Induction of DNA breaks in SV40 by heavy ions.

Simian virus (SV40) DNA was used to study the induction of DNA strandbreaks by heavy ions varying in LET. DNA was exposed to X-rays and to accelerated particles either in dilute solution or in the presence of different radical scavengers. Relative proportions of the intact supercoiled DNA, nicked form arising from single strand breaks (SSB) and linear molecules produced by double strandbreaks (DSB) were quantified on the base of their electrophoretic mobility in agarose gels. Cross sections for the induction of SSBs and DSBs were calculated from the slope of dose effect curves. Mercaptoethanol was found to protect more efficiently against DNA strand breakage than Tris. When the biological efficiency, i.e. the number of strand breaks per unit dose and molecule weight was evaluated as a function of LET, curves for SSB induction always showed a continuous decrease. For DSB induction, an increase in the yield of DSBs with a maximum around 500 keV/micrometer was observed in the presence of radical scavenger. This peak of biological efficiency gradually disappeared when the radiosensitivity of the system was increased, and was no longer apparent in the dilute buffer system, where DNA showed a high susceptibility to strand breakage. When the relative biological efficiency was plotted versus LET, the curve for DSB induction observed in a low radical scavenging environment paralleled the curve obtained for SSB induction.     

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.     

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.     

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.     

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 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.     

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.     

Correlation between cell death and induction of non-rejoining PCC breaks by carbon-ion beams.

We have shown a correlation between cell death and induction of non-rejoining chromatin breaks in two normal human cells and three human tumor cell lines irradiated by carbon-ion beams and X rays. Non-rejoining chromatin breaks were measured by counting the number of remaining chromatin fragments detected by the premature chromosome condensation (PCC) technique. Carbon-ion beams were accelerated by the Heavy Ion Medical Accelerator in Chiba (HIMAC). The cells were irradiated by two different mono-LET beams (LET = 13 keV/micrometer and 77 keV/micrometer ) and 200 kV X rays. The RBE values of cell death for carbon-ion beams relative to X rays were 1.1 to 1.4 for 13 keV/micrometer beams and 2.5 to 2.9 for 77 keV/micrometer beams. The induction rate of non-rejoining PCC breaks per cell per Gy was found to be highest for the 77 keV/micrometer beams for all of the cell lines.The results found in this study show that there is a good correlation between cell death and induction of non-rejoining PCC breaks for these human cell lines.     

LET dependence of cell death, mutation induction and chromatin damage in human cells irradiated with accelerated carbon ions.

We investigated the LET dependence of cell death, mutation induction and chromatin break induction in human embryo (HE) cells irradiated by accelerated carbon-ion beams. The results showed that cell death, mutation induction and induction of non-rejoining chromatin breaks detected by the premature chromosome condensation (PCC) technique had the same LET dependence. Carbon ions of 110 to 124keV/micrometer were the most effective at all endpoints. However, the number of initially induced chromatin breaks was independent of LET. About 10 to 15 chromatin breaks per Gy per cell were induced in the LET range of 22 to 230 keV/micrometer. The deletion pattern of exons in the HPRT locus, analyzed by the polymerase chain reaction (PCR), was LET-specific. Almost all of the mutants induced by 124 keV/micrometer beams showed deletion of the entire gene, while all mutants induced by 230keV/micrometer carbon-ion beams showed no deletion. These results suggest that the difference in the density distribution of carbon-ion track and secondary electron with various LET is responsible for the LET dependency of biological effects.     

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.     

Mutation induction in human lymphoid cells by energetic heavy ions.

One of the concerns for extended space flight outside the magnetosphere is exposure to galactic cosmic radiation. In the series of studies presented herein, the mutagenic effectiveness of high energy heavy ions is examined using human B-lymphoblastoid cells across an LET range from 32keV/micrometer to 190 keV/micrometer. Mutations were scored for an autosomal locus, thymidine kinase (tk), and for an X-linked locus, hypoxanthine phosphoribosyltransferase (hprt). For each of the radiations studied, the autosomal locus is more sensitive to mutation induction than is the X-linked locus. When mutational yields are expressed in terms of particle fluence, the two loci respond quite differently across the range of LET. The action cross section for mutation induction peaks at 61 keV/micrometer for the tk locus and then declines for particles of higher LET, including Fe ions. For the hprt locus, the action cross section for mutation is maximal at 95 keV/micrometer but is relatively constant across the range from 61 keV/micrometer to 190 keV/micrometer. The yields of hprt-deficient mutants obtained after HZE exposure to TK6 lymphoblasts may be compared directly with published data on the induction of hprt-deficient mutants in human neonatal fibroblasts exposed to similar ions. The action cross section for induction of hprt-deficient mutants by energetic Fe ions is more than 10-fold lower for lymphoblastoid cells than for fibroblasts.     

DNA fragmentation by charged particle tracks.

High-LET (linear energy transfer) charged particles induce DNA double-strand breaks (DSB) in a non-random fashion in mammalian cells. The clustering of DSB, probably determined by track structure as well as chromatin conformation, results in an excess of small- and intermediate-sized DNA fragments. DNA fragmentation in normal human fibroblasts (GM5758) was analyzed by pulsed-field gel electrophoresis after irradiation with photons (60Co) or 125 keV/micrometers nitrogen ions. Compared to conventional DSB analysis, i.e. assays only measuring the fraction of DNA smaller than a single threshold, the relative biological effectiveness (RBE) for DSB induction increased with 100%. Further, the size distribution of DNA fragments showed a significant dependence on radiation quality, with an excess of fragments up to 1 Mbp. Irradiation of naked genomic DNA without histone proteins increased the DSB yields 25 and 13 times for photons and nitrogen ions, respectively. The results suggest possible roles of both track structure and chromatin organization in the distribution of DNA double-strand breaks along the chromosome.     

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.     

Neoplastic cell transformation by energetic heavy ions and its modification with chemical agents.

For many years we have been interested in understanding the potential carcinogenic effects of cosmic rays. We have studied the oncogenic effects of cosmic rays with accelerator-produced heavy particle radiation and with a cultured mammalian cell system--C3H10T1/2 cells. Our quantitative data obtained with carbon, neon, silicon, and iron particles showed that RBE is both dose and LET dependent for neoplastic cell transformation. RBE is higher at lower dose, and RBE increases with LET up to about 200 keV/micrometer. In nonproliferation confluent cells, heavy-ion induced transformation damage may not be repairable, although a dose modifying factor of about 1.7 was observed for X-ray radiation. Our recent studies with super-heavy high-energy particles, e.g., 960 MeV/U U235 ions (LET = 1900 keV/micrometer), indicate that these ions with a high inactivation cross-section can cause neoplastic cell transformation. The induction of cell transformation by radiation can be modified with various chemicals. We have found that the presence of DMSO (either during or many days after irradiation) decreased the transformation frequency significantly. It is, therefore, potentially possible to reduce the oncogenic effect of cosmic rays in space through some chemical protection.     

Radiation quality and risk estimation in relation to space missions.

While Q is specified as a function of linear energy transfer (LET) in practice the Q for neutrons has been selected by a judgment decision based on the relative biological effectiveness (RBE) to induce stochastic effects. There are no RBE values for tumor induction by heavy ions or protons in humans. Thus, selection of Q values has been based either on LET (or lineal energy) or RBEs from animal experiments. Estimates of Q for heavy ions in low earth orbit (LEO) range from about 5 to 14. The average Q value of all radiation in LEO has been estimated to be about 1.3. There is a lack of experimental data for RBEs for heavy ions but RBE increases as a function of LET. In the case of the Harderian gland the RBE reaches a maximum of 25-30 between about 100-200 keV/micrometer but does not appear to decrease at higher LETs. The International Commission of Radiological Protection have proposed the use of radiation weighting factors in lieu of quality factors. The weighting factors will range from 1 to 20.     

Fluence-based relative biological effectiveness for charged particle carcinogenesis in mouse Harderian gland.

Neoplasia in the rodent Harderian gland has been used to determine the carcinogenic potential of irradiation by HZE particles. Ions from protons to lanthanum at energies up to 670 MeV/a have been used to irradiate mice, and prevalence of Harderian gland tumors has been measured 16 months after irradiation. The RBE for tumor induction has been expressed as the RBEmax, which is the ratio of the initial slopes of the dose vs prevalence curve. The RBEmax has been found to be approximately 30 for ions with LET values in excess of 100 keV/micrometer. Analysis on the basis of fluence as a substitute for dose has shown that on a per particle basis all of the ions with LET values in excess of 100 keV/micrometer have equal effectiveness. An analysis of the probabilities of ion traversals of the nucleus has shown that for these high stopping powers that a single hit is effective in producing neoplastic transformation.     

DNA damage and repair in oncogenic transformation by heavy ion radiation.

Energetic heavy ions are present in galactic cosmic rays and solar particle events. One of the most important late effects in risk assessment is carcinogenesis. We have studied the carcinogenic effects of heavy ions at the cellular and molecular levels and have obtained quantitative data on dose-response curves and on the repair of oncogenic lesions for heavy particles with various charges and energies. Studies with repair inhibitors and restriction endonucleases indicated that for oncogenic transformation DNA is the primary target. Results from heavy ion experiments showed that the cross section increased with LET and reached a maximum value of about 0.02 micrometer2 at about 500 keV/micrometer. This limited size of cross section suggests that only a fraction of cellular genomic DNA is important in radiogenic transformation. Free radical scavengers, such as DMSO, do not give any effect on induction of oncogenic transformation by 600 MeV/u iron particles, suggesting most oncogenic damage induced by high-LET heavy ions is through direct action. Repair studies with stationary phase cells showed that the amount of reparable oncogenic lesions decreased with an increase of LET and that heavy ions with LET greater than 200 keV/micrometer produced only irreparable oncogenic damage. An enhancement effect for oncogenic transformation was observed in cells irradiated by low-dose-rate argon ions (400 MeV/u; 120 keV/micrometer). Chromosomal aberrations, such as translocation and deletion, but not sister chromatid exchange, are essential for heavy-ion-induced oncogenic transformation. The basic mechanism(s) of misrepair of DNA damage, which form oncogenic lesions, is unknown.     

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.     

Chromosome instability of HPRT-mutant subclones induced by ionising radiation of various LET.

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20-360 keV/micrometers), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100-200 keV/micrometers and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT-mutant subclones that arose after exposure to gamma-rays, 1 GeV protons and 14N-ions (LET-77 keV/micrometers), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.