Hiroaki Terato

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm−1, respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process.

Oxidative DNA damage caused by pulsed discharge with cavitation on the bactericidal function

Plasma-based techniques are expected to have practical use for wastewater purification with a potential for killing contaminated microorganisms and degrading recalcitrant materials. In the present study, we analysed oxidative DNA damage in bacterial cells treated by the plasma to unveil its mechanisms in the bactericidal process. Escherichia coli cell suspension was exposed to the plasma induced by applying an alternating-current voltage of about 1 kV with bubbling formed by water-cavitation, termed pulsed discharge with cavitation. Chromosomal DNA damage, such as double strand break (DSB) and oxidative base lesions, increased proportionally with the applied energy, as determined by electrophoretic and mass spectrometric analyses. Among the base lesions identified, the yields of 8-hydroxyguanine (8-OH-G) and 5-hydroxycytosine (5-OH-C) in chromosomal DNA increased by up to 4- and 15-fold, respectively, compared to untreated samples. The progeny DNA sequences, derived from plasmid DNA exposed to the plasma, indicated that the production rate of 5-OH-C exceeded that of 8-OH-G, as G:C to A:T transitions accounted for 65% of all base changes, but only a few G:C to T:A transversions were observed. The cell viabilities of E. coli cells decreased in direct proportion to increases in the applied energy. Therefore, the plasma-induced bactericidal mechanism appears to relate to oxidative damage caused to bacterial DNA. These results were confirmed by observing the generation of hydroxyl radicals and hydrogen peroxide molecules following the plasma exposure. We also compared our results with the plasma to those obtained with 137Cs γ-rays, as a well-known ROS generator to confirm the DNA-damaging mechanism involved.

Quantitative characteristics of clustered DNA damage in irradiated cells by heavy ion beams

Heavy ion beam as typical high linear energy transfer (LET) radiation produces more expanding ionization domain around their tracks than low LET radiation such as X-rays and gamma rays. Thus, heavy ion beam can cause more densely accumulated damage cluster in the target DNA, termed clustered DNA damage. This damage exhibits difficulty for repair and inhibition of DNA replication with its complex structure [ 1]. So, clustered DNA damage is thought to be strongly involved in the biological effectiveness of heavy ion beam. However, a lot of studies have presented no certain correlation between yields of clustered DNA damage and severity of radiation effect. We previously indicated that the yields of clustered DNA damage decreased with increasing LET in the DNA molecules irradiated in test tubes with gamma rays, and carbon and iron ion beams whose showed different LET, respectively [ 2]. In this study, we aimed to reveal correlation between clustered DNA damage and the LET of heavy ion beam in the irradiated cells. In the experiments, Chinese hamster ovary AA8 cells growing exponentially were irradiated by carbon, silicon, argon and iron ion beams from Heavy Ion Medical Accelerator in Chiba (HIMAC) of the National Institute of Radiological Sciences, Japan. These LETs were 13, 55, 90 and 200 keV/µm, respectively. For comparison, we used gamma rays from 137Cs-gamma source, Gammacell 40 (Atomic Energy of Canada Ltd), at Saga University. The irradiated cells were subjected by static-field gel electrophoresis to quantify clustered DNA damage of the genomic DNA. For this analysis, we used Fpg and endonuclease III for clustered DNA damage including oxidative purine and pyrimidine lesions, respectively. We also analysed the corresponding isolated DNA damages by aldehyde reactive probe method [ 3], and the surviving fractions of the irradiated cells in this study. The electrophoretic results indicated that total yields of clustered DNA damage in the irradiated cells decreased with increasing LET, including the double-strand break (DSB) and the respective clustered base damages (Fig. 1). This result conforms to our previous study with the irradiated DNA molecules [ 2]. The damage kinetics is thought to be mainly derived from two reasons: decreasing fluxes and increasing reaction with reactive oxygen species each other in increase in LET. In the clustered DNA damage induced by each radiation, the most decremental fraction was clustered base damage, but not DSB. The isolated DNA damages decreased with increasing LET like clustered DNA damage in this study (data not shown). These results make us realize the degree of contribution of direct and indirect effects of ionizing radiation. The certain amount of DSB were derived from the direct effect and showed less reactivity to LET. In contrast, oxidative base lesions were mainly generated by indirect effect with reactive oxygen species, which sensitively responded to LET change. We also found seemingly conflicted result of the relationship between LET and RBE (data not shown). We need further study to elucidate act of clustered DNA damage in radiobiological effect with heavy ion beams. Fig. 1. The yields of clustered DNA damages in the cells irradiated with respective ionizing radiations. Each clustered DNA damage consists of DSB (open bar) and clustered base damage (closed bar), and calculated from the strength of released band on electrophoretic gel. Clinical trial registration number if required: None.