The role of repair in the survival of mammalian cells from heavy ion irradiation: approximation to the ideal case of target theory.

Theories of cellular radiation sensitivity that preclude a significant role for cellular repair processes in the final biological expression of cellular damage induced by ionizing radiation are unsound. Experiments are discussed here in which the cell-cycle dependency of the repair deficiency of the S/S variant, of the L5178Y murine leukemic lymphoblast was examined by treatment with the heavy ions, 20Ne, 28Si, 40Ar, 56Fe and 93Nb. Evidence from those studies, which will be described in detail elsewhere, provide support for the notion that as the linear energy transfer (LET infinity) of the incident radiation increases the ability of the S/S cell to repair radiation damage decreases until effectively it is eliminated around 500 keV/micrometer. In the region of the latter LET infinity value, the behavior of the S/S cell approximates the ideal case of target theory where post-irradiation metabolism (repair) does not influence cell survival. The expression of this phenomenon among different cell types and tissues will depend upon the actual repair systems involved and other considerations.     

Cell inactivation, repair and mutation induction in bacteria after heavy ion exposure: results from experiments at accelerators and in space.

To understand the mechanisms of accelerated heavy ions on biological matter, the responses of spores of B. subtilis to this structured high LET radiation was investigated applying two different approaches. 1) By the use of the Biostack concept, the inactivation probability as a function of radial distance to single particles' trajectory (i.e. impact parameter) was determined in space experiments as well as at accelerators using low fluences of heavy ions. It was found that spores can survive even a central hit and that the effective range of inactivation extends far beyond impact parameters where inactivation by delta-ray dose would be effective. Concerning the space experiment, the inactivation cross section exceeds those from comparable accelerator experiments by roughly a factor of 20. 2) From fluence effect curves, cross sections for inactivation and mutation induction, and the efficiency of repair processes were determined. They are influenced by the ions characteristics in a complex manner. According to dependence on LET, at least 3 LET ranges can be differentiated: A low LET range (app. < 200 keV/micrometers), where cross sections for inactivation and mutation induction follow a common curve for different ions and where repair processes are effective; an intermediate LET range of the so-called saturation cross section with negligible mutagenic and repair efficiency; and a high LET range (>1000 keV/micrometers) where the biological endpoints are majorly dependent on atomic mass and energy of the ion under consideration.     

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.     

Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation.

The lens epithelium is the initiation site for the development of radiation induced cataracts. Radiation in the cortex and nucleus interacts with proteins, while in the epithelium, experimental results reveal mutagenic and cytotoxic effects. It is suggested that incorrectly repaired DNA damage may be lethal in terms of cellular reproduction and also may initiate the development of mutations or transformations in surviving cells. The occurrence of such genetically modified cells may lead to lens opacification. For a quantitative risk estimation for astronauts and space travelers it is necessary to know the relative biological effectiveness (RBE), because the spacial and temporal distribution of initial physical damage induced by cosmic radiation differ significantly from that of X-rays. RBEs for the induction of DNA strand breaks and the efficiency of repair of these breaks were measured in cultured diploid bovine lens epithelial cells exposed to different LET irradiation to either 300 kV X-rays or to heavy ions at the UNILAC accelerator at GSI. Accelerated ions from Z=8 (O) to Z=92 (U) were used. Strand breaks were measured by hydroxyapatite chromatography of alkaline unwound DNA (overall strand breaks). Results showed that DNA damage occurs as a function of dose, of kinetic energy and of LET. For particles having the same LET the severity of the DNA damage increases with dose. For a given particle dose, as the LET rises, the numbers of DNA strand breaks increase to a maximum and then reach a plateau or decrease. Repair kinetics depend on the fluence (irradiation dose). At any LET value, repair is much slower after heavy ion exposure than after X-irradiation. For ions with an LET of less than 10,000 keV micrometers-1 more than 90 percent of the strand breaks induced are repaired within 24 hours. At higher particle fluences, especially for low energetic particles with a very high local density of energy deposition within the particle track, a higher proportion of non-rejoined breaks is found, even after prolonged periods of incubation. At the highest LET value (16,300 keV micrometers-1) no significant repair is observed. These LET-dependencies are consistent with the current mechanistic model for radiation induced cataractogenesis which postulates that genomic damage to the surviving fraction of epithelial cells is responsible for lens opacification.     

Mutagenic effects of heavy ions in bacteria.

The peculiarities and mechanisms of the mutagenic action of gamma-rays and heavy ions on bacterial cells have been investigated. Direct mutations in the lac-operon of E. coli in wild type cells and repair deficient strains have been detected. Furthermore, the induction of revertants in Salmonella tester strains was measured. It was found that the mutation rate was a linear-quadratic function of dose in the case of both gamma-rays and heavy ions with LET up to 200 keV/micrometer. The relative biological effectiveness (RBE) increased with LET up to 20 keV/micrometer. Low mutation rates were observed in repair deficient mutants with a block of SOS-induction. The induction of SOS-repair by ionizing radiation has been investigated by means of the "SOS-chromotest" and lambda-prophage induction. It was shown that the intensity of the SOS-induction in E. coli increased with increasing LET up to 40-60 keV/micrometer.     

Radiation measured during ISS-Expedition 13 with different dosimeters

Radiation in low Earth orbit (LEO) is mainly composed of galactic cosmic rays (GCR), solar energetic particles and particles in SAA (South Atlantic Anomaly). The biological impact of space radiation to astronauts depends strongly on the particles’ linear energy transfer (LET) and is dominated by high LET radiation. It is important to measure the LET spectrum for the space radiation field and to investigate the influence of radiation on astronauts. At present, the preferred active dosimeters sensitive to all LET are the tissue equivalent proportional counter (TEPC) and the silicon detectors in various configurations; the preferred passive dosimeters are CR-39 plastic nuclear track detectors (PNTDs) sensitive to high LET and thermoluminescence dosimeters (TLDs) as well as optically stimulated luminescence dosimeters (OSLDs) sensitive to low LET. The TEPC, CR-39 PNTDs, TLDs and OSLDs were used to investigate the radiation field for the ISS mission Expedition 13 (ISS-12S) in LEO. LET spectra and radiation quantities (fluence, absorbed dose, dose equivalent and quality factor) were measured for the space mission with different dosimeters. This paper introduces the role of high LET radiation in radiobiology, the operational principles for the different dosimeters, the LET spectrum method using CR-39 detectors, the method to combine the results measured with TLDs/OSLDs and CR-39 PNTDs, and presents the LET spectra and the radiation quantities measured and combined.     

Inactivation, mutation induction and repair in Bacillus subtilis spores irradiated with heavy ions.

Studies on the response of bacterial spores to accelerated heavy ions (HZE particles) help in understanding problems of space radiobiology and exobiology. Layers of spores of Bacillus subtilis strains, differing in repair capabilities, were irradiated with accelerated boron, carbon and neon ions of linear energy transfer (LET) values up to 14000 MeV cm2/g. Inactivation as measured by loss of colony forming ability and induction of mutations as measured by reversion to histidine prototrophy and resistance to 150 micrograms/ml sodium azide were tested, as well as the influence of repair processes on these effects. For inactivation, the cross-sectional values sigma plotted as a function of LET follow a saturation curve. The plateau, which is reached around a LET of 2000 MeV cm2/g, occurs at 2.5 x 10(-9) cm2, a value in good agreement with the dimensions of the spore protoplast. Lethal damage produced at LET values < 2000 MeV cm2/g is reparable. Recombination repair is more effective than excision repair. At higher LET values, lethal damage could not be reconstituted by the repair mechanisms studied. In addition, at these high LET values, the frequency of induced mutations was drastically decreased. The data support the assumption of at least two qualitatively different types of lesion, depending on the LET of the affecting heavy ion.     

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.     

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.     

Analysis of secondary electron emission spectra of equal-LET protons and alpha particles for purposes of radiation quality and spaceflight hazard assessment.

Amongst the great variety of heavy particles present in the galactic and solar cosmic ray spectra, hydrogen and helium nuclei are significantly more abundant than all other heavier ions and, as such, represent a major radiation hazard to humans in space. Experimental data have suggested that differences in relative biological effectiveness (RBE) exist between the two species at the same value of linear energy transfer (LET). This has consequences for heavily ionising radiation protection procedures, which currently still assume a simple dependence of radiation quality on LET. By analysing the secondary electron (delta-ray) emission spectra of protons and alpha particles, in terms of the spatial characteristics of energy deposition in cellular targets and the likelihood of complex lesion formation, a numerical quantity representing biological effectiveness is generated. When expressed relative to a reference radiation, this quantity is found to differ for protons and a particles of the same LET, demonstrating not only the ion-specific nature of RBE but also the inadequacy of specifying radiation quality as a function of LET only. Such a method for numerically assessing radiation quality may have implications for procedures for heavy ion protection in space at low doses and for understanding the initial mechanisms of radiation action.     

Biochemical mechanisms and clusters of damage for high-LET radiation.

Using mechanisms of indirect and direct radiation, a generalized theory has been developed to account for strand break yields by high-LET particles. The major assumptions of this theory are: (i) damage at deoxyribose sites results primarily in strand break formation and (2) damage to bases leads to a variety of base alterations. Results of the present theory compare well with cellular data without enzymatic repair. As an extension of this theory, we show that damage clusters are formed near each double strand break for high-LET radiation only. For 10 MeV/n (LET = 450 keV/micrometer) neon ions, the results show that on average there are approximately 3 additional breaks and approximately 3 damaged bases formed near each double strand break. For 100 MeV/n helium ions (LET = 3 keV/micrometer), less than 1% of the strand breaks have additional damage within 10 base pairs.     

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.     

Early and late mammalian responses to heavy charged particles.

This overview summarizes murine results on acute lethality responses, inactivation of marrow CFU-S and intestinal microcolonies, testes weight loss, life span shortening, and posterior lens opacification in mice irradiated with heavy charged particles. RBE-LET relationships for these mammalian responses are compared with results from in vitro studies. The trend is that the maximum RBE for in vivo responses tends to be lower and occurs at a lower LET than for inactivation of V79 and T-1 cells in culture. Based on inactivation cross sections, the response of CFU-S in vivo conforms to expectations from earlier studies with prokaryotic systems and mammalian cells in culture. Effects of heavy ions are compared with fission spectrum neutrons, and the results are consistent with the interpretation that RBEs are lower than for fission neutrons at about the same LET, probably due to differences in track structure. Issues discussed focus on challenges associated with assessments of early and late effects of charged particles based on dose, RBE and LET, and with the concordance or discordance of results obtained with in vivo and in vitro model systems. Models for radiation damage/repair and misrepair should consider effects observed with in vivo as well as in vitro model systems.     

Deoxyribonucleoprotein structure and radiation injury: cellular radiosensitivity is determined by LET infinity -dependent DNA damage in hydrated deoxyribonucleoproteins and the extent of its repair.

For decades, theories of cellular radiosensitivity relied upon the initial patterns of energy deposition to explain radiation lethality. Such theories are unsound: cellular (DNA) repair also underlies cellular radiosensitivity. For the charged particles encountered in deep space, both the types of DNA damage caused in cellular deoxyribonucleoproteins and the efficacies of their repair are dependent on linear energy transfer (LET infinity), and repair efficiency is also influenced by cell and tissue type, i.e., the actual recovery processes involved. Therefore, quality factors derived from radiation quality alone are inadequate parameters for assessing the radiation risks of space flight. Until recently, OH radicals formed in bulk nuclear water were believed to be the major causes of DNA damage that results in cell death, especially for sparsely ionizing radiations. That hypothesis has now been challenged, if not refuted. Lethal genomic DNA damage is determined mainly by energy deposition in deoxyribonucleoproteins, and their hydration shells, and charge (energy) transfer processes within those structures.     

Induction and repair of DNA double-strand breaks in human fibroblasts after particle irradiation.

Two assay were employed to study the induction and repair of DNA double-strand breaks (dsbs) in normal human fibroblasts after exposure to particle radiation covering an LET range from 1 to 350 keV/micrometer. The hybridization assay allows measurement of absolute induction frequencies in defined regions of the genome and quantitates rejoining of correct DNA ends while the FAR assay determines all rejoining events, correct and incorrect. Assuming Poisson statistics for the number of breaks per DNA fragment investigated, and thus neglecting any clustering of breaks, we found the induction rate to decrease with increasing LET of the particles. RBE values compared to 225 kVp X-rays dropped to 0.48 for the highest LETs. Repair studies of X-ray-induced dsbs showed that almost all breaks (>95%) are rejoined after incubation times of 24 h while the frequency for correct rejoining is only 70%. Thus about 25% of the initially induced breaks are rejoined by the connection of incorrect DNA ends. Postirradiation incubation after particle irradiation showed less efficient total rejoining with increasing LET and an impaired ability for correct rejoining. The frequency for rejoining of incorrect DNA ends was found to be independent of LET. The possible biological significance of the different rejoining events is discussed.     

RBE: mechanisms inferred from cytogenetics.

Cyclotron-accelerated heavy ion beams provide a fine degree of control over the physical parameters of radiation. Cytogenetics affords a view into the irradiated cell at the resolution of chromosomes. Combined they form a powerful means to probe the mechanisms of RBE. Cytogenetic studies with high energy heavy ion beams reveal three LET-dependent trends for 1) level of initial damage, 2) distribution of damage among cells, and 3) lesion severity. The number of initial breaks per unit dose increases from a low-LET plateau to a peak at approximately 180 keV/micrometer and declines thereafter. Overdispersion of breaks is significant above approximately 100 keV/micrometer. Lesion severity, indicated by the level of chromosomal fragments that have not restituted even after long repair times, increases with LET. Similar studies with very low energy 238Pu alpha particles (120 keV/micrometer) reveal higher levels of initial breakage per unit dose, fewer residual fragments and a higher level of misrepair when compared to high energy heavy ions at the same LET. These observations would suggest that track structure is an important factor in genetic damage in addition to LET.     

Induction of chromosomal damage in CHO-K1 cells and their repair-deficient mutant XRS5 by X-ray and particle irradiation.

The cytogenetic effects of X-rays and Au ions were investigated in repair-proficient CHO-K1 cells and their radiosensitive mutant strain xrs5, which shows a defect in the rejoining of DNA double-strand breaks. Both cell lines were synchronized by mitotic shake off, irradiated in G1-phase with either 250 kV X-rays or 780 MeV/u Au ions (LET: 1150 keV/micrometer) and chromosome aberrations were analyzed in first post-irradiation metaphases. Isoeffective doses of X-rays for the induction of aberrant cells and aberrations per cell were about 14 times lower for xrs5 than for CHO-K1 cells. After high LET radiation the difference in the cytogenetic response of both cell lines was drastically diminished. Furthermore, the analysis of the aberration types induced by sparsely and densely ionizing radiation showed for both cell lines specific changes in the spectrum of aberration types as LET increases. The experimental results are discussed with respect to the different types of lesions induced by sparsely and densely ionizing radiation.     

Radiobiology and photobiology on Earth and in space: points of encounter and protection considerations.

All radiations originate in space, and the spectrum of radiations reaching the troposphere is limited only because of their range and absorption by the ozone layer above the atmosphere. Ultraviolet-C and the very heavy ions are therefore produced on earth only artificially, by special lamps and in accelerators. The range of biological effects of the different UV radiations and low and high LET radiations have been studied extensively, yet only recently new facts such as the production of DNA strand breaks by long wave UV light were established, adding to the various points of encounter existing between ionizing and nonionizing radiations. There are some similarities in radiation products, and the resulting effects of insult by radiation on biological systems very often are similar, if not the same. A common phenomenon that exists in all healthy biological cells is the ability to repair damage to DNA and thus either survive or mutate, and although the specific mechanisms of repair are somewhat different, the end result is the same. Recently a mechanism of improved radioprotection was found to involve an effect of certain radioprotective compounds on DNA repair. It is suggested that improved, and nontoxic, modes of protection may be offered by employing such compounds as biological response modifiers and natural substances. Further research is needed and is under way.     

Cell cycle delays induced by heavy ion irradiation of synchronous mammalian cells.

Cell cycle effects of very high LET particles on synchronous V79 Chinese Hamster cells have been studied in a track segment experiment by means of flow cytometric methods. Cells were irradiated with 10 MeV/u Pb-ions (LET = 13500 keV/micrometers) at an average fluence of 2 particles per cell nucleus, corresponding to a survival level of about 25%. Instantaneous drastic reductions of cell proliferation in all cycle phases have been observed, which affect the cell cycle for at least 50 hours after exposure to heavy ions. These findings are in clear contrast to the results from low LET radiation experiments, where significant delays can only be observed in S-phase and G2M-phase and for comparatively short time intervals of a few hours. Additionally, high LET radiation gives rise to prolonged DNA synthesis bypassing cell division, which leads to cells with DNA content greater than that of G2M-cells.