DNA damage during aging of mouse myocardium

J Mol Cell Cardio118, 1317-1320 (1986)

DNA Damage During Agingof Mouse Myocardium (Received15 oTuly1986, acceptedin revisedform 28 August 1986) It is n o t k n o w n i f DNA lesions, s u c h a s c o v a l e n t l y modified nucleotides, c h a n g e q u a l i t a t i v e l y o r q u a n t i t a t i v e l y d u r i n g a g i n g o f p o s t - m i t o t i c cells [7]. I f g e n e t i c d a m a g e a c c u m u l a t e s f a s t e r t h a n c e l l u l a r s y s t e m s c a n r e p a i r it, a cell will e v e n t u a l l y b e c o m e defective in m a i n t a i n i n g h o m e o s t a t i s . T h i s s i t u a t i o n w o u l d be p a r t i c u l a r l y s e r i o u s f o r cells t h a t do not divide after they have differentiated to their t e r m i n a l f o r m s , for example, heart m u s c l e cells. T o e x a m i n e t h i s p o s s i b i l i t y a 32p. p o s t l a b e l i n g t e c h n i q u e [12, 13] w a s u s e d to m e a s u r e the relative level o f m o d i f i e d n u c l e o t i d e s in m o u s e m y o c a r d i a l DNA a s a f u n c t i o n o f age. T h e p o s t l a b e l l n g a n a l y s i s i n d i c a t e d t h a t a modified nucleotide changed in a n age-dependent m a n n e r . A n u c l e o t i d e w i t h s i m i l a r c h r o m a t o g r a p h i c properties w a s induced b y N - m e t h y l - N n i t r o s o u r e a (MNU) a l k y l a t i o n o f s y n t h e t i c p o l y d e o x y n u c l e o t i d e s c o n t a i n i n g the base g u a n i n e b u t n o t b y a l k y l a t i o n o f s y n t h e t i c DNAs l a c k i n g t h i s base. T h i s m o d i f i e d nucleotide w a s found to increase about 9-fold in heart DNA between 2 m o n t h s a n d 39 m o n t h s . These r e s u l t s s u g g e s t that the steady-state level o f t h i s t y p e o f g e n o m i c d a m a g e is greatly elevated in senescent m o u s e heart tissue.

The animals used in this investigation were the highly inbred mouse strain C57BL/ 6NNia. These mice are used extensively in aging research, one reason being to minimize genetic fluctuations. The mice were obtained from the colony maintained by the National Institute on Aging where they are Caesareanoriginated and maintained behind a barrier to exclude microbial pathogens. Immediately after receiving the mice, they were killed, and hearts were frozen at --70~ until such time as the DNA was extracted. DNA was prepared [5] from 3 to 5 pooled heart tissues of male mice at 2, 6, 10, 15, 17, 29 and 39 months of age. Two samples were prepared at each age, and DNA concentrations were determined by the diphenylamine assay [3]. The [32p]-postlabeling technique of Randerath and associates [12, 13] was then used to examine alkylated nucleotides that exist in mouse DNAs in the absence of any experimental treatment that would induce such modified nucleotides. Therefore, any alkylated nucleotides observed in these DNAs 0022-2828/86/121317 + 04 $03.00/0

would presumably arise as a consequence of 'normal' environmental factors and biological processes during aging. Nucleotides from 32p-labeled digests were separated by two-dimensional, thin-layer chromatography (TLC) carried out on polyethyleneimine cellulose. Under the solvent conditions employed in this separation, some of the major alkylated species can be resolved from normal nucleotides on the basis of reduced charge and increased hydrophobicity [12]. This system does not separate the naturally-occurring methylated nucleotide, 5methyldeoxycytosine-5'-monophosphate (5medCMP), from d C M P [13]. As shown in Figure 1, this analysis revealed a labeled nucleotide (indicated by arrows) that appeared to increase substantially with age. A series of control experiments were performed to characterize the postlabeling assay. These controls included T L C analyses of [-y_32p]. ATP alone, reactions minus DNA, and reactions labeling a variety of synthetic deoxypolymers. As represented by labeling of 9 1986AcademicPress Inc. (London)Limited

1318

Rapid Conmaunicatlon: J. W. Gaubatz

FIGURE 1. Autoradiograms of a2P-labeled nucleotides obtained from digests of mouse hearts DNA and poly (dA - dC) 9 poly(dG - d 7-) and mapped by two-dimensional, thin-layer chromatography, a2p-postlabeling analysis of methylated adducts was performed as described by Reddy et al. [13]. Autoradiography was performed for 10 min at room temperature for standard nucleotides (left side of plates) or for 62 to 78 h at --70~ for modified nucleotides (right side of plates). (a) Poly(dA- dC) - poly(dG-dT); (b) DNA from 2-month-old mouse heart; (c) DNA from 17-month-old mouse heart; (d) DNA from 29-month-old mouse heart; (e) DNA from 39-month-old mouse heart; (f) Poly(dA) -- dC) - poly(dG - dT) treated with 80 mM N-methyl-N-nitrosourea as described by Randerath et al. [12]. Plate (f) is a 13 h exposure at room temperature. OR, origin; ATP, y-a2p-ATP; P, inorganic-a2P; X, unknown spot produced during kinase reaction; dGMP, dTMP, dAMP, and dCMP are 32p-labeled deoxyguanosine-5'monophosphate, etc.; 7-meGMP, 10 pg of 7-methylguanosine-5'-monophosphate (Aldrich) identified by uvfluorescence. p o l y ( d A - dC) 9 p o l y ( d G -- d T ) , shown in Figure 1 (a), these reactions d i d not p r o d u c e a distinct a u t o r a d i o g r a p h i c spot t h a t corresponded to the labeled nucleotide observed with h e a r t D N A . O n the o t h e r h a n d , the a u t o r a d i o g r a p h i c intensity of one spot was clearly greater in D N A samples from senescent h e a r t tissue. This is seen b y c o m p a r i n g a2P-fingerprints of D N A from y o u n g mice [Fig. l ( b ) ] with m y o c a r d i a l D N A from m a t u r e animals [-Fig. 1 (c)] or those samples o b t a i n e d from old animals [-Fig. l ( d ) a n d l(e)]. Studies h a v e shown t h a t v i r t u a l l y all nucleophilic sites on D N A are susceptible to alkylation [9, 15]. T h e relative yield of alkyladducts d e p e n d s u p o n m a n y factors, b u t in general, the most reactive site on D N A is the N - 7 position of the base g u a n i n e which lies

exposed in the m a j o r groove o f the helix [15]. T o a t t e m p t to identify the modified nucleotide found in h e a r t D N A , various synthetic D N A s were treated with the alkylating a g e n t M N U . N-methyl-Nonitrosourea t r e a t m e n t of p o l y ( d A -- dC) 9 p o l y ( d G - dT), p o l y ( d G dC) 9 p o l y ( d G - dC), or oligo(dG) p r o d u c e d a m e t h y l a t e d nucleotide t h a t h a d the same or similar mobilities as the modified nucleotide [-Fig. l ( f ) ] . I n contrast, M N U - t r e a t m e n t of poly(dA - dT) 9 poly(dA - dT) did not induce a m e t h y l a t e d nucleotide with mobilities similar to 7-methylguanosine-5'monophosphate (7-meGMP). Furthermore, u n l a b e l e d 7 - m e G M P was c h r o m a t o g r a p h e d with 32p-labeled nucleotides as i n d i c a t e d in F i g u r e 1 (a). T h e modified nucleotide, identified in a u t o r a d i o g r a m s of mouse h e a r t 32p. maps, c o c h r o m a t o g r a p h e d with 7 - m e G M P in

Cardiac DNA During Aging both the first and second dimensions. T h u s the results are consistent with the modified nucleotide of m y o c a r d i u m being an in vivo m e t h y l a t i o n a d d u c t o f guanine. T h e r a d i o a c t i v i t y associated with the m o d i fied a n d n o r m a l nucleotides was d e t e r m i n e d by scraping a n d c o u n t i n g the a p p r o p r i a t e areas of the c h r o m a t o g r a m s . T h e relative level of modified nucleotide ( R M N ) is an estimate o f the a m o u n t o f modified nucleotide in a sample. T h e values are corrected for the labeling efficiency o f i n d i v i d u a l reactions a n d b a c k g r o u n d levels o f r a d i o a c t i v i t y associated with such reactions. W i t h i n one e x p e r i m e n t all assays were performed u n d e r identical conditions with the same reagents, a n d the m a j o r v a r i a b l e was the D N A sample. F o r a c o m p a r ative study a n a l y z i n g a single modification, one can c o m p a r e the relative a b u n d a n c e , b u t not the absolute q u a n t i t y , of an a d d u c t between samples. T h e postlabeling analysis of mouse m y o c a r d i u m , s u m m a r i z e d in F i g u r e 2, shows the relative level of modified nucleotide at different ages. I n y o u n g mice, 2 to 10 m o n t h s old, the R M N v a r i e d from 7 to 11 modifications p e r 106 nucleotides. T h e level of modified nucleotide increased a b o u t 2-fold in m a t u r e mice, 15 a n d 17 months old, a n d reached a 4-fold i n c r e m e n t of 40 a d d u c t s p e r 106 nucleotides at 29 months of age. I n the terminal p o r t i o n o f lifespan between 29 months a n d 39 months, there was a n o t h e r d o u b l i n g in the level of this modified nucleotide. T h e differences between age-groups were significant at the following levels: l 0 a n d 15 months, P < 0.01; 15 and 29 months, P < 0.05; a n d 29 a n d 39 months, P < 0.001 (Student's t statistic). T h e d a t a of F i g u r e 2 suggest an exponential rise in this modified nucleotide past m a t u r i t y . T h e present results i n d i c a t e t h a t a l k y l a t e d nucleotides exist at some steady-state level in m y o c a r d i a l D N A in vivo a n d t h a t there is an increase in this form o f d a m a g e d u r i n g aging. A m o n g c o m p o u n d s t h a t a r e c o m m o n in o u r external a n d internal environments are p o t e n t alkylating agents which are c a p a b l e of d a m aging cellular D N A in vivo [1]. N u m e r o u s studies have d e m o n s t r a t e d t h a t some alkyla d d u c t s can persist for e x t e n d e d periods in postmitotic tissues [6, 10, 11]. T h e a m o u n t o f 7-meG usually correlates well with the overall level of i n d u c e d in vivo D N A alkylation, b u t

1319

I00

80

T~ X

z n.*

60

IO

20

1 30

,

i

40

Age in months

FIGURE 2. Relative level of a modified nucleotide (RMN) in mouse heart DNA at different ages. Radioactivity associated with modified and normal nuclentides was determined by aligning a TLC sheet over an autoradiogram on a light box; the various spots were circled and labeled ; a sharp spatula was used to scrape off the PEI-cellulose corresponding to a labeled nucleotide; the contents were then placed in a 7 ml scintillation vial and 4 ml of Aquasol-2 (New England Nuclear) was added for scintillation counting. The cpm for control reactions lacking DNA and for poly(dA - dG) 9 poly(dG -- dT) were obtained from scraping the area corresponding to the reference compound, 7-meGMP. The relative level of modified nucleotide (RMN) present in a DNA sample was calculated from the formula: RMN =

cpm in modified nueleotide-background cpm in normal nucleotides

Background radioactivity was taken from the modified nucleotide region of thin layers of the poly(dA -- dG) 9 poly(dG -- dT) reaction as described above. The data are the mean of two experiments for 6-month (RMNs, 5 and 9) and 17-month (RMNs, 26 and 31) heart, and the mean and standard deviation (bars) of three experiments for 2-month, 10-month, 15-month, 29month and 39-month heart samples. persistence o f i n d i v i d u a l adducts varies with D N A r e p a i r capabilities t h a t d e p e n d u p o n species a n d cell type [2, 4]. T h e a p p r o a c h taken in this s t u d y does not give information a b o u t the persistence of d a m a g e . T h u s it is not known w h e t h e r the nucleotide modifications are recent events or have a c c u m u l a t e d from an earlier age. A n age-related increase in the steady-state level o f D N A d a m a g e implies that the rate of d a m a g e or the rate o f r e p a i r (or both) have changed. T h e r e are several ways the level o f

1320

R a p i d C o m m u n i c a t i o n : J. W. G a u b a t z

d a m a g e m i g h t increase w i t h a g e : (a) there m a y be a decrease in r e p a i r capabilities; (b) r e p a i r enzymes m a y lose their fidelity; (c) D N A m a y become less accessible to r e p a i r enzymes; or (d) changes in the i n t r a c e l l u l a r e n v i r o n m e n t a n d m e t a b o l i s m m a y increase the extent of D N A alkylation. Studies have d e m o n s t r a t e d an a g e - r e l a t e d loss of D N A r e p a i r synthesis in isolated r o d e n t m y o c a r d i a l cells [8] a n d mouse h e a r t cell function, as i n d i c a t e d by R N A a n d protein synthesis, a p p e a r s to decline w i t h age [14]. I n fact, most n o r m a l b o d y homeostasis processes begin to fail d u r i n g the last o n e - t h i r d of a species lifespan where there is a clustering o f degenerative diseases i n c l u d i n g c a r d i o m y o p a t h y . This w o u l d be a reasonable p a r t of the lifespan in which to expect a significant c h a n g e in the level of D N A d a m a g e . Such changes could likely be an effect of aging a n d not necessarily a cause o f aging. H o w e v e r , a s h a r p increase in the a m o u n t of genomic d a m a g e m i g h t accelerate the physiological decline associated with senescence. T h e postlabeling analysis represents a new w a y to e x a m i n e possible links between D N A d a m a g e , r e p a i r and function.

Acknowledgements I t h a n k S. W o o d l e y for advice a n d help on the 32p-postlabeling p r o c e d u r e a n d E. Hughes, N. Flodin, J . U m b r e i t , S. W o o d l e y a n d S. Flores for reviewing the manuscript. T h e technical assistance o f J u l i a D a n n e l l e y a n d m a n u s c r i p t p r e p a r a t i o n by M a r y Burns are gratefully acknowledged. T h e mice used in this study were supplied b y the N a t i o n a l Institute on Aging. This work was s u p p o r t e d by grants from the Council for T o b a c c o Research (GN 1611), the H e a r s t F o u n d a t i o n , the A m e r i c a n F e d e r a t i o n for A g i n g Research, and the A m e r i c a n H e a r t Association, A l a b a m a Affiliate.

Jalnes W. GaulQ~tz Department of Biochemistry University of South Alabama Collegeof Medicine Mobile, AL. 36688 USA. KEY WORDS: DNA; Damage; Aging; Postlabeling; Alkylation ; Nucleotides.

References 1 BARTSCH,H., MONTESANO,R. Relevance ofnitrosoamincs to human cancer, Carcinogenesis 5, 1381-1393 (1984). 2 BECKER,R. A., SHANK,R. C. Kinetics of formation and persistence of ethylguanines in DNA of rats and hamsters

treated with diethylnitrosoamine. Cancer Res 45, 2076-2084 (1985). 3 BURTON,K. A study of the conditions and mechanism of the diphenylamine reactions for the colorimetric estimation ofDNA. BiochemJ 62, 315-322 (1956). 4 FREX,J. V., SWENSON,D. H., WARaEN,W., P. D. Alkylation of deoxyribonucleic acid in various organs of C57BL mice by the carcinogen N-methyl-N-nitrosourea, N-ethyl-N-nitrnsourea, and ethyl methanesulphonate in relation to induction of thymic tumors. BiochemJ 174, 1031-1044 (1978). 5 GAU~ATZ,J., PP.ASH~, N., CUTLER,R. G. Ribosomal RNA gene dosage as a function of tissue and age for mouse and human. Biochem Biophys Acta 418, 358-375 (1976). 6 GOTH,R., RAJEWSKY,M. F. Persistence of Or-ethylguanine in rat brain DNA: correlation with nervous system-specificcarcinogenesis by ethylnitrosourea. Proc Natl Acad Sci USA 71,639-643 (1974). 7 HAYFUCK,L. Theoriesof BiologicalAging. Exp Geronto120, 145-159 (1985). 8 LAbIPIDIS,T. J., SCnAIBESOER,G. E. Age-related loss of DNA repair synthesis in isolated rat myocardial cells. Exptl Cell R e s t , 412416 (1985). 9 MARGISON,G. P., O'CoNNoR, P.J. Nucleic acid modification by N-nitroso compounds. In ChemicalCarcinogensand DNA. Grover, P. L. (Ed.) vol. I pp. 112-150, Boca Raton: CRC Press, (1979). 10 MARGISON,G. P., KLEIHUES,P. Chemical carcinogenesis in the nervous system: Preferential accumulation of or-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-Nnitrosourea. BiochemJ 148, 521-525 (1975). 11 NICrIOLL,J. W., SWANt~,P. F., P~no, A. E. Effect ofdimethylnitrosoamine on persistance of methylated guanines in rat liver and kidney DNA. Nature (Lond) 254, 264-265 (t975). 12 RANDERATH,K., REDDY,M. V., GUPTA, R. C. 32p-labeling test for DNA damage. Proc Nail Acad Sci USA 78, 6126-6129 (1981). 13 RgDDY,M. V., GUPTA,R. C., RANDERATIt,E., RANDERATH,K. s2P-postlabeling test for covalent DNA binding of chemical in vivo: application to a variety of aromatic carcinogens and methylating agents. Carcinogenesis 5, 231-243 (1984). 14 RmHAgDSON,A., ROnER'rS,M. S., RUTHERFORO,M. S. Aging and gene expression In Reviewof BiologicalResearchin Aging. Rothstein, M. (Ed.) vol. 2 pp. 395-419. New York: Alan R. Liss, Inc., (1985). 15 SAFFHILL,R., MARmSON,G. P., O'CoNt~OR, P. J. Mechanisms of carcinogenesis induced by alkylation agents. Biochem Biophys Acta 823, 111 145.