali plants (0.025-0.05 mg/m 3) [15]. Furthermore, the exposure periods were relatively short (1 or 4 h/day) resulting in a quite low exposure level even of the high dose group compared to previously reported exposure levels required to cause hypoactivity after oral dosing of animals prenatally with MeHg (2-6 mg/kg body weight) [25] or prenatal exposure for mercury vapour (1.8 mg/m 3, 3 h/day) [26]. The present study showed that both doses of Hg ° induced the same pattern of disturbed spontaneous motor activity in the adult animal. Of special interest is the
159
fact that in rats exposed to the high dose the disturbed motor activity appeared already in the test at 2 months. This finding corroborates our previous report on the relation between neonatal exposure and behaviour changes in the animals when reaching adult age [13]. In this study, no behavioural deviation was seen 1 week after exposure but developed with advancing age to a permanent state. Our results demonstrate that Hg present in CNS during the critical period of brain development generates changes in the adult brain function. The influence of Hg on the brain may lead to dysfunctions contributing to an earlier onset of aging and/or degenerative processes. Although the mechanism is far from clear, it may be mentioned that the implication on the association between body burden mercury and idiopathic Parkinson's disease [27] as well as regional CNS elevation of mercury in Alzheimer's disease [28] have been stated. The role of mercury, if any, in these age-related diseases has not yet been elucidated. Mercury may, together with other toxic agents in the environment contribute to a more rapid aging process and to cell death in CNS.
Acknowledgement We are grateful to E. Wallin for technical assistance. This work was financially supported by grants from the Swedish Medical Research Council, the Swedish Work Environment Fund, the Swedish Environmental Protection Board and the Swedish Council for the Humanities and Social Sciences.
References 1
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160 deltamethrin, on immature and adult mice: changes in behavioral and muscarinic receptor variables. Toxicol. Appl. Pharmacol, 15 (1991) 78. 14 P. Eriksson, U. Lundkvist and A. Fredriksson, Neonatal exposure to 3,3',4,4'-tetrachlorobiphenyl. Changes in cholinergic muscarinic receptor and behaviour variables. Toxicology, 69 (1991) 27. 15 P. Eriksson, L. Nilsson-H~kansson, A. Nordberg, A. Aspberg and A. Fredriksson, Neonatal exposure to DDT and its fatty acid conjugate - - Effects on cholinergic and behavioural variables in the adult mouse. Neurotoxicology, 11 (1990) 345. 16 P. Eriksson, J. Ahlbom and A. Fredriksson, Exposure to DDT during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice. Brain Res., 582 (1992) 277. 17 A.N. Davison and J. Dobbing, Applied Neurochemistry, Blackwelk Oxford, 1968, pp. 178-221, 253-316. 18 O. Giorgi, G. De Monteys, M.L. Porceddu, S. Mele, G. Calderini and G. Toffano, Developmental and age-related changes in Dl-dopamine receptors and dopamine content in the rat striatum. Dev. Brain Res., 35 (1987) 283. 19 R.G. Bolles and P.J. Woods, The ontogeny of behaviour in the albino rat. Anim. Behav., 12 (1964) 427. 20 B.A. Campbell, L.D. Lytle and H.C. Fibiger, Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science, 166 (1969) 635. 21 D.S. Olton, J.T. Becker and G.E. Handelmann, Hippocampus, space and memory. Behav. Brain Sci., 2 (1979) 313. 22 D.S. Olton and M.A. Werz, Hippocampal function and behaviour: Spatial discrimination and response inhibition. Physiol. Behav., 20 (1978) 597. 23 T. Archer, A.J. Hiltunen, T.U. Jarbe, M.R. Kamkar, J. Luthman, E. Sundstrom and A. Teiling, Hyperactivity and instrumental learning deficits in methylazoxymethanol-treated rat offspring. Neurotoxicol. Teratol., 10 (1988) 341. 24 R.E. Kirk, Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole, Belmont, CA, 1968. 25 C.V. Vorhees, Behavioral effects of prenatal methylmercury in rats: a parallel trial to the collaborative behavioral teratology study. Neurobehav. Toxicol. Teratol., 7 (1985) 717. 26 B.R.G. Danielsson, A. Fredriksson, L. Dahlgren, A. Teiling-G~rdlund, L. Olsson, L. Dencker and T. Archer, Behavioural effects of prenatal metallic mercury exposure in rats. Neurotoxicol. Teratol., (1992) in press. 27 C.H. Ngim and G. Devathasan, Epidemiologic study on the association between body burden mercury level and idiopathic Parkinson's disease. Neuroepidemiology, 8 (1989) 128. 28 C.M. Thompson, W.R. Markesbery, W.R. Ehmann, Y.X. Mao and D.E. Vance, Regional brain trace-element studies in Alzheimer's disease. Neurotoxicology, 9 (1988) 1. -
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Behavioural effects of neonatal metallic mercury exposure in rats A. F r e d r i k s s o n a, L. D a h l g r e n b, B. D a n i e l s s o n a, P. Eriksson c, L. D e n c k e r a a n d T. Archer b aDepartment of Toxicology, Uppsala University, Box 594, S-75124 Uppsala, bDepartment of Psychology, University of Gothenburg, Box 14158, S-400 20 Gothenburg and CDepartment of Zoophysiology, Uppsala University, Box 560, S-75122 Uppsala (Sweden) (Received December 1 lth, 1991; accepted June 9th, 1992)
Summary The effect of neonatal exposure of rats to mercury vapour (Hg°), at the concentration 0.05 mg/m 3, 1 h (low dose) or 4 h (high dose), on the behaviour in adulthood were studied. Exposure occurred on days 11-17 (the period of rapid brain growth). Tests for spontaneous motor activity were performed at the ages of 2 and 4 months. Rats exposed to the high dose Hg ° showed a marked increase in variables locomotion and total activity but a decrease for rearing when tested at 2 months of age. At 4 months of age these rats showed a marked hypoactivity with respect to all three variables. Rats exposed to the low dose showed no significant differences at 2 months compared to controls. However, at the age of 4 months the same pattern (increase in variables locomotion and total activity but a decrease for rearing) already noticed in the high dose group at 2 months was observed. In the spatial learning tasks applied, the radial arm maze and circular swim maze, neonatally exposed pups showed a retarded acquisition to the former, while there was no difference compared to controls in the latter. These data indicate that neonatal exposure to mercury vapour results in similar behaviour changes as reported from offspring prenatally exposed to mercury vapour or methylmercury. Furthermore, exposure for 1 week to concentrations around Swedish threshold values (TLV) for 1 or 4 h resulted in dose and age-related behavioural changes.
Key words: Mercury; Neuro behaviour; Neuro development; Behavioural teratology
Introduction Exposure to inorganic mercury may originate from occupational work [1-3], setting, removing and polishing amalgam restorations [4,5] or else in the environment. While inorganic mercury in the ionic form does not enter the brain in high concentrations [6,7], the more lipid soluble metallic mercury passes the blood brain barrier rapidly. Metallic mercury is oxidized by catalase to the inorganic form followed by its binding to intracellular proteins [8-11]. Metallic mercury, like Correspondence to. M.K. Anders Fredriksson, Department of Toxicology, Uppsala University, Box 594, S-75124 Uppsala, Sweden. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
152 methylmercury is an established neurotoxicant. The sensitivity of the developing central nervous system to metallic mercury has not been studied in detail and is thus largely unknown. In recently reported experiments [12-15] we have observed that neonatal exposure to environmental toxic agents such as DDT, PCBs and pyrethroids, potently induces permanent aberrations in spontaneous behaviour and cholinergic system in CNS of the adult animal. The changes were induced even with low doses to neonatal mice provided that the toxic agent was given during the period of rapid development of the brain [16], 'the brain growth spurt' [17]. This species-related period of development (around partus in humans and 10 days after birth in rats) is characterized by the maturation of axonal and dendritic outgrowth and establishment of neural connections, synaptogenesis. This stage of development is also associated with a large number of biochemical changes that will transform the feto-neonatal brain into that of the mature adult. The development of the dopaminergic system in the striatum [18] belongs to these biochemical changes. During this period an animal acquires new motor and sensory qualities [19] and the spontaneous motor behaviour peaks [20]. The present study was undertaken to investigate whether neonatal rats exposed to low levels of mercury (Hg °) vapour during 'the brain growth spurt' would alter physical development or spontaneous behaviour and learning capacity of the adult. Method
Animals Thirty pregnant Sprague-Dawley rats, - 3 months old, were purchased from ALAB, Sollentuna, Sweden. They were housed in plastic cages in a room with a 12-h light-dark cycle, thermostatically maintained at 22 ± I°C. Rats were allowed free access to food (R3, Ewos, S6dertfilje, Sweden) and tap water. The day of birth was designated day 0.
Study design Three treatment groups, 8 litters per group normalized to 8 individuals on Day 3, were exposed to metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) or air (2 h in a chamber without Hg vapour in control group). Daily exposure occurred in the chambers from Day 11 to Day 17 of age. Body weights and general appearance were monitored during the pre-weaning period. On Day 23 the pups were separated from their mothers and placed in plastic cages (6 males in each cage) according to treatment group. On Day 25, 6 pups from each group were sacrificed for quantitative analysis of Hg in brain, liver and kidney. All treatment groups were tested for spontaneous motor activity, learning in the radial arm maze and in a circular swim maze.
Test procedures and apparatus Hg exposure and analysis Hg ° vapour was generated into the exposure chamber by blowing a stream of
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nitrogen over a surface of metallic Hg. Air was aspirated through the chamber with a vacuum pump, resulting in a pressure slightly below atmospheric. Air samples were taken every 10 rain with a gas-tight syringe and bubbled into an acidified KMnO4 solution for Hg analysis. Flameless atomic absorption spectrophotometry was used to quantify the content of Hg in the samples (detection limit 0.1 ng Hg/sample). To permit analysis organic samples were digested under pressure in metallic bombs (MeAna - - Konsult, S-740 22 B/ilinge, Sweden) under the influence of acid and heat (2 ml conc. HNO3, 1 h at 175°C).
Spontaneous motor activity An automated device, consisting of cages (40 x 25 x 15 cm) placed within two series of IR beams (high and low level), was used to measure spontaneous motor behaviour (Rat-O-Matic, ADEA Elektronik AB, Uppsala, Sweden). Each rat was alone in its cage. In each group 10 animals were tested. The following variables were measured: Locomotion. Registered by the low level grid of invisible IR beams. Counts occurred only when the rat moved horizontally, showing predominantly locomotion behaviour. Rearing. Registered by the high level IR beams when the rat raised their front legs and/or rested on their haunches. Counts (at a rate of 4 per s) occurred as long as a single high level beam remained interrupted, i.e., the number of rearing counts was proportional to the amount of time the animal reared and interrupted the beam. Total activity. Registered by a pick-up, mounted on a lever with a counter-weight, resting on the wall of the cage. The pick-up registered all types of vibrations within the test cage, e.g., particular rat movements (tremors, headshakes or grooming behavior) as well as rearing and locomotion.
Radial arm maze The radial arm maze test, a procedure sensitive to deficits in learning performance [21,22], was adapted both to evaluate spontaneous motor activity and cognitive function [231. The maze had 8 arms (each 54 cm long, 10 cm wide and 25 cm high) which extended radially from a central hub, 25 × 25 cm. At the extremity of each arm an extra, removable, back wall concealed food cups. For the activity testing the arms were marked into 3 units each, giving a total of 25 units. Ambulation and rearing were measured for 10 min by direct observation. Ambulation is defined as the passage of a rat's body from one unit to another and rearing was scored each time a rat raised itself onto its hindlegs. The maze was carefully cleaned after each test. After the activity testing, rats were placed on total food deprivation for 48 h. For the learning tests, the back walls were removed, exposing the 8 food cups (one at the distal end of each arm) into which a food pellet was placed. At the start of each test the rat was placed in the central hub and then monitored for its performance, i.e., the latency until all pellets were collected and the number of arms visited during the session. Immediately after the second test the rats were given free access to food. Testing was performed when the offspring were about 6 months old.
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Swim maze The swim maze was a circular bath (diameter 140 cm) thermostatically controlled to 25°C. The bath included a platform, placed at a constant position, 1 cm below the water level. Before testing, all rats were given swim training, for which the platform was removed and the animals allowed to swim for 60 s. After the platform had been placed at a set position in the pool, each animal had one test session on each of 3 consecutive days. On each test session, five trials were presented to the rat. For all trials, the rat was placed at the same point in the pool and allowed to swim around to find the submerged platform and escape from the water. On reaching the platform the rat was allowed to remain upon it for 30 s before being placed in the water again for the next trial. If a rat failed to locate the platform within 65 s it was placed on the platform for its 30-s orientation period. Statistical analysis Locomotion, rearing and total activity measures of spontaneous motor activity over three consecutive 20-rain periods at 2 and 4 months of age were submitted to a split-plot A N O V A [24]. The variables from the radial arm maze (latency and error data) were all subjected to one-way A N O V A analyses based on a completely randomized design. The latency data in the water maze was also subjected to split-plot ANOVA. Pairwise testing was performed using the Tukey HSD test. The results from the analysis of mercury were subjected to one-way A N O V A analyses based on a completely randomized design.
Results Chamber concentrations o f Hg ° To evaluate the conditions during the exposure samples were drawn every l0 rain and the concentration of Hg ° was estimated. Figure 1 shows the mean concentration of Hg ° + SD during the exposure for the low Hg ° dose group.
7503 tO
45 E 30.
o 0
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0 0
10
20
30
40
50
60
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155 TABLE I THE AMOUNT OF Hg IN THE BRAIN, LIVER AND KIDNEY OF 25-DAY-OLD RATS EXPOSED TO Hg ° BETWEEN DAYS 11 AND 17a OF AGE
Control Hg-low Hg-high
Brain
Liver
Kidney
0.002 0.017 b 0.063 b
0.002 0.084 b 0.219 b
0.002 1.247b 6.734 b
aThe rats were subjected to daily exposure of metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days l I to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg vapour. The amount of Hg is expressed as mg/kg organ. blndicates significant differences between means (P < 0.01) versus control and the other dose group. One-way ANOVA based on a completely randomized design [24] was significant for all three organs (F(2,15) > 90.30). Pairwise testing using Tukey HSD tests [24] showed a dose-dependent increase of the amount of Hg °.
Analysis of Hg All three g r o u p s were a n a l y z e d for contents o f H g in animals s a c r i f i c e d on D a y 25. Thus, o n e - w a y A N O V A b a s e d on a c o m p l e t e l y r a n d o m i z e d design was significant for all three o r g a n s (F(2,15) >_ 90.30, for brain, liver a n d kidney). Pairwise testing using T u k e y H S D tests showed a d o s e - d e p e n d e n t increase o f the a m o u n t o f H g (Table I).
Clinical observations of the litters N o changes in b o d y weight o f the pups were seen as a result o f the H g exposure, n o r were any clinical signs o f d y s f u n c t i o n o b s e r v e d in any o f the groups.
Spontaneous motor activity H g ° - t r e a t e d rats s h o w e d m a r k e d changes in the different variables o f m o t o r activity that were to some extent d e p e n d e n t u p o n the age at which they were rested (Fig. 2). Two-month-old rats. R a t s e x p o s e d to the high H g dose showed increases in the l o c o m o t i o n a n d t o t a l activity variables but a decrease in rearing b e h a v i o u r at the age o f 2 m o n t h s . There were significant G r o u p s x Test Periods interaction (F(4,42) = 8.49, F(4,42) = 3.79, F(4,42) = 5.90) for l o c o m o t i o n , rearing and total activity, respectively. These rats h a d significantly ( P _ 0.01) m o r e counts at the 20-, 40- a n d 60-rain p e r i o d s for l o c o m o t i o n a n d total activity, whereas rearing counts were significantly ( P _ 0.05) decreased at the 20- a n d 40-rain periods, c o m p a r e d with controls. A t the age o f 2 m o n t h s no significant changes in m o t o r activity were observed in rats e x p o s e d to the low H g dose. Four-month-old rats. R a t s exposed to the high H g dose showed decreases in all three variables o f activity at the age o f 4 m o n t h s , whereas rats exposed to the low H g dose showed increases in the l o c o m o t i o n a n d total activity variables a n d
156
2 MONTHS
4 MONTHS
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o
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20-40 TIME (rain)
40-60
itt
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20 - 40
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Fig. 2. Spontaneous motor activity in 2- and 4-month-old rats exposed to Hg ° as neonates. The rats were subjected to daily exposure of Hg ° vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Day 11-17 of age. Control rats were exposed to air for 2 h in a chamber without Hg ° vapour. Each treatment group comprised of 10 rats from at least 3 different litters. Regarding measurement of motor activity, see methods section. Statistical analysis of behavioural data was performed using ANOVA with a split-plot design [24]. There were significant Groups x Periods interaction (F(4,42)= 11.0, F(4,42) = 16.96, F(4,42) = 20.73) for the locomotion, rearing and total activity variables, respectively. Pairwise testing between Hg-low, Hg-high and control groups was performed with the Tukey HSD test [24]. The statistical difference from the control is indicated by *P -< 0.05; **P _< 0.01. rl, Control; k~ Hglow; I , Hg-high.
decreases in the r e a r i n g v a r i a b l e . T h e r e were s i g n i f i c a n t G r o u p s x Test P e r i o d s i n t e r a c t i o n (F(4,42) = l l . 0 , F(4,42) = 16.96, F(4,42) --- 20.73) for l o c o m o t i o n , reari n g a n d total activity, respectively. R a t s c o n s t i t u t i n g the high H g dose g r o u p h a d significantly ( P _< 0.05, P _< 0.01) fewer c o u n t s at the first 20-rain p e r i o d for the v a r i a b l e s l o c o m o t i o n , r e a r i n g a n d total activity a n d also at the 40-rain p e r i o d for the r e a r i n g a n d total activity v a r i a b l e , c o m p a r e d with c o n t r o l s . R a t s c o n s t i t u t i n g the low H g dose g r o u p s h o w e d s i g n i f i c a n t l y ( P _< 0.05, P _< 0.01) m o r e c o u n t s for the
157
600
450
NEONATAL, Hg ° ** xx xx
124 -18 m ,m
>-
300-
z uJ ,-I
-12 XX
150.
-9 NN XN
Fig. 3. Radial arm maze performance in 6-month-old rats exposed to Hg ° as neonates. The rats were subjected to daily exposure of Hg ° vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days 11 to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg ° vapour. Each treatment group comprised of 8 rats from at least 3 different litters. Regarding measurement of radial arm maze performance, see methods section. Statistical analysis of behavioural data was performed using one-way ANOVA based on a completely randomized design [24]. There were significant changes for both latency to acquire all 8 pellets (F(2,24)= 7.13) and number of errors made in acquiring all 8 pellets (F(2,24) = 3.68). Pairwise testing between Hg-low, Hg-high and control groups was performed with the Tukey HSD test [24]. The statistical difference from the control is indicated by *P-< 0.05; **P _< 0.01. n, Control; ~1, Hg-low; •, Hg-high.
locomotion and total activity variables and fewer counts for rearing variable at the 20- and 40-min intervals, compared with controls. Radial arm maze
Rats treated neonatally with Hg ° showed at 6 months of age dose-related impairment in radial arm maze learning. Thus, one-way ANOVA based on a completely TABLE II SWIM MAZE L E A R N I N G IN 5-MONTH-OLD RATS EXPOSED TO Hg ° AS NEONATES a Day
Control Hg-low Hg-high
Latency to find platform (s) 1
2
3
44.2 45.7 39.4
26.6 25.9 23.5
20.2 18.8 24.6
aThe rats were subjected to daily exposure of metallic Hg vapour (0.05 mg/m 3, 1 h in low dose group; 4 h in high dose group) from Days 11 to 17 of age. Control rats were exposed to air for 2 h in a chamber without Hg vapour. The result is expressed in seconds in latency to find platform. Split-plot ANOVA [24] showed neither a Groups nor a Groups x Days interaction effect. Significant days effect (F(2,52) = 32.3) was observed indicating that all three groups learned the task of locating the platform within the circular pool from day to day.
158 randomized design was significant for both latency to acquire all 8 pellets (F(2,24) = 7.13, P < 0.01) and number of errors made in acquiring all 8 pellets (F(2,24) = 3.68, P _ 0.05). Pairwise testing using Tukey HSD tests indicated that the high Hg dose group showed significantly longer latencies (q = 5.33, P _ 0.01) and more errors (q = 4.28, P _< 0.05) than the control group. Figure 3 shows the performance of control and Hg-exposed groups in the radial arm maze. The low Hg dose group showed higher latencies and more errors than the control group but did not reach significantly different values. Swim maze learning
Control and Hg-exposed rats were tested for swim maze learning performance at 5 months of age. No difference among the three groups was obtained. Thus, splitplot ANOVA showed neither a Groups (F(4,52) < 0.06) nor a Groups x Days interaction effect (F(4,52) ___ 0.89), but only a Days effect (F(2,52) = 32.3) indicating that all three groups learned the task of locating the platform within the circular pool. Table II presents the latencies to reach the platform by the control and Hgexposed groups. Discussion Neonatal exposure of rat pups to metallic mercury, during Day 11-17 of age, 'the period of brain growth spurt' caused behavioural changes in these animals when they were tested at adult ages. The results may be summarized as follows: (i) There was a dose-dependent concentration of mercury in the organs of animals sacrificed 1 week after exposure to Hg °. For example, the brain concentration of Hg in the high dose group was about 4 times higher than in the low dose group; 63/~g/kg and 17 #g/kg, respectively. (ii) Rats exposed to the high dose Hg ° showed a marked increase in locomotion and total activity but a decrease for rearing when tested at 2 months of age. At 4 months of age these rats showed a marked hypoactivity for all three variables. Rats exposed to the low dose showed no difference at 2 months compared to controls. However, at the age of 4 months the same pattern (increase in variables locomotion and total activity but a decrease for rearing) already noticed in the high dose group at 2 months was observed. (iii) Spatial learning ability was measured in the radial arm maze and a dose-related retardation of the Hg°-exposed offspring was observed when tested at 6 months of age. (iv) Spatial navigation was studied in a Morris-type circular swim maze at 5 months of age. No significant difference was obtained. The exposure concentration used (0.05 mg/m 3) corresponds to levels of mercury in the air found in chloralkali plants (0.025-0.05 mg/m 3) [15]. Furthermore, the exposure periods were relatively short (1 or 4 h/day) resulting in a quite low exposure level even of the high dose group compared to previously reported exposure levels required to cause hypoactivity after oral dosing of animals prenatally with MeHg (2-6 mg/kg body weight) [25] or prenatal exposure for mercury vapour (1.8 mg/m 3, 3 h/day) [26]. The present study showed that both doses of Hg ° induced the same pattern of disturbed spontaneous motor activity in the adult animal. Of special interest is the
159
fact that in rats exposed to the high dose the disturbed motor activity appeared already in the test at 2 months. This finding corroborates our previous report on the relation between neonatal exposure and behaviour changes in the animals when reaching adult age [13]. In this study, no behavioural deviation was seen 1 week after exposure but developed with advancing age to a permanent state. Our results demonstrate that Hg present in CNS during the critical period of brain development generates changes in the adult brain function. The influence of Hg on the brain may lead to dysfunctions contributing to an earlier onset of aging and/or degenerative processes. Although the mechanism is far from clear, it may be mentioned that the implication on the association between body burden mercury and idiopathic Parkinson's disease [27] as well as regional CNS elevation of mercury in Alzheimer's disease [28] have been stated. The role of mercury, if any, in these age-related diseases has not yet been elucidated. Mercury may, together with other toxic agents in the environment contribute to a more rapid aging process and to cell death in CNS.
Acknowledgement We are grateful to E. Wallin for technical assistance. This work was financially supported by grants from the Swedish Medical Research Council, the Swedish Work Environment Fund, the Swedish Environmental Protection Board and the Swedish Council for the Humanities and Social Sciences.
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