Residual chromatin breaks as biodosimetry for cell killing by carbon ions

We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET= 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour postirradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.     

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.     

Repair and misrepair of heavy-ion-induced chromosomal damage.

The premature chromosome condensation (PCC) technique was used to investigate chromosomal damage, repair, and misrepair in the G phase of a human/hamster hybrid cell line that contains a single human chromosome. Plateau-phase cell cultures were exposed to either x-rays or a 425 MeV/u beam of neon ions near the Bragg peak where the LET is 183 kev/micrometers. An in situ hybridization technique coupled to fluorescent staining of PCC spreads confirmed the linearity of the dose response for initial chromatin breakage in the human chromosome to high doses (1600 cGy x-ray or 1062 cGy Ne). On Giemsa-stained slides, initial chromatin breakage in the total genome and the rejoining kinetics of these breaks were determined. As a measure of chromosomal misrepair, ring PCC aberrations were also scored. Ne ions were about 1.5 x more effective per unit dose compared to x-rays at producing the initially measured chromatin breakage. 90% of the x-ray-induced breaks rejoined in cells incubated at 37 degrees C after exposure. In contrast, only 50% of Ne-ion-induced breaks rejoined. In the irradiated G1 cells, ring PCC aberrations increased with time apparently by first order kinetics after either x-ray or Ne exposures. However, far fewer rings formed in Ne-irradiated cells after a dose giving a comparable initial number of chromatin breaks. Following x-ray exposures, the yield of rings formed after long repair times (6 to 9 hrs) fit a quadratic dose-response curve. These results indicate quantitative and qualitative differences in the chromosomal lesions induced by low- and high-LET radiations.     

Cytogenetic effects of energetic ions with shielding

In order to understand the effects of shielding on the induction of biological damages by charged particles, we conducted experiments with accelerated protons (250 MeV) and iron particles (1 GeV/u). Human lymphocytes in vitro were exposed to particle beams through polyethylene with various thickness, and chromosomal aberrations were determined using FISH technique. Dose response curves for chromosome aberrations were obtained and compared for various particle types. Experimental results indicated that for a given absorbed dose at the cell, the effectiveness of protons and iron particles in the induction of chromosomal aberrations was not significantly altered by polyethylene with thickness up to 30-cm and 15-cm respectively. Comparing with gamma rays, charged particles were very effective in producing complex chromosomal damages, which may be an important mechanism in alterating functions in nondividing tissues, such as nervous systems.     

Neoplastic transformation of hamster embryo cells by heavy ions

We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/μm. Exponentially growing cells were irradiated with ¹²C or ²⁸Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/μm, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.     

Theoretical consideration of the chemical pathways for radiation-induced strand breaks.

A theoretical approach to the understanding of the biochemical mechanisms of indirect action of ionizing radiation on SV40 DNA in aqueous solution is presented. The extent of OH attack on the sugar moiety and bases has been calculated. A realistic model for the DNA (in B form) based on available X-ray diffraction data is used and specific reaction sites for the OH radicals are obtained. A Monte Carlo scheme is used to follow the diffusion and reaction of the OH radicals. Effects of track structure have been considered and the single strand break D37 values for 14 MeV electrons (low-LET) and 670 MeV/u and 40 MeV/u neon particles are presented. Calculated results are in agreement with available experimental data. It has been found that regardless of the qualities of radiation, 80% of the OH attack on DNA is on the bases and 20% is on the deoxyribose. From probability considerations only, it appears that the number of double strand breaks varies linearly with dose.     

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.     

Numerical simulations of the radiance from the comet 46P/Wirtanen in the various configurations of the measurements during “Rosetta” mission

The work we present deals with the spectrometric measurements of VIRTIS instrument of the Comet P/Wirtanen planned for the Rosetta mission. This spectrometer can monitor (VIRTIS M channel: 0.250μm – 0.980μm; Δκ=20cm⁻¹; 0.980 – 5.0 μm; Δκ = 5cm⁻¹; VIRTIS H channel: 2.0 μm – 5.0 μm; Δκ=2cm⁻¹) the nucleus and the coma in order to provide a general picture of coma's composition, the production of gas and dust, the relationship of coma production to surface composition and the structure and variation of mineralogy of the nucleus surface. During the mission the observation conditions of the spectroscopic investigation change due to different relative positions spacecraft/comet, and to the different illumination conditions of the surface at various distances of the comet to the Sun. The nucleus surface is continuously modified by the ice sublimation accompanied by gas and dust emission. Consequently the surface also its spectrophotometric properties changes and their monitoring can give a new insight. The important role of simulations is to predict the results of measurements in various experimental condition what, in the future, can help in interpretation of the measured data.

In this paper the first results of our simulation the radiance from the comet in the 0.25–5.0μm spectral range at two distances from the Sun (1AU and 3AU) are shown. The distance between the Rosetta orbiter and the nucleus surface as well as the sun zenith angles are taken into account according to the Rosetta mission phases. In fact the surface and coma properties vary along the comet orbit, and should be taken into account in our calculations. The optical parameters of the dust on the surface (e.g. reflectance) and in the coma (e.g. Q_ext) were calculated from optical constants of possible comet analogues. The thermodynamic parameters of the comet are taken from the models of comet evolution. Through this kind of modelling it is possible to identify the surface characteristics in spectra of the radiation from the surface of nucleus transmitted through the coma loaded with dust and gases.

Even if the “Rosetta mission” is postponed, with the consequence of a target change, we think that our idea and the method used for the simulations can be useful also for the new Rosetta target - the comet 67P/Churyumov Gerasimenko.     

Effects of heavy ion radiation on the brain vascular system and embryonic development.

Using neonatal rats as a model system, we investigated the response of the brain vascular system to ionizing radiation and found that distinct petechial hemorrhages developed in the cerebral cortex within a few hours after irradiation, reached a maximum about 13 to 24 hours, and decreased exponentially with time. No brain hemorrhage was found in neonatal rats 12 days after irradiation. Our experimental results indicate that a dose of a few hundred rad of X rays can induce a significant number of hemorrhages in the brain, and the number of lesions increases exponentially with dose. Heavy ions induce more hemorrhages than X rays for a given dose, and the RBE for 670 MeV/u neon particles ranges from about 2.0 for low doses to about 1.4 for high doses. A histological study of the hemorrhages indicates that a large number of red blood cells leak from the blood vessels. The radiation-induced hemorrhages may be a result of some capillary membrane damages or reproductive death of some blood vessel epithelial cells. The fast onset of hemorrhage after irradiation suggest that some membrane damage may be involved. The effect of heavy-ion radiation on the embryonic development was studied with energetic iron particles. Pregnant mice were whole-body irradiated with 600 MeV/u iron particles on day 6 of gestation and were sacrificed 12 days after irradiation. Various physical abnormalities were observed, and embryos irradiated with 1 rad iron particles showed retardation of body development.     

HZE effects on mammalian cells.

In track segment experiments cell survival and chromosome aberrations of mammalian cells have been measured for various heavy ion beams between helium and uranium in the energy range between 0.5 and 960 MeV/u, corresponding to a velocity range of 0.03 to 0.87 C, and an LET spectrum from 10 to 15 000 keV/micrometers. At low LET, the cross section (sigma) for cell killing increases with increasing LET and shows a common curve for all ions regardless of the atomic number. This indicates that in this region the track structure of the different ions is of only a minor influence, and it is rather the total energy transfer, which is important for cell killing. At higher LET values, deviations from a common sigma-LET curve can be observed which indicate a saturation effect. The saturation of the lighter ions occurs at lower LET values than for the heavier ions. These findings are also confirmed by the chromosome data, where the efficiency for the induction of chromosomal aberrations for high LET particles depends on the track structure and is nearly independent of LET. In the heavier beams (Z > or = 10) individual particles cause multiple chromosome breaks in mitotic cells.     

Mutagenic effects of heavy ion radiation in plants.

Genetic and developmental effects of heavy ions in maize and rice were investigated. Heavy particles with various charges and energies were accelerated at the BEVALAC. The frequency of occurrence of white-yellow stripes on leaves of plants developed from irradiated maize seeds increased linearly with dose, and high-LET heavy charged particles, e.g., neon, argon, and iron, were 2-12 times as effective as gamma rays in inducing this type of mutation. The effectiveness of high-LET heavy ion in (1) inhibiting rice seedling growth, (2) reducing plant fertility, (3) inducing chromosome aberration and micronuclei in root tip cells and pollen mother cells of the first generation plants developed from exposed seeds, and (4) inducing mutation in the second generation, were greater than that of low-LET gamma rays. All effects observed were dose-dependent; however, there appeared to be an optimal range of doses for inducing certain types of mutation, for example, for argon ions (400 MeV/u) at 90-100 Gy, several valuable mutant lines with favorable characters, such as semidwarf, early maturity and high yield ability, were obtained. Experimental results suggest that the potential application of heavy ions in crop improvement is promising. RFLP analysis of two semidwarf mutants induced by argon particles revealed that large DNA alterations might be involved in these mutants.     

DNA-lesion and cell death by alpha-particles and nitrogen ions.

When the natural logarithm of the surviving fraction is plotted against the dose of radiation, curves with shoulders at relatively high survival levels are obtained after gamma-rays. The curves were practically linear in case of HMV-I and HA-1 cells irradiated by charged particle beams. These cells were derived from human malignant melanoma and Chinese hamster cells, respectively. The amount of DNA single strand breaks (ssb) by gamma-rays or nitrogen-ions (LET=530KeV/micrometers) in HMV-I cells increases linearly with increment in dose, when the ssb is detected using the alkaline elution technique. There is no close relationship between the dose-response curve of the ssb and the dose-survival curves after gamma-rays or N-ions. The amount of DNA double strand breaks (dsb) by gamma-rays increases quadratically with increment of dose, in both HMV-I cells and HA-1 cells, when the dsb is detected using the neutral elution technique. The survival fraction for HA-1 cells is slightly higher than that for HMV-I cells, at the same dose, and the amount of dsb for HA-1 cells is considerably greater than that for HMV-I cells. These results suggest that the radiosensitivities to gamma-rays in different cell lines do not correspond to the number of DNA strand breaks. The amount of both non-repairable ssb and dsb also increases quadratically with increment of dose for gamma-rays and almost linearly with increment of dose for N-ions and alpha-particles (LET=36keV/micrometers for HA-1 cells and LET=77keV/micrometers for HMV-I cells). The dose-response curves for non-repairable dsb in case of these radiations seemed to mirror image the dose-survival curves for these radiations, in both cell lines. The number of non-repairable DNA strand breaks in the two cell lines, at the same level of survival was much the same. These results show the close relationship between the induction of non-repairable DNA strand breaks and cell killing.     

Results of cosmic radiation dose field measurements aboard the “Salyut-6” orbital station

During the 3rd main expedition on board the “Salyut-6” orbital station in 1979 the integral characteristics of cosmic radiation were measured in various positions inside the manned modules (experiment “Integral”). Measurements were performed with thermoluminescent dosimeters, photographic films and solid state plastic detectors supplied for the experiment by specialists of the USSR, Bulgaria, Hungary, GDR and Romania. The dose gradient inside the manned modules of the station amounted to 70 % for long intervals of time. During the experimental period the dose rate inside the station was 15 to 30 mrad per day. The mean flux of particles with z 6 and LET 200 keV/μm was found to be 0.22 cm⁻² day⁻¹.     

Energetic particle fluxes data base of “CORONAS-I” satellite observations

The data base DB SCR uses data obtained by the SCR instrument package on the CORONAS-I satellite. DB SCR contains information about fluxes of relativistic electrons (0.5–124 MeV), protons (1–300 MeV), nuclei (1–19 MeV/nucl) and γ-radiation (0.1–7 MeV) in the low altitude region (500 km). The time resolution of the data is 2.5 s. Magnetic field parameters (B, magnetic latitude and longitude), L-shell and local times (LT and MLT) are included in DB SCR. Since all parameters are equivalents it is possible to perform the multidimensional analysis for any set of DB SCR parameters. The additional DB SCR software packages may be used to develop different semi-empirical models.     

The INFRARED SPACE OBSERVATORY (ISO)

The INFRARED SPACE OBSERVATORY (ISO), a project of the European Space Agency (ESA), will make various astronomical observations in the wavelength range of 2 to 200 μm. Two-thirds of its observing time will be available to guest observers via the traditional route of proposal submission. ISO will be launched in 1995 by an Ariane 4 from Kourou (Guyana) and be brought into a very elongated orbit with a 24hr period of which 16 hr can be used for astronomical observations. The payload module is essentially a large cryostat with a tank that will be filled with over 2000 litres of superfluid helium to keep the instruments cold (2 to 4 K) during an expected lifetime of 18 months. Inside the dewar, there is a Ritchey-Chretien telescope with a primary mirror of diameter 60cm. Four instruments, each provided by a different PI consortium, share the ISO focal plane. These instruments are : a photometer, a camera and two spectrometers — one for the wavelength range 2–45 μm and the other for 45–180 μm.     

Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions.

We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.     

A preliminary look at the dust in comet Halley

Highlights of infrared observations of the dust are discussed and compared with first results from the space probes. An emission feature was detected at 3.4 μm; the 10 and 20 μm silicate features were well-observed; and far-infrared data out to 160 μm were obtained. Organic material seems to be abundant in grains and may explain the 3.4 μm emission. Calculations are presented for one example of organic material. A component of the grains may volatize at temperatures around 300 K.     

Near infrared observations of comet Halley from Vega 2

Spatial distribution of the continuum radiation in the range of 0.95–1.9 μm presumes total dust production rate of the comet of 10ρ tonne s⁻¹ (ρ is the dust material density) and its angular distribution proportional cos . Observations of the water vapor band at 1.38 μ m reveal strong jets, their time shift from the dust jet measured in situ is consistent with gas velocity of 0.82±0.1 km s⁻¹ and dust velocity of 0.55±0.08 km s⁻¹. The OH vibrational-rotational bands observed are excided directly via photolysis of water vapor. Water vapor production rate deduced from the H₂O band and OH band intensities is 8×10²⁹ s⁻¹. Intensity of the CN(0,0) band result in the CN column density of 9×10¹² cm⁻², i.e. larger by a factor of 3 than given by the violet band.     

Energy content of accelerated electrons and ions in intense solar flares

The energy content of nonthermal particles in solar flares is shared between accelerated electrons and ions. It isimportant for understanding the particle acceleration mechanism in solar flares. Yohkoh observed a few intense flares which produced both strong gamma-ray lines and electron bremsstrahlung continuum. We analyze energy spectra of X-class solar flares on October 27, 1991(X6.1), November 6, 1997 (X9.4), July 14, 2000 (X5.7) and November 24, 2000 (X2.3). The accelerated electron and proton spectra are derived from a spectral analysis of their high-energy photon emission and the energy contents in >1 MeV electrons and >10 MeV protons are estimated to be 6×l0²⁸ – 4×10³⁰ and 2×10²⁸ – 5×10²⁹ erg, respectively. We study the flare to flare variation in the energy content of >1 MeV electrons and >10 MeV protons for the four Yohkoh gamma-ray flares. Ratios of >1 MeV electron energy content to >10 MeV proton energy content are roughly within an order of magnitude.     

Simulation of depth–dose distributions for various ions in polyethylene medium

Study of depth–dose distributions for intermediate energy ion beams in tissue-like media such as polyethylene (CH₂)_n provides a good platform for further improvements in the fields of hadrontherapy and space radiation shielding. The depth–dose distributions for ¹²C ions at various energies and for light and intermediate ion beams (³He, ¹⁶O, ²⁰Ne and ²⁸Si) as well as for heavy ions ⁵⁶Fe in polyethylene were estimated by using simulation toolkit: Geant4. Calculations were performed mainly by considering two different combinations of standard electromagnetic (EM), binary cascade (BIC), statistical multifragmentation (SMF) and Fermi breakup (FB) models. The energies of the ion beams were selected to achieve the Bragg peaks at predefined position (∼60 mm) and as per their availability. Variations of peak-to-entrance ratio (from 7.44 ± 0.05 to 8.87 ± 0.05), entrance dose (from 2.89 ± 0.01 to 203.71 ± 0.63 MeV/mm) and entrance stopping power (from 3.608 to 208.858 MeV/mm, calculated by SRIM) with atomic number (Z) were presented in a systematic manner. The better peak-to-entrance ratio and less entrance dose in the region Z = 2 to 8 (i.e. ³He to ¹⁶O) may provide the suitability of the ion beams for hadrontherapy.