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Long-term effects of aluminium on the fetal mouse brain
Life Sciences, Vol. Printed in the USA
51, pp. 1921-1928
Pergamon Press
LONG-TERM EFFECTS OF ALUMINIUM ON THE FETAL MOUSE BRAIN R.M. Clayton,
S.K.A. Sedowofia,
J.M. Rankin and A. Manning.
Division of Biological Sciences, I n s t i t u t e of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JN. (Received in final form October 7, 1992) Summary
Potentially n o x i o u s s u b s t a n c e s may a c t a s f e t a l t e r a t o g e n s at levels far lower than those required to produce detectable effects in adults, and behavioural teratogenicity may occur at levels lower than those which produce morphological teratogenesis. Aluminium (A1) is a potential neurotoxin in adults. S i n c e p r e g n a n t women may b e e x p o s e d t o u n t o w a r d l e v e l s o f A1 c o m p o u n d s u n d e r c e r t a i n conditions, we h a v e e x a m i n e d t h e long-term effects of treating the pregnant mouse with intraperitoneal o r o r a l a l u m i n i u m s u l p h a t e on b r a i n b i o c h e m i s t r y and behaviour of the offspring. The cholinergic system, as evaluated by t h e a c t i v i t y of choline acetyltransferase (CHAT), was a f f e c t e d differentially in different regions of the brain, and still showed significant effects in the adult. Differences between the intraperitoneal and oral series in the magnitude of effect seen in the regions of the brain probably reflect differences in the effective level of exposure. Growth rate and psychomotor maturation i n t h e p r e - w e a n i n g mouse w e r e a f f e c t e d in the intraperitoneal series only, showing a marked post-natal
maternal effect. The p r e s e n c e o f a l u m i n i u m (A1) compounds in the brains of sufferers of senile d e m e n t i a o f t h e A l z h e i m e r t y p e (SDAT) h a s l e d t o t h e s u g g e s t i o n that A1 a c c u m u l a t i o n in the brain leads to degenerative processes(l). Although A1 i s t h e t h i r d most abundant element, most of it exists in insoluble forms. The limited passage o f A1 s a l t s across the gut, the efficient excretion by t h e k i d n e y s , a n d t h e b l o o d - b r a i n barrier diminish the access of soluble A1 c o m p o u n d s t o t h e b r a i n ( 2 ) . However a c u t e o r c h r o n i c administration o f A1 c o m p o u n d s t o e x p e r i m e n t a l animals by various routes, including the oral(3-5), has established the neurotoxicity of soluble aluminium compounds, as do human conditions such as dialysis encephalopathy(6,7). Many o f t h e n e u r o l o g i c a l diseases i n w h i c h A1 h a s b e e n implicated a r e t h o s e i n w h i c h t h e r e i s damage t o t h e c h o l i n e r g i c system(8), and there is evidence of cholinergic damage i n c o n d i t i o n s where aluminium has been introduced experimentally into the brain(5,9,10). A1 c o m p o u n d s a r e more readily t a k e n up a t a c i d pH o r i n o r g a n i f i e d f o r m . F o r e x a m p l e A1 u p t a k e i s increased by citrate(ll,12) or by complexing with sugars(13,14). Such organification may p e r m i t s o l u b i l i t y in cell membranes, thus affecting permeability and the blood-brain barrier(15). A l u m i n i u m may b i n d t o r e c e p t o r .
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Correspondence: R.M. Clayton, Division of Biological Sciences, I n s t i t u t e of Cell, Animal and Population Biology (Genetics B u i l d i n g ) , U n i v e r s i t y of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JN.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
1922
Aluminium
Effects
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sites for several neurotransmitters, to membrane phospholipids, or to calmodulin, thus affecting calcium homeostasis(16). P r e g n a n t women may b e exposed to aluminium compounds in several industries(8), or from environmental pollution in the vicinity of aluminium smelters, or following accidents (several of which have been reported in the U.K.) with aluminium sulphate added to water supplies. The f e t u s i s i n g e n e r a l s u s c e p t i b l e to far lower concentrations of noxious substances than adult tissues(17), and some s u b s t a n c e s may a c t a s b e h a v i o u r a l teratogens, producing long-term or permanent deficits in brain function, often at even lower levels than those required to produce morphological teratogenesis(18). We h a v e u s e d m i c e which, like humans, have a haemochorial placenta, to study the effects of prenatal e x p o s u r e t o a l u m i n i u m s u l p h a t e on b r a i n b i o c h e m i s t r y and b e h a v i o u r a l development, a n d r e p o r t h e r e on l o n g - t e r m e f f e c t s on g r o w t h , b e h a v i o u r , and the cholinergic s y s t e m . The e f f e c t on t h e c h o l i n e r g i c s y s t e m , a s a s s e s s e d by measuring the activity of choline acetyltransferase (CHAT) was c h o s e n b e c a u s e o f t h e known e f f e c t s o f A1 on t h i s n e u r o t r a n s m i t t e r system(9,10). Materials
and methods
Administration of aluminium Aluminium was administered to pregnant CBA mice by two routes. One group of mice was i n j e c t e d i n t r a p e r i t o n e a l l y ( i . p ) with 200mg/kg aluminium sulphate (Alz(SO4)3.18H20, pH 2.5-2.7) or s a l i n e a c i d i f i e d to the same pH as t h e aluminium, from days I0-13 of g e s t a t i o n inclusive. The second group was given aluminium sulphate (750mg/L, pH 4 . ] - 4 . 3 ) as t h e i r s o l e d r i n k i n g w a t e r from days I0-17 of g e s t a t i o n inclusive. Control mice in t h i s group were given water a c i d i f i e d to the same pH as the aluminium sulphate. Determination of choline acetyltransferase a c t i v i t y Control and aluminium exposed m i c e w e r e k i l l e d a t ages ranging from 3 - 4 4 weeks. The brains w e r e dissected into d i f f e r e n t regions including cerebral cortex, cerebellum, hippocampus, midbrain, hypothalamus, nucleus magnocellularis, striatum and the brainstem. Pooled samples from 2-4 mice (not separated by sex), were stored a t -70°C u n t i l used for the determination of ChAT a c t i v i t y by the method of Glover and Green (19). B r i e f l y , samples were homogenised on ice in s a l i n e containing I% b u t a n - l - o l , and assayed with an assay mixture containing 14C acetylCoA a t 37°C for 20 mins. 14C acetylcholine formed was extracted into octan-2-one containing potassium mercuric iodide, and a l i q u o t s counted in a s c i n t i l l a t i o n counter. Each group of age-matched control and t r e a t e d brains was dissected and analysed together. However, the oral s e r i e s were begun a year a f t e r the i n t r a p e r i t o n e a l s e r i e s , and d i s s e c t i o n s were done by a d i f f e r e n t operator. Thus while each control and t r e a t e d group i s wholly comparable, the controls for the i n t r a p e r i t o n e a ] and oral s e r i e s are not comparable. Behavioural and developmental studies On the day of b i r t h ( p o s t - n a t a l day zero, PdO) l i t t e r s i z e s were recorded. The following day p u p s were weighed, sexed, marked by toe c l i p p i n g and c r o s s - f o s t e r e d u s i n g a t o t a l s p l i t - l i t t e r c r o s s - f o s t e r design, and weighed on every t h i r d day from Pd3 u n t i l weaning (see r e f . 20). Thereafter, body weights were recorded weekly. Male and female weights were analysed separately from weaning onwards. Tests for sensori-motor coordination and development were also performed every t h i r d day using a modified version of the Fox b a t t e r y of t e s t s (21). Subjects were t e s t e d for slow r i g h t i n g , c l i f f aversion, grasping with forelimbs, pole grasping, climbing on a wire mesh and eye opening. All other behavioural
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Aluminium Effects
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tests were performed as previously described (20), except for the test of learning ability. In these experiments, 10 week o l d m a l e m i c e w e r e t e s t e d in a n 8 - a r m r a d i a l maze f o r 12 d a y s a s d e s c r i b e d by Pick and Yanai (22). Adult activity tests on t h r e e s u c c e s s i v e days were carried o u t a t 22 weeks ( s e e
20). The f o l l o w i n g f o u r e x p e r i m e n t a l groups of mice were established and tested:(1) pups from control litters fostered to control mothers (Cc), (2) pups from control litters fostered t o A1 t r e a t e d m o t h e r s ( C t ) , ( 3 ) p u p s f r o m A1 t r e a t e d litters fostered to control mothers (Tc), a n d ( 4 ) p u p s f r o m A1 t r e a t e d litters f o s t e r e d t o A1 t r e a t e d m o t h e r s ( T t ) . A l l t h e b e h a v i o u r a l studies were c a r r i e d o u t by t h e same o b s e r v e r who was b l i n d t o t h e a l l o c a t i o n of treatment g r o u p s . The s a m p l e s i z e f o r e a c h g r o u p , i n c l u d i n g males and females, in the i . p s e r i e s was a s f o l l o w s : Cc n = 1 3 , Ct n = 1 4 , T t n = 1 2 , Tc n = l l ; a n d f o r t h e o r a l s e r i e s Cc n = 8 , C t n = 9 , T t n=10 a n d Tc n=8. Statistical analysis Pup weights at birth were analysed using Student's t-test. A n a l y s i s o f V a r i a n c e (ANOVA) was e m p l o y e d f o r t h e w e i g h t s and activity scores thereafter (23). Adult activity d a t a was a n a l y s e d u s i n g the Kruskal-Wallis o n e - w a y ANOVA. The c a t e g o r i c a l data generated from the Fox t e s t s was a s s e s s e d b y C h i - s q u a r e t e s t s on e a c h d a y o f o b s e r v a t i o n . ANOVA f o l l o w e d by t - t e s t was u s e d t o a n a l y s e ChAT a c t i v i t y data. Differences at p<0.05 were taken as significant.
Results Levels of aluminium consumed or injected With an average weight of 25g, pregnant mice injected intraperitoneally with aluminium sulphate received a daily dose of 0.41mg of e l e m e n t a l a l u m i n i u m . The mean a m o u n t o f f l u i d c o n s u m e d d a i l y b y c o n t r o l m i c e in the orally e x p o s e d g r o u p was 6 . 0 m l . T h i s c o m p a r e s w i t h a mean o f 4 . 4 ml consumed by the treated mice, and is equivalent to a daily intake of 0.27mg of elemental aluminium. Over a four-day treatment period, i.p mice received a t o t a l o f 1.64mg o f a l u m i n i u m , c o m p a r e d w i t h 1.89mg c o n s u m e d by o r a l l y e x p o s e d mice in seven days. Neurochemistry Our data indicate that pre-natal exposure to aluminium sulphate affects the cholinergic system, as judged by effects on choline acetyltransferase activity. T a b l e 1 s h o w s ChAT a c t i v i t y in different brain regions from mice whose mothers were administered aluminium sulphate by intraperitoneal injection. The d a t a i n t a b l e 2 show t h e a c t i v i t y in mice whose mothers were exposed orally to aluminium. B o t h t a b l e s show s i g n i f i c a n t effects on t h e c h o l i n e r g i c system, and also that the effects are differential between brain regions. It is also clear from the data that, with the exception of the hypothalamus and striatum, the effects persist into adu 1 t h o o d . Developmental and behavioural effects T h e r e w e r e no e f f e c t s on t h e l e n g t h o f g e s t a t i o n , litter size or sex ratio of either A1 t r e a t m e n t . The b i r t h weights of exposed pups in the intraperitoneal series were about 5 per cent below controls (df=l,47 t=2.63 p=0.011) but this difference persisted o n l y i n t h e m i c e r e a r e d by t r e a t e d mothers. Fig. 1 shows this clear maternal effect, and the increasing
1924
Aluminium
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Vol.
51, No. 25, 1992
d i v e r g e n c e o f pup w e i g h t s r e a r e d by c o n t r o l o r t r e a t e d m o t h e r s from Pd3 up t o w e a n i n g ( d f = 1 , 4 2 F = ] 6 . 4 3 p = O . 0 0 0 2 ) . The m a t e r n a l e f f e c t on b o d y w e i g h t p e r s i s t e d i n t o a d u l t h o o d f o r f e m a l e mice o n l y ( d r = l , 1 8 F=8.42 p < 0 . 0 1 ) . There were no s i g n i f i c a n t e f f e c t s on body w e i g h t o f pups whose m o t h e r s were exposed t o aluminium s u l p h a t e o r a l l y d u r i n g g e s t a t i o n . Pups exposed t o aluminium v i a t h e i . p r o u t e were s l o w e r t o a t t a i n a d u l t s c o r e s on s e v e r a l o f t h e Fox s c a l e m e a s u r e s o f r e f l e x development(21): i n c l u d i n g slow r i g h t i n g , f o r e l i m b g r a s p i n g ( F i g . 2) and p o l e g r a s p i n g ( F i g . 3 ) , a l l p<0.05. In a d d i t i o n , we have found a m a t e r n a l e f f e c t on s e n s o r i - m o t o r performance. F i g s . 2 and 3 show t h a t t r e a t e d pups r e a r e d by t r e a t e d m o t h er s ( T t ) a r e more s e v e r e l y a f f e c t e d than t r e a t e d pups r e a r e d by c o n t r o l m o t h e r s (Tc) o r c o n t r o l pups r e a r e d by t r e a t e d m o t h e r s ( C t ) , b u t a l l t h e s e groups a r e affected as c o m p a r e d t o c o n t r o l s r e a r e d by c o n t r o l mothers (Cc). The d i v e r g e n c e between t h e two e x t r e m e g r o u p s , c o n t r o ] pups r e a r e d by c o n t r o l m o t h e r s , and t r e a t e d pups r e a r e d by t r e a t e d m o t h er s i s t h e w i d e s t . The s i g n i f i c a n c e f o r p o l e g r a s p i n g , f o r example, i s p<0.001. The o t h e r t e s t s were n o t f o u n d t o be s i g n i f i c a n t . Open f i e l d activity levels were largely u n a f f e c t e d up t o weaning.
TABLE I CHOLINE ACETYLTRANSFERASE ACTIVITY IN CBA MICE EXPOSED PRE-NATALLY TO ALUMINIUM SULPHATE 3 weeks
CEREBRAL CORTEX CEREBELLUM HIPPOCAMPUS MIDBRAIN HYPOTHALAMUS STRIATUM
CONTROL
TREATED
213.4± 43.3± 744.4± N.D. N.D. 459.6±
2 ] 0 . 9 ± 4.8 50.7± 3.1 580.2± 8.2* N.D. N.D. 251.5± 5.2*
9.3 0.7 7.6
9.7
34 weeks CONTROL
17 weeks
CONTROL
TREATED
440.3± 2.6 328.9± 8.3* 98.5± 2.7 64.9± 1 . 9 ' I015.6±31.0 917.9± 3.6 793.3±13.1 856.6± 3.9* 665.7±19.9 573.9±26.7 2141.7&52.7 1871.4±43.9'
44 weeks TREATED
CEREBRAL CORTEX 3 0 9 . 4 ± 2 2 . 0 180.6± 1 . 4 . CEREBELLUM 50.9± 1.1 46.5± 0 . 9 * HIPPOCAMPUS 381.1±18.1 306.7± 1 . 4 . MIDBRAIN 649.4± 7.8 552.1± 6.3* HYPOTHALAMUS 530.9+2.1 433.8± 6.3* STRIATUM 1047.7±24.3995.0± 7.0
CONTROL
TREATED
247.0± 5.3 73.2± 1.1 567.2± 6 .0 505.7±15.0 247.5± 2.4 431.7± 8.4
215.4± 2.5* 45.6± 1 . 0 . 545.5±10.0 439.8± 3.8* 342.2± 9.0* 728.2±16.0.
Choline acetyltransferase activity in mouse b r a i n . B r a i n s were dissected from offspring whose mothers were injected intraperitoneally w i t h 200mg/kg aluminium s u l p h a t e o r s a l i n e on g e s t a t i o n days 10-13. Enzyme a c t i v i t y e x p r e s s e d as p i c o m o l e s / m g protein/minute. Each r e s u l t represents t h e Mean ± SE o f 4 d e t e r m i n a t i o n s . Values *p<0.05; N.D.= Not D e t e r m i n e d .
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Measurements of activity a t w e a n i n g a n d i n a d u l t l i f e show p e r s i s t e n t trends towards a maternal treatment effect. At 22 w e e k s , a f i v e m i n u t e a c t i v i t y t e s t was p e r f o r m e d . T h e r e was n o s i g n i f i c a n t difference b e t w e e n g r o u p s on d a y 1 when t h e y w e r e i n t r o d u c e d t o t h e f i e l d . However, there were marginally significant differences between treatment groups in the total number of s q u a r e s c r o s s e d on d a y s 2 a n d 3, p u p s r e a r e d by t r e a t e d mothers had lower activity levels.
TABLE II ChAT ACTIVITY IN CBA MICE EXPOSED TO ALUMINIUM SULPHATE PRE-NATALLY (ORALLY ADMINISTERED TO MOTHERS FROMDAY 10-17 OF PREGNANCY) 3.5 Weeks CONTROL CEREBRAL CORTEX CEREBELLUM HIPPOCAMPUS MIDBRAIN HYPOTHALAMUS NUCLEUS STRIATUM BRAINSTEM
16 Weeks
TREATED
CONTROL
TREATED
197.3± 3.8 322.9± 2.5* 516.5±12.1 3 5 8 . 2 ± 5 O* 105.5± 1.2 61.4± 4.1' 97.6± 2.3 8 7 . 3 ± 0 8* 568.9± 8.5 661.5±26.6' 678.7± 5.8 7 2 5 . 9 ± 1 3 O* 7 3 3 . 9 ± 1 8 . 6 498.1± 9.1, 542.7± 6.1 562.8± 6 7* 3 3 8 . 4 ±9.0 442.7± 2.7* 564.4± 8.7 478.8± 3 6* ]289.1± 5.3 1173.9±14.6, 1695.2±13.5 1415.1± 4 5* 831.6± 7.7 751.2± 9.9* 1426.4± 5.4 868.3±10.5, 8 7 0 . 7 ± 1 2 . 8 890.9± 6.2 8 4 1 . 1 ±7.1 977.6±45.9,
Choline a c e t y l t r a n s f e r a s e a c t i v i t y in mouse b r a i n . Brain dissections and measurement of ChAT a c t i v i t y were as in table I. Values *p<0.05 were s i g n i f i c a n t l y d i f f e r e n t from controls. T e s t s a t 10 w e e k s o f a g e f o r l e a r n i n g a b i l i t y and retention in an 8arm r a d i a l maze c o u l d b e made w i t h s m a l l n u m b e r s o n l y . 10 o u t o f 13 c o n t r o l a n i m a l s (77%) r e a c h e d c r i t e r i o n [two consecutive errorless trials (22)] and 6 o u t o f 11 (55%) o f t h e A ] - e x p o s e d g r o u p . The e x p o s e d m i c e w h i c h d i d r e a c h criterion w e r e s l o w e r t o do s o ( a m e d i a n o f 7 . 5 d a y s , c o m p a r e d t o 6 d a y s f o r controls), but this difference did not reach statistical significance.
Discussion In both series the effects on ChAT l e v e l s f o l l o w i n g p r e n a t a l exposure t o A1 a r e d i f f e r e n t i a l between brain regions: a finding also reported for the neurotoxic effects i n A1 e n c e p h a l o p a t h y in adult rats (24). Table 2 shows that significant effects are found i n ChAT a c t i v i t y following administration by t h e o r a l r o u t e , implying sufficient access of aluminium to the fetal brain for neurotoxic effects to be obtained. I t i s c l e a r f r o m t h e d a t a on ChAT t h a t t h e e f f e c t s o f a l u m i n i u m on t h e developing brain are not transient, but persist into adult life, although there are deviations from this trend in the striatum and hypothalamus in the oldest animals. The d i f f e r e n c e s in the extent t o w h i c h ChAT a c t i v i t y was affected in the different r e g i o n s o f t h e b r a i n may b e d u e t o t h e l e v e l s o f aluminium reaching each region, or to differences in the distribution of cholinergic neurons in the brain.
regions
There were also differences of the brain are compared
in the effects on ChAT when t h e same in the intraperitoneal and oral series.
1926
Aluminium
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on Fetal B r a i n
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Effects on t h e body w e i g h t from b i r t h to weaning of mice pups w h o s e m o t h e r s w e r e e x p o s e d by i n t r a p e r i t o n e a l injections of aluminium sulphate ( 2 0 0 m g / k g ) on g e s t a t i o n days 10-13. Cc C o n t r o l p u p s r e a r e d by c o n t r o l m o t h e r s ; Ct - C o n t r o l p u p s r e a r e d by t r e a t e d m o t h e r s ; Tt - T r e a t e d p u p s r e a r e d by t r e a t e d m o t h e r s ; Tc - T r e a t e d p u p s r e a r e d by c o n t r o l m o t h e r s .
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Effects of prenatal exposure to g r a s p i n g (2) a n d on p o l e g r a s p i n g
aluminium sulphate on f o r e l i m b (3) on p o s t - n a t a l d a y 15.
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51, No. 25,
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Aluminium Effects on Fetal
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T h i s may r e f l e c t a dose effect. I t h a s b e e n shown t h a t a l u m i n i u m l e v e l s in the placentas of pregnant female mice injected intraperitoneally w e r e 10 times higher than in controls, while they were only three times higher than controls in orally treated females(25). Also a rise in performance in a classically conditioned reflex test(26) has been reported in rabbits f o l l o w i n g low l e v e l s o f h l a d m i n i s t r a t i o n with a fall following high levels. Effects of prenatal a l u m i n i u m on p o s t - n a t a l growth have also been reported by several other workers (27-31), and other investigators have reported similar results to ours, in that hl treatment in utero affects the development of certain but not all sensori-motor skills (32,33). Treatment of infant mice similarly affects motor skills (27), and regular clinical administration o f A1 t o human i n f a n t s is neurotoxic (34). The i n c r e a s i n g d i v e r g e n c e b e t w e e n t h e pup w e i g h t s o f o f f s p r i n g reared by control or treated m o t h e r s up t o w e a n i n g , may b e d u e t o r e t e n t i o n of a l n m i n i u m by t h e m o t h e r a n d i t s r e l e a s e into the milk, to effects on m i l k output(26), or to effects on m a t e r n a l b e h a v i o u r ( 3 5 ) , but the neurochemical sequelae would imply that the pups received A1 i n t h e m i l k . Although Yokel(26) found relatively low l e v e l s o f A1 i n t h e m i l k o f t r e a t e d dams, it may b e i n o r g a n i f i e d c o m p l e x e s a n d t h u s r e a d i l y a b s o r b e d by t h e n e o n a t e . A l t h o u g h some b e h a v i o u r a l m e a s u r e s show e f f e c t s , others do n o t , and overall the behavioural effects do n o t seem c o m m e n s u r a t e w i t h t h e d e g r e e o f ChAT d e f i c i t . Such d i s p a r i t i e s c a n a l s o o c c u r i n SDAT b r a i n s (36,37). It m u s t b e p o i n t e d o u t t h a t r e s p o n s e s t o d e p r e s s e d l e v e l s o f ChAT may b e h i g h l y selective amongst different t y p e s o f memory ( 3 8 ) s o t h a t o u r b a t t e r y o f t e s t s (20) and the effects on m i c e do n o t p e r m i t u s t o p r e d i c t any particular loss of mental functions i n man. H o w e v e r , Adams ( 3 9 ) h a s p o i n t e d t o t h e s i m i l a r i t y of behavio,~ral responses i n humans and r o d e n t s after exposure to several different types of behavioural teratogens, s u g g e s t i n g t h a t m i c e may p r o v i d e a very suitable model for behavioural teratogenicity in humans. I t may a l s o b e t h a t r e d u c e d ChAT a c t i v i t y increases the liability of the system to other forms of stress, a possibility which requires investigation.
F i n a l l y we h a v e c l e a r e v i d e n c e f o r g e n e t i c d i f f e r e n c e s in response to teratogens,(17,20,40) and for sensitivity to aluminium. P r e g n a n t C57 m i c e , g i v e n t h e same i n t r a p e r i t o n e a l d o s e l e v e l s a s w e r e u s e d f o r t h e CBA g e n o t y p e and reported here, failed to continue to term. Acknowledgements
to
an
We a r e g r a t e f u l to the anonymous referee for
UK H e a l t h a n d S a f e t y helpful c o m m e n t s on
Executive for support and an earlier draft of this
manuscript. References 1. 2.
D.R. CRAPPER MCLACHLAN, W.J. LUEIW a n d T . P . A . KRUCE, Can. J . N e u r o l . S c i . 16 490-497 (1989). G.H. MAJOR, J . A . KEISER, D. MAKDAIN, a n d P.K. KU, S c i e n c e 197 1 1 8 7 - 1 1 8 9
(1977). 3. G.M. BERLYNE, J. BEN AVI, G. KNOPF, I.R. YAGI, G. WIENBERG and G.M DANOVICH, L a n c e ti 564-567 (1972). 4. N.C. BOWDLER, D.S. BEASLEY, C. ERITZE, A.M. GOULETTE, J . D . HATTON, J. HESSION, D.L. OSTMAN, D.J. RUGGand C.J. SCHMITTDIEL, Pharmacol. Biochem. Behav. I0 505-512 (1979). 5. R.J. BOEGMAN and L.A. BATES, Can. J. Physiol. Pharmacol. 62 1010-1013 6.
(1984). J . A . FLENDRIG, H. KRUIS a n d H.A. DAS, L a n c e t i
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S. SIDEMANN and D. MANOR, Nephron 311-10 (1982). T.P.A. KRUCKand D.R. MCLACHLAN, Aluminiumand i t s role in biology, In: Metal Ions in Biological Systems, V o l . 24, eds. H. Sigel and A. Sigel, pp. 285-314, Marcell Dekker Inc. Publ. NY & Basel (1988). C.M. YATES, J. SIMPSON, D. RUSSELL and A. GORDON, Brain Res. 197269-274 (1980). K.S. KOSIK, W.G BRADLEY, P.F. GOOD, C.G. RASOOL and D.J. SELKOE, J. Neuropathol. Exp. Neurol. 42365-375 (1983). P. SLANINA, W. FRECH, L-G. EKSTROM, L. LOOF, S. SLORACH and C. ANDERS, Olin. Chem. 32539-541 (1986). R.B. MARTIN, Clin. Chem. 321797-1806 (1986). T.P.A. KRUCK and D.R. CRAPPER-MCLACHLAN, Alzheimer Disease and Related Disorders, pp. ]144-1167, Alan R. Liss Inc. Publ. (1989). G. LEBLONDEL and P. ALLAIN, R e s . Comm. Chem. Path. & Pharm. 27579-586 (1980). W.A. BANKS, A . J . KASTIN and M.A. FASOLD, J . Pharmacol. Exp. T h e r a p e u t . 244 579-585 (1988). B.J. FARNELL, D.R. CRAPPER MCLACHAN, K. BAINBRIDGE, U. DE BONI, L. WONG and P.L. WOOD, Exp. Neurol. 88 68-83 (1985). R.M. CLAYTON, Alternatives in Drug Research, eds A. Rowan and C. Stratman, pp. 153-173, Macmillan, London (1980). C.V. VOORHEES and R.E. BUTCHER, Developmental Toxicology, ed. K. Snell, pp. 13-29, Croom Helm Publ. (1982). V. GLOVER and D.P.L. Green, J. Neurochem. 19 2465-2466. (1972) S.K.A. SEDOWOFIA, J. INNES, A. PETER, E. ALLEVA, A. MANNING and R.M. CLAYTON, Psychopharmacology 97 123-130 (1989). W.M. FOX, Anim. Behav. 13 234-241 (1965). C.G. PICK and J. YANAI, Intern. J. Neurosci. 21 63-66 (1983). W.J. DIXON, BMDP Statistical Software. University of California Press, Berkeley (1985). K. GULYA, Z. RAKONCZY and P. KASA, J. Neur. Chem. 54 1020-1026 (1990). J.M. CRANMER, J.D. WILKINS, D.J. CANNON and L. SMITH, Neurotoxicology
60]-608 (1986). 26. R.A. YOKEL, Toxicol. Appl. Pharmacol. 7__99121-133 (]985). 27. V. BERNUZZl, D. DESOR and P.R. LEHR, Neurobehav. Toxicol and Teratol 8 115-119 (]986). 28. M.S. GOLUB, M.E. GERSCHWIN, J.M. DONALD, S. NEGRI and C.L. KEEN, Fundam. Appl. Toxicol. 8346-357 (1987). 29. J.L. DOMINGO, J.L. PATERNAIN, J.M. LLOBET and J. CORBELLA, Res. Comm. Chem. Path. & Pharmacol. 5_77129-132 (1987). 30. V. BERNUZZI, D. DESOR and P.R. LEHR. Terato14021-27 (1989). 31. G. MULLER, V. BERNUZZI, D. DESOR, M-F. HUTIN, D. BURNEL and P.R. LEHR. Terato142253-261 (1990). 32. H. TSUJII and K. HOSHISHIMA, J. Fac. Agric. Shinsu Univ. I__6613-27 (1979). 33. J . M . DONALD, M.S. GOLUB, M,E. GERSHWIN and C.L. KEEN, Neurotoxicol. & Terato]. 11345-35] (1989). 34. L. FINBERG, H.S. DWECK, F. HOLMES, N. KRETCHMER, A.M. MANER, J.W. REYNOLDS, R.M. SUSKIND and S. HELLERSTAIN, P e d i a t r i c s 78 i150-I153
(1986). 35. G. LAVIOLA, S.K.A. SEDOWOFIA, J. INNES, R.M. CLAYTON and A. MANNING, Psychopharmacology 102383-390 (1990). 36. M.N. ROSSOR, N.J. GARRETT, A.L. JOHNSON, C.G. MOUNTJOY, M. ROTH and L.L. IVERSEN, Brain 105313-330 (1982). 37. D. COLLERTON, Neuroscience 1_99, 1-28 (1986). 38. R.T. BARTUS, L. FLICKER, R.L. DEAN, M. PONTECERVO, J.C. FIGUEIREDO and S.K. FISHER, Pharmacol. Biochem. & Behav. 23125-133 (1985). 39. J. ADAMS, Neurotoxicology 719-34 (1986). 40. R.M. CLAYTON and A. ZEHIR, In: Developmental Toxicology, ed K. Snell, pp. 59-92, Croom Helm Publ. Lond. (1982).
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Pergamon Press
LONG-TERM EFFECTS OF ALUMINIUM ON THE FETAL MOUSE BRAIN R.M. Clayton,
S.K.A. Sedowofia,
J.M. Rankin and A. Manning.
Division of Biological Sciences, I n s t i t u t e of Cell, Animal and Population Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JN. (Received in final form October 7, 1992) Summary
Potentially n o x i o u s s u b s t a n c e s may a c t a s f e t a l t e r a t o g e n s at levels far lower than those required to produce detectable effects in adults, and behavioural teratogenicity may occur at levels lower than those which produce morphological teratogenesis. Aluminium (A1) is a potential neurotoxin in adults. S i n c e p r e g n a n t women may b e e x p o s e d t o u n t o w a r d l e v e l s o f A1 c o m p o u n d s u n d e r c e r t a i n conditions, we h a v e e x a m i n e d t h e long-term effects of treating the pregnant mouse with intraperitoneal o r o r a l a l u m i n i u m s u l p h a t e on b r a i n b i o c h e m i s t r y and behaviour of the offspring. The cholinergic system, as evaluated by t h e a c t i v i t y of choline acetyltransferase (CHAT), was a f f e c t e d differentially in different regions of the brain, and still showed significant effects in the adult. Differences between the intraperitoneal and oral series in the magnitude of effect seen in the regions of the brain probably reflect differences in the effective level of exposure. Growth rate and psychomotor maturation i n t h e p r e - w e a n i n g mouse w e r e a f f e c t e d in the intraperitoneal series only, showing a marked post-natal
maternal effect. The p r e s e n c e o f a l u m i n i u m (A1) compounds in the brains of sufferers of senile d e m e n t i a o f t h e A l z h e i m e r t y p e (SDAT) h a s l e d t o t h e s u g g e s t i o n that A1 a c c u m u l a t i o n in the brain leads to degenerative processes(l). Although A1 i s t h e t h i r d most abundant element, most of it exists in insoluble forms. The limited passage o f A1 s a l t s across the gut, the efficient excretion by t h e k i d n e y s , a n d t h e b l o o d - b r a i n barrier diminish the access of soluble A1 c o m p o u n d s t o t h e b r a i n ( 2 ) . However a c u t e o r c h r o n i c administration o f A1 c o m p o u n d s t o e x p e r i m e n t a l animals by various routes, including the oral(3-5), has established the neurotoxicity of soluble aluminium compounds, as do human conditions such as dialysis encephalopathy(6,7). Many o f t h e n e u r o l o g i c a l diseases i n w h i c h A1 h a s b e e n implicated a r e t h o s e i n w h i c h t h e r e i s damage t o t h e c h o l i n e r g i c system(8), and there is evidence of cholinergic damage i n c o n d i t i o n s where aluminium has been introduced experimentally into the brain(5,9,10). A1 c o m p o u n d s a r e more readily t a k e n up a t a c i d pH o r i n o r g a n i f i e d f o r m . F o r e x a m p l e A1 u p t a k e i s increased by citrate(ll,12) or by complexing with sugars(13,14). Such organification may p e r m i t s o l u b i l i t y in cell membranes, thus affecting permeability and the blood-brain barrier(15). A l u m i n i u m may b i n d t o r e c e p t o r .
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Correspondence: R.M. Clayton, Division of Biological Sciences, I n s t i t u t e of Cell, Animal and Population Biology (Genetics B u i l d i n g ) , U n i v e r s i t y of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JN.
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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sites for several neurotransmitters, to membrane phospholipids, or to calmodulin, thus affecting calcium homeostasis(16). P r e g n a n t women may b e exposed to aluminium compounds in several industries(8), or from environmental pollution in the vicinity of aluminium smelters, or following accidents (several of which have been reported in the U.K.) with aluminium sulphate added to water supplies. The f e t u s i s i n g e n e r a l s u s c e p t i b l e to far lower concentrations of noxious substances than adult tissues(17), and some s u b s t a n c e s may a c t a s b e h a v i o u r a l teratogens, producing long-term or permanent deficits in brain function, often at even lower levels than those required to produce morphological teratogenesis(18). We h a v e u s e d m i c e which, like humans, have a haemochorial placenta, to study the effects of prenatal e x p o s u r e t o a l u m i n i u m s u l p h a t e on b r a i n b i o c h e m i s t r y and b e h a v i o u r a l development, a n d r e p o r t h e r e on l o n g - t e r m e f f e c t s on g r o w t h , b e h a v i o u r , and the cholinergic s y s t e m . The e f f e c t on t h e c h o l i n e r g i c s y s t e m , a s a s s e s s e d by measuring the activity of choline acetyltransferase (CHAT) was c h o s e n b e c a u s e o f t h e known e f f e c t s o f A1 on t h i s n e u r o t r a n s m i t t e r system(9,10). Materials
and methods
Administration of aluminium Aluminium was administered to pregnant CBA mice by two routes. One group of mice was i n j e c t e d i n t r a p e r i t o n e a l l y ( i . p ) with 200mg/kg aluminium sulphate (Alz(SO4)3.18H20, pH 2.5-2.7) or s a l i n e a c i d i f i e d to the same pH as t h e aluminium, from days I0-13 of g e s t a t i o n inclusive. The second group was given aluminium sulphate (750mg/L, pH 4 . ] - 4 . 3 ) as t h e i r s o l e d r i n k i n g w a t e r from days I0-17 of g e s t a t i o n inclusive. Control mice in t h i s group were given water a c i d i f i e d to the same pH as the aluminium sulphate. Determination of choline acetyltransferase a c t i v i t y Control and aluminium exposed m i c e w e r e k i l l e d a t ages ranging from 3 - 4 4 weeks. The brains w e r e dissected into d i f f e r e n t regions including cerebral cortex, cerebellum, hippocampus, midbrain, hypothalamus, nucleus magnocellularis, striatum and the brainstem. Pooled samples from 2-4 mice (not separated by sex), were stored a t -70°C u n t i l used for the determination of ChAT a c t i v i t y by the method of Glover and Green (19). B r i e f l y , samples were homogenised on ice in s a l i n e containing I% b u t a n - l - o l , and assayed with an assay mixture containing 14C acetylCoA a t 37°C for 20 mins. 14C acetylcholine formed was extracted into octan-2-one containing potassium mercuric iodide, and a l i q u o t s counted in a s c i n t i l l a t i o n counter. Each group of age-matched control and t r e a t e d brains was dissected and analysed together. However, the oral s e r i e s were begun a year a f t e r the i n t r a p e r i t o n e a l s e r i e s , and d i s s e c t i o n s were done by a d i f f e r e n t operator. Thus while each control and t r e a t e d group i s wholly comparable, the controls for the i n t r a p e r i t o n e a ] and oral s e r i e s are not comparable. Behavioural and developmental studies On the day of b i r t h ( p o s t - n a t a l day zero, PdO) l i t t e r s i z e s were recorded. The following day p u p s were weighed, sexed, marked by toe c l i p p i n g and c r o s s - f o s t e r e d u s i n g a t o t a l s p l i t - l i t t e r c r o s s - f o s t e r design, and weighed on every t h i r d day from Pd3 u n t i l weaning (see r e f . 20). Thereafter, body weights were recorded weekly. Male and female weights were analysed separately from weaning onwards. Tests for sensori-motor coordination and development were also performed every t h i r d day using a modified version of the Fox b a t t e r y of t e s t s (21). Subjects were t e s t e d for slow r i g h t i n g , c l i f f aversion, grasping with forelimbs, pole grasping, climbing on a wire mesh and eye opening. All other behavioural
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tests were performed as previously described (20), except for the test of learning ability. In these experiments, 10 week o l d m a l e m i c e w e r e t e s t e d in a n 8 - a r m r a d i a l maze f o r 12 d a y s a s d e s c r i b e d by Pick and Yanai (22). Adult activity tests on t h r e e s u c c e s s i v e days were carried o u t a t 22 weeks ( s e e
20). The f o l l o w i n g f o u r e x p e r i m e n t a l groups of mice were established and tested:(1) pups from control litters fostered to control mothers (Cc), (2) pups from control litters fostered t o A1 t r e a t e d m o t h e r s ( C t ) , ( 3 ) p u p s f r o m A1 t r e a t e d litters fostered to control mothers (Tc), a n d ( 4 ) p u p s f r o m A1 t r e a t e d litters f o s t e r e d t o A1 t r e a t e d m o t h e r s ( T t ) . A l l t h e b e h a v i o u r a l studies were c a r r i e d o u t by t h e same o b s e r v e r who was b l i n d t o t h e a l l o c a t i o n of treatment g r o u p s . The s a m p l e s i z e f o r e a c h g r o u p , i n c l u d i n g males and females, in the i . p s e r i e s was a s f o l l o w s : Cc n = 1 3 , Ct n = 1 4 , T t n = 1 2 , Tc n = l l ; a n d f o r t h e o r a l s e r i e s Cc n = 8 , C t n = 9 , T t n=10 a n d Tc n=8. Statistical analysis Pup weights at birth were analysed using Student's t-test. A n a l y s i s o f V a r i a n c e (ANOVA) was e m p l o y e d f o r t h e w e i g h t s and activity scores thereafter (23). Adult activity d a t a was a n a l y s e d u s i n g the Kruskal-Wallis o n e - w a y ANOVA. The c a t e g o r i c a l data generated from the Fox t e s t s was a s s e s s e d b y C h i - s q u a r e t e s t s on e a c h d a y o f o b s e r v a t i o n . ANOVA f o l l o w e d by t - t e s t was u s e d t o a n a l y s e ChAT a c t i v i t y data. Differences at p<0.05 were taken as significant.
Results Levels of aluminium consumed or injected With an average weight of 25g, pregnant mice injected intraperitoneally with aluminium sulphate received a daily dose of 0.41mg of e l e m e n t a l a l u m i n i u m . The mean a m o u n t o f f l u i d c o n s u m e d d a i l y b y c o n t r o l m i c e in the orally e x p o s e d g r o u p was 6 . 0 m l . T h i s c o m p a r e s w i t h a mean o f 4 . 4 ml consumed by the treated mice, and is equivalent to a daily intake of 0.27mg of elemental aluminium. Over a four-day treatment period, i.p mice received a t o t a l o f 1.64mg o f a l u m i n i u m , c o m p a r e d w i t h 1.89mg c o n s u m e d by o r a l l y e x p o s e d mice in seven days. Neurochemistry Our data indicate that pre-natal exposure to aluminium sulphate affects the cholinergic system, as judged by effects on choline acetyltransferase activity. T a b l e 1 s h o w s ChAT a c t i v i t y in different brain regions from mice whose mothers were administered aluminium sulphate by intraperitoneal injection. The d a t a i n t a b l e 2 show t h e a c t i v i t y in mice whose mothers were exposed orally to aluminium. B o t h t a b l e s show s i g n i f i c a n t effects on t h e c h o l i n e r g i c system, and also that the effects are differential between brain regions. It is also clear from the data that, with the exception of the hypothalamus and striatum, the effects persist into adu 1 t h o o d . Developmental and behavioural effects T h e r e w e r e no e f f e c t s on t h e l e n g t h o f g e s t a t i o n , litter size or sex ratio of either A1 t r e a t m e n t . The b i r t h weights of exposed pups in the intraperitoneal series were about 5 per cent below controls (df=l,47 t=2.63 p=0.011) but this difference persisted o n l y i n t h e m i c e r e a r e d by t r e a t e d mothers. Fig. 1 shows this clear maternal effect, and the increasing
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d i v e r g e n c e o f pup w e i g h t s r e a r e d by c o n t r o l o r t r e a t e d m o t h e r s from Pd3 up t o w e a n i n g ( d f = 1 , 4 2 F = ] 6 . 4 3 p = O . 0 0 0 2 ) . The m a t e r n a l e f f e c t on b o d y w e i g h t p e r s i s t e d i n t o a d u l t h o o d f o r f e m a l e mice o n l y ( d r = l , 1 8 F=8.42 p < 0 . 0 1 ) . There were no s i g n i f i c a n t e f f e c t s on body w e i g h t o f pups whose m o t h e r s were exposed t o aluminium s u l p h a t e o r a l l y d u r i n g g e s t a t i o n . Pups exposed t o aluminium v i a t h e i . p r o u t e were s l o w e r t o a t t a i n a d u l t s c o r e s on s e v e r a l o f t h e Fox s c a l e m e a s u r e s o f r e f l e x development(21): i n c l u d i n g slow r i g h t i n g , f o r e l i m b g r a s p i n g ( F i g . 2) and p o l e g r a s p i n g ( F i g . 3 ) , a l l p<0.05. In a d d i t i o n , we have found a m a t e r n a l e f f e c t on s e n s o r i - m o t o r performance. F i g s . 2 and 3 show t h a t t r e a t e d pups r e a r e d by t r e a t e d m o t h er s ( T t ) a r e more s e v e r e l y a f f e c t e d than t r e a t e d pups r e a r e d by c o n t r o l m o t h e r s (Tc) o r c o n t r o l pups r e a r e d by t r e a t e d m o t h e r s ( C t ) , b u t a l l t h e s e groups a r e affected as c o m p a r e d t o c o n t r o l s r e a r e d by c o n t r o l mothers (Cc). The d i v e r g e n c e between t h e two e x t r e m e g r o u p s , c o n t r o ] pups r e a r e d by c o n t r o l m o t h e r s , and t r e a t e d pups r e a r e d by t r e a t e d m o t h er s i s t h e w i d e s t . The s i g n i f i c a n c e f o r p o l e g r a s p i n g , f o r example, i s p<0.001. The o t h e r t e s t s were n o t f o u n d t o be s i g n i f i c a n t . Open f i e l d activity levels were largely u n a f f e c t e d up t o weaning.
TABLE I CHOLINE ACETYLTRANSFERASE ACTIVITY IN CBA MICE EXPOSED PRE-NATALLY TO ALUMINIUM SULPHATE 3 weeks
CEREBRAL CORTEX CEREBELLUM HIPPOCAMPUS MIDBRAIN HYPOTHALAMUS STRIATUM
CONTROL
TREATED
213.4± 43.3± 744.4± N.D. N.D. 459.6±
2 ] 0 . 9 ± 4.8 50.7± 3.1 580.2± 8.2* N.D. N.D. 251.5± 5.2*
9.3 0.7 7.6
9.7
34 weeks CONTROL
17 weeks
CONTROL
TREATED
440.3± 2.6 328.9± 8.3* 98.5± 2.7 64.9± 1 . 9 ' I015.6±31.0 917.9± 3.6 793.3±13.1 856.6± 3.9* 665.7±19.9 573.9±26.7 2141.7&52.7 1871.4±43.9'
44 weeks TREATED
CEREBRAL CORTEX 3 0 9 . 4 ± 2 2 . 0 180.6± 1 . 4 . CEREBELLUM 50.9± 1.1 46.5± 0 . 9 * HIPPOCAMPUS 381.1±18.1 306.7± 1 . 4 . MIDBRAIN 649.4± 7.8 552.1± 6.3* HYPOTHALAMUS 530.9+2.1 433.8± 6.3* STRIATUM 1047.7±24.3995.0± 7.0
CONTROL
TREATED
247.0± 5.3 73.2± 1.1 567.2± 6 .0 505.7±15.0 247.5± 2.4 431.7± 8.4
215.4± 2.5* 45.6± 1 . 0 . 545.5±10.0 439.8± 3.8* 342.2± 9.0* 728.2±16.0.
Choline acetyltransferase activity in mouse b r a i n . B r a i n s were dissected from offspring whose mothers were injected intraperitoneally w i t h 200mg/kg aluminium s u l p h a t e o r s a l i n e on g e s t a t i o n days 10-13. Enzyme a c t i v i t y e x p r e s s e d as p i c o m o l e s / m g protein/minute. Each r e s u l t represents t h e Mean ± SE o f 4 d e t e r m i n a t i o n s . Values *p<0.05; N.D.= Not D e t e r m i n e d .
Vol.
51, No.
25,
1992
Aluminium
Effects
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1925
Measurements of activity a t w e a n i n g a n d i n a d u l t l i f e show p e r s i s t e n t trends towards a maternal treatment effect. At 22 w e e k s , a f i v e m i n u t e a c t i v i t y t e s t was p e r f o r m e d . T h e r e was n o s i g n i f i c a n t difference b e t w e e n g r o u p s on d a y 1 when t h e y w e r e i n t r o d u c e d t o t h e f i e l d . However, there were marginally significant differences between treatment groups in the total number of s q u a r e s c r o s s e d on d a y s 2 a n d 3, p u p s r e a r e d by t r e a t e d mothers had lower activity levels.
TABLE II ChAT ACTIVITY IN CBA MICE EXPOSED TO ALUMINIUM SULPHATE PRE-NATALLY (ORALLY ADMINISTERED TO MOTHERS FROMDAY 10-17 OF PREGNANCY) 3.5 Weeks CONTROL CEREBRAL CORTEX CEREBELLUM HIPPOCAMPUS MIDBRAIN HYPOTHALAMUS NUCLEUS STRIATUM BRAINSTEM
16 Weeks
TREATED
CONTROL
TREATED
197.3± 3.8 322.9± 2.5* 516.5±12.1 3 5 8 . 2 ± 5 O* 105.5± 1.2 61.4± 4.1' 97.6± 2.3 8 7 . 3 ± 0 8* 568.9± 8.5 661.5±26.6' 678.7± 5.8 7 2 5 . 9 ± 1 3 O* 7 3 3 . 9 ± 1 8 . 6 498.1± 9.1, 542.7± 6.1 562.8± 6 7* 3 3 8 . 4 ±9.0 442.7± 2.7* 564.4± 8.7 478.8± 3 6* ]289.1± 5.3 1173.9±14.6, 1695.2±13.5 1415.1± 4 5* 831.6± 7.7 751.2± 9.9* 1426.4± 5.4 868.3±10.5, 8 7 0 . 7 ± 1 2 . 8 890.9± 6.2 8 4 1 . 1 ±7.1 977.6±45.9,
Choline a c e t y l t r a n s f e r a s e a c t i v i t y in mouse b r a i n . Brain dissections and measurement of ChAT a c t i v i t y were as in table I. Values *p<0.05 were s i g n i f i c a n t l y d i f f e r e n t from controls. T e s t s a t 10 w e e k s o f a g e f o r l e a r n i n g a b i l i t y and retention in an 8arm r a d i a l maze c o u l d b e made w i t h s m a l l n u m b e r s o n l y . 10 o u t o f 13 c o n t r o l a n i m a l s (77%) r e a c h e d c r i t e r i o n [two consecutive errorless trials (22)] and 6 o u t o f 11 (55%) o f t h e A ] - e x p o s e d g r o u p . The e x p o s e d m i c e w h i c h d i d r e a c h criterion w e r e s l o w e r t o do s o ( a m e d i a n o f 7 . 5 d a y s , c o m p a r e d t o 6 d a y s f o r controls), but this difference did not reach statistical significance.
Discussion In both series the effects on ChAT l e v e l s f o l l o w i n g p r e n a t a l exposure t o A1 a r e d i f f e r e n t i a l between brain regions: a finding also reported for the neurotoxic effects i n A1 e n c e p h a l o p a t h y in adult rats (24). Table 2 shows that significant effects are found i n ChAT a c t i v i t y following administration by t h e o r a l r o u t e , implying sufficient access of aluminium to the fetal brain for neurotoxic effects to be obtained. I t i s c l e a r f r o m t h e d a t a on ChAT t h a t t h e e f f e c t s o f a l u m i n i u m on t h e developing brain are not transient, but persist into adult life, although there are deviations from this trend in the striatum and hypothalamus in the oldest animals. The d i f f e r e n c e s in the extent t o w h i c h ChAT a c t i v i t y was affected in the different r e g i o n s o f t h e b r a i n may b e d u e t o t h e l e v e l s o f aluminium reaching each region, or to differences in the distribution of cholinergic neurons in the brain.
regions
There were also differences of the brain are compared
in the effects on ChAT when t h e same in the intraperitoneal and oral series.
1926
Aluminium
Effects
on Fetal B r a i n
CBA i v" ~
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Effects of prenatal exposure to g r a s p i n g (2) a n d on p o l e g r a s p i n g
aluminium sulphate on f o r e l i m b (3) on p o s t - n a t a l d a y 15.
25,
1992
Vol.
51, No. 25,
1992
Aluminium Effects on Fetal
Brain
1927
T h i s may r e f l e c t a dose effect. I t h a s b e e n shown t h a t a l u m i n i u m l e v e l s in the placentas of pregnant female mice injected intraperitoneally w e r e 10 times higher than in controls, while they were only three times higher than controls in orally treated females(25). Also a rise in performance in a classically conditioned reflex test(26) has been reported in rabbits f o l l o w i n g low l e v e l s o f h l a d m i n i s t r a t i o n with a fall following high levels. Effects of prenatal a l u m i n i u m on p o s t - n a t a l growth have also been reported by several other workers (27-31), and other investigators have reported similar results to ours, in that hl treatment in utero affects the development of certain but not all sensori-motor skills (32,33). Treatment of infant mice similarly affects motor skills (27), and regular clinical administration o f A1 t o human i n f a n t s is neurotoxic (34). The i n c r e a s i n g d i v e r g e n c e b e t w e e n t h e pup w e i g h t s o f o f f s p r i n g reared by control or treated m o t h e r s up t o w e a n i n g , may b e d u e t o r e t e n t i o n of a l n m i n i u m by t h e m o t h e r a n d i t s r e l e a s e into the milk, to effects on m i l k output(26), or to effects on m a t e r n a l b e h a v i o u r ( 3 5 ) , but the neurochemical sequelae would imply that the pups received A1 i n t h e m i l k . Although Yokel(26) found relatively low l e v e l s o f A1 i n t h e m i l k o f t r e a t e d dams, it may b e i n o r g a n i f i e d c o m p l e x e s a n d t h u s r e a d i l y a b s o r b e d by t h e n e o n a t e . A l t h o u g h some b e h a v i o u r a l m e a s u r e s show e f f e c t s , others do n o t , and overall the behavioural effects do n o t seem c o m m e n s u r a t e w i t h t h e d e g r e e o f ChAT d e f i c i t . Such d i s p a r i t i e s c a n a l s o o c c u r i n SDAT b r a i n s (36,37). It m u s t b e p o i n t e d o u t t h a t r e s p o n s e s t o d e p r e s s e d l e v e l s o f ChAT may b e h i g h l y selective amongst different t y p e s o f memory ( 3 8 ) s o t h a t o u r b a t t e r y o f t e s t s (20) and the effects on m i c e do n o t p e r m i t u s t o p r e d i c t any particular loss of mental functions i n man. H o w e v e r , Adams ( 3 9 ) h a s p o i n t e d t o t h e s i m i l a r i t y of behavio,~ral responses i n humans and r o d e n t s after exposure to several different types of behavioural teratogens, s u g g e s t i n g t h a t m i c e may p r o v i d e a very suitable model for behavioural teratogenicity in humans. I t may a l s o b e t h a t r e d u c e d ChAT a c t i v i t y increases the liability of the system to other forms of stress, a possibility which requires investigation.
F i n a l l y we h a v e c l e a r e v i d e n c e f o r g e n e t i c d i f f e r e n c e s in response to teratogens,(17,20,40) and for sensitivity to aluminium. P r e g n a n t C57 m i c e , g i v e n t h e same i n t r a p e r i t o n e a l d o s e l e v e l s a s w e r e u s e d f o r t h e CBA g e n o t y p e and reported here, failed to continue to term. Acknowledgements
to
an
We a r e g r a t e f u l to the anonymous referee for
UK H e a l t h a n d S a f e t y helpful c o m m e n t s on
Executive for support and an earlier draft of this
manuscript. References 1. 2.
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