Infrared spectral change in 2-deoxy-DD-riboseby irradiation with monochromatic photons

around oxygen K-edge

Ken Akamatsu a,*, Kentaro Fujii b, Akinari Yokoya b

a Japan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi, Saitama 332-0012, Japanb Japan Atomic Energy Research Institute, SPring-8, 1-1-1 Koto, Mikazuki, Sayo, Hyogo 679-5148, Japan

Abstract

Analyses of chemical changes in DNA by energy deposition from ionizing radiation are quite important to strictly

know characteristics of radiobiological effects. Monochromatic photons from synchrotron radiation are one of the

powerful probes to investigate the effects. As a step for the aim, chemical analyses by Fourier-transform infrared

spectroscopy of the samples irradiated with the monochromatic photons were performed. It appeared that 2-deoxy-DD-

ribose irradiated around the energy of oxygen K-edge contained C@O or C@C, which would be responsible for a direct

strand break of DNA. These data are noteworthy to find not only the strand scission at 2-deoxy-DD-ribose moiety by the

direct energy deposition by photon but also the following radiobiological responses such as cell killing or mutation.

� 2002 Elsevier Science B.V. All rights reserved.

Keywords: Fourier-transform infrared spectroscopy; 2-Deoxy-DD-ribose; Synchrotron radiation

1. Introduction

Three-dimensional structure of DNA is kept by

a couple of phosphate-2-deoxy-DD-ribose (DR)

backbones and stacking force between four bases.

Disruptions of this system by chemicals and radi-

ations lead to mutagenesis, carcinogenesis and

cell death. One of the disruptions is the breakage

of a phosphate–DR backbone, which can be led

by restriction or excision repair enzymes, reactiveoxygen species (ROS) such as OH� and direct

energy deposition from ionizing radiations. The

former enzymatic strand scissions are induced by

hydrolysis of the phosphodiester linkages on theassumption that resultant nicks should be closed

by dehydration condensation reaction by ligases,

whereas, the cut-ends produced by ROS and ion-

izing radiation would be too diverse to be con-

nected directly by the enzymes. In particular, little

is known about the effects of the direct energy

deposition by radiations except for results ob-

tained by electron paramagnetic resonance (EPR)spectroscopy [1], while those of ROS has been in-

vestigated to identify final stable cut-ends by high-

performance liquid chromatography (HPLC),

polyacrylamide gel electrophoresis (PAGE) and a

reaction with thiobarbituric acid [2].

Approximately 50% of whole DNA damages by

ionizing radiations in a cell nucleus are believed to

* Corresponding author. Tel.: +81-791-58-0802x3916; fax:

+81-791-58-2620.

E-mail address: kakamatu@spring8.or.jp (K. Akamatsu).

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PII: S0168 -583X(02)01534 -3

Nuclear Instruments and Methods in Physics Research B 199 (2003) 328–331

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be responsible for the excitation and ionization of

DNA components by the direct energy deposition,

so-called �direct effect�, while other damages are

caused mainly by diffusible OH� and H� etc. pro-duced by water radiolysis, �indirect effect� [3]. In

general, the direct effect is likely to occur in order

of absorption cross-section: P > O > N > C � H

in DNA. Then, there should be differences in re-

sulting chemical changes and their dose–response

profiles by irradiated photon around edges of these

atoms in DNA. One of successful methods to

investigate the direct effect is the use of synchro-tron radiation (SR). It has high photon flux over

a wide energy range (from infrared to c-region),

enough to give sufficient high-density monochro-

matic photons, which are essential to obtain high-

resolution X-ray absorption near edge structures

(XANES). With SR, it is possible to study the

selective excitation of an inner shell electron to a

specific transition state. In the last decade, severalstudies of radiobiological effects using SR have

been reported [4–6]. In fact, these studies demon-

strated that cell inactivation, transformation, and

DNA strand breaks frequently occurred at the

K-edge resonance absorption peak energy ofphosphorus in DNA (2.153 keV). These results

should be an evidence of direct strand scissions

produced on phosphate–DR backbones in DNA.

As a next step of the study on the direct strand

scissions on the phosphate–DR backbone, we have

aimed at chemical changes in DR by irradiation at

the energy of around oxygen K-edge because a DR

moiety (Fig. 1) in DNA has three oxygen atoms.Every XANES spectrum of irradiated DR samples

had a 1s ! p� absorption peak, suggesting that

C@O bonds might be produced in DR by the di-

rect energy deposition on oxygen [7,8]. However,

the XANES spectral changes would be not enough

for proving the production of the carbonyl groups

in DR. In this report, we will indicate a result

supporting the chemical changes using Fourier-transform infrared spectroscopy (FT-IR) and

predicted pathways of them triggered by some

excitation or ionizing patterns.

2. Materials and methods

2-Deoxy-DD-ribose (DR) was purchased fromTokyo Kasei Ltd. The sample solution (10 mg/ml)

was prepared in distilled water, and the aqueous

solution (5 ll) was spread on a gold-coated Be–Cu

plate to obtain a film sample by drying at room

temperature.

XANES measurements for determining pho-

ton energies around oxygen K-edge and sample

irradiation were performed using a soft X-raybeamline with a variably polarizing undulator

(BL23SU) in SPring-8 [9]. A high-resolution-type

monochromator equipped with a plane grating

having 600 line/mm was used to scan the proper

energy range. The pressure in the sample chamber

was of the order of 10�6 Pa. The photon flux at the

energy around oxygen K-edge was estimated on

the order of 3 � 1010 photons/s using an ionizationchamber [10]. The beam size on the sample was

about 0:5 � 2 mm. Irradiations were performed for

25, 50 and 100 min at the energies of 526 eV (oxy-

gen K pre-edge), 538 eV (at O 1s ! r� resonance)Fig. 1. Structures of DR and a phosphate–DR backbone in

DNA.

K. Akamatsu et al. / Nucl. Instr. and Meth. in Phys. Res. B 199 (2003) 328–331 329

and 553 eV (above oxygen K-shell ionization po-

tential) [8].

FT-IR study was performed using an IR spec-

trometer (FT/IR-615, JASCO Co., Japan) equip-ped with an infrared microscope (IRT-30, JASCO

Co., Japan). The spectra of the irradiated DR

samples were obtained by measuring relative re-

flectivity (% R) to background air within a range

of 700–4000 cm�1.

3. Results and discussion

Figs. 2(b)–(d) show the spectral changes of DR

irradiated by the three energies of photons. Al-

though it is difficult to compare them quantita-

tively because of the secession of irradiated sample

molecules from the plate to the chamber, the dif-

ference of sample thickness or IR photon scatter-

ing, it is noteworthy that every IR spectrum had abroad band within a range of 1600–1800 cm�1

corresponding to C@C and/or C@O stretching.

The productions of these unsaturated bonds imply

that a bond around these atoms was dissoci-

ated. On the basis of these data and XANES

spectral changes previously reported [8], we con-cluded that C@C and C@O bonds should be

produced by energy deposition mainly to non-

bonding electron pair (n-electrons) on oxygen

atoms from the secondary electrons such as photo-

and/or Auger electron. In fact, 70 eV electron

beam, which is used for electron impact mass

spectrometry, is capable to ionize n-electrons of

oxygen atoms in alcohols and ethers to producefragments containing C@O [11]. Probably, the

magnitude of the deposited energy might be a few

tens eV per a molecule. The C@C bond could be

yielded by a dehydration condensation rearrange-

ment. These chemical changes were also identi-

fied in the case of indirect effect [2]. Owing to

these considerations, a series of patterns for the

DNA strand scission triggered by ionizing oxy-gen n-electrons of DR in DNA was expected

(Fig. 3).

Fig. 2. FT-IR spectra of naked (a) and irradiated DR, (b: for 100 min at 526 eV, c: for 50 min at 538 eV, d: for 553 eV). Solid and

dotted arrows in these figures (b, c and d) indicate IR absorption by C@O and C@C, respectively. The experiments were performed at

25, 50 and 100 min in the every photon energy. But we could not find any irradiation-time-dependencies within the range of 1500–1700

cm�1 on a series of the spectra.

330 K. Akamatsu et al. / Nucl. Instr. and Meth. in Phys. Res. B 199 (2003) 328–331

On the other hand, behaviors of inner shell-

excited molecules produced by direct photo-

absorption should be considered. Although ourdata are not clear enough to discuss them from the

spectral shapes and intensities, multi-positive-

charged molecules followed by the Auger process

would produce more severe cut-ends on the

phosphate–DR backbone and these species might

not be detected by the FT-IR method. Indepen-

dencies of the FT-IR spectral changes on irradia-

tion time and energy shown in Fig. 2 could beresponsible for differences in the production fre-

quency of K-shell-excited DR and subsequent

bond degradation followed by vaporization of

some of resultant fragments from the sample plate.

Acknowledgements

We gratefully acknowledge valuable help of

Drs. Akane Agui and Akitaka Yoshigoe for the

operation of the beamline.

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Fig. 3. Predicted degradation patterns for DR irradiated with the SR monochromatic photons.

K. Akamatsu et al. / Nucl. Instr. and Meth. in Phys. Res. B 199 (2003) 328–331 331