Metal IonâNucleic Acid Interactions - ACS Symposium Series (ACS

4 Metal Ion-Nucleic Acid Interactions Aging and Alzheimer's Disease G. L. EICHHORN, J. J. BUTZOW, P. CLARK, H. P. VON HAHN, G. RAO, J. M. HEIM, and E. TARIEN

Downloaded by UNIV OF NORTH CAROLINA on June 21, 2013 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch004

Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore City Hospitals, Baltimore, MD 21224 D. R. CRAPPER and S. J. KARLIK

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University of Toronto, Ontario M5S 1A8, Canada In t h i s paper we d i s c u s s some s t u d i e s on the i n t e r a c t i o n of aluminum with DNA that were c a r r i e d out because of the apparent r e l a t i o n s h i p of aluminum with Alzheimer's d i s e a s e . We then consider how metal ions are i n v o l v e d i n genetic i n f o r m a t i o n t r a n s f e r , and may i n f l u e n c e the aging process, and f i n a l l y we d i s c u s s the use of metal ions i n probing the aging process. Aluminum, DNA and Alzheimer's Disease Alzheimer's disease i s one of the s e n i l e dementias; i n f a c t , i t i s estimated that 70% of the people who have s e n i l e dementia have a form of Alzheimer's d i s e a s e . The cause and treatment of Alzheimer's disease i s t h e r e f o r e o f utmost importance. Crapper and h i s c o l l a b o r a t o r s a t the U n i v e r s i t y of Toronto have reported that autopsies of Alzheimer's p a t i e n t s r e v e a l an accumulation of aluminum ions i n l o c a l i z e d areas of the b r a i n ( 1 ) . They a l s o s t u d i e d the e f f e c t o f i n t r a c r a n i a l l y i n j e c t i n g experimental animals with aluminum, and they found that cats so t r e a t e d accumul a t e aluminum i n b r a i n c e l l s i n concentrations s i m i l a r to those found i n Alzheimer's disease (2). These animals a l s o e x h i b i t s t r u c t u r a l a l t e r a t i o n s i n b r a i n c e l l s that are s i m i l a r but not i d e n t i c a l t o the a l t e r a t i o n s i n Alzheimer's d i s e a s e . DeBoni and Crapper (3) have demonstrated that aluminum accumulates i n the chromatin o f c e l l s . F l u o r e s c e n t microscopy of c e l l s i n m i t o s i s , s t a i n e d with aluminum-staining morin dye, shows aluminum bound to chromatin. I t i s t h e r e f o r e of some p o t e n t i a l relevance to Alzheimer's disease to i n v e s t i g a t e the i n t e r a c t i o n of aluminum and DNA. Let us f i r s t consider what kinds of e f f e c t s metal ions g e n e r a l l y have on DNA. Metal ions b i n d p r i m a r i l y a t two p o s i t i o n s on DNA. They can b i n d to the bases, and i n so doing they can des t r o y the hydrogen-bonded s t r u c t u r e . Therefore, they d e s t a b i l i z e i

Current Address:

Gerontology Research Center, NIA, NIH, B a l t o . C i t y H o s p i t a l s , B a l t o . , MD 21224.

0-8412-05 8 8-4/ 80/47-140-075 $05.00/0 © 1980 American Chemical Society In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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the DNA double h e l i x . On the other hand, metal ions b i n d i n g t o phosphate s t a b i l i z e the double h e l i x . The reason f o r t h i s s t a b i l i z a t i o n i s that the metal ions n e u t r a l i z e the n e g a t i v e l y charged phosphate groups on the s u r f a c e o f the molecule; these would r e p e l each other and cause the molecule t o unwind (4). The two d i f f e r e n t e f f e c t s that metal ions have on the s t a b i l i t y o f DNA are d r a m a t i c a l l y i l l u s t r a t e d by the e f f e c t s of magnesium and copper ions on the DNA "melting" curves, which show the t r a n s i t i o n s between double h e l i c a l DNA, which has a r e l a t i v e l y low absorbance, and s i n g l e stranded DNA, which has a high absorbance (5). An absorbance-temperature p l o t t h e r e f o r e f o l l o w s the unwinding of DNA; the midpoint i n the t r a n s i t i o n i s c a l l e d the melting temperature ( T ) . M g , which binds to phosphate, r a i s e s t h i s T , while C u , which binds to the bases, lowers i t . Mg s t a b i l i z e s the double h e l i x , and C u d e s t a b i l i z e s i t . The e f f e c t s of these two metals demonstrate that metals can s t a b i l i z e DNA by binding to phosphate o r d e s t a b i l i z e i t by b i n d i n g to bases. The melting curves o f DNA i n the presence of these two metal i o n s , and metal ions g e n e r a l l y , are r e l a t i v e l y simple: they produce a monophasic t r a n s i t i o n .


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Aluminum turned out to produce more complicated e f f e c t s . This was perhaps t o be expected, s i n c e A l has a complex chemistry; i n aqueous s o l u t i o n i t e x i s t s i n a l a r g e v a r i e t y o f species (6). In a d d i t i o n to hydrated aluminum i o n . A l or [ A 1 ( H 0 ) ] , there are A1(0H) +, A 1 ( 0 H ) , A1(0H) , A1(0H>4", as w e l l as [Al-j^O^ (OH) 24 ( H 2 0 ) ] ^ The r e l a t i v e amounts o f these s p e c i e s v a r i e s with pH. DNA melting curves were obtained t h e r e f o r e a t d i f f e r e n t pH values and a t d i f f e r e n t aluminum c o n c e n t r a t i o n s . Some o f the m e l t i n g curves e x h i b i t b i p h a s i c t r a n s i t i o n s ; i . e . , part o f the DNA complex melts out i n one temperature region and another p a r t melts out i n another r e g i o n . M e l t i n g curves are presented as d e r i v a t i v e curves, i n which t r a n s i t i o n s become peaks ( F i g . 1 ) . Note the e x i s t e n c e of a high melting aluminum-DNA complex even above 100°C, e.g. a t pH 7.5 and 0.6 Al/DNA as w e l l as a low melting aluminum-DNA complex, as a t pH 5.0 and 0.4 Al/DNA. A t h i r d aluminum-DNA complex melts out i n an intermediate temperature range, e.g. a t pH 6.0 and 0.6 Al/DNA. A n a l y s i s o f the data over a pH range from 5.0 to 7.5 and an Al/DNA concentrat i o n range o f from 0 to 0.7 leads to the c o n c l u s i o n that a l l the melting areas a r e accounted f o r by these three complexes and uncomplexed DNA. We propose the s t r u c t u r e s shown i n Figure 2 f o r the three Al-DNA complexes. We consider that the high m e l t i n g complex I, s t a b l e a t r e l a t i v e l y high pH, contains hydroxylated A l , perhaps A 1 ( 0 H ) i o n . The metal c o n c e n t r a t i o n dependence of the m e l t i n g temperature i s that produced by a d i v a l e n t i o n , and the b i n d i n g o f t h i s i o n to the phosphate would s t a b i l i z e the DNA molecules. The low melting complex I I , s t a b l e i n the a c i d i c r e g i o n , presumably i n v o l v e s A l , hydrated aluminum i o n s , b i n d i n g to the bases o f the DNA and thereby d e s t a b i l i z i n g the DNA double h e l i x . The t h i r d complex, which occurs a t high aluminum concen3 +

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In Inorganic Chemistry in Biology and Medicine; Martell, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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EICHHORN E T A L .

Aging

& Alzheimer's

Disease

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T,«C Figure 1. Derivative melting curves of solutions containing 6 X 10~ M DNA (residue), 5 X 10~ M NON0 , and a mole ratio of Al/DNA residue indicated on top of the columns. pH is shown to the left of the curves for DNA without AL 5

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A l|3*. A l (OH) - A l AI(OH)' ii

AI(OH)' hAl H 3+

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AH Figure 2.

Proposed structures of DNA complexes


Al-

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t r a t i o n s , probably contains both phosphate b i n d i n g A1(0H) and base b i n d i n g A l . 2+ I t has p r e v i o u s l y been shown that Cu ions produce c r o s s l i n k s between DNA strands (12, 13, 14, 15). A l a l s o produces such c r o s s l i n k s , as demonstrated i n the f o l l o w i n g way. When calf-thymus DNA i s heat-denatured, and then cooled, the absorbance does not decrease to the l e v e l c h a r a c t e r i s t i c of d o u b l e - h e l i c a l DNA ( F i g . 3A). The double h e l i x i s not regenerated because the bases i n the denatured s t a t e are out of r e g i s t e r . The s l i g h t decrease i n absorbance on c o o l i n g i s a t t r i b u t e d to l i m i t e d i n t r a strand hydrogen bonding, or h a i r p i n formation. The low-melting Al-DNA complex, on the other hand, does not even form these h a i r p i n s on c o o l i n g - the absorbance remains constant ( F i g . 3B). However, removal of A l by EDTA or by the i n t r o d u c t i o n of a high e l e c t r o l y t e concentration b r i n g s the absorbance back to that of n a t i v e DNA. The e x p l a n a t i o n of t h i s r e v e r s i b i l i t y of DNA denatura t i o n i s that the aluminum ions c r o s s l i n k the n u c l e o t i d e s of the DNA strands during the unwinding of these strands; when the s o l u t i o n has been cooled the DNA strands are h e l d together i n such a way that i t i s now impossible to form h a i r p i n s , and when the aluminum i s then removed with EDTA or with h i g h s a l t , the double h e l i x i s reformed, because the c r o s s l i n k i n g A l ions are able to maintain the complementary bases i n r e g i s t e r . Crossl i n k i n g of the DNA strands could of course account f o r d e l e t e r i o u s b i o l o g i c a l e f f e c t s , and i t i s tempting to speculate that d e f e c t s i n b r a i n s t r u c t u r e c h a r a c t e r i s t i c of Alzheimer's disease could be due to such s t r u c t u r e s . At t h i s p o i n t there i s no evidence that such s t r u c t u r e s e x i s t i n diseased b r a i n . 3 +


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Metal Ions, Genetic Information T r a n s f e r and Aging The p o s s i b l e involvement of aluminum i n Alzheimer's disease i s of i n t e r e s t i n aging because s e n i l e dementia i s sometimes a s s o c i a t e d with aging. Metal ions may be i n v o l v e d i n the aging process i n more general ways, as we s h a l l t r y to demonstrate. I t i s g e n e r a l l y accepted that aging i s g e n e t i c a l l y determined. The dependence of l o n g e v i t y on species and sex, f o r example, cannot be r e a d i l y explained i n any other way. I f aging i s g e n e t i c a l l y determined, there must be changes i n genetic i n f o r m a t i o n t r a n s f e r , which i n v o l v e s the r e p l i c a t i o n of DNA i n the c e l l nucleus, t r a n s c r i p t i o n of the i n f o r m a t i o n contained i n DNA onto messenger RNA, which moves from the nucleus to the cytoplasm, where i t s n u c l e o t i d e sequence i s t r a n s l a t e d i n t o the amino a c i d sequence of p r o t e i n s . Many l a b o r a t o r i e s have demonstrated that age changes do occur i n genetic information t r a n s f e r . C l a r k and Eichhorn have r e c e n t l y shown that there i s an age d i f f e r e n c e i n the a c c e s s i b i l i t y of DNA from the chromatin of o l d and young r a t l i v e r c e l l s to the a c t i o n of m i c r o c o c c a l nuclease, which s p l i t s i n t e r n u c l e o t i d e bonds of DNA (11). As already i n d i c a t e d , t h i s i s only one of many examples of age changes i n genetic i n f o r m a t i o n


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Figure 3. Melting (O) and cooling-reheating (% A) curves for 6 X 10~ M DNA (residue) in 5 X 10 ' M N,NO,: (A) pH 6.3, without Al; (B) pH 5.1, 0.6 Al/DNA (residue) 5

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t r a n s f e r . None of these s t u d i e s have l e d t o an understanding o f the b a s i c cause of aging. I t i s u s e f u l to consider what, i f anything could be done about aging, i f the b a s i c cause (or causes) o f the aging process were ever discovered. Perhaps some form o f genetic engineering could become f e a s i b l e , but genetic engineering i s a s s o c i a t e d with d i f f i c u l t moral problems. I f there i s an impact from the environment onto genetic i n f o r m a t i o n t r a n s f e r , i t could be e a s i e r t o d e a l with such an environmental impact, and i t would be morally l e s s d i f f i c u l t (11). Metal ions enter c e l l s o f l i v i n g organisms from the e n v i r o n ment. Some o f these are e s s e n t i a l metal ions and others are none s s e n t i a l . Metal ions are i n v o l v e d i n every step o f genetic i n f o r m a t i o n t r a n s f e r . They a f f e c t the s t r u c t u r e o f chromatin; i t has been demonstrated by e l e c t r o n microscopy that the concent r a t i o n of magnesium ions i n c e l l n u c l e i determines the packing of the chromatin (12). Some s t u d i e s c a r r i e d out i n our l a b o r a t o r y i n d i c a t e that metal ions may be i n v o l v e d i n age changes i n the s t r u c t u r e o f chromatin (13). C e l l n u c l e i were i s o l a t e d from the l i v e r o f mature (12 mo.) and o l d (26 mo.) r a t s , and from the chromatin obtained from these n u c l e i , the h i s t o n e s were chromatographed on a Sephadex column. Four peaks were produced from mature r a t l i v e r chromatin ( F i g . 4A); two of these peaks were s u b s t a n t i a l l y diminished i n the chromatogram from the o l d r a t s ( F i g . 4B). The n u c l e i i n both instances had been i s o l a t e d i n the presence o f magnesium. I f the h i s t o n e s from mature r a t l i v e r chromatin were obtained from n u c l e i i s o l a t e d i n the absence o f magnesium, o r even i n the presence o f EDTA, the same peaks were diminished as i n the case o f the m a t e r i a l from the o l d n u c l e i ( F i g . 4C). Thus, the absence o f metal ions i n the i s o l a t i o n o f the n u c l e i produces a s i m i l a r a f f e c t as aging. I t seems that metal ions a r e i n v o l v e d i n the o r g a n i z a t i o n o f the nuclear matter, and something i n t h i s o r g a n i z a t i o n changes with age. As has been i n d i c a t e d above, metal ions are e s s e n t i a l i n every aspect o f genetic i n f o r m a t i o n t r a n s f e r . Nevertheless, metal ions can a l s o cause d e l e t e r i o u s e f f e c t s i n i n f o r m a t i o n t r a n s f e r e i t h e r i f they are present i n the wrong k i n d or i n the wrong c o n c e n t r a t i o n . L e t us consider an example of each o f these p o s s i b l i t i e s ; f i r s t , that i n which metals are present i n the wrong k i n d . In RNA s y n t h e s i s , the RNA polymerase enzyme must be capable of d i f f e r e n t i a t i n g between a r i b o n u c l e o t i d e and a deoxynucleotide; i . e . , i t must i n s e r t only those n u c l e o t i d e s that have an OH group i n the 2 - r i b o s y l p o s i t i o n . One o f the f o l l o w i n g metal i o n s , M g , C o , o r Mn +, i s r e q u i r e d f o r the a c t i v i t y o f RNA polymerase. Manganese i s the most e f f e c t i v e f o r the c o r r e c t i n c o r p o r a t i o n o f the r i b o n u c l e o t i d e s i n t o RNA. However, manganese i s the only one of these three metal ions t h a t causes s u b s t a n t i a l i n c o r r e c t i n t r o d u c t i o n o f deoxynucleotides i n t o RNA (14, 15). Thus, even though magnesium i s l e s s e f f e c t i v e than manganese f o r the c o r r e c t ,

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Metal IonâNucleic Acid Interactions - ACS Symposium Series (ACS

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