Temporally distinct response of irradiated normal human fibroblasts and their bystander cells to energetic heavy ions

Ionizing radiation-induced bystander effects have been documented for a multitude of endpoints such as mutations, chromosome aberrations and cell death, which arise in nonirradiated bystander cells having received signals from directly irradiated cells; however, energetic heavy ion-induced bystander response is incompletely characterized. To address this, we employed precise microbeams of carbon and neon ions for targeting only a very small fraction of cells in confluent fibroblast cultures. Conventional broadfield irradiation was conducted in parallel to see the effects in irradiated cells. Exposure of 0.00026% of cells led to nearly 10% reductions in the clonogenic survival and twofold rises in the apoptotic incidence regardless of ion species. Whilst apoptotic frequency increased with time up to 72 h postirradiation in irradiated cells, its frequency escalated up to 24h postirradiation but declined at 48 h postirradiation in bystander cells, indicating that bystander cells exhibit transient commitment to apoptosis. Carbon- and neon-ion microbeam irradiation similarly caused almost twofold increments in the levels of serine 15-phosphorylated p53 proteins, irrespective of whether 0.00026, 0.0013 or 0.0066% of cells were targeted. Whereas the levels of phosphorylated p53 were elevated and remained unchanged at 2h and 6h postirradiation in irradiated cells, its levels rose at 6h postirradiation but not at 2h postirradiation in bystander cells, suggesting that bystander cells manifest delayed p53 phosphorylation. Collectively, our results indicate that heavy ions inactivate clonogenic potential of bystander cells, and that the time course of the response to heavy ions differs between irradiated and bystander cells. These induced bystander responses could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Effects of heavy ions and energetic protons on normal human fibroblasts

At the low particle fluences of radiation to which astronauts are exposed in space, "non-targeted" effects such as the bystander response may have increased significance. The radiation-induced bystander effect is the occurrence of biological responses in unirradiated cells near to or sharing medium with cells traversed by radiation. The objectives of this study were to establish the responses of AG01522 diploid human fibroblasts after exposure to several heavy ions and energetic protons, as compared to X-rays, and to obtain initial information on the bystander effect in terms of cell clonogenic survival after Fe ion irradiation. Using a clonogenic survival assay, relative biological effectiveness (RBE) values at 10% survival were 2.5, 2.3, 1.0 and 1.2 for 1 GeV/amu Fe, 1 GeV/amu Ti, 290 MeV/amu C and 1 GeV/amu protons, respectively, compared to 250 kVp X-rays. For induction of micronuclei (MN), compared to the low LET protons, Fe and Ti are very effective inducers of damage, although C ions are similar to protons. Using a transwell insert system in which irradiated and unirradiated bystander cells share medium but are not touching each other, it was found that clonogenic survival in unirradiated bystander cells was decreased when irradiated cells were exposed to Fe ions or X-rays. The magnitude of the decrease in bystander survival was similar with both radiation types, reaching a plateau of about 80% survival at doses of about 0.5 Gy or larger.     

Activation of thick targets by energetic heavy ions and the resultant radiation levels

This study completes data collected for thick targets exposed to carbon and oxygen ions accelerated at 86 MeV/u. The radioactivity induced in carbon and tungsten targets bombarded by argon projectiles at 95 MeV/u has been studied in order to assess the relative contributions of the incoming heavy ion and the mass number of the bombarded nuclei to the consequent radiation hazards related to the production of radioactive ion beams. Induced radioactivity measurements are only rarely made under controlled irradiation conditions, in order to derive from the measured activites the dose rates after beam bombardment and a prediction of radiation protection constraints.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Induction of micronuclei in human fibroblasts across the Bragg curve of energetic heavy ions

The space environment consists of a varying field of radiation particles including high-energy ions, with spacecraft shielding material providing the major protection to astronauts from harmful exposure. Unlike low-LEpsilonTau gamma or X rays, the presence of shielding does not always reduce the radiation risks for energetic charged-particle exposure. The dose delivered by the charged particle increases sharply as the particle approaches the end of its range, a position known as the Bragg peak. However, the Bragg curve does not necessarily represent the biological damage along the particle path since biological effects are influenced by the track structures of both primary and secondary particles. Therefore, the "biological Bragg curve" is dependent on the energy and the type of the primary particle and may vary for different biological end points. Here we report measurements of the biological response across the Bragg curve in human fibroblasts exposed to energetic silicon and iron ions in vitro at two different energies, 300 MeV/nucleon and 1 GeV/nucleon. A quantitative biological response curve generated for micronuclei per binucleated cell across the Bragg curve did not reveal an increased yield of micronuclei at the location of the Bragg peak. However, the ratio of mono- to binucleated cells, which indicates inhibition of cell progression, increased at the Bragg peak location. These results confirm the hypothesis that severely damaged cells at the Bragg peak are more likely to go through reproductive death and not be evaluated for micronuclei.     

Treatment planning with heavy ions

The use of heavy charged particles in radiotherapy potentially represents an advance towards better local tumour control and a decrease in morbidity related to radiation injury of healthy tissues surrounding the target volume. This assertion only holds, however, if treatment planning systems give a real representation of the three-dimensional dose distribution, including physical and biological aspects, especially for heavier ions. The influence of linear energy transfer on the biological effects, its variations related to depth, particle, target tissue, position in the Bragg peak, etc. make the possible models for treatment planning extremely complex. A brief review of the problems to be addressed and some solutions is presented from the radiation oncologistàs point of view.     

Mutagenic action of heavy ions on Escherichia coli cells

Induction of direct mutations in the lactose operon of E. coli cells by gamma-radiation and accelerated heavy ions with different LET was studied. The experiments were performed with the wild-type PolA and LexA strains. A quadratic dependence of the mutation rate on the dose of different radiations for the wild-type strain and the PolA mutant was observed. However, different types of radiation showed different relative genetic effectivenesses (RGE). The dependence of RGE on LET for the wild-type and PolA strain has a maximum. A LexA strain showed much reduced mutation rates and a linear dose response. The RGE decreased with increasing LET of ionizing radiation.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Induction of proline prototrophs in CHO-K1 cells by heavy ions

Using an established mammalian cell line, Chinese hamster ovary cells (CHO-K1), we have observed the induction of prototrophs by various heavy ions. This cell line requires proline for normal growth in medium with low serum concentration. X-rays, three types of heavy particles (600 MeV/u iron, 670 MeV/u neon, and 320 MeV/u silicon ions), ethylmethane sulphonate and 5-azacytidine were used to induce revertants which were proline independent. Log-phase cells treated with 5-azacytidine showed a very high reversion frequency. The induction frequency per viable cell appears to be dose dependent for these four types of radiation, and the dose-response curves are approximately linear. Our results also indicate that the effectiveness of high-LET particles in inducing proline prototrophs is much greater than that of low-LET radiation. The RBE value for the induction of prototrophs was calculated for neon, silicon, and iron particles and found to be about 1.3, 1.7 and 4.5, respectively. At equal survival level, the reversion frequency for X-rays and EMS was about the same.     

Track structure in DNA irradiated with heavy ions

The spatial properties of trapped radicals produced in heavy-ion-irradiated solid DNA at 77 K have been probed using pulsed electron paramagnetic double resonance (PELDOR or DEER) techniques. Salmon testes DNA hydrated to 12 water molecules per nucleotide was irradiated with 40Ar ions of energy 100 MeV/nucleon and LET ranging from 300 to 400 keV/microm. Irradiated samples were maintained at cryogenic temperature at all times. PELDOR measurements were made using a refocused echo detection sequence that allows dipolar interaction between trapped radicals to be observed. The EPR spectrum is attributed to electron loss/gain DNA base radicals and neutral carbon-centered radicals that likely arise from sugar damage. We find a radical concentration of 13.5 x 10(18) cm(-3) in the tracks and a track radius of 6.79 nm. The cross section of these tracks is 144 nm2, yielding a lineal radical density of 2.6 radicals/nm. Based on the yields determined previously for particles having calculated LET values of 300-400 keV/microm and our measured lineal density, we obtain an LET of 270 keV/microm, which is in good agreement with the calculated range of values. These measurements of radical density and spatial extent provide the first direct experimental determination of track characteristics in irradiated DNA.     

The mutagenic effect of accelerated heavy ions on E. coli cells

In studying E. coli mutation rate as a function of dose of different types of ionizing radiation it was found that mutagenic efficiency of helium ions (LET-22, 54 and 72 keV/microns) was higher than that of gamma-rays. As LET increased the mutagenic efficiency decreased. The mutation rate for all types of radiation under study was both a power function and a linear-quadratic function of dose.     

Electron paramagnetic resonance investigation of sucrose irradiated with heavy ions

We investigated the potential use of sucrose to estimate linear energy transfer (LET) for heavy-ion irradiation. We also made a quantitative comparison between heavy-ion and gamma irradiation in terms of spin concentration. Heavy-ion irradiation of sucrose produces stable free radicals. Based on the electron paramagnetic resonance (EPR) spectra obtained, the stable sucrose radicals are the same among helium ions, carbon ions and gamma rays. The EPR spectrum was approximately 70 G wide and was composed of several hyperfine structures. The total spin concentration obtained after the heavy-ion irradiation increased linearly as the absorbed dose increased and decreased logarithmically as LET increased. Production of the spin concentration of helium ions was two times more dependent on LET than that for carbon-ion irradiation. The empirical relationships obtained imply that LET at a certain dose can be determined by the spin concentration. Furthermore, the results of gamma irradiation of deuterated sucrose suggest that one of the persistent radicals is a carbon-centered radical.     

Antibody-based sensors for heavy metal ions

Competitive immunoassays for Cd(II), Co(II), Pb(II) and U(VI) were developed using identical reagents in two different assay formats, a competitive microwell format and an immunosensor format with the KinExA™ 3000. Four different monoclonal antibodies specific for complexes of EDTA–Cd(II), DTPA–Co(II), 2,9-dicarboxyl-1,10-phenanthroline–U(VI), or cyclohexyl–DTPA–Pb(II) were incubated with the appropriate soluble metal–chelate complex. In the microwell assay format, the immobilized version of the metal–chelate complex was present simultaneously in the assay mixture. In the KinExA format, the antibody was allowed to pre-equilibrate with the soluble metal-chelate complex, then the incubation mixture was rapidly passed through a microcolumn containing the immobilized metal-chelate complex. In all four assays, the KinExA format yielded an assay with 10–1000-fold greater sensitivity. The enhanced sensitivity of the KinExA format is most likely due to the differences in the affinity of the monoclonal antibodies for the soluble versus the immobilized metal–chelate complex. The KinExA 3000 instrument and the Cd(II)-specific antibody were used to construct a prototype assay that could correctly assess the concentration of cadmium spiked into a groundwater sample. Mean analytical recovery of added Cd(II) was 114.25±11.37%. The precision of the assay was satisfactory; coefficients of variation were 0.81–7.77% and 3.62–14.16% for within run and between run precision, respectively.     

Neoplastic cell transformation by heavy ions

We have studied the induction of morphological transformation by heavy ions. Golden hamster embryo cells were irradiated with 95 MeV 14N ions (530 keV/microns), 22 MeV 4He ions (36 keV/microns), and 22 MeV 4He ions with a 100-microns Al absorber (77 keV/microns) which were generated by a cyclotron at the Institute of Physical and Chemical Research in Japan. Colonies were considered to contain neoplastically transformed cells when the cells were densely stacked and made a crisscross pattern. It was shown that the induction of transformation was much more effective with 14N and 4He ions than with gamma or X rays. The relative biological effectiveness (RBE) relative to 60Co gamma rays was 3.3 for 14N ions, 2.4 for 4He ions, and 3.3 for 4He ions with a 100-microns Al absorber. The relationship between RBE and linear energy transfer was qualitatively similar for both cell death and transformation.     

Homologous recombination in Arabidopsis seeds along the track of energetic carbon ions

Nanomaterials are already used today and offer even greater use and benefits in the future. The progress of nanotechnology must be accompanied by investigations of their potential harmful effects. For airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. While standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. We have investigated two genotoxic endpoints - alkaline Comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow - in a combined study 72 h after a single instillation of 18 μg gold nanoparticles (NP) into the trachea of male adult Wistar rats. The administration of three test materials differing only in their primary particle size (2, 20 and 200 nm) did not lead to relevant DNA damage in the mentioned tests. The measurement of clinical pathology parameters in bronchoalveolar lavage fluid (BALF) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. Minor histopathology findings occurred in the lung of the animals exposed to 20 nm and 200 nm sized nanomaterials. In conclusion, under the conditions of this study the different sized gold NP tested were non-genotoxic and showed no systemic and local adverse effects at the given dose.     

Chromatid-type aberrations following irradiation in G0 lymphocytes with heavy ions

We describe a low level of chromatid-type aberrations which included the relatively rare isochromatid/chromatid triradial in peripheral blood lymphocytes that were irradiated, ostensibly in G0, with accelerated heavy (12)C ions. These were produced only at the energies of 69 MeV/n (34.6 keV/microm), almost absent at the energy of either 58.6 MeV/n (46.07 keV/ microm) or 19.3 MeV/n (97 keV/microm), nor were they found after low-LET X-rays. Mechanisms potentially responsible for their formation are discussed.