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.

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.

Contributions of various types of damage to inactivation of T4 bacteriophage by protons.

Mean doses for damage induced by 3.7-MeV protons in T4 phage were measured for the following effects: inactivation, killing, adsorption, DNA injection, capsid rupture with DNA release, and single- and double-strand DNA breaks. These effects have been related to phage survival in the same experiment because of the variability inherent in such measurements. The experiments were carried out in nutrient broth, phosphate buffer, and phosphate buffer plus histidine as suspension media. The following conclusions can be drawn: (i) DNA double-strand breakage is the dominant cause of inactivation in nutrient broth; (ii) scavengers protect the DNA inside the capsid to only a small degree; (iii) indirect actions affect functions associated with proteins; (iv) DNA release, as measured by capsid rupture, accounts for only a small percentage of the loss of viability; (v) essentially all DNA from adsorbed phage is injected even though a large proportion of the DNA contains double-strand breaks.

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.

Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells.

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.

Inactivation of C3H 10T1/2 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 alpha-particles in plateau-phase C3H 10T1/2 cells.

Chromosome aberrations induced by X-rays and alpha-particles (LET = 177 keV/microns) 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 alpha-irradiation. A linear-quadratic dose-response relationship was obtained for X-rays, while that for alpha-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 (approximately 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 at the mean lethal dose was about 1.4. This number was reduced to 1.0 when asymmetrical interchanges, which generally result in very small deletions, were subtracted from acentrics. It could be hypothesized that very small deletions could not impair cell survival. However, an alternative hypothesis related to the aneuploid state of C3H 10T1/2 cells can be formulated. Robertsonian translocations were also observed at the first mitosis. The dose-response curve of these translocations appears to be very similar to the dose-response curve for induction of sister chromatid exchanges (observed at the second mitosis) reported by other authors studying the same cell line. This similarity could indicate a general mechanism of action of radiation on the process of recombination of genetic material.

Radiation-induced chromosomal aberrations in mouse 10T1/2 cells: dependence on the cell-cycle stage at the time of irradiation.

Cell-cycle stage radiosensitivity for the induction of chromosome aberrations has been investigated in C3H 10T1/2 cells. Exponentially growing cells were irradiated with 3 Gy X-rays (80 kVp) or 0.6 Gy alpha-particles (LET = 101 keV/micron). The two doses produce the same survival level (37%) in the asynchronous population. Cells were harvested at four different times following irradiation and cell-cycle phase at the time of irradiation was assessed by using the differential replication staining technique. The frequency of chromosome aberrations produced in a given stage of the cell cycle was not constant as a function of the sampling time, but this could not be simply related to the existence of subphases exhibiting different radiosensitivity, because of cell-cycle perturbation introduced by radiation. X-radiation induced more exchanges than deletions, whereas a predominance of isochromatid deletions was observed after alpha-irradiation. This can be interpreted on the basis of the different patterns of energy deposition of densely- and sparsely-ionizing radiation. Both X- and alpha-rays produced a significant increase in the frequency of Robertsonian translocations when cells were exposed in G1 or S phase, but not in G2 phase.

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.

Inactivation of individual mammalian cells by single alpha-particles.

PURPOSE: To measure clonogenic death of Chinese hamster V79 cells following exposure to a defined number of 4.3 MeV alpha-particles (track-averaged LET = 105 keV/micron). MATERIALS AND METHODS: Cells were irradiated at the radiobiological facility installed at the TTT-3 Tandem accelerator in Naples by using a 'Biostack' approach, which allows the positions of incident tracks relative to cells to be carefully determined. Subcellular structure was identified by fluorescence microscopy, while tracks were visualized by LR-115 solid state nuclear track detectors. RESULTS: Particle hits in the cytoplasm did not significantly affect cell survival, yet survival probability decreased exponentially as a function of the number of nuclear traversals. Measured probability of surviving to exactly one 4.3 MeV alpha-particle traversal in the cell nucleus was 0.67 +/- 0.10. Inactivation cross-section was substantially higher than expected from conventional survival curves. However, folding of the data with Poisson statistics showed that survival level expected if a mean of one alpha-particle goes through a nucleus is higher than the measured value after exactly one particle traversal. CONCLUSIONS: V79 cells have about 67% probability to survive a single alpha-particle traversal in the cell nucleus. Single-particle survival curves are consistent with conventional dose-survival relationships, once Poisson distribution of traversals is taken into account.

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.

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.

The induction of Robertsonian translocations by X-rays and mitomycin C in mouse cells.

The induction of Robertsonian translocations in murine C3H 10T1/2 embryo fibroblasts after exposure to X-rays and mitomycin C has been investigated. Cells were irradiated in log-phase and harvested at different times for chromosome analysis. The stage of the cell cycle of individual cells at the time of irradiation could be determined by differential replication staining. A dose-dependent delay in the progression through S- and G2-phase has been observed. X-rays produced an increase in the frequency of Robertsonian translocations when cells were exposed in G1- or S-phase, but not in G2. The dose-response curve for the induction of Robertsonian translocations both in G1 and S peaked at 2 Gy and slightly declined at higher doses. For G2 cells, an increase compared to the control level was observed only after 1 Gy. Mitomycin C induced chromosomal aberrations and Robertsonian translocations in 10T1/2 cells, but no significant interaction between ionizing radiation and the alkylating agent was observed for these two endpoints. However, the combined exposure caused satellite associations of chromosomes. Both the number of satellite associations/metaphase (five times the frequency observed after mitomycin C alone) and the number of chromosomes/satellite (up to 10 chromosomes were observed in satellite associations) were greatly enhanced compared to X-rays and mitomycin C alone.

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.

Trace elements and chronic liver diseases.

The relationships between chronic liver diseases and trace element (TE) contents are debated. Particularly, no defined data are available about the TE levels in viral liver disease patients with or without malnutrition. In this study we evaluated blood and plasma levels of various trace elements in patients with HCV-related chronic liver disease, at different stages of liver damage (8 patients with chronic hepatitis and 32 with liver cirrhosis) with or without malnutrition. We also studied 10 healthy volunteers as control group. We found that cirrhotic subjects had a significant decrease of blood levels of Zn and Se, independently on the nutritional status, whereas plasma levels of Fe were significantly reduced only in malnourished cirrhotic patients. Our data indicate that liver impairment is the main cause of the blood decrease of Se and Zn levels in patients with non alcoholic liver disease, whereas the malnutrition affects Fe levels only.

Trace elements in hair of healthy children sampled by age and sex.

Hair trace element (TE) (Cr, Mn, Fe, Zn, Cu, Br, Rb, Sr, Pb) levels from 336 healthy subjects were measured by the Proton-Induced X-ray Emission (PIXE) method. The subjects were divided in three groups: 157 full-term neonates (75 male and 82 female), 86 children (41 male and 45 female) ages 6 to 11 yr, and 93 adolescents (51 male and 42 female) 11 to 16 yr old. Cu, Zn, Cr, and Br show an increase from birth to 8 yr and then decrease. Fe, Mn, and Sr strongly decrease up to 8 yr and then remain almost stable. Sex differences are present in Fe, Zn, and Br of children and in Cu, Cr, and Br of adolescents.

Interaction of trace elements in a longitudinal study of human milk from full-term and preterm mothers.

Concentrations of 8 trace elements (Fe, Cu, Zn, Se, Br, Pb, Rb, and Sr) at different lactation time were measured by the PIXE multi-elemental technique. Time dependence and interelement correlations were studied. A total of 200 milk samples from 32 lactating mothers were supplied from 2 to 120 d after delivery of 26 full-term and 6 preterm infants. All elements showed a lognormal frequency-distribution. The Fe, Cu, Zn, and Se contents in preterm milk were found to be somewhat different with respect to full-term milk. Cu, Zn, Se, Br, Pb, and Rb concentrations declined with lactation time, both in pre- and full-term samples. Sr and Fe contents did not show any change with time. Detailed analysis of data by partial correlation and multiple regression methods was performed. No substantial differences between preterm and full-term samples were found in the results of partial correlation analysis. Cu and Zn were found to be correlated with lactation time, whereas the measured time dependence for the other elements has to be attributed to the effect of the existing interelement correlation. All the measured elements appeared to be correlated with at least one other element. In particular, Se was inversely correlated with Zn and directly with Cu. The zinc and copper contents in milk can therefore depend on the variation in the mother selenium intake.