NOVEL ANALYTICAL STUDY FOR REACTION INTERMEDIATES IN THE PRIMARY RADIATION INTERACTION OF DNA USING A SYNCHROTRON RADIATION-INDUCED LUMINESCENCE SPECTROSCOPY.

To identify the precise molecular processes to induce DNA lesions, we attempt a novel spectroscopy of X-ray induced luminescence (XIL) using soft X-ray synchrotron radiation, which is a non-destructive analysis of the reaction intermediates in the elementary reaction pathway of damage induction and self-organized restoration. Using a liquid micro-jet technique to introduce aqueous samples in a vacuum chamber, we measure UV-visible luminescence from nucleotide solution as a function of the soft X-ray energy from the nitrogen to oxygen K-edge region. The XIL intensities for the nucleotide solutions are significantly enhanced in the soft X-ray region (410-530 eV) which is ascribed to the K-shell excitation/ionization of nitrogen atoms in the nucleobases. Furthermore, the XIL spectra do not show any signature of X-ray absorption near-edge structure (XANES) of the nucleobases. This is because the luminescence intensities collected from the integral area of the micro-jet only reflect the quantum yield of luminescence of the absorbed X-ray into UV-visible light irrespective of the absorption cross sections, i.e. of XANES. Thus the present result is the first evidence of luminescence as a result of X-ray absorption of aqueous nucleotides.

MITOCHONDRIAL MEMBRANE POTENTIAL, MORPHOLOGY AND ATP PRODUCTION IN MAMMALIAN CELLS EXPOSED TO X-RAYS.

This study aimed to reveal the effect of X-irradiation on mitochondrial function in terms of mitochondrial membrane potential (ΔΨm) and ATP productivity. At the cellular level, ΔΨm was quantified using JC-1, a mitochondrial probe that emits red or green fluorescence at high or low ΔΨm sites, respectively. The fluorescence area was quantified for both colors together relative to the whole-cell area of the same cell. The fluorescence areas versus whole-cell areas varied widely among the irradiated cells depending on the X-ray doses received (6 and 10 Gy) and incubation time, although the relative red area to total mitochondrial area was rather constant. Average ATP concentrations temporarily increased and showed a maximum at 48 h after irradiation for largely G1-arrested cells. These results indicate that an increase of mitochondrial volume per cell, not simply an increase in their active sites, is induced by irradiation, resulting in enhanced ATP production.

Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators.

To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells.

Real-time observation of irradiated HeLa-cell modified by fluorescent ubiquitination-based cell-cycle indicator using synchrotron X-ray microbeam.

Fluorescent ubiquitination-based cell-cycle indicator (FUCCI) human cancer (HeLa) cells (red indicates G1; green, S/G2) were exposed to a synchrotron X-ray microbeam. Cells in either G1 or S/G2 were irradiated selectively according to their colour in the same microscopic field. Time-lapse micrographs of the irradiated cells were acquired for 24 h after irradiation. For fluorescent immunostaining, phosphorylated histone proteins (γ-H2AX) indicated the induction of DNA double-strand breaks. The cell cycle was arrested by irradiation at S/G2. In contrast, cells irradiated at G1 progressed to S/G2. The foci were induced in cells irradiated at both G1 and S/G2, suggesting that the G1-S (or S) checkpoint pathway does not function in HeLa cells due to the fact that the cells are functionally p53 deficient, even though X-ray microbeam irradiation significantly induces double-strand breaks. These results demonstrate that single FUCCI cell exposure and live cell imaging are powerful methods for studying the effects of radiation on the cell cycle.

Live-cell imaging study of mitochondrial morphology in mammalian cells exposed to X-rays.

Morphological changes in mitochondria induced by X-irradiation in normal murine mammary gland cells were studied with a live-cell microscopic imaging technique. Mitochondria were visualised by staining with a specific fluorescent probe in the cells, which express fluorescent ubiquitination-based cell-cycle indicator 2 (Fucci2) probes to visualise cell cycle. In unirradiated cells, the number of cells with fragmented mitochondria was about 20 % of the total cells through observation period (96 h). In irradiated cells, the population with fragmented mitochondria significantly increased depending on the absorbed dose. Particularly, for 8 Gy irradiation, the accumulation of fragmentation persists even in the cells whose cell cycle came to a stand (80 % in G1 (G0-like) phase). The fraction reached to a maximum at 96 h after irradiation. The kinetics of the fraction with fragmented mitochondria was similar to that for cells in S/G2/M phase (20 %) through the observation period (120 h). The evidences show that, in irradiated cells, some signals are continually released from a nucleus or cytoplasm even in the G0-like cells to operate some sort of protein machineries involved in mitochondrial fission. It is inferred that this delayed mitochondrial fragmentation is strongly related to their dysfunction, and hence might modulate radiobiological effects such as mutation or cell death.

Unpaired electron species in thin films of calf-thymus DNA molecules induced by nitrogen and oxygen K-shell photoabsorption.

The mechanism of DNA modification induced by K-shell photoabsorption of nitrogen and oxygen atoms was investigated by electron paramagnetic resonance and x-ray absorption near edge structure measurements of calf thymus DNA. A g factor of 2.000 for the unpaired electron species, which only arises during irradiation, was measured. The EPR intensities for DNA zwere twofold times larger than those estimated based on the photoabsorption cross section. This suggests that the DNA film itself forms unpaired electron species through the excitation of enhanced electron recapturing, known as the postcollision interaction process.

A model for analysis of the yield and the level of clustering of radiation-induced DNA-strand breaks in hydrated plasmids.

Although it is widely accepted that the spatial distribution of strand breaks is highly relevant to the biological consequences of radiation, the extent to which strand breaks are clustered is not usually demonstrated directly from the experimental data. To evaluate the spatial distribution of radiation-induced strand breaks, the authors have developed a model for the generation of strand breaks after irradiation. The model assumes that (1) a radiation track has a certain probability of 'hitting' a plasmid, (2) the radiation generates strand break(s) by chance within a relatively small region that could produce double-strand breaks and (3) the number of strand breaks generated within the region follows a Poisson distribution. To find out whether the model is valid, the authors compared the calculated values with the experimental data obtained by a plasmid DNA assay. Taking into account the inherent bias of the plasmid assay, the model described well the experimental results of hydrated plasmids exposed to radiation.

Studies of soft X-ray-induced Auger effect on the induction of DNA damage.

PURPOSE: To understand the characteristics of DNA damage induced by Auger effect in DNA by ultrasoft X-irradiation. In situ electron paramagnetic resonance (EPR) spectroscopy as well as biochemical analysis has been applied to examine the DNA damage induction in both viewpoints of intermediate species and final products. MATERIALS AND METHODS: Unpaired electron species induced in a calf thymus DNA film irradiated with monochromatic ultrasoft X-rays (270-580 eV) was observed using an X-band EPR spectrometer installed in a synchrotron beamline. To determine the yield of single strand break (SSB), pUC18 plasmid DNA was irradiated and then analyzed by agarose gel electrophoresis. To analyze molecular change in a single strand DNA, a new technique using DNA-denaturation-treatment has been applied to quantify multiple SSB arising in both DNA strands. RESULTS: Short-lived EPR spectra were observed during irradiation. The intensity of transient EPR spectrum shows the similar energy dependence with that of the SSB yield around oxygen K-edge in particular. The fraction of the single-strand plasmid DNA (SS-DNA) after irradiation could be determined using a low-temperature-denaturation condition. The obtained slope of the dose-response for SS-DNA shows half of that of closed circular DNA as expected under the diluted solution condition. CONCLUSION: The availability of an EPR apparatus installed in a synchrotron beamline is demonstrated by detecting very short-lived unpaired electron species. Transient EPR spectra of DNA show the similar energy dependence to that of the SSB yield. The proposed DNA-denaturation assay works as expected using the low-temperature-denaturation condition.

Yields of strand breaks and base lesions induced by soft X-rays in plasmid DNA.

The yields of soft-X-ray-induced DNA damages have been measured by using closed-circular plasmid DNA. Several DNA solutions with three kinds of radical scavenger capacity and also fully hydrated DNA samples were irradiated to compare the contribution by indirect reaction of diffusible water radicals, such as OH*, with those by direct action of secondary electrons. The yields of prompt single- (SSBs) and double-strand breaks (DSBs) decrease with increasing scavenging capacity. The SSB yields for soft X-rays are approximately midway those between gamma-ray and ultrasoft X-ray data previously reported. Heat labile sites are observed only in the low scavenger condition. The yields of the base lesions revealed by post irradiation treatment with base excision repair enzymes showed a similar value for Nth and Fpg protein except in the hydrated sample. These results indicate that the direct effect of soft X-rays induces the damages with different efficiency from those by indirect effect.

X-ray absorption near-edge structure (XANES) spectral changes of 2-deoxy-D-ribose by irradiation within the energy region around the oxygen K-shell absorption edge.

The physicochemical characteristics of 2-deoxy-D-ribose moieties in DNA strands are important to understand biological radiation stress. So, the X-ray absorption near edge structure (XANES) of 2-deoxy-D-ribose within the energy region around the oxygen K-shell absorption edge was measured. 2-deoxy-D-ribose was exposed to 3 energies of X-rays, i.e., 526.3 eV (below O 1s-->pi*), 537.8 eV (at the absorption peak of O 1s-->sigma*) and 552.6 eV (above O 1s-->sigma*) for given periods. Slight differences in spectral changes were seen in the each irradiation energy, suggesting in fact that the chemical state and following rearranged chemical structure of 2-deoxy-D-ribose may be different between the 3 irradiation energies.

Oxygen K-edge X-ray absorption near edge structures (XANES) of sublimated films of amino acids.

Oxygen K-edge X-ray absorption near edge structures (XANES) spectra of amino acids (glycine, L-alpha-alanine, beta-alanine, L-serine, L-asparic acid and L-tyrosine) were measured. Several peaks of XANES spectra were successfully assigned on the basis of DV-Xalpha calculation.

A grating monochromator of BL23SU at SPring-8 covering silicon and oxygen K-edges.

We report the present performance of a grating monochromator at the newly constructed soft x-ray beamline (BL23SU at the SPring-8), which can measure both silicon and oxygen K-edges. That provides new opportunities for XAFS measurements of solids up to 2100 eV.

X-ray absorption near edge structures of DNA or its components around the oxygen K-shell edge.

The initial process of radiation damage in DNA was investigated by measuring the X-ray absorption near edge structures (XANES) within the energy region around the oxygen K-shell absorption edge for DNA, cytosine and 2-deoxy-d-ribose. Irradiation and XANES experiments were performed with the BL23SU soft X-ray beamline, using synchrotron radiation from the 8 GeV electron storage ring at SPring-8. Samples were mounted on gold-coated plates in a vacuum chamber. The XANES spectra were obtained by measuring the photoelectron current of the samples. 2-Deoxy-d-ribose was exposed to X rays at the absorption peak corresponding to the oxygen (O) 1s-->sigma* transition energy (538 eV); the XANES spectra were obtained after each irradiation. DNA and cytosine, possessing characteristic XANES spectra, both had two major energy bands corresponding to the O 1s-->pi* and 1s-->sigma* transitions. Two new peaks appeared and gradually increased in the XANES spectra of 2-deoxy-d-ribose during irradiation. These results suggest that C-O bonds in 2-deoxy-d-ribose are transformed to C=O bonds by O 1s-->sigma* transition, suggesting that the molecules undergo chemical changes into carbonyl-containing compounds.

Reparability of lethal lesions produced by phosphorus photoabsorption in yeast cells.

The characteristics of DNA lesions produced by the photoabsorption of phosphorus in yeast cells were studied using monochromatized soft X-rays tuned to the absorption peak of the phosphorus K-edge (2153 eV) and below the peak energy (2147 eV). The repaired fractions of DNA double-strand breaks (dsb) were measured relatively by using both a mutant, rad 54-3, which shows the temperature-sensitive dsb repair-deficient phenotype, and a wild-type strain. The repaired fraction of lesion in rad 54-3, which corresponds to the relative yield of dsb reparable by the RAD 54 pathway, was not affected by the phosphorus photoabsorption. Repair of the produced lesions in the wild-type cells was also measured by comparing the surviving fraction of the immediately plated cells to that of those cells plated after holding in a non-nutrient medium for 80 hrs. The recovery of the surviving fraction after the holding treatment was dependent upon the irradiated X-ray energy. These results suggest that irreparable lesions are produced by the inner-shell photoabsorption of phosphorus in DNA, although its yield is small.

Single- and double-strand breaks in solid pBR322 DNA induced by ultrasoft X-rays at photon energies of 388, 435 and 573 eV.

We measured strand breaks of pBR322 plasmid DNA irradiated with ultrasoft X-rays using monochromatic synchrotron radiation as a light source. Three photon energies, 388, 435 and 573 eV, a value below and above the nitrogen K-edge and above the oxygen K-edge, respectively, were chosen for the irradiation experiments as they have an equivalent photon transmittance of the sample. Irradiated DNA was analyzed by agarose gel electrophoresis and the numbers of single- and double-strand breaks (ssb and dsb) were determined by measuring the band intensity on the gel after ethidium bromide staining. The action cross-sections for the ssb and dsb slightly increased with the photon energy. The ratio between 388 and 573 eV was about 1.5 for both forms of strand breaks. The absorbed energy required for a strand break was about 60 eV for ssb and 1 keV for dsb, less than one fifth of the values obtained previously in the 2 keV region. On the other hand, the absorbed energies per strand break, as well as the ratio of the action cross-section for the ssb to that for the dsb, were constant regardless of the photon energy used. The K-shell photoabsorption on carbon, nitrogen and oxygen atoms in the DNA molecule, followed by an Auger cascade, induced DNA strand breaks with a constant efficiency in terms of the absorbed energy. These results indicate that the strand breaks of the DNA molecule in the solid state are mainly caused by the photo- and Auger-electrons and the efficiency of the strand breaks little depends on the atoms ejecting these secondary electrons.

Construction of the JAERI soft X-ray beamline for actinide material sciences.

An undulator beamline for spectroscopy studies focusing on the electronic structure of actinide materials is under construction. Linearly or circularly polarized soft X-rays are provided by employing a variably polarizing undulator. Varied-line-spacing plane gratings and a sagittal-focusing system are used to monochromatize the undulator beam, whose energy ranges from 0.3 to 1.5 keV. A resolving power of 10(4) is expected in the whole energy region. These components are methodically operated by the SPring-8 beamline control system. There are three experimental stations in the beamline. In one of the stations the photoemission spectroscopy experiments are carried out at a radioisotope-controlled area where actinide compounds as well as unsealed radioactive materials are usable. Other experimental stations are planned in the beamline for surface photochemical reactions and biological applications.

Soft X-ray beamline specialized for actinides and radioactive materials equipped with a variably polarizing undulator.

This report presents the design of an undulator beamline at SPring-8 to be used for soft X-ray spectroscopy focused on radioactive materials. Photoemission spectroscopy experiments are carried out in a radioisotope (RI)-controlled area where actinide compounds as well as unsealed radioactive materials are usable. Intrusion of the radioactive materials into the electron storage ring or to the outside of the evacuated beamline components can be avoided by a specially devised RI protection/inspection mechanism. The combination of a variably polarizing undulator and a varied-line-spacing plane-grating monochromator provides linearly or circularly polarized soft X-rays with a high resolving power in the energy range 0.28-1.5 keV. The beamline will become operational in December 1997.

Single- and double-strand breaks in pBR322 plasmid DNA by monochromatic X-rays on and off the K-absorption peak of phosphorus.

Using a synchrotron irradiation system pBR322 plasmid DNA was irradiated under vacuum by monochromatic X-rays having five specific photon energies (2.147, 2.153, 2.159, 2.168 and 2.199 keV) both on and off the K-absorption peak (2.153 keV) of phosphorus. The single- and double-strand breaks (ssb and dsb) were measured as conversions of the closed circular form of DNA (form I) to open circular (form II) and linear (form III) forms respectively. Exposures to induce one strand break per molecule were lowest at the peak (2.153 keV), and highest at 2.147 keV; the ratios were 2.7 for ssb and 3.0 for dsb. The exposures for dsb were 21-26 times higher than those for ssb. When the exposures were converted to absorbed doses in grays the absorbed doses per ssb were almost independent of photon energy. This result indicates that a certain absorbed dose was necessary to induce a ssb, regardless of whether photons were absorbed by the K-shell of phosphorus or by other shells, or by other atoms. However, the absorbed dose per dsb at 2.147 keV was 1.17 times higher than that averaged over four X-ray energies above 2-153 keV, indicating that the K-shell absorption, and the subsequent Auger event, efficiently induce dsb. The results are also discussed concerning the number of photo-absorptions of the constituent atoms per DNA strand break.

Lethal effect of K-shell absorption of intracellular phosphorus on wild-type and radiation sensitive mutants of Escherichia coli.

The present study was conducted to clarify the lethality of Auger cascades induced by the K-shell photoabsorption of phosphorus in Escherichia coli. Killing of wild-type and radiation-sensitive mutants of E. coli was examined. Three x-ray energies were chosen for irradiation; at 2.153 keV: the resonance peak of K-shell photoabsorption of phosphorus; at 2.146 and 2.160 keV: off-peak. Enhancement ratio, which was defined as the ratio of the killing sensitivity of 2.153 keV to that at 2.146 keV, were 1.32 to 1.54 for examined strains. Increment of absorbed energy calculated in entire cells for 2.153 keV radiation could not explain the degree of observed enhancement of killing. Lethality of Auger cascades depended on the killing sensitivity with x-rays which did not induce Auger cascades. The lethality for wild-type was lower than that for recombination repair-deficient mutants. It was concluded that one part of damages produced by Auger cascades was repaired in wild-type strains.

Inactivation action spectra of Bacillus subtilis spores with monochromatic soft X rays (0.1-0.6 nm) of synchrotron radiation.

Five types of Bacillus subtilis spores differing in DNA repair and recombinational capacities were exposed in vacuum to monochromatic soft X rays from synchrotron radiation. The inactivation rate constants were obtained from exposure-survival curves upon irradiations at 12 wavelengths in the range of 0.1000 nm (12.40 keV) to 0.6000 nm (2.066 keV). Spores of two repair-deficient strains, UVS (uvrA ssp) and UVP (uvrA ssp polA), exhibited almost equal sensitivities to those of wild-type UVR+, while those of two recombination-deficient strains, RCE (recE) and RCF (recF), exhibited higher sensitivities in the whole wavelength range. This suggested that the repair of DNA damage produced by soft X rays was dependent on the recombinational capabilities. Inactivation action spectra based on photon fluence showed that the effectiveness of the radiation increased as the wavelengths became longer. Abrupt changes in the effectiveness occurred around the wavelengths corresponding to the absorption edges of K-shell electrons of phosphorus and calcium. In both cases, the sensitivity was the highest at the wavelengths of the resonance absorption peak, the next highest at those of the higher energy, and the lowest at the lower energy. Mass energy absorption coefficients of spores were obtained from the transmission of a flake made of spores. They were used to derive inactivation action spectra based on absorbed doses. In these spectra, basal levels of the sensitivity seemed constant, and enhancements of the sensitivity were observed consistent with the absorption by calcium and phosphorus. Thus calcium and phosphorus atoms were the predominant targets for the absorption events leading to the inactivation of spores in the wavelength range examined.