Chromosome inter- and intrachanges detected by arm-specific DNA probes in the progeny of human lymphocytes exposed to energetic heavy ions.

We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or gamma rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to gamma rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

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 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.

Shielding of relativistic protons.

Protons are the most abundant element in the galactic cosmic radiation, and the energy spectrum peaks around 1 GeV. Shielding of relativistic protons is therefore a key problem in the radiation protection strategy of crewmembers involved in long-term missions in deep space. Hydrogen ions were accelerated up to 1 GeV at the NASA Space Radiation Laboratory, Brookhaven National Laboratory, New York. The proton beam was also shielded with thick (about 20 g/cm2) blocks of lucite (PMMA) or aluminium (Al). We found that the dose rate was increased 40-60% by the shielding and decreased as a function of the distance along the axis. Simulations using the General-Purpose Particle and Heavy-Ion Transport code System (PHITS) show that the dose increase is mostly caused by secondary protons emitted by the target. The modified radiation field after the shield has been characterized for its biological effectiveness by measuring chromosomal aberrations in human peripheral blood lymphocytes exposed just behind the shield block, or to the direct beam, in the dose range 0.5-3 Gy. Notwithstanding the increased dose per incident proton, the fraction of aberrant cells at the same dose in the sample position was not significantly modified by the shield. The PHITS code simulations show that, albeit secondary protons are slower than incident nuclei, the LET spectrum is still contained in the low-LET range (<10 keV/microm), which explains the approximately unitary value measured for the relative biological effectiveness.

Cytogenetic effects of high-energy iron ions: dependence on shielding thickness and material.

We report results for chromosomal aberrations in human peripheral blood lymphocytes after they were exposed to high-energy iron ions with or without shielding at the HIMAC, AGS and NSRL accelerators. Isolated lymphocytes were exposed to iron ions with energies between 200 and 5000 MeV/nucleon in the 0.1-1-Gy dose range. Shielding materials consisted of polyethylene, lucite (PMMA), carbon, aluminum and lead, with mass thickness ranging from 2 to 30 g/cm2. After exposure, lymphocytes were stimulated to grow in vitro, and chromosomes were prematurely condensed using a phosphatase inhibitor (calyculin A). Aberrations were scored using FISH painting. The yield of total interchromosomal exchanges (including dicentrics, translocations and complex rearrangements) increased linearly with dose or fluence in the range studied. Shielding decreased the effectiveness per unit dose of iron ions. The highest RBE value was measured with the 1 GeV/nucleon iron-ion beam at NSRL. However, the RBE for the induction of aberrations apparently is not well correlated with the mean LET. When shielding thickness was increased, the frequency of aberrations per particle incident on the shield increased for the 500 MeV/nucleon ions and decreased for the 1 GeV/nucleon ions. Maximum variation at equal mass thickness was obtained with light materials (polyethylene, carbon or PMMA). Variations in the yield of chromosomal aberrations per iron particle incident on the shield follow variations in the dose per incident particle behind the shield but can be modified by the different RBE of the mixed radiation field produced by nuclear fragmentation. The results suggest that shielding design models should be benchmarked using both physics and biological data.

Modelled microgravity does not modify the yield of chromosome aberrations induced by high-energy protons in human lymphocytes.

The aim was to evaluate the effect of modelled microgravity on radiation-induced chromosome aberrations (CAs). G0 peripheral blood lymphocytes were exposed to 60 MeV protons or 250 kVp X-rays in the dose range 0-6 Gy, and allowed to repair DNA damage for 24 h under either normal gravity or microgravity modelled by the NASA-designed rotating-wall bioreactor. Cells were then stimulated to proliferate by phytohaemagglutinin (PHA) under normal gravity conditions and prematurely condensed chromosomes were harvested after 48 h. CAs were scored in chromosomes 1 and 2 by fluorescence in-situ hybridization. Proliferation gravisensitivity was examined by cell growth curves and by morphological evaluation of mitogen-induced activation. Cell replication rounds were monitored by bromodeoxyuridine labelling. Modelled microgravity markedly reduced PHA-mediated lymphocyte blastogenesis and cell growth. However, no significant differences between normal gravity and modelled microgravity were found in the dose-response curves for the induction of aberrant cells or total interchromosomal exchange frequency. Rotating-wall bioreactor-based microgravity reproduced space-related alterations of mitogen stimulation in human lymphocytes but did not affect the yield of CAs induced by low-linear energy transfer radiation.

Fragmentation studies of relativistic iron ions using plastic nuclear track detectors.

We measured fluence and fragmentation of high-energy (1 or 5 A GeV) 56Fe ions accelerated at the Alternating Gradient Synchrotron or at the NASA Space Radiation Laboratory (Brookhaven National Laboratory, NY, USA) using solid-state CR-39 nuclear track detectors. Different targets (polyethylene, PMMA, C, Al, Pb) were used to produce a large spectrum of charged fragments. CR-39 plastics were exposed both in front and behind the shielding block (thickness ranging from 5 to 30 g/cm2) at a normal incidence and low fluence. The radiation dose deposited by surviving Fe ions and charged fragments was measured behind the shield using an ionization chamber. The distribution of the measured track size was exploited to distinguish the primary 56Fe ions tracks from the lighter fragments. Measurements of projectile's fluence in front of the shield were used to determine the dose per incident particle behind the block. Simultaneous measurements of primary 56Fe ion tracks in front and behind the shield were used to evaluate the fraction of surviving iron projectiles and the total charge-changing fragmentation cross-section. These physical measurements will be used to characterize the beam used in parallel biological experiments.

Chromosomal aberrations induced by high-energy iron ions with shielding.

Biophysical models are commonly used to evaluate the effectiveness of shielding in reducing the biological damage caused by cosmic radiation in space flights. To improve and validate these codes biophysical experiments are needed. We have measured the induction of chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to 500 MeV/n iron ion beams (dose range 0.1-1 Gy) after traversing shields of different material (lucite, aluminium, or lead) and thickness (0-11.3 g/cm2). For comparison, cells were exposed to 200 MeV/n iron ions and to X-rays. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid cell-cycle selection produced by the exposure to high-LET heavy-ion beams. Aberrations were scored in chromosomes 1, 2, and 4 following fluorescence in situ hybridization. The fraction of aberrant lymphocytes has been evaluated as a function of the dose at the sample position, and of the fluence of primary 56Fe ions hitting the shield. The influence of shield thickness on the action cross-section for the induction of exchange-type aberrations has been analyzed, and the dose average-LET measured as a function of the shield thickness. These preliminary results prove that the effectiveness of heavy ions is modified by shielding, and the biological damage is dependent upon shield thickness and material.

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.

On the radiosensitivity of man in space.

Astronauts' radiation exposure limits are based on experimental and epidemiological data obtained on Earth. It is assumed that radiation sensitivity remains the same in the extraterrestrial space. However, human radiosensitivity is dependent upon the response of the hematopoietic tissue to the radiation insult. It is well known that the immune system is affected by microgravity. We have developed a mathematical model of radiation-induced myelopoiesis which includes the effect of microgravity on bone marrow kinetics. It is assumed that cellular radiosensitivity is not modified by the space environment, but repopulation rates of stem and stromal cells are reduced as a function of time in weightlessness. A realistic model of the space radiation environment, including the HZE component, is used to simulate the radiation damage. A dedicated computer code was written and applied to solar particle events and to the mission to Mars. The results suggest that altered myelopoiesis and lymphopoiesis in microgravity might increase human radiosensitivity in space.

Relationship of blood trace elements to liver damage, nutritional status, and oxidative stress in chronic nonalcoholic liver disease.

Trace elements are involved in chronic liver diseases because these elements may have a direct hepatic toxicity or may be decreased as a consequence of the impaired liver function, particularly in patients with alcoholic cirrhosis and/or malnutrition. In this study, we determined plasma and erythrocytes trace elements in 50 inpatients with nonalcoholic chronic liver disease (11 with biopsy-proven chronic hepatitis, 39 with cirrhosis [16 in stage A according to Child-Pugh criteria, 23 Child B+C]), and in a control group of 10 healthy subjects by the proton induced x-ray emission method. The relationship between trace element concentration and the extent of liver damage, the nutritional status (by anthropometric evaluations), and various blood markers of oxidative stress--reduced glutathione, total lipoperoxides and malonyldialdehyde--was investigated. We found that cirrhotics had a significant decrease of Fe, Zn, Se, and GSH levels in the plasma and of GSH and Se in the erythrocytes with respect to the control and chronic hepatitis groups. GSH levels were related to the degree of liver damage; a significant direct correlation was observed among Se, Zn, and GSH plasma values and between GSH and Se in the erythrocytes. The trace element decrease was, on the contrary, independent of the degree of liver function impairment and only partially affected by the nutritional status. Data indicate that liver cirrhosis, even if not alcohol related, induces a decrease of Se and Zn and that, in these patients, an oxidative stress is present, as documented by the significant correlation between Se and GSH. The plasma Br level was higher in cirrhotics with respect to the control and chronic hepatitis groups.

Calibration curves for biological dosimetry by fluorescence in situ hybridisation.

Dose-response curves were measured for the induction of chromosomal aberrations in peripheral blood lymphocytes after acute exposure in vitro to 60Co gamma rays. Blood was obtained from four different healthy donors, and chromosomes were either observed at metaphase, following colcemid accumulation, or prematurely condensed by calyculin A. Cells were analysed in three different Italian laboratories. Chromosomes 1, 2, and 4 were painted, and simple-type interchanges between painted and non-painted chromosomes were scored in cells exposed in the dose range 0.1-3.0 Gy. The chemical-induced premature chromosome condensation method was also used combined with chromosome painting (chromosome 4 only) to determine calibration curves for high dose exposures (up to 20 Gy X rays). Calibration curves described in this paper will be used in our laboratories for biological dosimetry by fluorescence in situ hybridisation.

Cell inactivation by beryllium, boron and carbon ions at the low-energy irradiation facility of the Naples University.

The 3MV HVEC TTT-3 Tandem accelerator at the University of Naples, already used for radiobiological studies with protons and alpha particles, was set up for irradiation of biological samples with low energy carbon, boron, and beryllium beams. Radiobiological characterisation and study of these ion beams is essential in hadrontherapy (correction of hadrotherapy) to understand, for example, the possible biological effect of the target fragmentation products. Furthermore in space radiation biology we need to know the biological effects of heavy ions, a component of cosmic radiation that can contribute to the radiobiological risk when long sojourns in space are concerned. V79 Chinese hamster cells were irradiated with the different ions and the resulting cell inactivation data are reported.

A model of radiation-induced myelopoiesis in space.

Astronauts' radiation exposure limits are based on experimental and epidemiological data obtained on Earth. It is assumed that radiation sensitivity remains the same in the extraterrestrial space. However, human radiosensitivity is dependent upon the response of the hematopoietic tissue to the radiation insult. It is well known that the immune system is affected by microgravity. We have developed a mathematical model of radiation-induced myelopoiesis which includes the effect of microgravity on bone marrow kinetics. It is assumed that cellular radiosensitivity is not modified by the space environment, but repopulation rates of stem and stromal cells are reduced as a function of time in weightlessness. A realistic model of the space radiation environment, including the HZE component, is used to simulate the radiation damage. A dedicated computer code was written and applied to solar particle events and to the mission to Mars. The results suggest that altered myelopoiesis and lymphopoiesis in microgravity might increase human radiosensitivity in space.

Effect of space radiation on expression of apoptosis-related genes in endometrial cells: a preliminary study.

In this paper we present some preliminary results on alteration of gene expression caused by radiation on human endometrial cells. To this purpose, we have studied the modulation of the expression of the bcl-2 gene family in two cell lines following irradiations with low energy protons and gamma-rays from a 60Co. The two epithelial cell strains, namely AN3Ca and HEC1B cells, both obtained from human neoplastic endometrial tissues, grow in culture and continue to maintain some differentiated functions typical of the original tissue. Indeed, these cells, that can be considered as representative of different stages of cellular transformation of endometrium. Because their epithelial nature and rapid growth, the expression of genes related to the maintenance of the cellular homeostasis (correction of omeostasis), as the pro and anti-apoptotic ones, is expected to be susceptible to changes in environment, including radiation. The effects have been evaluated in terms of both cell survival and changes in the expression of pro- and anti apoptotic proteins. Even though the data reported above can not be considered complete and/or definitive, nevertheless, in whole, they confirm that these cells may constitute a suitable model system to study, at molecular level, the effects of cosmic radiation on endometrium. Further observation, ensuing from these preliminary data, is that endometrial cells present different sensitivity to radiation in regard to its 'quality' and 'dosage', in accord to the original stage of differentiation.

Inactivation of human normal and tumour cells irradiated with low energy protons.

PURPOSE: To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation. MATERIALS AND METHODS: Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed. RESULTS: Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively. CONCLUSIONS: RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

Long-term zinc and iron supplementation in children of short stature: effect of growth and on trace element content in tissues.

We evaluated the effect of one year of supplementation with iron plus zinc (12 mg/day of Fe+++ and 12.5 mg/day of Zn++), zinc alone (12.5 mg/day of Zn++) and placebo on growth and on the iron, zinc, copper and selenium tissue contents in 30 well-selected children of short stature (16 M and 14 F; 4-11 years old). Before and after supplementation, we measured the concentrations of iron, transferrin, ferritin, zinc and copper in serum, of zinc in erythrocytes and leukocytes, and of zinc, copper and selenium in hair, as well as glutathione peroxidase activity in erythrocytes. Before supplementation, ferritin and serum, erythrocyte and hair zinc contents were significantly lower than in age-matched controls, while the other measured indices were in the normal range. Iron plus zinc supplementation caused an improvement in growth rate in all subjects, i.e., the median Z-score increased from -2.22 +/- 0.45 to -0.64 +/- 0.55; (p < 0.01). In the zinc-supplemented group, only the subjects whose ferritin levels were higher than 20 ng/L before supplementation showed a similar improvement of growth rate. Iron plus zinc supplementation could be a reasonable treatment in short, prepubertal children affected by marginal zinc and iron deficiency.

Chromosome aberrations induced by light ions: Monte Carlo simulations based on a mechanistic model.

PURPOSE: To investigate the mechanisms underlying the induction of chromosome aberrations by ionizing radiation, focusing attention on DNA damage severity, interphase chromosome geometry and the distribution of DNA strand breaks. METHODS: An ab initio biophysical model of aberration induction in human lymphocytes specific for light ions was developed, based on the assumption that 'complex lesions' (clustered DNA breaks) produce aberrations, whereas less severe breaks are repaired. It was assumed that interphase chromosomes are spatially localized and that chromosome break free-ends rejoin pairwise randomly; the unrejoining of a certain fraction of free-ends was assumed to be possible, and small fragments were neglected in order to reproduce experimental conditions. The yield of different aberrations was calculated and compared with some data obtained using Giemsa or FISH techniques. RESULTS: Dose-response curves for dicentrics and centric rings (Giemsa) and for reciprocal, complex and incomplete exchanges (FISH) were simulated; the ratio between complex and reciprocal exchanges was also calculated as a function of particle type and LET. The results showed agreement with data from lymphocyte irradiation with light ions. CONCLUSIONS: The results suggest that clustered DNA breaks are a critical damage type for aberration induction and that interphase chromosome localization plays an important role. Moreover, the effect of a given particle type is related both to the number of induced complex lesions and to their spatial distribution.

Effects of melanin on high- and low- linear energy transfer (LET) radiation response of human epithelial cells.

The search for effective radioprotectors is of major concern in the medical, military, environmental, and space sciences. Conventional radioprotectors are generally effective only during a single irradiation and display their radioprotective properties only at high, toxic concentrations. In addition, they reduce somatic radiation effects but are poorly efficient in protecting from hereditary stochastic radiation effects. In this respect, the pigment melanin merits attention. Experiments referring to potential melanin effects on the ionising radiation response have been carried out with different biological systems, both in vivo and in vitro. In this paper, we present results on the response to high- and low-linear energy transfer (LET) radiation of a human mammary epithelial cell line, H184B5 F5-1 M/10, supplemented by melanin. The incorporation of auto-oxidative (L-dopa) melanin was linear for concentrations from 3 to 10 micrograms/ml in the growth medium. Concentrations of up to 250 micrograms/ml did not significantly impair the cells proliferative ability. No significant protective effect of melanin on the survival of cultured cells after exposure to alpha-particles (130 keV/micron) or x-rays was observed.

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.

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.