Katsumi Kobayashi

Induction of chromosome aberrations in human lymphocytes by monochromatic X-rays of quantum energy between 4.8 and 14.6 keV

The induction of chromosome aberrations was studied in human peripheral blood lymphocytes irradiated in vitro with synchrotron-produced monochromatic soft X-rays of quantum energy in a range between 4.8 and 14.6 keV. These X-rays were more effective in producing chromosome aberrations (dicentrics and rings) than 60Co gamma-rays. The efficiency increased with increasing LET of the photoelectrons and their associated Auger electrons, reaching a maximum at a track average LET (L delta = 100, T) of around 4 keV/microns, and tended to decrease or become rather refractory with further increase of LET. This unique LET dependency was consistent with the dual nature of chromosome aberration formation, and interpreted as a reflection of a limited range of photoelectrons as compared with the size and intranuclear geometry of the elemental chromatin fibres as vehicles of damage interaction.

Effects of K-shell X-ray absorption of intracellular phosphorus on yeast cells.

The effects of K-shell absorption of phosphorus atoms on yeast cells were investigated using synchrotron X-rays that were tuned to the resonance absorption peak (2153 eV). Three types of cellular effect (cell inactivation, induction of gene conversion at the trp-5 locus, and cell membrane impairment (changes in the permeability] were measured. It was demonstrated that the enhancement factor was 1.4 at the resonance peak regarding both lethality and the induction of gene conversion in reference of off-peak irradiation (2146 and 2160 eV). No difference was found between the two off-peak irradiation energies. No cell membrane impairment was detected, irrespective of the X-ray photon energies employed within the fluence range tested. These results strongly suggest that K-shell X-ray absorption in the resonance mode by cellular phosphorus atoms causes significantly more cellular effects than the off-resonance mode of absorption, probably via some specific changes induced in the phosphates of the DNA strand. Calculations using the number of phosphorus atoms in a defined size of the trp locus (2127 base pairs) on the DNA and the absorption cross-section of the resonance mode of phosphorus showed that gene conversion is inducible at a rate of 0.13 per X-ray photon absorption per locus. These results are discussed regarding the modes of K-shell photoabsorption.

Mammalian cells loaded with platinum-containing molecules are sensitized to fast atomic ions

Purpose: This work investigates whether a synergy in cell death induction exists in combining atomic ions irradiation and addition of platinum salts. Such a synergy could be of interest in view of new cancer therapy protocol based on atomic ions – hadrontherapy – with the addition of radiosensitizing agents containing high-Z atoms. The experiment consists in irradiating by fast ions cultured cells previously exposed to dichloroterpyridine Platinum (PtTC) and analyzing cell survival by a colony-forming assay. Materials and methods: Chinese Hamster Ovary (CHO) cells were incubated for six hours in medium containing 350 μM PtTC, and then irradiated by fast ions C6+ and He2+, with Linear Energy Transfer (LET) within range 2–70 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added to investigate the role of free radicals. The intracellular localization of platinum was determined by Nano Secondary Ion Mass Spectroscopy (Nano-SIMS). Results: For all LET examined, cell death rate is largely enhanced when irradiating in presence of PtTC. At fixed irradiation dose, cell death rate increases with increasing LET, while the platinum relative effect is larger at low LET. Conclusion: This finding suggests that hadrontherapy or protontherapy therapeutic index could be improved by combining irradiation procedure with concomitant chemotherapy protocols using platinum salts.

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.

ACTION SPECTRA IN ULTRAVIOLET WAVELENGTHS (150‐250 nm) FOR INACTIVATION AND MUTAGENESIS OF Bacillus subtilis SPORES OBTAINED WITH SYNCHROTRON RADIATION

Bacillus subtilis spores were exposed in vacuo to monochromatic UV radiation from synchrotron radiation in the wavelength range of 150 nm to 250 nm. Survival and frequency of mutation to histidine‐independent reversion were analysed for three types of spores differing in DNA‐repair capabilities. UVR spores (wild‐type DNA repair capability) exhibited nearly equal sensitivity to the lethal effects of far‐UV (220 nm and 250 nm) and of vacuum‐UV radiation (150 and 165 nm), but showed marked resistance to 190 nm radiation. UVS spores (excision‐repair and spore‐repair deficient) and UVP spores (a DNA polymerase I‐defective derivative of UVS) exhibited similar action spectra; pronounced sensitivity at 250 and 220 nm, insensitivity at 190 nm and a gradual increase of the sensitivity as the wavelength decreased to 165 nm. In all strains, the action spectra for mutation induction paralleled those for the inactivation, indicating that vacuum‐UV radiation induced lethal and mutagenic damages in the spore DNA. The insensitivity of the spores to wavelengths around 190 nm may be explicable by assuming that radiation is absorbed by materials surrounding the core in which DNA is situated.

Strand Break Induction by Photoabsorption in DNA-Bound Molecules

Abstract Le Sech, C., Takakura, K., Saint-Marc, C., Frohlich, H., Charlier, M., Usami, N. and Kobayashi, K. Strand Break Induction by Photoabsorption in DNA-Bound Molecules. Dried samples of a DNA–chloroterpyridine platinum complex were irradiated with monochromatic X rays tuned to the photoabsorption resonance of the LIII inner shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) triggered by the Auger effect in supercoiled DNA plasmids was measured by the production of circular nicked and linear forms. To probe the specific contribution of the LIII inner-shell excitation of the platinum atom, photon wavelengths were tuned on the resonance energy (on peak) and below (off peak). The quantum yields of the resonance radiation were typically found to be 11 for the SSBs and 1 for the DSBs. The DSB-to-SSB ratio increased by 20% when switching from off-resonance to on-resonance irradiation.

Oxidation yield of the ferrous ion in a Fricke solution irradiated with monochromatic synchrotron soft X-rays in the 1.8-10 keV region

The oxidation yield of ferrous ion in a Fricke solution was measured in the soft X-ray region from 1.8 to 10 keV. The standard Fricke solution was irradiated with monochromatic X-rays from synchrotron radiation. The yield decreased with decreasing X-ray energy, as several theoretical calculations have predicted. No significant changes were observed in the K-shell photo-absorption of the iron and sulphur contained in the standard Fricke solution. The high-LET nature of soft X-rays is discussed.

Effects of Monoenergetic X-rays with Resonance Energy of Bromine K-absorption Edge on Bromouracil-labelled E. Coli Cells

In order to examine enhanced killing that might be induced by Auger cascades in the incorporated atoms in cells, bromouracil(BrU)-labelled E. coli cells were irradiated with monoenergetic X-rays at 13.49 and 12.40keV, just above and below the K-absorption edge of bromine. In both cases BrU-labelled cells were more sensitive for killing than were normal cells. However, when the degree of BrU-sensitization was compared between the two energies of X-rays, the enhanced killing at 13.49 keV was only small, 2 +/- 8 per cent based on the D0 value in saline. By the addition of DMSO, which is believed to suppress radical-mediated effects, killing of BrU-labelled cells was enhanced at 13.49 keV by 8 +/- 4 per cent as compared with 12.40 keV, based on D0. These results have been examined in terms of absorbed energy in BrU-labelled cells and in terms of the number of induced Auger events.

SINGLE‐STRAND BREAKS IN SUPERCOILED DNA INDUCED BY VACUUM‐UV RADIATION IN AQUEOUS SOLUTION

We studied the induction of single‐strand breaks in the DNA of plasmid pBR 322 by vacuum‐UV radiation above 145 nm in aqueous solutions in relation to the production of OH‐radicals in water. The similarity and dissimilarity were examined of the wavelength dependence between the two effects. The maximum of single strand breaks at 150 nm could be explained by the action of OH‐radicals derived from direct water photolysis: the maximum at 180 nm remains unexplained. There was no indication that the direct absorption of photon by the DNA molecule plays an important role in the production of single‐strand breaks.

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C6+ and Fe26+

Purpose:u2003In order to study the role of the Linear Energy Transfer (LET) of fast atomic ions in platinum-DNA complexes inducing breaks, DNA Plasmids were irradiated by C6+ and Fe26+ ions. Material and methods:u2003DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C6+ ions and Fe26+ ions. The LET values ranged between 13.4 keV/μm and 550 keV/μm. In some experiments, dimethyl sulfoxide (DMSO) was added. Results:u2003In all experiments, a significant increase in DNA strand breaks was observed when platinum was present. The yield of breaks induced per Gray decreased when the LET increased. The yield of single and double strand breaks per plasmid per track increased with the LET, indicating that the number of DNA breaks per Gray was related to the number of tracks through the medium. Conclusions:u2003These findings show that more DNA breaks are induced by atomic ions when platinum is present. This effect increases for low LET heavy atoms. As DSB induction may induce cell death, these results could open new perspectives with the association of hadrontherapy and chemotherapy. Thus the therapeutic index might be improved by loading the tumour with platinum salts.