A ct a Neuropathologica

Acta Neuropathol (Berl) (1982) 57:249 254

9 Springer-Verlag 1982

Inhibition of Corticosterone Binding in Vitro, in Rabbit Hippocampus, by Chromatin Bound Aluminum* C. Sanderson 1,2, D. R. Crapper McLachlan 1, and U. De Boni 1, 2 Dept. of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S IA8 2 Surrey Place Centre, Ministry of Community and Social Services, Province of Ontario, Canada

Summary. Brains of patients with senile dementia of the Alzheimer type and with the dialysis encephalopathy syndrome, exhibit elevated aluminum levels. In Alzheimer's disease and in the experimental aluminum induced encephalopathy~ intracellular aluminum is associated with nuclear chromatin. The work reported here was undertaken to test whether chromatin bound aluminum in hippoccampus of the rabbit interferes with nuclear binding of corticosterone-receptor complexes. The results showed that the mean binding of corticosterone decreased from 460 + 71 fmol/mg D N A in controls, to 343 _+ 81 fmol/mg D N A in hippocampal nuclei from aluminum treated rabbits, representing a decrease of 25%. This reduction occurred in the absence of aluminum induced neurofibrillary degeneration and indicates a possible functional consequence of the presence of aluminum on chromatin, and importantly, in the absence of morphological changes.

Key

words: Aluminum Hippocampus - Rabbit

-

Steroid

binding

-

Introduction Aluminum is an extremely neurotoxic element which induces a lethal encephalopathy with neurofibrillary degeneration in experimental animals (Klatzo et al. 1965; Crapper and Dalton 1973; De Boni et al. 1976). The observation of elevated aluminum levels in whole brain tissue of patients with Alzheimer's disease, in Alzheimer's disease associated with Down's syndrome, as well as in the dialysis encephalopathy syndrome (Crapper et al. 1973; Crapper et al. 1976; Crapper et al. 1980; Alfrey et al. 1976; Arieff et al. * Supported by the Ontario Mental Health Foundation and by Surrey Place Center Oj]))rint requests to: U. De Boni, PhD, Dept. of Physiology, Medical Sciences Building, Universityof Toronto, Toronto, Ontario, Canada, M5S l A8

1979; M c D e r m o t t et al. 1978), has precipitated recent interest in the mechanism of action of this cytotoxic element. De Boni et al. (1974), established nuclear chromatin as the intranuclear locus of aluminum accumulation in a number of cell types, including neurons and glia of experimental animals. Similarly, analysis of nuclear fractions of Alzheimer affected tissues (Crapper and De Boni 1977) showed aluminum to be associated with nuclear chromatin. Recent Work by Perl and Brody (1980) further confirmed this locus of accumulation in brain cells of patients with senile dementia of the Alzheimer type, using wavelength X-ray spectrometry. In addition, studies in our laboratories, employing such sensitive techniques as sister chromatid exchange and unscheduled D N A synthesis (De Boni et al. 1980) have demonstrated aluminum induced functional consequences of this association. Moreover, work on chromosomes of a dipteran species clearly showed that chromatin bound aluminum interacts functionally with its binding sites by inhibiting steroid induced puffing (Sanderson et al. 1979, 1981). The work reported here was undertaken to test whether changes in gene modulation are possible mechanisms by which this element mediates its cytotoxic effects. In the work reported here, intranuclear binding of corticosterone receptor complexes was used as a marker; employing rabbit hippocampus from controls and from aluminum treated animals.

Material and Methods Animal Preparation

Four week old female New Zealand white rabbits were used in this study. Aluminum (21 gmol in 0.1 ml, sterile distilled water, pH 7.2) was infused as the lactate salt directly into the ventricles. Controls were infused with an equimolar amount of sodium lactate in distilled water (pH = 7.2). Both, experimental and control animals were kept for 10 days post injection.

/

/

0001-6322/82/0057/0249/$1.20

250

Acta Neuropathol (Berl) 57 (1982)

Tissue Preparationand Incubation

Histology

Stock solutions of 1.039 • 10 - 6 M 3H-corticosterone (NEN; 1,2,6,73H, 82.1Ci/mmol) were prepared as a benzene:ethanol solution (9 : 1, V/V). The non-radioactive corticosterone (Sigma)solution was made fresh on the day of each experiment (1.075 • 10 z M). The total volume was brought to 5 ml with a balanced salt solution (pH 7.4) (Yamamoto 1972), resulting in a final concentration of 3Hcorticosterone of 1.039 x 10-7M and of non'radioactive corticosterone of 1.975 • 10-4 M, in the incubation vial. For each experiment 3 or 4 animals were sedated with acepromazine maleate (2.0 mg/kg, Atrovet, Ayerst) and killed 1.5 h later by decapitation. At this time, citrated plasma was prepared for the determination, by the method of Etches (1976), of endogenous corticosterone levels. The brains were removed, placed on a plastic dish on ice and the hippocampi isolated. The two hippocampi of each animal were combined, minced and dividedevenlyby weight (approx. 0.25g) into the incubation vials prepared as described above. Incubation was carried out for 3h with continuous, vigorous agitation in a water bath at 37~C. At the end of incubation the vials were placed on ice.

To test for the presence in hippocampus of neurofibrillary degeneration, sections were stained with Bielschowsky stain. In addition, electronmicroscopy was carried out on selected areas of the hippocampus, including specificallythe regions CA-l, CA-4 and the dentate cells, using standard electronmicroscopy techniques.

Nuclear Isolation and Extraction The following procedures were performed at 0~ to 4~ unless otherwise noted. After incubation the tissue was washed three times in 20 ml fresh balanced salt solution followed by centrifugation at 2,000 • g for 5 rain. Nuclei were isolated in sucrose solutions using methods modified from McEwen and Zigmend (1972). The washed tissue was suspended in 10ml of phosphate buffer with 0.32M sucrose (1 mM KH2PO4, 3 mM MgCI2, 0.32M sucrose, pH 6.5 Solution A) and homogenized for 2.5rain in a Potter-Elvehjem homogenizer. The homogenate was filtered through a 100 ~tm nylon mesh to remove blood vessels and connective tissue, followed by centrifugation at 2,000 x g for 5 rain. The supernatant was decanted and discarded, the pellet resuspended in 10ml of solution A with 0.25 % (V/V) Triton X-100, then centrifuged as before. The resulting nuclear pellet was washed again in 10ml of solutionA and centrifuged. The nuclear pellet was then transferred in J.5 ml solution A to a "Biorad", conical micro test tube, and centrifuged at 2,000 • g for 5 rain. The receptor-steroid complex was extracted from this final nuclear pellet into i ml of 25 mM Tris, 0.6 M KCI, pH 8.5 for 1 h and vortexed every 10 rain Following centrifugation for 10 min at 20,000 x g, 400 gl aliquots of the supernatent containing the steroid receptor complex were transferred to scintillation vials, containing 10ml Aquasol (NEN) and counted in a Nuclear Chicago Corporation, Mark II scintillation counter at 40 ~ efficiency as determined by standard quench curves. The 20,000 • g pellet was resuspended in 1 N perchloric acid for DNA determination by the Burton method (1956). Specific binding was expressed as fmol bound/mg DNA and defined as the difference between total binding and the nondisplaceable or nonspecific binding measured in the presence of 1,000-fold excess cold steroid. The results were analyzed using ANOVA statistics for unequal N.

Optimization of Conditionsfor Incubation The time course of binding was studied by incubation of the tissue with labelled corticosterone with and without 1,000-fold excess of non-labelled corticosterone for 30, 60, 120, 180, 240 min and at 15, 21, 30 and 37~C, to determine optimal incubation time and temperature respectively. Saturation of binding was determined using 5 concentrations of 3H-corticosterone between 10-v and 10 - 9 M. Parallel incubations were carried out with the addition of 1,000-foldexcess cold corticosterone, to measure nonspecific binding, according to the method of McEwen and Wallach (1973).

Results Optimization o f Incubation Parameters M i n c e d h i p p o c a m p i from n o n a d r e n a l e c t o m i z e d rabbits were i n c u b a t e d with increasing c o n c e n t r a t i o n s of 3H-Corticosterone (see Methods) at 37~ for 3 h. As s h o w n in Fig. 1 a, b i n d i n g reached a plateau, i n d i c a t i n g s a t u r a t i o n of specific b i n d i n g sites, at 1.039 • 1 0 - 7 M 3H-Corticosterone. The results of studies m e a s u r i n g the b i n d i n g o f steroid as a f u n c t i o n o f time (Fig. 1 b) a n d t e m p e r a t u r e (Fig. l c ) also clearly indicated that inc u b a t i o n of 3 h at 37 ~C were the c o n d i t i o n s required for m a x i m u m exchange of e n d o g e n o u s steroid to occur.

Effects o f Aluminum Treatment H a v i n g established o p t i m a l c o n d i t i o n s for exchange a n d r e p r o d u c i b l e b i n d i n g , studies were u n d e r t a k e n to c o m p a r e corticosterone b i n d i n g in c o n t r o l a n d alum i n u m treated rabbits. A two-way analysis of variance test ( A N O V A ) of the results clearly d e m o n s t r a t e d a decrease in corticosterone b i n d i n g in a l u m i n u m in~ jected versus c o n t r o l rabbits, which was treatment, b u t n o t age d e p e n d e n t [F0.975, (1, 3)]. T h e significance of the above statistical test permitted the b i n d i n g data to be pooled into two large t r e a t m e n t groups, c o n t r o l a n d a l u m i n u m treated, irrespective of r a b b i t age. A N O V A o f the data of these two groups, indicated a statistically significant difference [F0.999 (1, 35)] between the m e a n o f 460 f m o l / m g D N A for controls a n d of 343 f m o l / m g D N A for a l u m i n u m treated r a b b i t s (Table 1 a n d Fig. 2). This represents a definite depression of 25 ~ in corticosterone b i n d i n g in those a n i m a l s injected with a l u m i n u m lactate. This decrease occurred in the presence of u n a l t e r e d e n d o g e n o u s corticosterone levels in controls a n d a l u m i n u m treated animals. Specifically, s e r u m corticosterone levels from controls a n d treated a n i m a l s were 5.6 + 3.15 n g / m l ( N = 4) a n d 5.89 + 2 . 6 6 n g / m l ( N = 3) respectively. Therefore the reduced b i n d i n g o f 3H-corticosterone in the treated g r o u p c a n n o t be a t t r i b u t e d to competitive i n h i b i t i o n b y e n d o g e n o u s steroid. As s h o w n in T a b l e 2 , t r e a t m e n t with a l u m i n u m decreased b o t h the total a n d n o n specific b i n d i n g by 30 ~ a n d 28 ~ respectively. This decrease in b i n d i n g was associated with a n increase in D N A recovery of 33 ~o a n d 35 ~ respectively in these assays. T h e d a t a thus s u p p o r t s the hypothesis that

C. Sanderson et al. : A l u m i n u m and HippocampaI Steroid Binding

< Z r~

600

25t 600

A

-

400 -

~

E \ 200

<(

-

400

Z

0

/'

E

t1" 0

I 50

I 100

I 150

I 200

I 250

X 10- 9

< Z r"h

'N=2

600

E

\ E 200

B

-

9

Control

9

AI

treated

400 -

E \

-S

0

37

200 -

9 |

I

I

I

I

50

100

150

200

250

N 600-

40

mean of all expts.

Table 1. Corticosterone binding in control and a l u m i n u m treated rabbits

rain

< Z

39

in d a y s

Fig. 2. Composite diagram showing decreased binding of corticosterone in the presence of aluminum. Note absence of age effect

E 0

38

Age

Age (days)

Mean fmol/ mg DNA

•

S.D.

Range fmol/mg DNA

8 6 4 2

37 38 39 40

448 458 499 439

+ _+ + +

72 96 33 28

376-520 362-554 466-532 411-467

20

-

460 a

_+ 71

6 6 5

38 39 40

350 305 381

• 86 +_ 77 _+ 75

17

-

343 a

_+ 81

C

400 =

E \ 200

=

9

E

Pooled data

'4.--

I 15

I 21

30

3

0C Fig. I A - - C . Results of optimization experiments. A Binding as a function of steroid concentration. B Binding as a function of time of incubation. C Binding as a function of incubation temperature. See text for additional detail

aluminum treatment decreases the number of available chromatin receptor sites for corticosterone.

Histology Both light and electronmicroscopic examination revealed an absence of neurofibrillary degeneration in the hippocampal cells employed in this study. Specifically, the hippocampal structures CA-1 to CA-4 and the granule cells of the dentate gyrus were entirely free of neurofibrillary degeneration (Fig. 3). A few scattered

Pooled data

264-436 228-382 306-456

a Significantly different means at F0.999 (I, 35)

cells only in the subiculum exhibited neurofibrillary degeneration. The cells so affected were present in such low numbers, that the changes in steroid binding are unlikely to result from changes in cells exhibiting neurofibrillary degeneration. Moreover, there was no histological evidence of gliosis and no loss of neurons, as assessed by light and electronmicroscopy. Therefore, dilution of the neuronal nuclear fraction by glial cell nuclei, which do not bind corticosterone, may be excluded. Therefore, it. is important, t o note that aluminum alters steroid binding to hippocampal cells

Fig. 3A, B

253

C. Sanderson et al. : Aluminum and Hippocampal Steroid Binding Table 2. Total and nonspecific corticosterone binding and DNA recovery Nonspecific corticosterone binding data

Total Corticosterone binding data N

Control 20 950.3

fmol/mg DNA

,+ 76.8

Aluminum 17

Difference

Control 20

682.5

-30

487.8

+ 129.5

Whole tissue wet 2 weight (g)

0.2406 + 0.027

0.2243 _+

DNA recovered/ whole tissue weight mg/g

0.527 +__ 0.07

0.814 __+ 0.18

2

0.043

independent of metabolic changes associated with neurofibrillary degeneration. This observation is important when considering the possible contribution of aluminum to the pathophysiology of such conditions as dialysis dementia, where elevated aluminum is not associated with morphological changes. Discussion

The data shows that corticosterone binding in aluminum treated animals is reduced by 25 ~. While there was an increase of 33 ~ and 35 ~ in total and nonspecific binding assays respectively in the DNA yield from aluminum treated tissue, this increase was not paralleled by an increase in chromatin bound steroid. This supports the hypothesis that aluminum interferes with normal steroid-chromatin interactions. The intranuclear acceptors for cytosol-steroid complexes are thought to be a class of nuclear acidic proteins (Spelsberg et al. 1971). Evidence is available to show that intranuclear aluminum also is associated with acidic nuclear proteins in neurons and glial cells (Crapper and De Boni 1977). It may therefore be postulated that aluminum induced conformational changes involving these proteins in particular and in chromatin in general may directly alter nuclear binding of steroids. The finding that intranuclear aluminum does not interfere with access of the cytosol-steroid complex to its nuclear acceptor site in at least one cell system (Sanderson et al. 1979, 1981) supports the hypothesis that altered steroid binding as shown in this work is a direct consequence of the presence of aluminum on nuclear chromatin. Nevertheless, the possibility exists that aluminum either reversibly or irreversibly interacts with the cytosol receptors to

+_ 79.0

Aluminum 17

Difference

339.6

-28

,+ 131.1

-

0.2245 _+ 0.04

0 . 2 1 9 9 _ + 0.04

-

+33

0.541 +__ 0.11

0.810 __+ 0.23

+35

directly alter their affinity coefficient for corticosterone. Alternatively, it must be considered that intracellular aluminum may non specifically, yet sufficiently interfere with either transcription or translation. Changes in these processes may result in decreased numbers of acceptor proteins in either cytosol or nuclear fractions or in altered acceptor activity and consequently in the observed reduction in nuclear binding. It is important to note that the inhibition of binding occurs in the absence of neurofibrillary degeneration. This in turn indicates one possible mode of toxic action of aluminum in cells which do not exhibit morphological changes. Acknowledgements. We wish to thank Dr. G. E. Mobbs, Ivy Johnson and A. Rodriguez for their enthusiastic technical support in this project, and Yvonne Nease for assembling the manuscript.

References Alfrey AC, Le Gendre GR, Kaehny WD (1976) The dialysis encephalopathy syndrome: Possible aluminum intoxication. N Engl J Med 294:184-188 Arieff AI, Cooper JD, Armstrong D, Lazaromitz BS (1979) Dementia, renal failure and brain aluminum. Ann Intern Med 90: 741 - 747 Burton K (1956) A study of the conditions and mechanisms of diphanylamine reaction for the colourimetric estimation of deoxyribonucleic acid. Biochem J 62:315 - 323 Crapper DR, Krishnan SS, Dalton AJ (t973) Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science 180 : 511 - 513 Crapper DR, Dalton AJ (i973) Alterations in short term retention, conditioned avoidance response acquisition and motivation following aluminum induced neurofibrillary degeneration. Physiol Behav 10: 925 - 933 Crapper DR, Krishnan SS, Quittkat S (1976) Aluminum, neurofibrillary degeneration and Alzheimer's disease. Brain 99: 6 7 - 80 Crapper DR, De Boni U (1977) Aluminum and the genetic apparatus in Alzheimer's disease. In: Nandy K, Sherwin I (eds) The aging

Fig. 3A, B. Hippocampal region CA-I, from rabbit injected intracerebrally with aluminum lactate. Ten days post-injection, showing tissue typical of that employed in this study. A Light micrograph of 1 gm Epon section, stained with, toluidine btue. B Electronmicrograph of same area. Note absence of aluminum induced neurofibrillary degeneration. Magnification: A Bar = 10 gm, B Bar - 1 gm

254 brain and senile dementia. Plenum Press, New York, pp 2 2 9 246 Crapper DR, Quittkat S, Krishnan SS, Dalton A J, De Boni U (1980) Intranuclear aluminum content in Alzheimer's disease dialysis encephalopathy and experimental aluminum encephalopathy. Acta Neuropathol (Berl) 50:19 - 24 De Boni U, Scott JW, Crapper DR (1976) Intracellular aluminum binding: A histochemical study. Histochemie 40: 31 - 34 De Boni U, Otvos A, Scott JW, Crapper DR (1976) Neurofibrillary degeneration induced by systemic aluminum. Acta Neuropathol (Berl) 35 : 285-- 294 De Boni U, Seger M, Crapper-McLachlan DR (1980) Functional consequences of chromatin bound aluminum in cultured human cells. Neurotoxicology 1 : 65 - 82 Etches RJ (1976) A radioimmunoassay for corticosterone and its application to the measurement of stress on poultry. Steroids 28 : 763 - 773 Klatzo I, Wisniewski H, Streicher E (1965) Experimental production ofneurofibrillary degeneration. I. Light microscope observation. J Neuropathol Exp Neurol 24:187-199 McEwen BS, Zigmend RE (1972) Isolation of brain cell nuclei. In: Marks N, Rodnight R (eds) Research methods in neurochemistry, vol 1. Plenum Press, New York

Acta Neuropathol (Berl) 57 (1982) McEwen BS, Wallach G (1973) Corticosterone binding to hippocampus: Nuclear and cytosol binding in vitro. Brain Res 57:373-386 Perl DP, Brody AE (1980) Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tanglebearing neurons. Science 208:297- 298 Sanderson CL, Crapper DR, De Boni U (1979) Altered response to ecdysterone by chromatin bound aluminum in a polytene chromosome of Simulium Vittatum. J Cell BioI 83:152a (Abstract) Sanderson CL, Crapper-McLachlan DR, De Boni U (1982) Altered steroid induced puffing by chromatin bound aluminum in a polytene chromosome of the black fly, Simulium Vittatum. Can J Genet Cytol 24: 2 7 - 36 Spelsberg TC, Steggles AW, O'Malley BW (1971) Progesterone binding components of chick oviduct. III. Chromatin acceptor sites. J Biol Chem 246:4188- 4197 Yamamoto C (1972) Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro. Exp Brain Res 14:423-435

Received January 31, 1982/Accepted March 22, 1982