LET Dependence of 8-Hydroxy-2'-deoxyguanosine (8-OHdG) Generation in Mammalian Cells under Air-Saturated and Hypoxic Conditions: A Possible Experimental Approach to the Mechanism of the Decreasing Oxygen Effect in the High-LET Region.

One of the most distinguished features in biological effects of heavy ions would be the decrease of oxygen effect in the high-LET region. This feature has been referred to as the radiobiological basis for the control of hypoxic fraction in cancer radiotherapy. However, mechanisms to explain this phenomenon have not been fully understood. One of the explanations was given by the oxygen in the track hypothesis, which proposes that oxygen is produced along ion tracks even in the hypoxic irradiation condition. In the present study, we designed an experimental approach to support this hypothesis by using 8-hydroxy-2'-deoxyguanosine (8-OHdG) as DNA damage requiring oxygen to produce. The LET dependence of 8-OHdG under hypoxic condition revealed that with increasing LET 8-OHdG yield seems to increase, despite that the yield of OH radical, which is also required for the production of 8-OHdG, decreases in the high-LET region. This result is consistent with the explanation that the local generation of oxygen along ion tracks contributes to the increase of 8-OHdG yield.

BYSTANDER WI-38 CELLS MODULATE DNA DOUBLE-STRAND BREAK REPAIR IN MICROBEAM-TARGETED A549 CELLS THROUGH GAP JUNCTION INTERCELLULAR COMMUNICATION.

Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.

DOSE-RATE AND CELL-KILLING SENSITIVITY OF HIGH-LINEAR ENERGY TRANSFER ION BEAM.

It is believed that the dose-rate of radiation will have an influence on cell sensitivity. The dose-rate effects on cell survival can be expressed by the change of the ß term in the linear quadratic model. The value at a high-dose-rate decreases below 60 Gy/h and reaches zero at 0.2 Gy/h or less for photons. However, the effect for a high-LET ion-beam is not well known. At HIMAC, cells were exposed to 70 keV/µm carbon-ion beams at different dose-rates between 0.5 and 600 Gy/h at room temperature. The ß values for all survival curves show no significant differences among the dose-rates tested for HSG, V79 and CHO cells. Changing the ion-beam dose-rate had no effect on cell survival. This suggests that high-LET particle beams, such as galactic cosmic rays, may not exhibit a dose-rate effect on cell survival. Low-dose-rate radiation showed an effect similar to high-dose-rate radiation.

In vivo radiobiological assessment of the new clinical carbon ion beams at CNAO.

In this article, the in vivo study performed to evaluate the uniformity of biological doses within an hypothetical target volume and calculate the values of relative biological effectiveness (RBE) at different depths in the spread-out Bragg peak (SOBP) of the new CNAO (National Centre for Oncological Hadrontherapy) carbon beams is presented, in the framework of a typical radiobiological beam calibration procedure. The RBE values (relative to (60)Co γ rays) of the CNAO active scanning carbon ion beams were determined using jejunal crypt regeneration in mice as biological system at the entrance, centre and distal end of a 6-cm SOBP. The RBE values calculated from the iso-effective doses to reduce crypt survival per circumference to 10, ranged from 1.52 at the middle of the SOBP to 1.75 at the distal position and are in agreement with those previously reported from other carbon ion facilities. In conclusion, this first set of in vivo experiments shows that the CNAO carbon beam is radiobiologically comparable with the NIRS (National Institute of Radiological Sciences, Chiba, Japan) and GSI (Helmholtzzentrum für Schwerionenforschung, Darmstadt, Germany) ones.

Delivery mechanism of (134)Cs and (137)Cs in seawater off the Sanriku Coast, Japan, following the Fukushima Dai-ichi NPP accident.

To assess the delivery mechanism of radiocesium emitted from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP), we examined vertical profiles of (134)Cs, (137)Cs, and (228)Ra concentrations and the (228)Ra/(226)Ra ratio in the water columns off the Sanriku Coast in the northwestern Pacific Ocean, in July 2012, along with their surface lateral variations in July 2009. Radiocesium concentrations exhibited maximum peaks (3-5 mBq/L for (134)Cs) at depths of 100-200 m, accompanied by high (228)Ra concentrations (0.6-0.8 mBq/L) in comparison with shallower depths (∼0.4 mBq/L). Taking the circulation patterns of currents in the area into account, it was inferred that radioactive depositions were supplied to the (228)Ra-rich Tsugaru Warm Current Water (TWCW) in the offshore area of the Sanriku Coast following the FDNPP accident, and that after the spring of 2011, this water (∼26.5σθ) was covered by lower density surface water, which helped intrude its way to depths of 100-200 m.

228Ra/226Ra ratio and 7Be concentration in the Sea of Japan as indicators for water transport: comparison with migration pattern of Fukushima Dai-ichi NPP-derived 134Cs and 137Cs.

To assess the migration patterns of radiocesium emitted from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP), we analyzed (228)Ra/(226)Ra ratios and (7)Be concentrations and compared them with (134)Cs and (137)Cs concentrations in seawater samples collected within the Sea of Japan before and after the FDNPP accident (i.e., during the period 2007-2012) using low-background γ-spectrometry. The (228)Ra/(226)Ra ratios in surface waters exhibited lateral and seasonal variations, reflecting the flow patterns of surface water. This indicates the transport patterns of the FDNPP-derived radiocesium by surface water. Cosmogenic (7)Be (half-life: 53.3 d) exhibited markedly high concentrations (5-10 mBq/L) at depths shallower than 50 m, with concentrations decreasing steeply (0.2-2 mBq/L) at depths of 50-250 m. The distribution of (7)Be concentrations suggests that the downward delivery of the FDNPP-derived radiocesium to below 50 m depth was negligible for a few months prior to its removal from the Sea of Japan.

Radiobiological description of the LET dependence of the cell survival of oxic and anoxic cells irradiated by carbon ions.

Light-ion radiation therapy against hypoxic tumors is highly curative due to reduced dependence on the presence of oxygen in the tumor at elevated linear energy transfer (LET) towards the Bragg peak. Clinical ion beams using spread-out Bragg peak (SOBP) are characterized by a wide spectrum of LET values. Accurate treatment optimization requires a method that can account for influence of the variation in response for a broad range of tumor hypoxia, absorbed doses and LETs. This paper presents a parameterization of the Repairable Conditionally-Repairable (RCR) cell survival model that can describe the survival of oxic and hypoxic cells over a wide range of LET values, and investigates the relationship between hypoxic radiation resistance and LET. The biological response model was tested by fitting cell survival data under oxic and anoxic conditions for V79 cells irradiated with LETs within the range of 30-500 keV/µm. The model provides good agreement with experimental cell survival data for the range of LET investigated, confirming the robustness of the parameterization method. This new version of the RCR model is suitable for describing the biological response of mixed populations of oxic and hypoxic cells and at the same time taking into account the distribution of doses and LETs in the incident beam and its variation with depth in tissue. The model offers a versatile tool for the selection of LET and dose required in the optimization of the therapeutic effect, without severely affecting normal tissue in realistic tumors presenting highly heterogeneous oxic and hypoxic regions.

Mutational analysis of the feline CLN3 gene and an ultrastructural evaluation of lysosomal storage materials in a cat with neuronal ceroid lipofuscinosis: an investigation into the molecular basis of the disease.

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL.

Induction of DNA DSB and its rejoining in clamped and non-clamped tumours after exposure to carbon ion beams in comparison to X rays.

We studied double-strand breaks (DSB) induction and rejoining in clamped and non-clamped transplanted tumours in mice leg after exposure to 80 keV µm(-1) carbon ions and X rays. The yields of DSB in the tumours were analysed by a static-field gel electrophoresis. The OER of DSB after X rays was 1.68±0.31, and this value was not changed after 1 h rejoining time (1.40±0.26). These damages in oxygenated conditions were rejoined 60-70% within 1 h in situ. No difference was found between the exposure to X rays and carbon ions for the induction and rejoining of DSB. Thus, the values of OER and rejoined fraction after exposure to carbon ions were similar to those after X rays, and the calculated relative biological effectivenesses of carbon ion were around 1 under both oxygen conditions. The yields of DSB in vivo depend on exposure doses, oxygen conditions and rejoining time, but not on the types of radiation quality.

Analysis of cell-survival fractions for heavy-ion irradiations based on microdosimetric kinetic model implemented in the particle and heavy ion transport code system.

It is considered that the linear energy transfer (LET) may not be the ideal index for expressing the relative biological effectiveness (RBE) of cell killing for heavy-ion irradiation, as the ion-species dependencies have clearly been observed in the relation between LET and RBE derived from cell-survival fraction data. The previously measured survival fractions of four cell lines irradiated by various ion species, employing the saturation-corrected dose-mean lineal energy, y*, instead of LET as the index of the RBE were therefore re-analysed. In the analysis, the initial slopes of the survival fractions, the so-called α-parameter in the linear-quadratic model, were plotted as a function of y*, which was calculated by the microdosimetric kinetic (MK) model implemented in the Particle and Heavy Ion Transport code System. It was found from the analysis that the ion-species dependencies observed in the relations between α and LET disappeared from those between α and y*, and their relations can be well reproduced by a simple equation derived from the MK model. These results clearly indicate the suitability of y* to be used in the estimation of the RBE of cell killing for heavy-ion irradiations, which is of great importance in the treatment planning of charged-particle therapy.

Carbon-ion radiotherapy: clinical aspects and related dosimetry.

The features of relativistic carbon-ion beams are attractive from the viewpoint of radiotherapy. They exhibit not only a superior physical dose distribution but also an increase in biological efficiency with depth, because energy loss of the beams increases as they penetrate the body. This paper reviews clinical aspects of carbon-beam radiotherapy using the experience at the National Institute of Radiological Sciences. The paper also outlines the dosimetry related to carbon-beam radiotherapy, including absolute dosimetry of the carbon beam, neutron measurements and radiation protection measurements.

DNA fragmentation induced in human fibroblasts by 56Fe ions: experimental data and Monte Carlo simulations.

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1-5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1-23 kbp and 23-5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1-5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since gamma rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

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 microM PtTC, and then irradiated by fast ions C(6+) and He(2+), with Linear Energy Transfer (LET) within range 2-70 keV/microm. 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.

Irradiation of DNA loaded with platinum containing molecules by fast atomic ions C(6+) and Fe(26+).

PURPOSE: In 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 C(6+) and Fe(26+) ions. MATERIAL AND METHODS: DNA Plasmids (pBR322) loaded with different amounts of platinum contained in a terpyridine-platinum molecule (PtTC) were irradiated by C(6+) ions and Fe(26+) ions. The LET values ranged between 13.4 keV/microm and 550 keV/microm. In some experiments, dimethyl sulfoxide (DMSO) was added. RESULTS: In 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: These 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.

DNA fragments induction in human fibroblasts by radiations of different qualities.

Experimental data on DNA double strand break (DSB) induction in human fibroblasts (AG1522), following irradiation with several radiation qualities, namely gamma rays, 0.84 MeV protons, 58.9 MeV u(-1) carbon ions, iron ions of 115 MeV u(-1), 414 MeV u(-1), 1 GeV u(-1), and 5 GeV u(-1), are presented. DSB yields were measured by calibrated Pulsed Field Gel Electrophoresis in the DNA fragment size range 0.023-5.7 Mbp. The DSB yields show little LET dependence, in spite of the large variation of the latter among the beams, and are slightly higher than that obtained using gamma rays. The highest yield was found for the 5 GeV u(-1) iron beam, that gave a value 30% higher than the 1 GeV u(-1) iron beam. A phenomenological method is used to parametrise deviation from randomness in fragment size spectra.

Fast He2+ ion irradiation of DNA loaded with platinum-containing molecules.

PURPOSE: The association of radiotherapy and chemotherapy is an attractive approach to improve the therapeutic index of the treatment of tumors. A lot of work has been devoted to investigate the effects of X-ray, gamma-ray and neutron irradiation of DNA or living cells loaded with different chemical compounds containing heavy atoms like platinum. No such studies exist presently when fast atomic ions are chosen as ionizing particles. In the present work, we investigate quantitatively the increase of DNA breaks in complexes of plasmid-DNA loaded with platinum atoms under irradiation by fast atomic He2+ ions. MATERIALS AND METHODS: DNA Plasmids (pBR322) are incubated in solutions containing different concentrations of terpyridine platinum (PtTC). In some preparations, dimethyl sulfoxide (DMSO), a free radical scavenger, has been added in order to investigate the role of the free radicals. The complexes of DNA plasmids loaded with high-Z atoms are irradiated under atmospheric conditions by He2+ ions at an energy of 143 MeV/amu and a linear energy transfert (LET) of 2.24 keV/microm. Analysis of DNA damage--single and double strand breaks--is made by electrophoresis on agarose gels. RESULTS: The results show a significant increase in DNA strand breaks when platinum is present, indicating a radiosensitization by the high Z atoms. The increase in DNA damages is attributed to inner-shell ionization of a platinum atom by secondary electrons emitted along the He2+ tracks followed by an Auger deexcitation, leading, thus, to a local amplification of the radiative effects close to the DNA. The contributions of scavengeable--solvant mediated--indirect effects and non-scavengeable effects (direct ionization) are quantitatively evaluated. CONCLUSION: Enhancement of DNA breaks in plasmids loaded with heavy atoms like platinum and irradiated by atomic ions are observed. This finding suggests an enhancement of cell death rate will occur under irradiation by atomic ions when the cells contain high-Z atoms located close to DNA due to the increase of the DNA breaks.

Apoptosis induced by high-LET radiations is not affected by cellular p53 gene status.

To learn more about the biological effects of high-linear energy transfer (LET) radiations, we examined radiation-induced apoptosis in response to high-LET radiations in cells with wild-type, mutated and null p53 gene. Three human lung cancer cell lines were used. These lines had identical genotypes, except for the p53 gene. Cells were exposed to X-rays or high-LET radiations (13 - 200 keV microm(-1)) using different nuclei ion beams. Cellular radiation sensitivities were determined with the use of colony-forming assays. Apoptosis was detected and quantified using Hoechst 33342 staining with fluorescence microscopy. It was found that (1) there was no significant difference in cellular sensitivity to high-LET radiation (>85 keV microm(-1)), although the sensitivity of wild-type p53 cells to X-rays was higher than that of mutated p53 or p53-null cells; (2) X-ray-induced apoptosis at higher frequencies in wild-type p53 cells when compared with mutated p53 and p53-null cells; and (3) Fe beams (200 keV microm(-1)) induced apoptosis in a p53-independent manner. The results indicate that high-LET radiations induces apoptosis in human lung cancer cells in a manner that does not seem to depend on the p53 gene status of the cells.

DNA DSB induced by iron ions in human fibroblasts: LET dependence and shielding efficiency.

This paper reports on DNA DSB induction in human fibroblasts by iron ions of different energies, namely 5, 1 GeV/u, 414 and 115 MeV/u, in absence or presence of different shields (PMMA, Al and Pb). Measure of DNA DSB was performed by calibrated Pulsed Field Gel Electrophoresis using the fragment counting method. The RBE-LET relationships for unshielded and shielded beams were obtained both in terms of dose average LET and of track average LET. Weak dependence on these parameters was observed for DSB induction. The shielding efficiency, evaluated by the ratio between the cross sections for unshielded and shielded beams, depends not only on the shield type and thickness, but also on the beam energy. Protection is only observed at high iron ions energy, especially at 5 GeV/u, where PMMA shield gives higher protection compared to Al or Pb shields of the same thickness expressed in g/cm2.

G2 chromatid damage and repair kinetics in normal human fibroblast cells exposed to low- or high-LET radiation.

Radiation-induced chromosome damage can be measured in interphase using the Premature Chromosome Condensation (PCC) technique. With the introduction of a new PCC technique using the potent phosphatase inhibitor calyculin-A, chromosomes can be condensed within five minutes, and it is now possible to examine the early damage induced by radiation. Using this method, it has been shown that high-LET radiation induces a higher frequency of chromatid breaks and a much higher frequency of isochromatid breaks than low-LET radiation. The kinetics of chromatid break rejoining consists of two exponential components representing a rapid and a slow time constant, which appears to be similar for low- and high-LET radiations. However, after high-LET radiation exposures, the rejoining process for isochromatid breaks influences the repair kinetics of chromatid-type breaks, and this plays an important role in the assessment of chromatid break rejoining in the G2 phase of the cell cycle.

Complex chromosomal rearrangements induced in vivo by heavy ions.

It has been suggested that the ratio complex/simple exchanges can be used as a biomarker of exposure to high-LET radiation. We tested this hypothesis in vivo, by considering data from several studies that measured complex exchanges in peripheral blood from humans exposed to mixed fields of low- and high-LET radiation. In particular, we studied data from astronauts involved in long-term missions in low-Earth-orbit, and uterus cancer patients treated with accelerated carbon ions. Data from two studies of chromosomal aberrations in astronauts used blood samples obtained before and after space flight, and a third study used blood samples from patients before and after radiotherapy course. Similar methods were used in each study, where lymphocytes were stimulated to grow in vitro, and collected after incubation in either colcemid or calyculin A. Slides were painted with whole-chromosome DNA fluorescent probes (FISH), and complex and simple chromosome exchanges in the painted genome were classified separately. Complex-type exchanges were observed at low frequencies in control subjects, and in our test subjects before the treatment. No statistically significant increase in the yield of complex-type exchanges was induced by the space flight. Radiation therapy induced a high fraction of complex exchanges, but no significant differences could be detected between patients treated with accelerated carbon ions or X-rays. Complex chromosomal rearrangements do not represent a practical biomarker of radiation quality in our test subjects.