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Aluminum(III) influences the permeability of the blood-brain barrier to [14C]sucrose in rats
330
Brain Research, 569 (1992) 330-335 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50
BRES 17332
Aluminum(III) influences the permeability of the blood-brain barrier t o [ 1 4 C ] s u c r o s e i n rats Mos6 Favarato 1, Paolo Zatta 1, Maurizio Perazzolo 1, Laura Fontana I and Marino Nicolini 2 I CNR-Unit for the study of Physiology and Biochemistry of Hemocyanin and other Metalloproteins, and Department of Biology, Padua (Italy) and 2Department of Pharmaceutical Sciences, University of Padua (haly) (Accepted 27 August 1991) Key words: Aluminum; Blood-brain barrier; Microvessel permeability; Metal speciation; Brain; Morin; Maltol; Acetylacetone
To determine the influence of the metal coordination sphere on the permeability of the blood-brain barrier (BBB), rats were injected intraperitoneally with aluminum lactate (Al(lact)3), aluminum acetylacetonate (Al(acac)a), aluminum maltolate (Al(malt)3) at pH 7.5, or with physiological saline, Two h after each treatment, [14C]sucrose physiological saline solution was injected in animals, and the radioactivity was measured in 5 brain regions (cerebral cortex, mesencephalon, diencephalon, medulla-pons, cerebellum). Radioactivity was significantly elevated in brains from animals treated with Al(malt)s (hydrolytically stable and hydrophilic), and with Al(acac)3 (hydrolytically stable and lipophilic) but not with Al(lact)3. Time-course study carried out at 2, 4 and 24 h with different aluminum compounds shbwed a persistent radioactivity 24 h after treatment only in the brain from animals treated with Al(acac)3. Morin stain localized AIm only in neurons from animals treated with Al(acac)s. These findings indicate that AIm alters the BBB function in the rat either permanently or transiently depending on the physicoehemical properties of the:metal g60rdination sphere. Implications of these results, in terms of AIm as a potential toxic factor in humans, are considered and discussed. INTRODUCTION In spite of its wide distribution aluminum (AI m) has not exhibited biological properties so far. On the contrary its neurotoxicity has been proved, as in the case of dialysis dementia 2. Although aluminum accumulation has been identified in the histopathological features of Alzheimer's disease ( A D ) , i.e. senile plaques and neurofibrillary tangles s'lS, at the present time evidences connecting AI In accumulation in the human brain with A D etiology are still tangentials. AI nI administered systemically, centrally or orally to experimental animals, produced impairment of learning, retention, motor coordination, etc. The main mechanism(s) by which AI nI causes neurotoxic effects has not been established yet. In mammals the neuronal environment is controlled by the exchange of solutes and water across the BBB, which is formed by epithelial-like tight junctions devised to separate brain from plasma. The default of this regulation may allow the entry of serum proteins and/or neurotoxins into the brain, thus influencing the homeostasis and modifying the metabolic activities of the central nervous system. Recently Banks and Kastin 4 suggested that A1m might not only affect the permeability of the BBB by the gen-
eralized mechanism of the alteration of the lipophilic characteristics of the BBB i n terms of its free diffusion through the endothelial plasma membranes, but it may also selectively alter the saturable transport system. AIm administered systemically in experimental animals at high concentration has been shown to accumulate in the cerebro-endothelial cell surface9:7. It has also been shown that AICI3 (ref. 5) and Al(lact)3 (ref. 12) injected i.p., reversibly increase the BBB permeability in rats. Wen and Wisniewski 17 proved that AI m is bound to endothelial cells of the BBB in rats and it has also been shown that the metal is deposited around the microvessels in patients affected by dementia 15. Endothelial cells seem to be more sensitive with respect to other mammalian cells to the modifications induced by A1m (ref. 3). Data herein reported provide evidence that AIm can modify the permeability of BBB depending on the physico-chemical properties of the metal coordination sphere. These results are in agreement with numerous findings from our research group which stress the relevance of the metal speciation with its toxic effect on specific biological targets 6'7:9. It is most pertinent to remark that Al(acac)3 and AI(malt)3 possess diverse lipophilic-hydrophilic characters, but quite similar hydrolytic stability. O n the other hand, Al(lact)3, which possesses a molecular structure very sim-
Correspondence: P. Zatta, Department of Biology, Via Trieste 75, 35131 Padua, Italy.
331 TABLE I
Summary of the treatment of rats" with aluminum complexes Two different concentrations of Al(malt)~ were used. Al(malt)3 60 mM and Al(acac)3 8.70 mM represent the maximum of solubility in water of these compounds.
Group Group Group Group
1 2 3 4
AI compound
AI mg/kg
Al(lact)3 Al(acac)3 Al(malt)31 Al(malt)3
100.0 2.2 2.2 16.2
mg mg mg mg
M Al/kg Al/kg Al/kg A1/kg
0.55 8.70 8.70 60.00
M mM mM mM
ilar to t h o s e o f A l ( a c a c ) 3 a n d A l ( m a l t ) 3 in t h e solid s t a t e h a s b e e n f o u n d to exist as t h e h y d r o p h i l i c h i g h l y m e t a s t a b l e species: A I ( O H ) 3 ( O H 2 ) 3 at p H 7.5. MATERIALS AND METHODS
Animals Adult male outbred Wistar rats (320-420 g) obtained from Morini (Bologna), Italy, were used in this study. Animals were injected intraperitoneally. Solutions Al(malt)3 (malt=maltolate=3-hydroxy,2-methyl,4-pyronate: hydrolytically stable and lipophilic (K1 = 10+225)) (ref. 11), and Al(acac) 3 (acac=acetylacetone=2,4 pentanedionate: hydrolytically stable and lipophilic (K 1 = 10+22'3)) (ref. 13) were prepared and purified as decribed elsewhere l°'ls. Hacac (acetylacetone; Janssen) was distilled before use. Al(lact)3 (lact=lactate: hydrophilic and hydrolytically metastable; Fluka) was used without further purification. Solutions of Al(lact)3, Al(malt)3 and Al(acac)3 were prepared in sterile physiological saline solution (NaC1, 0.9%) and when required, the pH was adjusted to 7.5 with 0.1 M NaOH. Tracer [14ClSucrose was obtained from New England Nuclear (1-5 mCi/ retool) and diluted with physiological solution to a final concentration of 10 gCi/ml. Radioactivity measurement was reported as dpm (disintegrations per minute). BBB permeability stud), A total number of 40 animals was injected with 8 ml/kg of the following solutions: saline (n = 5), Al(lact)3 (n = 5), Al(malt)31 (n = 5), Al(malt)32 (n = 5), Al(acac)3 (n = 5) as reported in Table I, and with free-aluminum ligands: Na-lact (n = 5), Hacac (n = 5), Hmalt (n = 5) as reported in Table II. Two h after injection, anaesthetized animals were catheterized in the left and right ventricle of the heart with heparinized polyethylene cannulas. Five ~Ci of [14C]sucrose in 0.5 ml of saline solution were injected and allowed to circulate in blood for 10 rain, during which a
blood sample of 50 pl was collected in heparinized microtubes to calculate total intravascular tracer in the brain. Animals were sacrificed by decapitation. The brain, without leptomeninges, was quickly removed, bisected in the sagittal plane, and each half part was dissected in 5 regions: cerebral cortex, medulla-pons, mesencephalon, diencephalon and cerebellum. All samples were individually placed in 20 ml pre-weighed glass-vials (Packard, high performance glass-vials) and weighed. Each sample was solubilized in 2 ml of protosol (New England Nuclear) in a low-speed shaking water bath at 55°C until completely dissolved. The whole blood samples were treated with 1 ml of protosol/ethanol (1:2 v/v) (New England Nuclear) mixture, and the vials were incubated for 2 h. Each vial was cooled at room temperature before adding 10 ml of Econofluor-2 (New England Nuclear). Samples were counted in a scintillation counter Packard 1500 TriCarb. To study possible modifications of the BBB to the tracer as a consequence of aluminum complexes administration, a time-course experiment was carried out. Therefore, a second group of 50 rats was injected i.p. with saline (n = 5), Al(acac)3 (n = 5), Al(malt)31 (n = 5) at the concentration as reported in Table I, and with Hacac (n = 5) and Hmalt (n = 5). Sampling was carried out at 4 and 24 h after the treatment.
Regional blood volume To calculate the regional blood volume (RBV), a third group of 15 animals was injected with physiological saline (n = 3), Al(lact)3 (n = 3), Al(acac)3 (n = 3), Al(malt)31 (n = 3), Al(malt)32 (n = 3) at the concentration as reported in Table I. Measurements of RBV were necessary to calculate the radioactivity in each brain region without the contribution of the radioactivity from the blood in brain capillaries. Animals belonging to this last group, were sacrificed 1 rain after [14C]sucrose injection. RBV measurement was carried out according to the method reported by Rapoport et al.~6 Histochemistry To study morphological changes of the BBB as the consequence of the A1tu complexes administration, and to specifically localize the metal in brain tissue, a fourth group of rats was injected as described in Tables I and II. Animals were sacrificed by decapitation after 2, 4 and 24 h from the exposure to aluminum compounds. Brains were fixed in 10% buffered formalin. Stain Five-/~m-thick paraffin sections were stained with morin (3,5,7,2",4"pentahydroxyflavone; Fluka) which localizes aluminum producing a yellow fluorescent complex. The detailed methodology has been described elsewhere9.
RESULTS T h e m e a n R B V of 5 b r a i n a r e a s f r o m a n i m a l s t r e a t e d w i t h a l u m i n u m c o m p l e x e s , a n d f r o m n o r m a l r a t s , is rep o r t e d in T a b l e III. T h i s p a r a m e t e r
is d e f i n e d as t h e
[14C]sucrose s p a c e at 1 m i n u t e a n d it is e x p r e s s e d in t h e following manner: Brain: dpm/g fresh weight RBV =
x 100 Whole blood: dpm/ml
TABLE II
Summary of the treatment of rats with free-aluminum ligands T h e a v e r a g e s u c r o s e s p a c e in n o r m a l r a t s w i t h i n t a c t Each solution is 3 times more concentrated with respect to its aluminum complex.
B B B r a n g e d f r o m 2.10 to 2.69%. T h e s e v a l u e s a r e similar to t h o s e r e p o r t e d b y o t h e r l a b o r a t o r i e s 12'14. A n i m a l s
Group 1 Na-lact Group 2 Hacac Group 3 Hmalt
989.0 mg/kg 25.0 mg/kg 75.7 mg/kg
1.65 M 26.0 mM 60.0 mM
t r e a t e d w i t h A l ( l a c t ) 3 s h o w e d a R B V s i m i l a r to t h e c o n trol, w h i l e v a l u e s o b t a i n e d f r o m r a t s t r e a t e d w i t h A l ( a cac)3 a n d A l ( m a l t ) 3 r a n g e d f r o m 3.52 to 4.16 a n d f r o m
332 k~I Saline solution Q AI {lact) s AI (acac) 3
1
20000[[
t~l AI (malt) 3 m AI (malt)32
tta
I
Na- lactate
[ ] Acetylacetone [ ] Maltol
20000
~-~ 10000~+.
L
1000C E 13"O
I
,
I
Cortex
,
Cerebellum
i
II
t
Cortex
C e r e b e num
i PansMedulla
, Mesencep- Diencephalon halon
Brain regions
Fig. 1. BBB permeability to [14C]sucrose in 5 brain regions: cerebral cortex, cerebellum, pons-medulla, mesencephalon, diencephalon. Rats were treated with saline solution, Al(lact)3, Al(acac)3, Al(malt)31 and Al(malt)32.
2.60 to 5.38 respectively, showing a small increase with respect to the normal rat. Results of the BBB permeability to [t4C]sucrose in 5 brain regions from rats injected with aluminum complexes and in the control are depicted in Fig. 1. Rats treated with Al(lact)3 did not show differences with respect to the control. On the contrary, a large increase on BBB permeability was observed in rats treated both with Al(acac)3 and Al(malt)3. The modification of the cerebrovascular permeability due to Al(acac)3 and Al(malt)3 is neither quantitatively nor qualitatively the same in all brain areas examined. Fig. 2 shows data of the permeability to [14C]sucrose in different brain areas examined from rats treated with free-aluminum ligands. Na-lactate and Hacac don't have appreciable effect on BBB permeability, while a small effect can be observed using Hmalt. Experiments on time-dependent modification of BBB
~ l
Mesencep- Diencepnalon halon
Brain reglons
Fig. 2. BBB permeability to [14C]sucrose in 5 brain regions, as in Fig. 1, from rats treated with free tigands" Na-lactate, acetylacetone and maltol.
permeability are reported in Fig. 3. For the calculation of the permeability the RBV values as reported in Table III were used. Data here presented show that the action of Al(malt)3 significantly affects the cerebrovascular permeability after 2 h. and this permeability returns to be normal after 4 h. In rats treated with Al(acac)3, the BBB permeability to [14C]sucrose increased, after 2 h. the relevant values being lower than those observed in animals treated with Al(malt)3. However. this modification is found to last 24 h after the injection of the metal toxin. Saline solution, Al(lact)3 and free-aluminum ligands did
13000
/ /
11000
/
\
/
\
x
\
T
'\
T
//l
,, :
)"
\\
7000
TABLE III
, PansMedulla
'
/
Regional blood volume (RBV) of 5 brain regions Values are means -+ S.D. n = 3 for each aluminum compound and the control.
.
...............................
AI . (malt) 3 --// . .
.
.... i g &
........
/ ........
30OC
Brain region
NaCl
Al(Lact)3
Al(acac)3
Al(malt)3
(Blood volume % = (dpm/g/w.wt.)/(dprn/ml whole blood) x 100) l
Cerebral cortex Cerebellum Pans-medulla Mesencephalon Diencephalon
2.31---0.11 2.59---0.12 2.62+-0.02 2.35+-0.14 2.10---0.06
1.95-*-0.31 2.92-+0.23 3.28+-0.72 2.51+-0.25 1.88-+0.17
3.95---0,06 4.08-+0.16 4.16-+0.12 3.73+--0.09 3.52-+0.10
3.66-+0.06 2.60+-0.05 2.93-+0.10 5.38-+0.04 5.31-+0,06
I
2
4
//
I
24
Hodt
Fig. 3. Cerebrovascular permeability for [14C]sucrose 2, 4 and 24 h after injection of NaCI (O), 2.2 mg A1/kg as Al(acac)3 ( A ) , 2.2 mg Al/kg as Al(malt) 3 (O). Mean --- S~E.M.
333
Fig. 4. A: morin staining: cerebral cortex field from rat treated with 2.2 mg Al/kg as Al(malt)3. B: 2.2 mg A1/kg as Al(acac)3 after 24 h treatment and C: control with saline solution.
334 not affect the BBB permeability 2 and 24 h after treatment. Morin when applied to brain sections from animals treated with AI H~ compounds showed a strong fluorescence on the wall of the endothelial cells. Fig. 4A shows a representative cerebral cortical field from rats treated with Al(malt)3 where fluorescence occurs only in the blood vessels. The same results were also observed in animals treated with Al(lact)3. In the case o f Al(acac)3 treatment, a strong fluorescence can also be seen inside some neurons of the cerebral cortex, as shown in Fig. 4B. DISCUSSION This study demonstrates that aluminum compounds injected i.p. affect the BBB permeability in the rat in a metal speciation-dependent manner. Therefore, the behavior expressed by distinct aluminum compounds has to be explained on the basis of the different physicochemical properties of the metal coordination sphere, being the most effective agent able to modify the BBB permeability and the most lipophilic one. The molecular mechanism responsible for this modification is not totally clear since our understanding of how aluminum penetrates the biological membranes is still limited. Wen and Wisniewski ~7 obtained an increased BBB permeability to [X4C]sucrose in the rat after treatment with AIC13 and Al(lact)3, while AI(OH)3 was found to be inactive. These data apparently contradict our findings, but it should be observed that these authors utilized an acidic solution (pH approx. 3.0-3.5) and not a neutral one. Moreover, in contrast with the differential changes observed by us in different brain areas, they found a general undifferentiated effect in all brain areas examined.
REFERENCES 1 Akeson, M.A. and Munns, D.N., Lipid bilayer permeation by neutral aluminum citrate and by three a-hydroxy carboxylic acid, Biochim. Biophys. Acta, 984 (1989) 200-206. 2 Alfrey, A.C., Mishell, J.M. and Burks, J., Syndrome of dyspraxia and multifocal seizures associated with chronic hemodialysis, Trans. Am. Soc. Artif. Intern. Organs, 18 (!972) 257-261. 3 Audus, K.L., Holthaus, S.R., van Bree, J.M.B.B. and Shinogle, J.A., Aluminum effect on growth of brain mierovesset endothelial cells in primary culture, Res. Commun. Chem: Pathol. Pharmacol., 60 (1988) 71-85. 4 Banks, W.A., Kastin, A.J. and Fasold, M.B., Differential effect of aluminum on the blood-brain barrier transport of peptides, technetium and albumin, J. Pharmacol. Exp. Ther., 214 (1988) 579-585. 5 Banks, W.A. and Kastin, A.J., Aluminum increases permeability of the blood-brain barrier to the labelled DSIP and /~-endorphin: possible implications for senile and dialysis dementia, Lancet, 2 (1983) 1227-1229.
A n o t h e r example o f effecl of AI tH on the permeability of the BBB to a physiologically relevant neuropeptide. i.e. N-Tyr DISP, has been documented by Banks and Kastin 5. Again, acidic solutions of AI(OH2)6 3+ are likely to have been used. Although the mechanism o f the differential action on BBB permeability changes cannot yet be completely interpreted, the overall action of Al(acac) 3 and of AI(matt)3 on the rat brain also appears to be strongly related to metal speciation. In fact, the lipophilic coordination sphere offered by 3 acetylacetonate ligands per metal ion makes A I m able to cross the BBB and to reach the interior of some neurons of the cerebral cortex. Biophysical evidence both supports and opposes a neutral aluminum c o m p o u n d permeation mechanism ~. It has been recently reported that lipid bilayer permeability by neutral aluminum salts such as citrate is low. Comparison with a-hydroxyl carboxylic acids and trimethyl-citrate suggests that the rate of permeation is limited by hydrogen bonding between water and citrate ligand and by dipole-dipole bonds between water and the bound aluminum 1. O f relevant importance in understanding and interpreting a toxicological study, is therefore ~ welldefined chemical form of the aluminum compounds employed in experiments at physiological pH. Although the present study confirms previous observations of the effect of AIm on the integrity of BBB in the rat, however, we raise some major mechanistically relevant aspects: (i) the effect is qualitatively dependent on the metal speciation, (ii) in the case of the lipophilic toxin, such as Al(acac)3, the effect ~s irreversible, and (iii) it is accompanied by the passage of A1m beyond the BBB and by its access in the interior of neurons. Acknowledgements. Authors thank Dr. B. Corain for helpful discussion. This project was partially supported by Parke Davis.
6 Bombi, G,G., Corain, B., Favarato, M., Giordano, R., Nicolini, M., Perazzolo, M. and ~ t t a , P,, Experimental aluminum dismetabolism in rabbits; effects~of hydrophilic and lipophilic compounds, Environ. Health Persp., 89 (1990) 217-223. 7 Corain, B., Bombi, G.G; and Zatta, P., Differential effects of covalent compounds in aluminum toxicology, Neurobiol. Aging. 9 (1988) 413-414. 8 Candy, J.MI, Oakley, A.E,, Ktimowski, J., Carpenter, T.A.. Perry, R,H., Ataek, J.R., Perry, E.K., Blessed, G., Fairbairn, A. and Edwardson, J.A., Aluminumsilicates and senile plaque formation in Alzheimer's disease, Lancet, 1 (1986) 354-357. 9 De Boni, U., Scott; J,W. and Crapper, D.R., Intracellular aluminum binding: a histoehemieal study, Histochemistrv, 40 (1974) 31-37. 10 Finnegan, M.M;, Steven,LR. and Orvig, C., A neutral watersoluble aluminum e o ~ ! ¢ x of neurological interest, J. Am. Cheml Soc., 108(1986)5033-5035. 11 Hedlund, T. and Oet~man, L.-O., Equilibrium and structural studies of silicon (IV) and aluminium (III) in aqueous solution. 19. Composition and stability of aluminium complexes with ko-
335 jic acid and maltol, Acta Chem. Scand., A42 (1988) 702-709. 12 Kim, Y.S., Lee, M.H. and Wisniewski, H.M., Aluminum induces reversible change in permeability of the blood-brain barrier to 14C-sucrose, Brain Research, 377 (1986) 286-291. 13 Martell, R.M. and Smith, R.M., Critical Stability Constant, Vol. 3, Other Organic Ligands, Plenum, New York, 1977, 246 pp. 14 Ohno, K., Pettigrew, K.D. and Rapoport, S.I., Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat, Am. J. Physiol., 235 (1978) H229-H317. 15 Perl, D.P. and Brody, P.R., Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle-bearing neurons, Science, 208 (1980) 297-299.
16 Rapoport, S.I., Ohno, K. and Pettigrew, K.D., Regional cerebrovascular permeability to [14C]sucrose after osmotic opening of the blood-brain barrier, Brain Research, 150 (1978) 653-657. 17 Wen, G.Y. and Wisniewski, H.M., Histochemical localisation of aluminum in the rabbit central nervous system, Acta Neuropathol., 68 (1985) 175-184. 18 Yong, R.C. and Reynolds, J.P., Aluminum acetylacetonate, lnorg. Synth., 2 (1946) 25-26. 19 Zatta, P., Perazzolo, M. and Corain, B., Tris acetylacetonate aluminum(III) induces osmotic fragility and acantocyte formation in suspended erythrocytes, Toxicol. Lett., 45 (1989) 15-21.
Brain Research, 569 (1992) 330-335 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50
BRES 17332
Aluminum(III) influences the permeability of the blood-brain barrier t o [ 1 4 C ] s u c r o s e i n rats Mos6 Favarato 1, Paolo Zatta 1, Maurizio Perazzolo 1, Laura Fontana I and Marino Nicolini 2 I CNR-Unit for the study of Physiology and Biochemistry of Hemocyanin and other Metalloproteins, and Department of Biology, Padua (Italy) and 2Department of Pharmaceutical Sciences, University of Padua (haly) (Accepted 27 August 1991) Key words: Aluminum; Blood-brain barrier; Microvessel permeability; Metal speciation; Brain; Morin; Maltol; Acetylacetone
To determine the influence of the metal coordination sphere on the permeability of the blood-brain barrier (BBB), rats were injected intraperitoneally with aluminum lactate (Al(lact)3), aluminum acetylacetonate (Al(acac)a), aluminum maltolate (Al(malt)3) at pH 7.5, or with physiological saline, Two h after each treatment, [14C]sucrose physiological saline solution was injected in animals, and the radioactivity was measured in 5 brain regions (cerebral cortex, mesencephalon, diencephalon, medulla-pons, cerebellum). Radioactivity was significantly elevated in brains from animals treated with Al(malt)s (hydrolytically stable and hydrophilic), and with Al(acac)3 (hydrolytically stable and lipophilic) but not with Al(lact)3. Time-course study carried out at 2, 4 and 24 h with different aluminum compounds shbwed a persistent radioactivity 24 h after treatment only in the brain from animals treated with Al(acac)3. Morin stain localized AIm only in neurons from animals treated with Al(acac)s. These findings indicate that AIm alters the BBB function in the rat either permanently or transiently depending on the physicoehemical properties of the:metal g60rdination sphere. Implications of these results, in terms of AIm as a potential toxic factor in humans, are considered and discussed. INTRODUCTION In spite of its wide distribution aluminum (AI m) has not exhibited biological properties so far. On the contrary its neurotoxicity has been proved, as in the case of dialysis dementia 2. Although aluminum accumulation has been identified in the histopathological features of Alzheimer's disease ( A D ) , i.e. senile plaques and neurofibrillary tangles s'lS, at the present time evidences connecting AI In accumulation in the human brain with A D etiology are still tangentials. AI nI administered systemically, centrally or orally to experimental animals, produced impairment of learning, retention, motor coordination, etc. The main mechanism(s) by which AI nI causes neurotoxic effects has not been established yet. In mammals the neuronal environment is controlled by the exchange of solutes and water across the BBB, which is formed by epithelial-like tight junctions devised to separate brain from plasma. The default of this regulation may allow the entry of serum proteins and/or neurotoxins into the brain, thus influencing the homeostasis and modifying the metabolic activities of the central nervous system. Recently Banks and Kastin 4 suggested that A1m might not only affect the permeability of the BBB by the gen-
eralized mechanism of the alteration of the lipophilic characteristics of the BBB i n terms of its free diffusion through the endothelial plasma membranes, but it may also selectively alter the saturable transport system. AIm administered systemically in experimental animals at high concentration has been shown to accumulate in the cerebro-endothelial cell surface9:7. It has also been shown that AICI3 (ref. 5) and Al(lact)3 (ref. 12) injected i.p., reversibly increase the BBB permeability in rats. Wen and Wisniewski 17 proved that AI m is bound to endothelial cells of the BBB in rats and it has also been shown that the metal is deposited around the microvessels in patients affected by dementia 15. Endothelial cells seem to be more sensitive with respect to other mammalian cells to the modifications induced by A1m (ref. 3). Data herein reported provide evidence that AIm can modify the permeability of BBB depending on the physico-chemical properties of the metal coordination sphere. These results are in agreement with numerous findings from our research group which stress the relevance of the metal speciation with its toxic effect on specific biological targets 6'7:9. It is most pertinent to remark that Al(acac)3 and AI(malt)3 possess diverse lipophilic-hydrophilic characters, but quite similar hydrolytic stability. O n the other hand, Al(lact)3, which possesses a molecular structure very sim-
Correspondence: P. Zatta, Department of Biology, Via Trieste 75, 35131 Padua, Italy.
331 TABLE I
Summary of the treatment of rats" with aluminum complexes Two different concentrations of Al(malt)~ were used. Al(malt)3 60 mM and Al(acac)3 8.70 mM represent the maximum of solubility in water of these compounds.
Group Group Group Group
1 2 3 4
AI compound
AI mg/kg
Al(lact)3 Al(acac)3 Al(malt)31 Al(malt)3
100.0 2.2 2.2 16.2
mg mg mg mg
M Al/kg Al/kg Al/kg A1/kg
0.55 8.70 8.70 60.00
M mM mM mM
ilar to t h o s e o f A l ( a c a c ) 3 a n d A l ( m a l t ) 3 in t h e solid s t a t e h a s b e e n f o u n d to exist as t h e h y d r o p h i l i c h i g h l y m e t a s t a b l e species: A I ( O H ) 3 ( O H 2 ) 3 at p H 7.5. MATERIALS AND METHODS
Animals Adult male outbred Wistar rats (320-420 g) obtained from Morini (Bologna), Italy, were used in this study. Animals were injected intraperitoneally. Solutions Al(malt)3 (malt=maltolate=3-hydroxy,2-methyl,4-pyronate: hydrolytically stable and lipophilic (K1 = 10+225)) (ref. 11), and Al(acac) 3 (acac=acetylacetone=2,4 pentanedionate: hydrolytically stable and lipophilic (K 1 = 10+22'3)) (ref. 13) were prepared and purified as decribed elsewhere l°'ls. Hacac (acetylacetone; Janssen) was distilled before use. Al(lact)3 (lact=lactate: hydrophilic and hydrolytically metastable; Fluka) was used without further purification. Solutions of Al(lact)3, Al(malt)3 and Al(acac)3 were prepared in sterile physiological saline solution (NaC1, 0.9%) and when required, the pH was adjusted to 7.5 with 0.1 M NaOH. Tracer [14ClSucrose was obtained from New England Nuclear (1-5 mCi/ retool) and diluted with physiological solution to a final concentration of 10 gCi/ml. Radioactivity measurement was reported as dpm (disintegrations per minute). BBB permeability stud), A total number of 40 animals was injected with 8 ml/kg of the following solutions: saline (n = 5), Al(lact)3 (n = 5), Al(malt)31 (n = 5), Al(malt)32 (n = 5), Al(acac)3 (n = 5) as reported in Table I, and with free-aluminum ligands: Na-lact (n = 5), Hacac (n = 5), Hmalt (n = 5) as reported in Table II. Two h after injection, anaesthetized animals were catheterized in the left and right ventricle of the heart with heparinized polyethylene cannulas. Five ~Ci of [14C]sucrose in 0.5 ml of saline solution were injected and allowed to circulate in blood for 10 rain, during which a
blood sample of 50 pl was collected in heparinized microtubes to calculate total intravascular tracer in the brain. Animals were sacrificed by decapitation. The brain, without leptomeninges, was quickly removed, bisected in the sagittal plane, and each half part was dissected in 5 regions: cerebral cortex, medulla-pons, mesencephalon, diencephalon and cerebellum. All samples were individually placed in 20 ml pre-weighed glass-vials (Packard, high performance glass-vials) and weighed. Each sample was solubilized in 2 ml of protosol (New England Nuclear) in a low-speed shaking water bath at 55°C until completely dissolved. The whole blood samples were treated with 1 ml of protosol/ethanol (1:2 v/v) (New England Nuclear) mixture, and the vials were incubated for 2 h. Each vial was cooled at room temperature before adding 10 ml of Econofluor-2 (New England Nuclear). Samples were counted in a scintillation counter Packard 1500 TriCarb. To study possible modifications of the BBB to the tracer as a consequence of aluminum complexes administration, a time-course experiment was carried out. Therefore, a second group of 50 rats was injected i.p. with saline (n = 5), Al(acac)3 (n = 5), Al(malt)31 (n = 5) at the concentration as reported in Table I, and with Hacac (n = 5) and Hmalt (n = 5). Sampling was carried out at 4 and 24 h after the treatment.
Regional blood volume To calculate the regional blood volume (RBV), a third group of 15 animals was injected with physiological saline (n = 3), Al(lact)3 (n = 3), Al(acac)3 (n = 3), Al(malt)31 (n = 3), Al(malt)32 (n = 3) at the concentration as reported in Table I. Measurements of RBV were necessary to calculate the radioactivity in each brain region without the contribution of the radioactivity from the blood in brain capillaries. Animals belonging to this last group, were sacrificed 1 rain after [14C]sucrose injection. RBV measurement was carried out according to the method reported by Rapoport et al.~6 Histochemistry To study morphological changes of the BBB as the consequence of the A1tu complexes administration, and to specifically localize the metal in brain tissue, a fourth group of rats was injected as described in Tables I and II. Animals were sacrificed by decapitation after 2, 4 and 24 h from the exposure to aluminum compounds. Brains were fixed in 10% buffered formalin. Stain Five-/~m-thick paraffin sections were stained with morin (3,5,7,2",4"pentahydroxyflavone; Fluka) which localizes aluminum producing a yellow fluorescent complex. The detailed methodology has been described elsewhere9.
RESULTS T h e m e a n R B V of 5 b r a i n a r e a s f r o m a n i m a l s t r e a t e d w i t h a l u m i n u m c o m p l e x e s , a n d f r o m n o r m a l r a t s , is rep o r t e d in T a b l e III. T h i s p a r a m e t e r
is d e f i n e d as t h e
[14C]sucrose s p a c e at 1 m i n u t e a n d it is e x p r e s s e d in t h e following manner: Brain: dpm/g fresh weight RBV =
x 100 Whole blood: dpm/ml
TABLE II
Summary of the treatment of rats with free-aluminum ligands T h e a v e r a g e s u c r o s e s p a c e in n o r m a l r a t s w i t h i n t a c t Each solution is 3 times more concentrated with respect to its aluminum complex.
B B B r a n g e d f r o m 2.10 to 2.69%. T h e s e v a l u e s a r e similar to t h o s e r e p o r t e d b y o t h e r l a b o r a t o r i e s 12'14. A n i m a l s
Group 1 Na-lact Group 2 Hacac Group 3 Hmalt
989.0 mg/kg 25.0 mg/kg 75.7 mg/kg
1.65 M 26.0 mM 60.0 mM
t r e a t e d w i t h A l ( l a c t ) 3 s h o w e d a R B V s i m i l a r to t h e c o n trol, w h i l e v a l u e s o b t a i n e d f r o m r a t s t r e a t e d w i t h A l ( a cac)3 a n d A l ( m a l t ) 3 r a n g e d f r o m 3.52 to 4.16 a n d f r o m
332 k~I Saline solution Q AI {lact) s AI (acac) 3
1
20000[[
t~l AI (malt) 3 m AI (malt)32
tta
I
Na- lactate
[ ] Acetylacetone [ ] Maltol
20000
~-~ 10000~+.
L
1000C E 13"O
I
,
I
Cortex
,
Cerebellum
i
II
t
Cortex
C e r e b e num
i PansMedulla
, Mesencep- Diencephalon halon
Brain regions
Fig. 1. BBB permeability to [14C]sucrose in 5 brain regions: cerebral cortex, cerebellum, pons-medulla, mesencephalon, diencephalon. Rats were treated with saline solution, Al(lact)3, Al(acac)3, Al(malt)31 and Al(malt)32.
2.60 to 5.38 respectively, showing a small increase with respect to the normal rat. Results of the BBB permeability to [t4C]sucrose in 5 brain regions from rats injected with aluminum complexes and in the control are depicted in Fig. 1. Rats treated with Al(lact)3 did not show differences with respect to the control. On the contrary, a large increase on BBB permeability was observed in rats treated both with Al(acac)3 and Al(malt)3. The modification of the cerebrovascular permeability due to Al(acac)3 and Al(malt)3 is neither quantitatively nor qualitatively the same in all brain areas examined. Fig. 2 shows data of the permeability to [14C]sucrose in different brain areas examined from rats treated with free-aluminum ligands. Na-lactate and Hacac don't have appreciable effect on BBB permeability, while a small effect can be observed using Hmalt. Experiments on time-dependent modification of BBB
~ l
Mesencep- Diencepnalon halon
Brain reglons
Fig. 2. BBB permeability to [14C]sucrose in 5 brain regions, as in Fig. 1, from rats treated with free tigands" Na-lactate, acetylacetone and maltol.
permeability are reported in Fig. 3. For the calculation of the permeability the RBV values as reported in Table III were used. Data here presented show that the action of Al(malt)3 significantly affects the cerebrovascular permeability after 2 h. and this permeability returns to be normal after 4 h. In rats treated with Al(acac)3, the BBB permeability to [14C]sucrose increased, after 2 h. the relevant values being lower than those observed in animals treated with Al(malt)3. However. this modification is found to last 24 h after the injection of the metal toxin. Saline solution, Al(lact)3 and free-aluminum ligands did
13000
/ /
11000
/
\
/
\
x
\
T
'\
T
//l
,, :
)"
\\
7000
TABLE III
, PansMedulla
'
/
Regional blood volume (RBV) of 5 brain regions Values are means -+ S.D. n = 3 for each aluminum compound and the control.
.
...............................
AI . (malt) 3 --// . .
.
.... i g &
........
/ ........
30OC
Brain region
NaCl
Al(Lact)3
Al(acac)3
Al(malt)3
(Blood volume % = (dpm/g/w.wt.)/(dprn/ml whole blood) x 100) l
Cerebral cortex Cerebellum Pans-medulla Mesencephalon Diencephalon
2.31---0.11 2.59---0.12 2.62+-0.02 2.35+-0.14 2.10---0.06
1.95-*-0.31 2.92-+0.23 3.28+-0.72 2.51+-0.25 1.88-+0.17
3.95---0,06 4.08-+0.16 4.16-+0.12 3.73+--0.09 3.52-+0.10
3.66-+0.06 2.60+-0.05 2.93-+0.10 5.38-+0.04 5.31-+0,06
I
2
4
//
I
24
Hodt
Fig. 3. Cerebrovascular permeability for [14C]sucrose 2, 4 and 24 h after injection of NaCI (O), 2.2 mg A1/kg as Al(acac)3 ( A ) , 2.2 mg Al/kg as Al(malt) 3 (O). Mean --- S~E.M.
333
Fig. 4. A: morin staining: cerebral cortex field from rat treated with 2.2 mg Al/kg as Al(malt)3. B: 2.2 mg A1/kg as Al(acac)3 after 24 h treatment and C: control with saline solution.
334 not affect the BBB permeability 2 and 24 h after treatment. Morin when applied to brain sections from animals treated with AI H~ compounds showed a strong fluorescence on the wall of the endothelial cells. Fig. 4A shows a representative cerebral cortical field from rats treated with Al(malt)3 where fluorescence occurs only in the blood vessels. The same results were also observed in animals treated with Al(lact)3. In the case o f Al(acac)3 treatment, a strong fluorescence can also be seen inside some neurons of the cerebral cortex, as shown in Fig. 4B. DISCUSSION This study demonstrates that aluminum compounds injected i.p. affect the BBB permeability in the rat in a metal speciation-dependent manner. Therefore, the behavior expressed by distinct aluminum compounds has to be explained on the basis of the different physicochemical properties of the metal coordination sphere, being the most effective agent able to modify the BBB permeability and the most lipophilic one. The molecular mechanism responsible for this modification is not totally clear since our understanding of how aluminum penetrates the biological membranes is still limited. Wen and Wisniewski ~7 obtained an increased BBB permeability to [X4C]sucrose in the rat after treatment with AIC13 and Al(lact)3, while AI(OH)3 was found to be inactive. These data apparently contradict our findings, but it should be observed that these authors utilized an acidic solution (pH approx. 3.0-3.5) and not a neutral one. Moreover, in contrast with the differential changes observed by us in different brain areas, they found a general undifferentiated effect in all brain areas examined.
REFERENCES 1 Akeson, M.A. and Munns, D.N., Lipid bilayer permeation by neutral aluminum citrate and by three a-hydroxy carboxylic acid, Biochim. Biophys. Acta, 984 (1989) 200-206. 2 Alfrey, A.C., Mishell, J.M. and Burks, J., Syndrome of dyspraxia and multifocal seizures associated with chronic hemodialysis, Trans. Am. Soc. Artif. Intern. Organs, 18 (!972) 257-261. 3 Audus, K.L., Holthaus, S.R., van Bree, J.M.B.B. and Shinogle, J.A., Aluminum effect on growth of brain mierovesset endothelial cells in primary culture, Res. Commun. Chem: Pathol. Pharmacol., 60 (1988) 71-85. 4 Banks, W.A., Kastin, A.J. and Fasold, M.B., Differential effect of aluminum on the blood-brain barrier transport of peptides, technetium and albumin, J. Pharmacol. Exp. Ther., 214 (1988) 579-585. 5 Banks, W.A. and Kastin, A.J., Aluminum increases permeability of the blood-brain barrier to the labelled DSIP and /~-endorphin: possible implications for senile and dialysis dementia, Lancet, 2 (1983) 1227-1229.
A n o t h e r example o f effecl of AI tH on the permeability of the BBB to a physiologically relevant neuropeptide. i.e. N-Tyr DISP, has been documented by Banks and Kastin 5. Again, acidic solutions of AI(OH2)6 3+ are likely to have been used. Although the mechanism o f the differential action on BBB permeability changes cannot yet be completely interpreted, the overall action of Al(acac) 3 and of AI(matt)3 on the rat brain also appears to be strongly related to metal speciation. In fact, the lipophilic coordination sphere offered by 3 acetylacetonate ligands per metal ion makes A I m able to cross the BBB and to reach the interior of some neurons of the cerebral cortex. Biophysical evidence both supports and opposes a neutral aluminum c o m p o u n d permeation mechanism ~. It has been recently reported that lipid bilayer permeability by neutral aluminum salts such as citrate is low. Comparison with a-hydroxyl carboxylic acids and trimethyl-citrate suggests that the rate of permeation is limited by hydrogen bonding between water and citrate ligand and by dipole-dipole bonds between water and the bound aluminum 1. O f relevant importance in understanding and interpreting a toxicological study, is therefore ~ welldefined chemical form of the aluminum compounds employed in experiments at physiological pH. Although the present study confirms previous observations of the effect of AIm on the integrity of BBB in the rat, however, we raise some major mechanistically relevant aspects: (i) the effect is qualitatively dependent on the metal speciation, (ii) in the case of the lipophilic toxin, such as Al(acac)3, the effect ~s irreversible, and (iii) it is accompanied by the passage of A1m beyond the BBB and by its access in the interior of neurons. Acknowledgements. Authors thank Dr. B. Corain for helpful discussion. This project was partially supported by Parke Davis.
6 Bombi, G,G., Corain, B., Favarato, M., Giordano, R., Nicolini, M., Perazzolo, M. and ~ t t a , P,, Experimental aluminum dismetabolism in rabbits; effects~of hydrophilic and lipophilic compounds, Environ. Health Persp., 89 (1990) 217-223. 7 Corain, B., Bombi, G.G; and Zatta, P., Differential effects of covalent compounds in aluminum toxicology, Neurobiol. Aging. 9 (1988) 413-414. 8 Candy, J.MI, Oakley, A.E,, Ktimowski, J., Carpenter, T.A.. Perry, R,H., Ataek, J.R., Perry, E.K., Blessed, G., Fairbairn, A. and Edwardson, J.A., Aluminumsilicates and senile plaque formation in Alzheimer's disease, Lancet, 1 (1986) 354-357. 9 De Boni, U., Scott; J,W. and Crapper, D.R., Intracellular aluminum binding: a histoehemieal study, Histochemistrv, 40 (1974) 31-37. 10 Finnegan, M.M;, Steven,LR. and Orvig, C., A neutral watersoluble aluminum e o ~ ! ¢ x of neurological interest, J. Am. Cheml Soc., 108(1986)5033-5035. 11 Hedlund, T. and Oet~man, L.-O., Equilibrium and structural studies of silicon (IV) and aluminium (III) in aqueous solution. 19. Composition and stability of aluminium complexes with ko-
335 jic acid and maltol, Acta Chem. Scand., A42 (1988) 702-709. 12 Kim, Y.S., Lee, M.H. and Wisniewski, H.M., Aluminum induces reversible change in permeability of the blood-brain barrier to 14C-sucrose, Brain Research, 377 (1986) 286-291. 13 Martell, R.M. and Smith, R.M., Critical Stability Constant, Vol. 3, Other Organic Ligands, Plenum, New York, 1977, 246 pp. 14 Ohno, K., Pettigrew, K.D. and Rapoport, S.I., Lower limits of cerebrovascular permeability to nonelectrolytes in the conscious rat, Am. J. Physiol., 235 (1978) H229-H317. 15 Perl, D.P. and Brody, P.R., Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle-bearing neurons, Science, 208 (1980) 297-299.
16 Rapoport, S.I., Ohno, K. and Pettigrew, K.D., Regional cerebrovascular permeability to [14C]sucrose after osmotic opening of the blood-brain barrier, Brain Research, 150 (1978) 653-657. 17 Wen, G.Y. and Wisniewski, H.M., Histochemical localisation of aluminum in the rabbit central nervous system, Acta Neuropathol., 68 (1985) 175-184. 18 Yong, R.C. and Reynolds, J.P., Aluminum acetylacetonate, lnorg. Synth., 2 (1946) 25-26. 19 Zatta, P., Perazzolo, M. and Corain, B., Tris acetylacetonate aluminum(III) induces osmotic fragility and acantocyte formation in suspended erythrocytes, Toxicol. Lett., 45 (1989) 15-21.