S t u d y o f S o f t X - r a y - S t i m u l a t e d F r e e R a d i c a l s I n d u c i n g D N A B a s e D a m a g e S t u d y o f S o f t X - r a y - S t i m u l a t e d F r e e R a d i c a l s I n d u c i n g D N A B a s e D a m a g e Fig. 1. The microwave cavity of th e EP R ap pa ra tu s mo un te d in the high vacuum chamber. The s a m p l e i s s e t o n a c o p p e r r o d and installed in the narrow gap be tw ee n fi el d mo du la ti on co il s. X- ba nd mi cr ow av es ar e gu id ed i n t o t h e c h a m b e r v i a c o p p e r w a v e - g u i d e s c o n n e c t e d w i t h a vacuum-sealed junction. Stu die s emp loy ing syn chr otr on sof t X-r ays as p r o b e s t o i n v e s t i g a t e g e n e t i c c h a n g e s h a v e highlighted the biological effects, such as mutation or carcinogenesis, related to the molecular process in DNA damage [1]. Site - selective photoabsorption i n a D N A m o l e c u l e i s o n e o f t h e m o s t p o w e r f u l te ch ni qu es fo r un de rs ta nd in g th e ph ys ic oc he mi ca l m e c h a n i s m o f D N A d a m a g e , w h i c h i n d u c e s t h e r a d i o b i o l o g i c a l e f f e c t s . W e h a v e d e v e l o p e d a n E P R ( E l e c t r o n P a r a m a g n e t i c R e s o n a n c e ) spectrometer ( Fig. 1 ) installed in a synchrotron soft X-ray beamline at SPring-8 to observe free radical s p e c i e s a s i n t e r m e d i a t e s l e a d i n g t h e c o m p l i c a t e d D N A d a m a g e [ 2 ] . T h i s s y s t e m e n a b l e s u s t o i n v e s t i g a t e t h e r a d i c a l p r o c e s s i n s i t u i n D N A m o l e c u l e s b y c o r e - e x c i t a t i o n w i t h i r r a d i a t i o n b y t h e soft X-rays [3]. T h e e x p e r i m e n t s w e r e p e r f o r m e d a t t h e Bi o l o g i ca l Ap p l i ca ti o n St a ti o n i n th e JA ER I so ft X- ray beamline BL23SU . The measured sample was a pellet of guanine and thymine bases, which were chosen as typical purine and pyrimidine bases in a D N A m o l e c u l e . T h e o x y g e n i n t h e b a s e s w a s sel ect ed as a tar get ato m for K -sh ell exc ita tio n in t h e m o l e c u l e s . T h e s a m p l e p e l l e t w a s i r r a d i a t e d w i t h s o f t X - r a y p h o t o n s i n a m i c r o w a v e c a v i t y mo un te d in a va cu um ch am be r (< 10 -6 Pa ). Th e sample temperature was controlled from 15 to 300 K, and r elatively low microwave power ranging 0.1 - 7 mW was used to avoid power saturation of EPR si gn al s. Mo no ch ro ma ti c so ft X- ra y ph ot on s we re provided using a grazing incidence monochromator e q u i p p e d w i t h v a r i e d - l i n e - s p a c i n g - p l a n e - g r a t i n g s ( VLSPGM ). The resolution power E/ ∆ E is ~ 10,000 in the 0.5 keV region, and a photon flux of the order o f 1 0 1 1 ( p h o t o n s / s e c / 1 0 0 m A r i n g c u r r e n t / 0 . 0 2 % band width) w as realized. Ob ta in ed EP R sp ec tr a sh ow th e in du ct io n of s h o r t - l i v e d u n s t a b l e r a d i c a l s c l e a r l y d i s t i n g u i s h e d from stable ones which still exist after exposing to s o f t X - r a y s . T h e s i g n a l i n t e n s i t y o f a s h o r t - l i v e d doublet signal observed in the guanine spectrum at 77 K ( Fig. 2(a) ) linearly increases with an increase of the photon flux density. The signal immediately Microwave cavity Vacuum chamber Soft X-rays Microwave guide Copper sample rod Field modulation coils Iris Power supply for modulation coils X-band microwave generator Closed-cycle cryogenic system Digital marker 83 Fig. 2. EPR spectra of guanine during irradiation by soft X-rays (536 eV ) at 77 K (a) , and just after exposure (b) . The microwave power was 200 μ W and the width of the magnetic field modulation of 100 kHz was 0.5 mT. d i s a p p e a r e d b y b e a m - o f f . O n l y a s i n g l e t s i g n a l ( Fi g. 2( b) ), wh ic h is co ns is te nt wi th pr ev io us da ta o n a g u a n i n e c a t i o n ( o n e e l e c t r o n o x i d a t i o n ) [ 4 ] re ma in ed on th e sp ec tr um . It is in fe rr ed th at th e lif eti me of the sho rt- liv ed rad ica l sho uld be lon ger than 10 μ sec due to the magnetic field modulation of 100 kHz for the EPR spectrometer. C onsidering the experimental time response limit , the presence o f a d o u b l e t s i g n a l w o u l d i n d i c a t e a t r a n s i e n t radical species following the Auger final state that promptly decays within a psec. Thus , we propose a p a t h w a y f o r f o r m i n g a f i n a l g u a n i n e l e s i o n i n DN A, su ch as 7, 8- di hy dr o- 8- ox o- gu an in e ( 8- ox o- G ), shown in Fig. 2 . In the case of thymine, a strong singlet signal a p p e a r e d d u r i n g i r r a d i a t i o n b y s o f t X - r a y s ( F i g . 3(a ) ). In con tra st to gua nin e, how eve r, ove r 10 16 photons were needed obtain the detectable signal of stable radical at room temperature. The stable d o u b l e t s i g n a l w i t h a n a d d i t i o n a l m i n o r s t r u c t u r e ( F i g . 3 ( b ) ) i s s i m i l a r t o t h y m i n e a n i o n r a d i c a l pr ev io us ly re po rt ed [5 ]. Ba se d on th es e as pe ct s, we propose a possible pathway for forming a final t h y m i n e l e s i o n a s s h o w n i n F i g . 3 . T h e a n i o n radical would be induced by a secondary reaction w i t h p h o t o o r A u g e r e l e c t r o n s t o t h y m i n e i n b u l k condition. These results strongly indicate that the in situ 330 335 340 345 350 Beam-off Photon Energy = 539 eV, Sample Temp = 77 K Beam-on mT (b) (a) NH N N H N O NH 2 N N H N O + ∑ NH 2 NH N O N NH 2 NH N H + ∑ N O O NH 2 NH N H H N guanine guanine cation transient guanine radical cation by σ *excitation 7,8-dihydro-8-oxo guanine h ν = 536 eV H 2 O 84 330 335 340 345 350 Photon Energy = 539 eV, Sample Temp = R.T. mT NH N H O H 3 C O N H O + ∑ NH H 3 C O – ∑ NH N H O H 3 C O thymine thymine anion radical transient thymine cation radical h ν = 536 eV e – Beam-off Beam-on (a) (b) Fig. 3. (a) EPR spectrum of thymine during irradiation of soft X-rays (536 eV ) at room temperature. The microwave power was 1 mW and width of the magnetic field modulation of 100 kHz was 0.5 mT. (b) EPR spectrum of thymine after exposure of a high flux of about 1016 photons. The microwave power was 7 mW and width of the magnetic field modulation of 100 kHz was 0.5 mT. The gain was two times larger than that for (a) . References [1] K. Hieda and T. Ito, in: S. Ebashi, M. Koch and E . R u b e n s t e i n ( E d s . ) , H a n d b o o k o n S y n c h r o t r o n Rad., North-Holland, Amsterdam 4 (1991) 431. [ 2 ] A . Y o k o y a a n d K . A k a m a t s u , N u c l . I n s t r u m . Meth. A 467-468 (2001) 1333. [ 3 ] A . Y o k o y a , K . A k a m a t s u a n d K . F u j i i , N u c l . Instrum. Meth. B 199 (2003) 366. [ 4 ] D . M . C l o s e , E . S a g s t u e n a n d W . N e l s o n , J . Chem. Phys. 32 (1985) 4386. [5] H.C. Box and E.E. Budzinski, J. Chem. Phys. 62 (1975) 197. E P R m e a s u r e m e n t p r o v i d e s e v i d e n c e o f t h e tr an si en t ra di ca l sp ec ie s in du ce d, pr es um ab ly , as s i n g l e - e l e c t r o n o x i d a t i o n b y c o r e l e v e l e x c i t a t i o n . The se are inf err ed to be pre cur sor s of che mic all y s t a b l e D N A b a s e l e s i o n s . O u r f i n d i n g s w i l l o p e n discussions on physicochemical processes of DNA damage induction by ionizing radiation. Akinari Yokoya, Ken Akamatsu and Kentaro Fujii SPring-8 / JAERI E-mail: yokoya @ spring8.or.jp 85
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