Induction of DNA double-strand breaks in mammalian cells and yeast.

Induction of DNA double-strand breaks (dsb) and their distribution are dependent on the energy deposition pattern within the cell nucleus (physical structure) and the ultrastructure of the chromosomes and its variation by the cell cycle and gene activities (biological structure). For electron radiation very similar RBE-values are observed for mammalian and yeast cells (AlK, 1.5 keV, 15 keV/micrometer: 2.6 in mammalian cells and 2.2 in yeast; CK 0.278 keV, 23 keV/micrometer: approx. 2.5 in mammalian cells and 3.8 in yeast). In contrast, the RBE-values for the induction of dsb of 4He2+ and light ions in the LET range from about 100 keV/micrometer up to 1000 keV/micrometer are significantly higher for yeast cells compared to mammalian cells. For example, the RBE-value of alpha-particles (120 keV/micrometer) is about 1.2 for mammalian cells whereas for yeast the RBE-value is about 2.5. The yeast chromatin has less condensed fibres compared with mammalian cells. Since a single CK photoelectron can induce only one dsb, the different condensation of the mammalian and yeast chromatin has no influence. However, particles may induce more than one dsb when traversing a chromatin fibre. The probability for the induction of closely neighboured dsb is higher the more condensed the chromatin fibres are. Since small DNA fragments (50 bp up to several kbp) are lost by standard methods of lysis, the underestimation of dsb yields increases with fibre condensation, which is in accordance with the observes dsb yields in mammalian cells and yeast. In order to obtain relevant yields of dsb (and corresponding RBE-values) the measurement of all DNA fragments down to about 50 bp are needed.     

DNA double strand break production and rejoining in V79 cells irradiated with light ions.

Low energy protons and other densely ionizing light ions are known to have RBE>1 for cellular end points relevant for stochastic and deterministic effects. The occurrence of a close relationship between them and induction of DNA dsb is still a matter of debate. We studied the production of DNA dsb in V79 cells irradiated with low energy protons having LET values ranging from 11 to 31 keV/micrometer, i.e. in the energy range characteristic of the Bragg peak, using the sedimentation technique. We found that the initial yield of dsb is quite insensitive to proton LET and not significantly higher than that observed with X-rays, in agreement with recent data on V79 cells irradiated with alpha particles of various LET up to 120 keV/micrometer. By contrast, RBE for cell inactivation and for mutation induction rises with the proton LET. In experiments aimed at evaluating the rejoining of dsb after proton irradiation we found that the amount of dsb left unrepaired after 120 min incubation is higher for protons than for sparsely ionizing radiation. These results indicate that dsb are not homogeneous with respect to repair and give support to the hypothesis that increasing LET leads to an increase in the complexity of DNA lesions with a consequent decrease in their repairability.     

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.     

The role of DNA repair on cell killing by charged particles.

It can be noted that it is not simple double strand breaks (dsb) but the non-reparable breaks that are associated with high biological effectiveness in the cell killing effect for high LET radiation. Here, we have examined the effectiveness of fast neutrons and low (initial energy = 12 MeV/u) or high (135 MeV/u) energy charged particles on cell death in 19 mammalian cell lines including radiosensitive mutants. Some of the radiosensitive lines were deficient in DNA dsb repair such as LX830, M10, V3, and L5178Y-S cells and showed lower values of relative biological effectiveness (RBE) for fast neutrons if compared with their parent cell lines. The other lines of human ataxia-telangiectasia fibroblasts, irs 1, irs 2, irs 3 and irs1SF cells, which were also radiosensitive but known as proficient in dsb repair, showed moderated RBEs. Dsb repair deficient mutants showed low RBE values for heavy ions. These experimental findings suggest that the DNA repair system does not play a major role against the attack of high linear energy transfer (LET) radiations. Therefore, we hypothesize that a main cause of cell death induced by high LET radiations is due to non-reparable dsb, which are produced at a higher rate compared to low LET radiations.     

Effect of the space environment on the induction of DNA-repair related proteins and recovery from radiation damage.

Recovery of bacterial cells from radiation damage and the effects of microgravity were examined in an STS-79 Shuttle/Mir Mission-4 experiment using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space-Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity. No difference was observed for the survival of radiosensitive mutant rec30 cells whether incubated in space or on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, the product of a newly found gene related to the DNA repair mechanism of D. radiodurans, was enhanced under microgravity compared with ground controls.     

Repair of DNA double-strand breaks and cell killing by charged particles.

It has been suggested that it is not simple double-strand breaks (dsb) but the non-reparable breaks which correlate well with the high biological effectiveness of high LET radiations for cell killing (Kelland et al., 1988; Radford, 1986). We have compared the effects of charged particles on cell death in 3 pairs of cell lines which are normal or defective in the repair of DNA dsbs. For the cell lines SL3-147, M10, and SX10 which are deficient in DNA dsb repair, RBE values were close to unity for cell killing induced by charged particles with linear energy transfer (LET) up to 200 keV/micrometer and were even smaller than unity for the LET region greater than 300 keV/micrometer. The inactivation cross section (ICS) increased with LET for all 3 pairs. The ICS of dsb repair deficient mutants was always larger than that of their parents for all the LET ranges, but with increasing LET the difference in ICS between the mutant and its parent became smaller. Since a small difference in ICS remained at LET of about 300 keV/micrometer, dsb repair may still take place at this high LET, even if its role is apparently small. These results suggest that the DNA repair system does not play a major role in protection against the attack of high LET radiations and that a main muse of cell death is non-reparable dsb which are produced at a higher yield compared with low LET radiations. No correlation was observed between DNA content or nuclear area and ICS.     

Induction of high grade astrocytoma (HGA) by protons: molecular mechanisms and RBE considerations.

Protons of a specific energy, 55 MeV, have been found to induce primary high grade astrocytomas (HGA) in the Rhesus monkey (Macaca mulatta). Brain tumors of this type were not induced by protons of other energies (32-2,300 MeV). Induction of HGA has been identified in human patients who have had radiation therapy to the head. We believe that the induction of HGA in the monkey is a consequence of dose distribution, not some unique "toxic" property of protons. Comparison of the human experience with the monkey data indicates the RBE for induction of brain tumors to be about one. It is unlikely that protons cause an unusual change in oncogenic expression, as compared to conventional electromagnetic radiation.     

G2-chromosome aberrations induced by high-LET radiations.

We report measurement of initial G2-chromatid breaks in normal human fibroblasts exposed to various types of high-LET particles. Exponentially growing AG 1522 cells were exposed to gamma rays or heavy ions. Chromosomes were prematurely condensed by calyculin A. Chromatid-type breaks and isochromatid-type breaks were scored separately. The dose response curves for the induction of total chromatid breaks (chromatid-type + isochromatid-type) and chromatid-type breaks were linear for each type of radiation. However, dose response curves for the induction of isochromatid-type breaks were linear for high-LET radiations and linear-quadratic for gamma rays. Relative biological effectiveness (RBE), calculated from total breaks, showed a LET dependent tendency with a peak at 55 keV/micrometer silicon (2.7) or 80 keV/micrometer carbon (2.7) and then decreased with LET (1.5 at 440 keV/micrometer). RBE for chromatid-type break peaked at 55 keV/micrometer (2.4) then decreased rapidly with LET. The RBE of 440 keV/micrometer iron particles was 0.7. The RBE calculated from induction of isochromatid-type breaks was much higher for high-LET radiations. It is concluded that the increased production of isochromatid-type breaks, induced by the densely ionizing track structure, is a signature of high-LET radiation exposure.     

Effect of secondary radiation produced by 70 GeV protons on DNA of mammalian cells.

It is shown that the RBE of the 70 GeV proton secondary radiation for the induction of single-strand break is 1.6-7.6 in Chinese hamster fibroblasts and 1.04-3.8 in limphoid cells and for the lethality of Chinese hamster cells 1.14-1.7. The RBE value increases with decreasing dose of the secondary radiation. On post-irradiation incubation of mammalian cells at 37 degrees C, single-strand breaks induced by the secondary radiation are repaired with the sane time course as those induced by gamma-rays. In our earlier works we have made an attempt to estimate the biological efficiency of radiation generated by the 70 GeV protons on bacteria, phage T4 and Vicia faba beans. The obtained values of the relative biological efficiency (RBE) of this radiation varied between 1.4 and 5.5, depending on the object, criterion of estimation, times of registration and other experimental conditions. The aim of the present work is to estimate the biological efficiency of synchrotron radiation by its effect on mammalian cells.     

Initial approach to comparative studies on the evolutionary potentials of space radiation effects in a plant system.

The role of cosmic ionizing radiation, including heavy ions (HZE-particles) in the induction of mutations at the molecule-, chromosome-, genome- and cell-level is discussed on the basis of different DNA organization in a pro- and eukaryotically compartmented plant system (Arabidopsis thaliana (L.) Heynh.). Data recently obtained on the biological effects of ionizing radiation make it timely to discuss comparatively the evolutionary potentials of space radiation effects in the pro- and eukaryotic genomes (plasmon, plastidom, chondriom, and nucleom) during long duration exposure on space flights.     

High-LET radiation carcinogenesis.

Recent results for neutron radiation-induced tumors are presented to illustrate the complexities of the dose-response curves for high-LET radiation. It is suggested that in order to derive an appropriate model for dose-response curves for the induction of tumors by high-LET radiation it is necessary to take into account dose distribution, cell killing and the susceptibility of the tissue under study. Preliminary results for the induction of Harderian gland tumors in mice exposed to various heavy ion beams are presented. The results suggest that the effectiveness of the heavy ion beams increases with increasing LET. The slopes of the dose-response curves for the different high-LET radiations decrease between 20 and 40 rads and therefore comparisons of the relative effectiveness should be made from data obtained at doses below about 20-30 rads.     

Heavy ion induced DNA double strand breaks in cells of E. coli.

Vegetative cells of E. coli differing in their radiosensitivity have been used in heavy ion irradiation experiment. Besides inactivation measurements also the induction of DNA double strand breaks (DSB) have been measured using the method of pulse-field gel electrophoresis. This method allows to separate linear DNA with length up to 8 Mio base pairs. After irradiation with heavy ions we find a higher amount of low molecular weight fragments when compared to sparsely ionizing radiation. This agrees with the idea that heavy ions as a structured radiation have a high probability to induce more than one strand break in a DNA molecule if the particle hits the DNA. The amount of intact DNA remaining in the agarose plugs decreases exponentially for increasing radiation doses or particle fluences. From these curves cross sections for the induction of DSB after heavy ion irradiation have been determined. These results will be discussed in comparison to the results for cell survival.     

Radiation effects in space.

The radiation protection guidelines of the National Aeronautics and Space Administration (NASA) are under review by Scientific Committee 75 of the National Council Protection and Measurements. The re-evaluation of the current guidelines is necessary, first, because of the increase in information about radiation risks since 1970 when the original recommendations were made and second, the population at risk has changed. For example, women have joined the ranks of the astronauts. Two types of radiation, protons and heavy ions, are of particular concern in space. Unfortunately, there is less information about the effects on tissues and cancer by these radiations than by other radiations. The choice of Quality Factors (Q) for obtaining dose equivalents for these radiations, is an important aspect of the risk estimate for space travel. There are not sufficient data for the induction of late effects by either protons or by heavy ions. The current information suggests a RBE for the relative protons of about 1, whereas, a RBE of 20 for tumor induction by heavy ions, such as iron-56, appears appropriate. The recommendations for the dose equivalent career limits for skin and the lens of the eye have been reduced but the 30-day and annual limits have been raised.     

Development of human epithelial cell systems for radiation risk assessment.

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-LET radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic transformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.     

The influence of radiation quality on the formation of DNA breaks.

We have aimed to present a comprehensive review of our understanding to date of the formation of DNA strand breaks induced by high LET radiation. We have discussed data obtained from DNA in solution as well as from the formation and "repair" of strand breaks in cell DNA. There is good agreement, qualitatively, between these two systems. Results were evaluated for two parameters: (1) effectivity per particle, the cross section (sigma) in micrometers 2/particle; and (2) the strand break induction frequency as number of breaks per Gy per unit DNA (bp or dalton). A series of biological effects curves (one for each Z-number) is obtained in effectivity versus LET plots. The relationships between induction frequencies of single-strand breaks, or double-strand breaks, or the residual "irrepairable" breaks and LET-values have been evaluated and discussed for a wide spectrum of heavy ions, both for DNA in solution and for DNA in the cell. For radiation induced total breaks in cell DNA, the RBE is less than one, while the RBE for the induction of DSBs can be greater than one in the 100-200 keV/micrometers range. The level of irrepairable strand breaks is highest in this same LET range and may reach 25 percent of the initial break yield. The data presented cover results obtained for helium to uranium particles, covering a particle incident energy range of about 2 to 900 MeV/u with a corresponding LET range of near 16 to 16000 keV/micrometers.     

The role of hydration and radiation quality in the induction of DNA damage--chemical aspects.

The mutagenic and lethal effects of ionising radiation are thought to result from chemical modifications induced within DNA. This DNA damage is significantly influenced by the chemical environment and the radiation quality (LET). Water closely associated with the DNA and its immediate environment is involved in the early chemical pathways which lead to the induction of DNA damage and is reflected in the cellular radiosensitivity. For instance, hydration of DNA influences hole migration leading to its localisation at guanine. Changes in the radiation quality are discussed in terms of the complexity of the radical clusters produced. It is inferred that at higher LET, the influence of the chemical environment (O2 etc) decreases with respect to DNA damage and cellular radiosensitivity. It is therefore important to include these effects of environment of the DNA upon the early chemical pathways in models of radiation action.     

In vivo and in vitro measurements of complex-type chromosomal exchanges induced by heavy ions.

Heavy ions are more efficient in producing complex-type chromosome exchanges than sparsely ionizing radiation, and this can potentially be used as a biomarker of radiation quality. We measured the induction of complex-type chromosomal aberrations in human peripheral blood lymphocytes exposed in vitro to accelerated H-, He-, C-, Ar-, Fe- and Au-ions in the LET range of approximately 0.4-1400 keV/micrometers. Chromosomes were analyzed either at the first post-irradiation mitosis, or in interphase, following premature condensation by phosphatase inhibitors. Selected chromosomes were then visualized after FISH-painting. The dose-response curve for the induction of complex-type exchanges by heavy ions was linear in the dose-range 0.2-1.5 Gy, while gamma-rays did not produce a significant increase in the yield of complex rearrangements in this dose range. The yield of complex aberrations after 1 Gy of heavy ions increased up to an LET around 100 keV/micrometers, and then declined at higher LET values. When mitotic cells were analyzed, the frequency of complex rearrangements after 1 Gy was about 10 times higher for Ar- or Fe- ions (the most effective ions, with LET around 100 keV/micrometers) than for 250 MeV protons, and values were about 35 times higher in prematurely condensed chromosomes. These results suggest that complex rearrangements may be detected in astronauts' blood lymphocytes after long-term space flight, because crews are exposed to HZE particles from galactic cosmic radiation. However, in a cytogenetic study of ten astronauts after long-term missions on the Mir or International Space Station, we found a very low frequency of complex rearrangements, and a significant post-flight increase was detected in only one out of the ten crewmembers. It appears that the use of complex-type exchanges as biomarker of radiation quality in vivo after low-dose chronic exposure in mixed radiation fields is hampered by statistical uncertainties.     

A biophysical model for estimating the frequency of radiation-induced mutations resulting from chromosomal translocations.

Gene mutations can be induced by radiation as a result of chromosomal translocations. A biophysical model is developed to estimate the frequency of this type of mutation induced by low-LET radiation. Mutations resulting from translocations are assumed to be formed by misrejoining of two DNA double strand breaks (DSB), one within the gene and one on a different chromosome. The chromosome containing the gene is assumed to occupy a spherical territory and does not overlap spatially with other chromosomes. Misrejoining between two DSB can occur only if the two DSB are closer than an interaction distance at the time of their induction. Applying the model to mutations of the hprt gene induced in G0 human lymphocyte cells by low-LET radiation, it is calculated that mutations resulting from translocations account for about 14% of the total mutations. The value of the interaction distance is determined to be 0.6 micrometers by comparing with the observed frequency of translocations in the X-chromosome.     

A new perspective of carcinogenesis from protracted high-LET radiation arises from the two-stage clonal expansion model.

When applied to the Colorado Plateau miner population, the two-stage clonal expansion (TSCE) model of radiation carcinogenesis predicts that radiation-induced promotion dominates radiation-induced initiation. Thus, according to the model, at least for alpha-particle radiation from inhaled radon daughters, lung cancer induction over long periods of protracted irradiation appears to be dominated by radiation-induced modification of the proliferation kinetics of already-initiated cells rather than by direct radiation-induced initiation (i.e., mutation) of normal cells. We explore the possible consequences of this result for radiation exposures to space travelers on long missions. Still unknown is the LET dependence of this effect. Speculations of the cause of this phenomenon include the suggestion that modification of cell kinetics is caused by a "bystander" effect, i.e., the traversal of normal cells by alpha particles, followed by the signaling of these cells to nearby initiated cells which then modify their proliferation kinetics.