Long-Term Oral Administration of Aluminum in Mice

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1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 24, No. 1 Copyright 1994, Institute for Clinical Science, Inc. Long-Term Oral Administration of Aluminum in Mice Aluminum Distribution in Tissues and Effects on Calcium Metabolism* LEOPOLD J. ANGHILERI, Ph.D.,t PHILIPPE MAINCENT, Ph.D.,* and PIERRE THOUVENOT, M.D. Biophysics Laboratory, Medicine Faculty,t University o f Nancy, Biopharmacy Department, Faculty o f Pharmacy,$ University o f Nancy, Nuclear Medicine Service, University o f Nancy Medical Center, Nancy, France ABSTRACT Six months of oral administration of aluminum lactate provokes an important accumulation of aluminum in various tissues of mice. The accumulation magnitude order is spleen > kidney > brain > liver > blood. No systemic toxic effects (weight loss or neurolytic effects) were observed. Values of calcium content and 45Ca-uptake by the different tissues showed no modifications of calcium metabolism. The lack of calcium homeostasis modification caused by a probable aluminum insolubilization, and the incidence of other individual factors on individual deviations from group behavior is discussed. Introduction * Address reprint requests to: Dr. Leopold J. Anghileri, Laboratories de Biophysique, 18 rue Lionnois, Nancy, France. Aluminum is not known to play any physiological role and is norm ally present in almost every human tissue in extremely low concentrations (up to 70 nmol/g), and among them brain tissue shows the smallest amount (1.4 nmol/g).6 This low concentration could be the result of a defensive exclusion of an element which seems involved in neurodegenerative disorders such as Parkinson s and Alzheimer s diseases.7,9 Studies have been reported on long-term administration effects and tissue distribution of aluminum given orally as citrate (5 mg Al/L) in the drinking water.17,18 That sodium citrate increases the low dietary aluminum absorption ( percent) has been demonstrated.8 In the case of aluminum citrate complex, the intestinal absorption appears as a concentrationdependent passive diffusion.15 In vitro modification of cellular Ca2+transport and homeostasis by aluminum lactate complex, as well as in vivo brain /94/ $00.90 Institute for Clinical Science, Inc.

2 LONG-TERM ORAL ADMINISTRATION OF ALUMINUM IN MICE 23 uptake of calcium enhancement after its parenteral adm inistration have been demonstrated.1,2 Aluminum-induced disruption of intraneural calcium homeostasis could be an important event leading to cell injury and death by calcium overloading.3 A lum inum interaction with molecules of the Ca2 +-transport sy stem m ig h t b e re s p o n s ib le for this phenomenon.16 The object of the present study was to investigate the long-term absorption and metabolism of orally administered aluminum lactate, as well as its effects on calcium metabolism of various tissues. Material and Methods Animals used were female Swiss mice, weighing 24 to 26 g, fed ad libitum a normal animal diet.* They were distributed in two groups of 10 animals each: (1) aluminum-treated group having access to drinking tap water containing 0.4 g Al/L (as aluminum lactate), and (2) a group of control animals drinking only tap water. After six months, the animals were weighed, intraperitoneally injected with 0.5 xci of 45CaCl2 (specific activity: 12 mci/mg) each, and sacrificed by carotid section 48 h thereafter. Samples of blood, and after dissection whole liver, spleen, kidney and brain were ashed and the residue dissolved in IN HC1. On aliquots of the solution aluminum and calcium were spectrophotometrically determined by the Aluminon method10 and Arsenazo III method,12 respectively. In addition to this, 45Ca radioactivity was counted with a liquid scintillation counter. Results During the six months of the experiment the aluminum-treated group did not present animals with toxic manifestations. The initial body weight of the control animals was 24.2 ± 1.2 g and of the * A04 from U.A.R. France.) aluminum-treated animals was 24.5 ± 0.8 g. After six months the weights were 37.1 ± 3.7 g and 35.7 ± 3.8 g, respectively. In table I, it is shown that in aluminum-treated animals the order of aluminum accumulation was spleen > kidney > brain > liver > blood. Calcium showed for both animal groups the same pattern of concentration: brain > kidney > spleen > blood > liver. The rate of 45Cauptake by the tissues, calculated as the ratio 45Ca-specific activity of tissue to 45Ca-specific activity of blood, showed the highest value in both groups for brain tissue. With the exception of liver, there was no difference in 45Ca-uptake for tissues from both groups (table II). No significant differences between calcium concentration in all tissues from both groups have been observed. However, one animal from aluminum-treated group has shown a much higher brain calcium (186 percent of the highest value for control animals) which was concomitant with a very high 45Ca-uptake rate by brain (3.5 times the highest value for control animals). Discussion The analytical methods used for aluminum and calcium determinations present a suitable sensitivity for this type of comparative study: the minimum aluminum concentration detectable is (xmol in 5 ml, and for calcium the value is (xmol in 5 ml. In our work, the analyzed tissues present concentrations higher than these sensitivity limits. After six months of intestinal absorption from drinking water containing a high alum inum concentration (0.4 g Al/L), the different tissues presented an aluminum concentration in the range of 50 to 610 times the values in control animals. In spite of this high aluminum concentration no toxic effects (loss of weight or neurolytic manifestations) have been observed. This lack of toxicity might be related to an in vivo molecular modi-

3 24 ANGHILERI, MAINCENT, AND THOUVENOT TABLE I Aluminum and Calcium Concentrations per Gram of Wet Tissue3 Control Group Aluminum-treated Group Aluminum1 Calcium Alumium Calcium Ixmol \imol\im ol \im ol Blood ± ± ± 0.97 (3.33)-= (0.37)o (3.58)= Liver ± ± ± (1.90) (0.77)= (1.83)= Spleen ± ± ± (6.28)= (18.48)= (7.35)= Kidney ± ± ± 0.85 (7.15) (2.52) (6.20)= Brain ± ± ± 3.31 (8.75) (1.41)= (16.33)= amean value + SD (n = 10). Aluminum in control group was determined on pooled samples of each tissue. Calcium concentration differences between both groups are not significant (unpaired Student's t-test). bvalue of pooled aliquots. =Hlghest value. TABLE II 46Ca2+-uptake Rate of Different Tissues Expressed as 45Ca-specific Activity of Tissue to 45Ca2+-specific Activity of Blood Ratio Control Group Aluminum-treated Group Liver 0.42 ± ± Spleen 1.80 ± ± 0.54 Kidney 2.03 ± ± Brain 2.09 ± ± 1.82 Mean value ± SD (n = 10). Only for liver is the difference significant between both groups: P < fica tion of the alum inum complex (presumably an insolubilization) which impairs the metal ion reactivity responsible for the effects on tissular calcium homeostasis. The fact that there are a number of factors (animal model, administered aluminum concentration, length of time of aluminum administration, anion or ligand combined to aluminum, aluminum content of the solid food, etc.) affecting aluminum intestinal absorption, makes difficult to compare as absolute values the data from various authors who have used different experimental protocols.13,17,18 On the other hand, in the in vivo toxicity of metal complexes orally administered, two different processes are involved: the first is the intestinal absorption which in the case of aluminum seems to be a concentration-dependent passive diffusion phenomenon15 that is also influenced by the nature of the anion or ligand complexing the aluminum,5 and the second process is the availability, at the tissue level, of ionic aluminum capable to inter

4 LONG-TERM ORAL ADMINISTRATION OF ALUMINUM IN MICE 25 act with molecular structures of the cell, and also in this case the chemical characteristics of the alum inum compound (anion or ligand) play an important role.11 The higher aluminum accumulation in tissues than the one obtained by other authors using aluminum citrate1718 might be the result of a greater in vivo stability of the citrate complex4 which after intestinal absorption can be excreted with a minimal in vivo breakdown. The high spleen accumulation of aluminum from lactate complex appears to corroborate the hypothesis of an important complex dissociation and aluminum insolubilization, followed by retention by the reticuloendothelial system. In our experiment, the lack of effects on tissular calcium homeostasis appears to support the concept of an in vivo breakdown of the complex subsequent to its intestinal absorption. This is followed by insolubilization of alum inum leading to the lost of its characteristic property of calcium homeostasis modifier observed after parenteral administration.2 In spite of the statistical lack of significance of the differences between the two groups, the fact that a drastic modification of brain calcium accumulation and 45Ca-uptake rate (the coincidence of both results exclude any possible experimental error in one or another determination) was observed in one animal from the alum inum -treated group, suggests that under identical experimental conditions th e p h en om en o n of b rain calcium metabolism modification by aluminum might not be a general one, but a process d eterm ined by biological responses which may differ from one individual to another, and depends on other individual factors. Among them, intestinal food absorption14 and hormonal status might play an important role.19 Acknowledgm ent Thanks are extended to the Association pour la Promotion des Recherches Apliquees en Biologie, Nancy, France, for the support to this work, and to Mr. P. Girard, Biochemistry Laboratory, Faculty of Medicine, University of Nancy, for his technical assistance. References 1. AnGHILERI, L. J.: Erlich tumour cells: Ca2+uptake modification by aluminum lactate. Cell Calcium 13: , A n g h i l e r i, L. J.: Effects of complexed iron and aluminum on brain calcium. Neurotoxicol. 13: , A n g h i l e r i, L. J.: Calcium and cell death. In: Anghileri, L. J. ed. The Role o f Calcium in Biological System s, vol. IV. Boca Raton, CRC Press, 1987, pp A n g h i l e r i, L. J., C o r d o v a M a r t i n e z, A., M a in c e n t, P., and R o b e r t, J.: In vivo behaviour of low molecular weight iron complexes. Eur. J. Drug Metabol. Pharmacokinetics 16: , B i l k e i-g o r z o, A.: Neurotoxic effects of enteral aluminum. Food Chem. Toxicol. 31: , D i e m, K.: Scientific tables. In: D ocum enta Geigy, 6th ed. New York, Geigy Pharmaceuticals, 1968, p G o o d, P. F., P e r l, D. P., Bi e r e r, L. M., and Sc h m e id l e r, J.: Selective accumulation of aluminum and iron in the neurofibrillary tangles of A lzheim er s disease: A laser m icroprobe (LAMMA) study. Ann. Neurol. 31: , G r e g e r, J. L. and POWERS, C. F.: Assessment of exposure to parenteral and oral aluminum with and without citrate using a desferrioxamine test in rats. Toxicology 76: , H i r s c h, E. C., B r a n d e l, J. P., G a l l e, P., Ja v o y -A g id, F., and A g id, Y.: Iron and aluminum increase in the Substantia nigra of patients with Parkinson s disease: An X-ray microanalysis. J. Neurochem. 56: , H o r t o n, A. D. and T h o m a s o n, P. F.: Ion exchange Spectrophotometric determination of aluminum. Anal. Chem. 28: , L e v in e, S., Sa l t z m a n, A., and D r a k o n t id e s, A. B.: Parenteral aluminum compounds produce local toxic myopathy in rats: Importance of the anion. Toxicol. Pathol. 20: , M ic h a l o v a, V. and I l k o v a, P.: Photometric determination of microscopic amounts of calcium with Arsenazo II I. Anal. Chim. Acta 53: , O t e i z a, P. L., G o l u b, M. S., G e r s h w i n, M. E., D o n a l d, J. M., and Ke e n, C. L.: The influence of high dietary aluminum on brain microtuble polymerization in mice. Toxicol. Lett. 47: , Partridge, N. A., Regnier, F. E., White, J. L., and H e m, S. L.: Influence of dietary constituents on intestinal absorption of aluminum. Kidney Int. 35: , 1989.

5 26 ANGHILERI, MAINCENT, AND THOUVENOT 15. P a r t r i d g e, N. A., R e g n i e r, F. E., R e e d, W. M., W h i t e, J. L., and H e m, S. L.: Contribution of soluble aluminum species to absorption of aluminum from the rat gut in situ. Clin. Sci. 83: , S i e g e l, N. and H a u g, A.: Aluminum interaction w ith calm odulin. Evidence for altered structure and function from optical and enzymatic studies. Biochim. Biophys. Acta 744:36-45, W i l l s, M. R., H e w i t t, C. D., St u r g i l l, B. C., Sa v o r y, J., and H e r m a n, M. M.: Long-term oral or intravenous aluminum administration in rabbits. I. Renal and hepatic changes. Ann. Clin. Lab. Sci. 23:1-16, W i l l s, M. R., H e w i t t, C. D., Sa v o r y, J., and H e r m a n, M. M.: Long-term oral administration of aluminum in rabbits. II. Brain and other organs. Ann. Clin. Lab. Sci. 23:17-23, Ya n a g h i h a r a, R., G a r r u t o, R. M., G a j- DUSEK, D. C., TOM ITA, A., UCHIDAW A, T., Kn o a g a y a, Y., C h e n, K. M., S o b u e, I., P l a t o, C. C., and GlBBS, C. J.: Calcium and vitamin D m etabolism in guam anian cham arrow with amyotropic lateral sclerosis and parkinsonismdementia. Ann. Neurol. 15:42-48, 1984.