M. Pugliese

Rejoining and Misrejoining of Radiation-Induced Chromatin Breaks. IV. Charged Particles

We have recently reported the kinetics of chromosome rejoining and exchange formation in human lymphocytes exposed to gamma rays using the techniques of fluorescence in situ hybridization (FISH) and premature chromosome condensation (PCC). In this paper, we have extended previous measurements to cells exposed to charged particles. Our goal was to determine differences in chromatin break rejoining and misrejoining after exposure to low- and high-linear energy transfer (LET) radiation. Cells were irradiated with hydrogen, neon, carbon or iron ions in the LET range 0.3-140 keV/microm and were incubated at 37 degrees C for various times after exposure. Little difference was observed in the yield of early prematurely condensed chromosome breaks for the different ions. The kinetics of break rejoining was exponential for all ions and had similar time constants, but the residual level of unrejoined breaks after prolonged incubation was higher for high-LET radiation. The kinetics of exchange formation was also similar for the different ions, but the yield of chromosome interchanges measured soon after exposure was higher for high-LET particles, suggesting that a higher fraction of DNA breaks are misrejoined quickly. On the other hand, the rate of formation of complete exchanges was slightly lower for densely ionizing radiation. The ratios between the yields of different types of aberrations observed at 10 h postirradiation in prematurely condensed chromosome preparations were dependent on LET. We found significant differences between the yields of aberrations measured in interphase (after repair) and metaphase for densely ionizing radiation. This difference might be caused by prolonged mitotic delay and/or interphase death. Overall, the results point out significant differences between low- and high-LET radiation for the formation of chromosome aberrations.

Accelerator-based tests of radiation shielding properties of materials used in human space infrastructures.

Shielding is the only practical countermeasure for the exposure to cosmic radiation during space travel. It is well known that light, hydrogenated materials, such as water and polyethylene, provide the best shielding against space radiation. Kevlar and Nextel are two materials of great interest for spacecraft shielding because of their known ability to protect human space infrastructures from meteoroids and debris. We measured the response to simulated heavy-ion cosmic radiation of these shielding materials and compared it to polyethylene, Lucite (PMMA), and aluminum. As proxy to galactic nuclei we used 1 GeV n−1 iron or titanium ions. Both physics and biology tests were performed. The results show that Kevlar, which is rich in carbon atoms (about 50% in number), is an excellent space radiation shielding material. Physics tests show that its effectiveness is close (80–90%) to that of polyethylene, and biology data suggest that it can reduce the chromosomal damage more efficiently than PMMA. Nextel is less efficient as a radiation shield, and the expected reduction on dose is roughly half that provided by the same mass of polyethylene. Both Kevlar and Nextel are more effective than aluminum in the attenuation of heavy-ion dose.

Biological dosimetry in Russian and Italian astronauts

Large uncertainties are associated with estimates of equivalent dose and cancer risk for crews of long-term space missions. Biological dosimetry in astronauts is emerging as a useful technique to compare predictions based on quality factors and risk coefficients with actual measurements of biological damage in-flight. In the present study, chromosomal aberrations were analyzed in one Italian and eight Russian cosmonauts following missions of different duration on the MIR and the international space station (ISS). We used the technique of fluorescence in situ hybridization (FISH) to visualize translocations in chromosomes 1 and 2. In some cases, an increase in chromosome damage was observed after flight, but no correlation could be found between chromosome damage and flight history, in terms of number of flights at the time of sampling, duration in space and extra-vehicular activity. Blood samples from one of the cosmonauts were exposed in vitro to 6 MeV X-rays both before and after the flight. An enhancement in radiosensitivity induced by the spaceflight was observed.

Inactivation of C3H 10T½ Cells by Monoenergetic High LET Alpha-particles

Inactivation of mouse C3H 10T1/2 cells in plateau-phase (7.8 x 10(4) cells/cm2) was studied by using alpha-particles from the irradiation facility installed for radiobiological experiments at the 3 MV Tandem accelerator, University of Naples. Silicon detectors and CR39 plastic track detectors were employed for dosimetric purposes. The cells were exposed to high LET monoenergetic alpha-particles (energy of 1.8 MeV at the centre of the cell nucleus, track-averaged LET of 177 keV/micron and dose-rate of 1.1 Gy/min) and low-LET 80 kVp X-rays. The X-ray survival curve showed a significant shoulder (alpha/beta = 9 Gy) while the survival curve for alpha-particles was close to exponential. The mean lethal dose of alpha-particles was 0.77 +/- 0.02 Gy and the RBE was 5.2 at 80% survival and 3.0 at 5% survival. Survival of exponentially growing cells (2 x 10(4) cells/cm2) following irradiation with the alpha-particle beam is also reported. The nuclear areas of 10T1/2 cells were measured as 299 +/- 9 micron 2 and 250 +/- 8 micron 2 for cells in log phase and plateau phase, respectively. The inactivation cross-section, obtained from the mean lethal dose, was 34 micron 2 and 37 micron 2 for cells in log phase and plateau phase, respectively. These values appear to be the maximum measured values for the inactivation cross-section of 10T1/2 cells as a function of the alpha-particle LET. This saturation cross-section is very similar to the saturation values reported in the literature for other mammalian cell lines.

Chromosome damage induced by high-LET α-particles in plateau-phase C3H 10T1/2 cells

Chromosome aberrations induced by X-rays and α-particles (LET = 177 keV/µm) were observed at the first mitosis in C3H 10T1/2 cells released from density-inhibited cultures. X-radiation induced more chromosome exchanges than breaks (71% vs 27% of total aberrations), while a predominance of breaks (63%) was observed after α-irradiation. A linear-quadratic dose-response relationship was obtained for X-rays, while that for α-particles was linear. The RBE values for total aberration induction (ranging from 5·1 at low doses to 4·4 at high doses) were very similar to the RBE for cell killing (from 5·2 to 4·3). The RBE for dicentric induction (≈2) was much lower than the RBE for the induction of both breaks (from 7 to 6) and interstitial deletions (from 9 to 7). This behaviour supports the hypothesis that chromosome deletions play a major role in the malignant transformation of 10T1/2 cells. A high correlation between cell killing and number of acentric fragments per cell was found. The number of acentrics/cell a...

Effects of alpha-particles on survival and chromosomal aberrations in human mammary epithelial cells

We have studied the radiation responses of a human mammary epithelial cell line, H184B5 F5-1 M/10. This cell line was derived from primary mammary cells after treatment with chemicals and heavy ions. The F5-1 M/10 cells are immortal, density-inhibited in growth, and non-tumorigenic in athymic nude mice and represent an in vitro model of the human epithelium for radiation studies. Because epithelial cells are the target of α-particles emitted from radon daughters, we concentrated our studies on the efficiency of α-particles. Confluent cultures of M/10 cells were exposed to accelerated α-particles [beam energy incident at the cell monolayer=3.85 MeV, incident linear energy transfer (LET) in cell= 109 keV/µm] and, for comparison, to 80 kVp x-rays. The following endpoints were studied: (1) survival, (2) chromosome aberrations at the first postirradiation mitosis, and (3) chromosome alterations at later passages following irradiation. The survival curve was exponential for α-particles (D0 = 0.73± 0.04 Gy), while a shoulder was observed for x-rays (α/β = 2.9 Gy; D0 = 2.5 Gy, extrapolation number 1.6). The relative biological effectiveness (RBE) of high-LET α-particles for human epithelial cell killing was 3.3 at 37% survival. Dose-response curves for the induction of chromosome aberrations were linear for cc-particles and linear-quadratic for x-rays. The RBE for the induction of chromosome aberrations varied with the type of aberration scored and was high (about 5) for chromosome breaks and low (about 2) for chromosome exchanges. The RBE for the induction of total chromosome aberrations (2.3 at 37% cell survival) was lower than that for cell survival, suggesting that chromosome damage at the first postirradiation mitosis is not sufficient to account for the increased efficiency of α-particles in the induction of lethal effects. However, measured cell survival after α-particle irradiation can be predicted from chromosome damage when cells at different population doubling numbers after irradiation are considered. In fact, a high percentage of α-irradiated cells carried unstable chromosomal aberrations up to population doubling number about 5. On the other hand, x-ray-induced damage disappeared rapidly. These results suggest that α-particle-induced reproductive death of human mammary epithelial cells is caused by chromosome damage in the first 5 generations following exposure, whereas the in-activation produced by low-LET radiation is mostly related to the aberrations at the first post-irradiation mitosis.

Depleted uranium residual radiological risk assessment for Kosovo sites

During the recent conflict in Yugoslavia, depleted uranium rounds were employed and were left in the battlefield. Health concern is related to the risk arising from contamination of areas in Kosovo with depleted uranium penetrators and dust. Although chemical toxicity is the most significant health risk related to uranium, radiation exposure has been allegedly related to cancers among veterans of the Balkan conflict. Uranium munitions are considered to be a source of radiological contamination of the environment. Based on measurements and estimates from the recent Balkan Task Force UNEP mission in Kosovo, we have estimated effective doses to resident populations using a well-established food-web mathematical model (RESRAD code). The UNEP mission did not find any evidence of widespread contamination in Kosovo. Rather than the actual measurements, we elected to use a desk assessment scenario (Reference Case) proposed by the UNEP group as the source term for computer simulations. Specific applications to two Kosovo sites (Planeja village and Vranovac hill) are described. Results of the simulations suggest that radiation doses from water-independent pathways are negligible (annual doses below 30 microSv). A small radiological risk is expected from contamination of the groundwater in conditions of effective leaching and low distribution coefficient of uranium metal. Under the assumptions of the Reference Case, significant radiological doses (>1 mSv/year) might be achieved after many years from the conflict through water-dependent pathways. Even in this worst-case scenario, DU radiological risk would be far overshadowed by its chemical toxicity.

The effect of track structure on the induction of chromosomal aberrations in murine cells.

PURPOSE To measure chromosome aberrations in C3H 10T1/2 mouse fibroblasts using FISH painting at the first mitosis following exposure to 30 keV/microm hydrogen or neon ions. MATERIALS AND METHODS Cells in plateau-phase were irradiated with 0.86 MeV protons at the TTT-3 Tandem accelerator in Naples (Italy), or with 400 MeV/n Ne ions at the HIMAC accelerator in Chiba (Japan). Colcemid-blocked cells were harvested at the first mitosis following exposure, and chromosome spreads were hybridized in situ with a fluorescein-labelled composite mouse DNA probe specific for chromosomes 2 and 8. RESULTS Protons were more efficient than neon ions at the same LET in the induction of chromosome interchanges and breaks. Yields of complex exchanges were similar for both particles at the same dose, but protons produced mostly insertions, while with Ne exposure non-reciprocal exchanges were the most frequent complex-type exchange. CONCLUSIONS Charged particles with the same LET produce different yields of chromosome aberrations, and some observed differences can be explained based on the available track-structure models.

Single charged-particle damage to living cells: a new method based on track-etch detectors

Abstract Biological effects of ionizing radiation are usually expressed as a function of the absorbed dose. Low doses of high-LET radiation correspond to one or few particle traversals through the cell. In order to study the biological effectiveness of single charged particles, we have developed a new method based on solid state nuclear track detectors. Cells are seeded on mylar and a LR-115 film is stuck below the mylar base. After irradiation, the LR-115 film is etched and cells observed at a phase contrast microscope connected to a video camera and an image analyzer. In this way, it is possible to measure the number of traversals through the cell nucleus or cytoplasm. Coordinates of each cell on the microscope bench are saved. After incubation for about one week, cells are fixed and stained and the colonies observed at the microscope. The fate of each irradiated cell is therefore correlated to the number of traversals. We have tested this method with two different rodent embryo fibroblast cell lines, C3H 10T1/2 and V79, exposed to 3.2 MeV accelerated α-particles (LET=124 keV/ μ m). The studied endpoint was cell killing. Preliminary biological results suggest that few α-particle tracks in V79 hamster cells are sufficient to reduce surviving fraction.

Nuclear track detectors in cellular radiation biology

Abstract Uses of track-etch detectors in cellular radiation biology are reviewed. These applications are described in three main areas. First, with regard to dosimetry, nuclear track detectors should be used to measure beam uniformity, divergence, and fluence. Second, living cells attach and grow on many different kinds of track detectors. This property can be used to measure cell size by using nuclear methods. Cell thickness can be determined by measuring the energy of charged particles impinging a cell monolayer, and behind it. Also, nuclear images have been obtained as replicas on mica; the replicas can then be visualized with scanning electron microscopy. Finally, solid state track detectors play an important role in studies concerning the effectiveness of single-ion traversals in the cell nucleus or cytoplasm. Plastic detectors have been used for this purpose in many space missions in order to assess the risk from space radiation exposure to the crews. Similar techniques have been used in accelerator ground-based experiments. Tracks and living cells can be visualized either simultaneously or at different times. For the latter case, images of the irradiated cells, surviving colonies, and etched detector, being taken at different times, must be superimposed. Solid state nuclear track detectors can also be used in combination with microbeams for these kinds of studies.