Developmental patterns of aluminum in mouse brain and effects of dietary aluminum excess on manganese deficiency

Toxicology, 81 (1993) 33-47 Elsevier Scientific Publishers Ireland Ltd.

33

Developmental patterns of aluminum in mouse brain and effects of dietary aluminum excess on manganese deficiency Mari S. Golub, Bin Han, Carl L. Keen and M. Eric Gershwin Department of lnternal Medicine, University of California, Davis, Davis, CA (USA) (Received July 31st, 1992; accepted February 12th, 1993)

Summary Previous studies have shown that excess dietary AI during development can affect neurobehavioral measures and decrease tissue Mn of 21-day-old weanling mice without a corresponding increase in tissue AI concentrations. AI and Mn have similar tissue concentrations and similar affinities for transferrin, which is the major plasma transport protein for AI and Mn as well as Fe. In the present study, brain AI, Mn and Fe were studied at 6, 12, 18 and 24 days of age in offspring of Swiss Webster mice fed a semipurified diet containing excess AI (All+l, 1000 #g Al/g diet, AI as AI lactate), marginal Mn (Mn[-], 3/~g Mn/g diet) or both excess AI and marginal Mn (AI[+]Mn[-]) from conception to day 24 postnatal (weaning on day 18). Brain AI concentrations were higher at 6 days of age than at later ages and were significantly elevated by the excess AI diet (P = 0.017) but returned to control levels by weaning. Brain Mn concentrations increased from day 6 to day 24 and were lower in the Mn deficient groups (P < 0.001) and also in the excess AI group (P = 0.024) than in controls. Brain Fe concentrations were not influenced by diet. Similar patterns were seen in liver as in brain. The marginal Mn diet led to postnatal growth retardation which was more severe in litters of dams fed AI[+IMn[-I diets than in litters fed Mn[-] diet. These data suggest that excess AI in diet can interact specifically with Mn metabolism during development.

Key words: Aluminum; Manganese; Mice; Diet; Development; Brain

Introduction In p r e v i o u s studies we d e m o n s t r a t e d n e u r o b e h a v i o r a l c h a n g e s in w e a n l i n g m i c e after d e v e l o p m e n t a l a l u m i n u m (AI) e x p o s u r e [1,2]. O t h e r s h a v e also d e m o n s t r a t e d A1 d e v e l o p m e n t a l t o x i c i t y s y n d r o m e s , i n c l u d i n g n e u r o b e h a v i o r a l effects [ 3 - 6 ] but little is k n o w n a b o u t p o s s i b l e m e c h a n i s m s . In o u r p r e v i o u s studies we did n o t find e l e v a t e d b r a i n AI c o n c e n t r a t i o n s in A l - e x p o s e d w e a n l i n g s ; h o w e v e r , m a n g a n e s e ( M n ) a n d i r o n (Fe) tissue levels w e r e altered. T h e s e effects o n essential trace m e t a l s did n o t o c c u r in a d u l t m i c e e x p o s e d to AI [7]. T h i s s u g g e s t e d t h a t a direct effect o f A1, if it o c c u r r e d , m a y h a v e i n v o l v e d e l e v a t e d b r a i n A1 d u r i n g d e v e l o p m e n t a l p e r i o d s

Correspondence to: Mari S. Golub, California Primate Research Center, University of California, Davis, Davis, CA 95616, USA. 0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

35

Diets and feeding Mice were fed control diet ad libitum for 1 week prior to mating and then were transferred to one of the four experimental diets from pregnancy detection. The pelleted semi-purified diet [1] was based on sprayed egg white as the protein source to avoid metal ligands that occur in grain based diets. Vitamins and minerals were added at levels recommended by the National Research Council (NRC) [24]. The minimal achievable A1 concentration using purified ingredients was 7/~g Al/g diet. AI lactate was added to achieve concentrations of 25/~g Al/g diet for the control diet and 1000/~g A1/g for the excess A1 diets. The A1 concentration of the control diet was below the concentration found in commercial mouse chow (200 ~g/g as determined in our laboratory). Mn concentration of the control diet was 35 /zg/g, as recommended by the NRC. Trace metal concentrations of each batch of diet were confirmed in our laboratory when received from the vendor (Dyets, Inc., Bethlehem, PA). AI excess and Mn deficient diets were based on previous studies in mice [2,22]. Distilled deionized water was provided in plastic bottles to minimize trace metal exposure via drinking. After weaning pups continued to receive the experimental diet assigned to their dams until they were killed for tissue sampling on day 24.

Study design Four treatment groups were formed based on maternal diet: control diet: 25/zg A1/g and 35/zg Mn/g; Mn[-] diet: 25 #g A1/g and 3/xg Mn/g; AI[+] diet: 1000 #g Al/g and 35 #g Mn/g: AI[+]Mn[-] diet: 1000/zg Al/g and 3 #g Mn/g. Animals were assigned to diet groups randomly at the time of pregnancy detection. When necessary, group assignments were changed to balance initial body weight of dams across groups (stratified randomization). Litters with < 6 pups at birth or litters in which all pups died postnatally were not included in group comparisons.

Weights, food intake and toxic signs Maternal food intake was determined at 3-day intervals beginning at pregnancy detection. Preweighed portions of the pelleted diet were provided and remaining diet (including partially eaten pellets retrieved from bedding) was weighed to determine intake. Dams were weighed at conception and day 16 gestation. Dam and pup weights and pup toxic signs [1] were obtained at 0, 6, 12 and 18 days postnatal. Tissue trace element concentrations On postnatal days 6, 12, 18 and 24, 6-8 pups/group were selected for tissue trace element concentration assays (A1, Mn, Fe) with the provision that all litters were maintained with at least 6 pups, and that no two pups of the same group came from the same litter. To obtain basic information about tissue AI levels at earlier time points, a smaller experiment was performed in which organs were pooled within litters for 0 and 3 day pups in pups receiving a low AI diet (7/~g A1/g diet). Organ weights were obtained prior to freezing and all tissue mineral concentrations were expressed as nmol/g wet weight. During dissection, precautions were taken to avoid aluminum contamination by using plastic instruments, bench coverings, freezer containers, etc. Tissues were frozen at -20°C until analysis.

36 Tissues were ashed and analyzed for concentrations of AI, Mn and Fe by graphite furnace atomic absorption spectrometry as previously described [7]. AI recovery was determined by the standard-addition method using tissue matrix and a commercial AI stock solution. The accuracy of the method was verified by the analysis of an NBS bovine liver sample. The experimentally determined A1 concentration was within 10% of the certified value. Mn and Fe assays were also validated against NBS standards.

Statistical analysis Most measures were analyzed with ANOVA using age and diet group (control vs. All+], Mn[-] or AI[+]Mn[-]) as factors. Because of unequal group sizes and variances occasioned by postnatal mortality in the AI[+]Mn[-] group, each of the diet groups was compared to the control group in separate ANOVAs. Group differences were evaluated at individual ages only if a significant interaction between the age and diet variables was indicated. Group data are expressed as mean 4- S.E.M. Maternal body weight and food intake and pup body weight (based on like-sexed litter means) analyses were conducted with repeated measures ANOVA with day as the repeated measure. ANOVAS for pup body weight were also conducted separately at each time point because the number of pups per litter changed due to use of pups for tissue trace metal analysis. Only one pup from each litter was used for tissue trace element assays at any one time point. Tissue weights and trace metals were examined in the same subsample of litter mates. Matched pair t-tests were used to compare brain vs. liver concentrations with trace elements. Effects were considered statistically significant at P < 0.05. Results

Breeding success Table I shows pregnancy completion data. There were no effects of diet group on mating, pregnancy completion or littersize. Postnatal mortality was greatest in the AI[+]Mn[-] group. Because pups were being culled for tissue analysis at intervals during the postnatal period, analysis of per cent mortality was not possible.

Dam body weights, food intake There were no diet group effects on dam body weight, body weight gain or food intake during either gestation or lactation (Fig. 1). Although dams fed the Mn deficient diet (Mn[-] and AI[+]Mn[-] groups) weighed less than controls throughout lactation (Fig. 1) differences were small (2 g or less) and did not reach statistical significance.

Offspring growth and toxic signs There were no group differences between pup body weights in the control and the AI[+] group at any age (Fig. 1). Postnatal body weights of pups in Mn deficient litters were lower than those of controls and this effect was greater in AI[+]Mn[-] pups than in Mn[-] pups (Fig. 1). Mean weight of pups in Mn[-] litters were lower than those in control litters on days 6 (F = 5.28, P = 0.027), 12(F = 6.24, P = 0.017), and

37 TABLE I BREEDING SUCCESS, POSTNATALMORTALITYIN THE FOUR DIET GROUPS. Diet group

Mateda Completed pregnancy 100% preweaning mortality Final sample Litter size at birth

Control

Mn-

AI÷

Al+Mn-

17b 11 0

17 12 0

19 13 2

14 9 1

11 8.7 ± 0.5c

12 8.7 ~ 0.2

11 9.1 ± 0.6

8 9.4 ± 0.4

aAs indicated by vaginal plug. bNumber of dams/litters. CNumber of pups (mean ± S.E.M.).

18 (F = 9.92, P = 0.002). Mean pup weights in AI[+]Mn[-] litters were significantly less than those of controls at each age (day 0 F = 4.11, P = 0.050; day 6, F = 5.29, P = 0.027; day 12, F = 10.61, P < 0.001; day 18, F = 20.21, P < 0.001; day 24, F = 7.43, P = 0.011). In addition, body weights of AI[+]Mn[-] litters were lower than those of Mn[-] litters at weaning (day 18) (F = 6.71, P = 0.014). There were no sex effects on body weights or interactions between sex and diet group. There were no group differences in reports of toxic signs in pups during lactation. Reduced activity and ataxia were the only toxic signs reported.

Brain and liver weights There was no effect of excess dietary AI on pup brain and liver weights (Table II). Dietary Mn deficiency was associated with lower organ weights, and this effect was greater in the AI[+]Mn[-] group than the Mn[-] group. Pup brain and liver weights in the Mn-deficient groups were less than those of controls across all ages during lactation (significant effect of diet, no significant interaction between diet and age; see Table II). Brain weight as a percent of body weight was somewhat higher in the Mndeficient groups than in controls indicating brain sparing, while liver weights as a percent of body weight were lower than controls, indicating a somewhat greater growth retarding effect in liver than in the body as a whole. Brain and liver trace metal concentrations Figure 2 presents trace metal data from the control group. This figure is helpful in comparing concentrations of the three trace metals in different organs at various ages without the influence of altered diet. Comparing concentrations of the three trace metals, Fe concentrations were at least an order of magnitude higher than those of AI and Mn in both brain and liver at all ages, while AI and Mn concentrations were similar. Comparing brain and liver, Fe and Mn concentrations were higher in liver than in brain across all ages (Fe, F = 72.01, P = 0 . 0 0 0 1 ; Mn,

38

Dam's

32

"-"

28

~"

26

e-

24

•~

22

N

20

>' "t3

18

a

16

..... e.-- -

Mn-

--

AI+

0

42

14

control

..... e,*.*

AI+Mn-

12 10

,

,

0

6

days

la

~

,

,

12

18

i"' 24

postnatal

Pup

12 11

~ ~

lO 9 8

°~

7

.

5 ~

o

J~***"

4. I ~ a lj"

a .....

*----

:

control

I

M.AJ+

I I

2

0

0

6

days

12

18

24

postnatal

Fig. 1. Body weights of dam and pups during the postnatal period. There were no statistically significant group differences between control dams and dams in other diet groups at any time point. The mean weight of pups in Mn[-] litters were less than controls at 6, 12 and 18 days of age. Pups in Al[+]Mn[-] litters weighed less than controls at 6, 12, 18 and 24 days postnatal (see text for statistical analysis).

TABLE 11 BODY A N D O R G A N WEIGHTS OF PUPS IN EACH OF THE F O U R DIET C O M P A R I S O N G R O U P S Weights are for the subsample of pups contributing to tissue trace metal analysis Age (days postnatal) 6

12

18

24

Mean

S.E.M.

Mean

S.E.M.

Mean

S.E.M.

Mean

Pup body weight (g) Control MnAI+ AI+Mn-

4.55 3.78 4.24 3.79.

0.29 0.13 0.18 0.21

7.09 6.25 7.34 4.87

0.28 0.28 0.24 0.47

9.33 8.90 9.19 7.54

0.30 0.24 0.30 0.42

14.32 13.4 16.07 11.54

Pup liver weight (g) Control MnAI+ AI+Mn-

0.168 0.130 0.158 0.118

0.012 0.007 0.008 0.007

0.326 0.246 0.297 0.182

0.021 0.014 0.013 0.023

0.511 0.448 0.446 0.380

0.017 0.018 0.020 0.026

0.890 0.807 0.993 0.754

Pup brain weight (g) Control MnAI+ AI+Mn-

0.247 0.230 0.232 0.235

0.008 0.005 0.008 0.010

0.354 0.335 0.350 0.302

0.004 0.004 0.005 0.016

0.374 0.369 0.361 0.354

0.004 0.004 0.008 0.004

0.394 0.375 0.412 0.371

Pup liver weight (%) Control MnAI+ AI+Mn-

3.67 3.42 3.72 3.11

0.089 0.118 0.1 04 0.107

4.64 3.90 4.04 3.68

0.31 0.08 0.07 0.11

5.51 5.00 4.85 4.99

0.14 0.09 0.15 0.10

6.17 6.05 6.17 6.82

Pup brain weight (%) Control MnAI+ AI+Mn-

5.53 6.13 5.50 6.29

0.156 0.156 0.105 0.198

4.64 3.90 4.04 3.68

0.31 0.08 0.07 0.11

4.09 4.20 4.03 4.88

0.12 0.09 0.16 0.25

2.81 2.85 2.58 3.40

ap value reported is for the group factor of a two factor A N O V A (group by age). For each endpoint, 3 ANOVAS were cor the group factor (control vs. Mn-, control vs. AI+, control vs. AI+Mn-). The effect of age was significant for each of the end[ interactions between group and age.

4O

Brain-control

diet

10000 A 1000

~

Fe

[]

Mn

O

AI

100

0

E c

0

I

I

I

I

6

12

18

24

30

days of a g e Liver-control

diet

10000

1000

f

¢~ "-o

13 O

Mn AI

100

E

¢-

10

0

I

I

I

I

6

12

18

24

30

days of age Fig. 2. Comparison of AI, Mn and Fe concentrations in brain and liver of pups in the control diet group. M n and Fe concentrations were lower in brain than liver while AI concentrations were higher in brain than liver. Mn and Fe concentrations increased significantly from days 6 to 24 postnatal in both brain and liver while AI concentrations did not (see text for statistical analysis).

41

Brain AI 45 40

a -

- ....

35

.

.

.

.

.

.

control

e----

Mn-

e-.-,

AI+ AI+Mn-

30 o

O E c

25 2015 lO 5 0

0

I

I

I

6

12

18

I

24

days of age Liver

AI

16 a ..... e----

14 •

Mn-

--

12

Al+

. . . . . e- . . . .

|

C

control

emlgse

AI+Mn-

Qe

6

o.', ,.""°'°°"

2 0

0

I

I

I

6

12

18

I

24

days of age Fig. 3. Brain and liver AI concentrations in pups from each of the four diet comparison groups. AI concentrations were significantly higher in AI[+] and AI[+]Mn[-] groups than in controls.

42

F = 93.74, P -- 0.0001), while AI concentrations were higher in brain than in liver (F--39.59, P = 0.0001). Comparing ages, Fe and Mn concentrations changed significantly in both brain and liver over the ages studied (Fe, brain, F-- 16.01, P--0.001; liver F = 8 . 8 7 , P--0.001: Mn brain F - - 57.95, P = 0 . 0 0 1 ; liver F -- 28.25, P -- 0.001). Mn increased more rapidly prior to weaning (day 18), particularly in liver, while Fe increased more rapidly after weaning (see also Figs. 4 and 5). AI concentrations did not show significant age-related effects (brain P = 0.053; liver P = 0.252). Excess dietary AI (control vs. AI[+] group) led to higher A1 and also lower Mn tissue concentrations in both brain and liver when Mn dietary levels were adequate. ANOVA demonstrated a significant effect of AI[+] diet on brain A1 (F-- 6.27, P -- 0.017), liver AI ( F = 5.06, P -- 0.031), brain Mn (F-- 5.49, P -- 0.024), and liver Mn (F -- 13.22, P < 0.001) (Figs. 3 and 4). There was a significant effect of age for brain and liver Mn (P < 0.001), but not for brain and liver A1. There were no significant interactions between diet group and age. Neither brain nor liver Fe concentrations were altered by excess dietary A1 (Fig. 5). Dietary Mn deficiency (control vs. Mn[-] group) was associated with lower brain and liver Mn concentrations (brain, F - - 134.69, P < 0.001; liver, F - - 23.23, P < 0.001) (Fig. 4). AI concentrations were greater at some ages in Mn[-] pups relative to controls, but differences were not statistically significant (0.10 > P > 0.05). Neither brain nor liver Fe concentrations were altered by dietary Mn deprivation (Fig. 5). Combined AI excess and Mn deficiency (control vs. AI[+]Mn[-] group) led to higher AI concentrations in both brain and liver (brain 15.93, P < 0.001; liver F = 21.73, P < 0.001) as well as lower Mn concentrations (brain F = 149.87, P < 0.001; liver F = 21.73, P < 0.001) as would be anticipated from the dietary content (Figs. 3 and 4). These effects were no greater in the combined group than in individual diet groups [AI[+] and Mn[-]]. There were no effects on Fe concentrations in either brain or liver (Fig. 5). Because organ weights were lower in Mn-deficient groups, statistical analyses of trace metal data were repeated using total organ content rather than concentration. These analyses had similar results to analyses based on concentration with the exception that the trend toward elevated AI concentrations in the Mn[-] group was not apparent on a whole organ basis. Pup brain and liver A1 levels on days 0 and 3 postnatal were determined using homogenates obtained by pooling organs within litters (8 litters/group). This experiment was conducted only with a low A1 diet (7 #g Al/g diet) to confirm the pattern of decreasing A1 tissue concentrations during lactation. AI concentrations decreased 53% in brain (t -- 9.12, P < 0.001) and 83% in liver (t = 7.07, P < 0.001) from day 0 to day 3. Discussion

A primary goal of this study was to obtain information on developmental changes in brain Al concentrations in mice. Brain Al concentrations were found to be higher at 6 days of age than later in the postnatal period (days 12, 18 and 24). In supplemen-

43

Brain Mn 16

•~

C

14

a control ..... e---* Mn-

10

. . . . . o-- o

/z~ / ~

~=

AI+Mn-

/

f

II

6

***'*'~°"

,

2

• .... .....g

0

0

I

I

6

12

I

I

18 of a g e

days

24

Liver Mn

1i

....

a Mncontrol ,,,T e---

...=.. ~E¢3}~ 40

= • ""

0

~,+

/,\

AI+Mn-~

~

i

I

I

I

6

12

18

24

days

of a g e

Fig, 4. Brain and liver Mn concentrations in pups from each of the four diet comparison groups. Mn concentrations were significantly lower in the All+], Mn[-], and AI[+]Mn[-] groups than in controls (see text for statistical analysis).

44

Brain Fe 0.34 t 0.32 1

a

control

0.30 "~

..... ~ " ' "

0.28 jl

..... e-.-*

~.

..~

Mn-

1" / Z.-'I'

A,,

AI+Mn-

**~o-"

_=P 0261 0 E

0.24 1 O.PP I 0.20

"""

:

i

0-18 1 0.161 0

,

,

,

6

12

18

,

24

days of age

Liver Fe 4.0 = ..... ~'--~-

3.5

lllllO=lll

3.0 ¢:~

2.5

0

2.0

E ..~

1.5

control MnAI+ AI+Mn-

.•

/

/

;~

1.0 0.50.0 0

6

12

18

24

days postnatal Fig. 5. Brain and liver Fe concentrations in pups from each of the four diet comparison groups. There were no significant differences between diet groups.

45 tal studies, brain AI concentrations were also found to be higher at birth than at 3 days of age. Thus brain AI concentrations decreased during the period of most rapid brain growth. A similar pattern was seen in liver. Also, brain and liver AI concentrations were higher in offspring of dams fed excess A1 than in controls. Notably, elevated brain and liver AI concentrations caused by excess dietary AI were no longer apparent by weaning in the mouse. Clearly, studies tracking brain AI at even earlier developmental periods are needed to understand the dynamics of early A1 accumulation and its potential influence on brain development. It is possible that previously reported neurobehavioral effects of excess dietary AI in weanling mice [1,2] are mediated by elevated brain AI concentrations during earlier developmental periods. Developmental A1 concentration changes did not resemble those of the essential elements Mn and Fe, which showed a several fold increase during the postnatal period as reported previously in mice [11]. Increased tissue concentrations of Fe and Mn from days 6 to 18 can be attributed primarily to uptake from maternal milk. A1 may not be obtained in substantial amounts from milk but may reach offspring primarily via maternal-fetal transfer during gestation. Relative concentrations of AI, Mn and Fe in mouse milk are not known. In guinea pig milk trace element concentrations have been reported as 0.81 /~g Al/g, 0.71 #g Fe/g and 0.019/xg Mn/g [25]. Lactoferrin may be important for Fe and Mn uptake from milk but little is known about AI in this regard. Brain AI concentrations were higher than liver AI concentrations at all ages. This result has been consistently obtained in both adult and developing mice fed diets similar to those used here [2,7,26] and has also been reported in humans [27]. However, other studies in which AI was administered via injection or in drinking water to rabbits or rats reported similar A1 concentrations in brain and liver [28-31]. A second goal of this study was to examine effects of excess dietary AI on tissue Mn and on Mn deficiency syndromes during development. Lower Mn concentrations were previously found in the brain and liver of weanling mice after exposure to excess dietary AI during development. In the present study, excess dietary AI also decreased brain and liver Mn concentrations in offspring of dams fed Mn sufficient diets, but did not further decrease Mn concentrations in offspring of dams fed Mn deficient diets. Lower Mn concentrations persisted through the time period studied (24 days postnatal). In addition, excess dietary A1 apparently exacerbated the effects of Mn deficiency on postnatal mortality and body and organ growth. Body weights, body weight gain and food intake of dams during lactation were not influenced by either excess dietary A1 or marginal dietary Mn. Other studies also indicate that similar dietary Mn deprivation does not produce an Mn deficiency syndrome in adult mice [32]. A growth retarding effect of developmental A1 was seen previously in an early study from this laboratory [33] which used a standard purified mouse diet (Lueke diet), but not in a second study [1] which used an optimal diet (met or exceeded NRC recommendations for all nutrients), similar to the diet as used for the present study. The difference between the results of these two studies as regards growth retardation may be partially due to the Mn content of the diets, which was 10 #g Mn/g diet for the Lueke diet and 55 #g Mn/g diet for the optimal NRC diet. The present study

46 suggests that while excess A1 does n o t cause M n deficiency it might exacerbate a marginal M n deficiency. N o effect of excess dietary AI or deficient dietary M n on Fe tissue c o n c e n t r a t i o n s were noted in this study a l t h o u g h such effects have been noted previously in older mice [2,7]. Longer periods of AI exposure, or exposures at later ages may be required. T a k e n together, these results suggest that excess dietary AI during development can produce a short term increase in b r a i n A1 a n d a longer term decrease in tissue M n concentrations. In addition, excess dietary A1 can exacerbate developmental M n deficiency syndromes. These results confirm a n d extend previous observations that dietary A1 can interact with M n m e t a b o l i s m d u r i n g developmental periods. These effects obtained at the whole a n i m a l a n d tissue level may be indicative of interference with M n m e t a b o l i s m of sensitive cell types or developmental processes that are of greater toxicologic consequence. T o investigate this further experiments tracking ontogeny a n d function of M n d e p e n d e n t enzymes are planned.

Acknowledgments The authors appreciate the technical assistance of C. June Savage. Supported by NIH-ES04190.

References 1 J.M. Donald, M.S. Golub, M.E. Gershwin and C.L. Keen, Neurobehavioral effects in offspring of mice given excess aluminum in diet during pregnancy and lactation. Neurotoxicol. Teratol., 11 (1989) 345. 2 M.S. Golub, C.L. Keen and M.E. Gershwin, Neurodevelopmental effect of aluminum in mice: Fostering studies. Neurotoxicol. Teratol., 14 (1992) 177. 3 G. Muller, V. Bernuzzi, D. Desor, M.F. Hutin, D. Burnel and P.R. Lehr, Developmentalalterations in offspring of female rats orally intoxicated by aluminum lactate at different gestation periods. Teratology, 42 (1990) 253. 4 V. Bernuzzi, D. Desor and P.R. Lehr, Developmental alterations in offspring of female rats orally intoxicated by aluminum chloride or lactate during gestation. Teratology, 40 (1989) 21. 5 V. Bernuzzi, D. Desor and P.R. Lehr, Effects of postnatal aluminum lactate exposure on neuromotor maturation in ~the rat. Bull. Environ. Contam. Toxicol., 42 (1989) 451. 6 M. Gomez, J.L. Domingo and J,M. Llobet, Developmental toxicity evaluation of oral aluminum in rats: Influence of citrate. Neurotoxicol. Teratol., 13 (1991) 323. 7 M.S. Golub, B. Han, C.L. Keen and M.E. Gershwin, Effects of dietary aluminum excess and manganese deficiencyon neurobehavioral endpoints in adult mice. Toxicol. Appl. Pharmacol., 112 (1992) 154. 8 R. Boegman and L.A. Bates, Neurotoxicity of aluminum. Can. J. Physiol. Pharmacol., 62 (1984) 1010. 9 D. Gawlik, P. Br/itter, P. Gardiner, W. Gatschke and W. Meyer-Sabellek,Aluminum in the maintenance diet of rats, in P. Br~itter and P. Schramel, (Eds.), Trace Element - - Analytical Chemistry in Medicine and Biology, Vol. 4, Walter de Gruyter & Co., New York, 1987. 10 J.L. Greger, Aluminum content of the American diet. Food Technol., 39 (1985) 73. 11 S. Zidenberg-Cherr, C.L. Keen, S.M. Casey and L.S. Hurley, Developmental changes affected by Mn deficiency. Biol. Trace Element Res., 7 (1985) 209. 12 R.B. Martin, The chemistry of aluminum as related to biology and medicine.Clin. Chem., 32 (1986) 1797.

47 13 14

15 16

17

18 19

20

21 22

23 24

25 26 27 28 29 30 31

32 33

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