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.     

Energy deposition rates by charged particles measured during the energy budget campaign

Measurements of the precipitation of electrons and positive ions (in the keV to MeV range) detected aboard eight rockets launched from Northern Scandinavia are reported together with corresponding satellite data. The downgoing integral fluxes indicate the temporal fluctuations during each flight. Height profiles of the energy deposition into the atmosphere at different levels of geomagnetic disturbance are given.     

Propagation modes of energetic charged particles in the heliosphere

Several years ago, the anisotropic diffusion and convective transport accompanied by adiabatic deceleration were considered as the principal means for cosmic ray propagation. Particles of relatively small energies (～ 1 MeV) can propagate along the force lines of the magnetic field without scattering at distances of several astronomical units in the quiet heliosphere. The theory describing the 11-year variation of galactic cosmic ray intensity and the propagation of solar cosmic rays was founded on this basis. However, the anomalies of the 11-year variation of galactic cosmic ray intensity in 1969–1971 revealed the necessity to take into account the influence of the general electromagnetic field of the heliosphere giving rise to a rapid magnetic drift of particles. The particles drift either from the magnetic axis to the ecliptic plane (in the cycle of 1969–1980) or in the opposite direction depending on the sign of the general magnetic field of the sun. The neutral layers along which the drift velocity is comparable to the particle velocity is of great significance. However, in the presence of sector structure, the time of particle propagation along the neutral layer from the boundary of the modulation region to the earth orbit is substantially increased. Thus a marked adiabatic deceleration is here possible. The time delay observed in the recovery of proton intensities at various energies can be explained in terms of a transient phase of the interplanetary field following the polarity reversal.     

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.     

Residual chromatin breaks as biodosimetry for cell killing by carbon ions

We have studied the relationship between cell killing and the induction of residual chromatin breaks on various human cell lines and primary cultured cells obtained by biopsy from patients irradiated with either X-rays or heavy-ion beams to identify potential bio-marker of radiosensitivity for radiation-induced cell killing. The carbon-ion beams were accelerated with the Heavy Ion Medical Accelerator in Chiba (HIMAC). Six primary cultures obtained by biopsy from 6 patients with carcinoma of the cervix were irradiated with two different mono-LET beams (LET= 13 keV/μm, 76 keV/μm) and 200kV X rays. Residual chromatin breaks were measured by counting the number of non-rejoining chromatin fragments detected by the premature chromosome condensation (PCC) technique after a 24 hour postirradiation incubation period. The induction rate of residual chromatin breaks per cell per Gy was the highest for 76 keV/μm beams on all of the cells. Our results indicated that cell which was more sensitive to the cell killing was similarly more susceptible to induction of residual chromatin breaks. Furthermore there is a good correlation between these two end points in various cell lines and primary cultured cells. This suggests that the detection of residual chromatin breaks by the PCC technique may be useful as a predictive assay of tumor response to cancer radiotherapy.     

Dose rate and repair effects on cell damage in Earth orbit.

Radiobiology experiments performed in space will encounter continuous exposures to the cosmic rays and fractionated exposures to trapped protons which accumulate to several hundred dose fractions in a few weeks. Using models of track structure and cellular kinetics combined with models of the radiation environment and radiation transport, we consider calculations of damage rates for cell cultures. Analysis of the role of repair mechanisms for space exposures for the endpoints of survival and transformation is emphasized.     

Dictyostelium discoideum, a lower eukaryote model for the study of DNA repair: implications for the role of DNA-damaging chemicals in the evolution of repair proficient cells.

The evolution of the ability of living cells to cope with stress is crucial for the maintenance of their genetic integrity. Yet low levels of mutation must remain to allow adaptation to environmental changes. The cellular slime mold D. discoideum is a good system for studying molecular aspects of the repair of lethal and mutagenic damage to DNA by radiation and chemicals. The wild-type strains of this soil microorganism are extremely resistant to DNA damaging agents. In nature the amoeboid cells in their replicative stage feed on soil bacteria and are exposed to numerous DNA-damaging chemicals produced by various soil microorganisms. It is probable that the evolution of repair systems in this organism and perhaps in others is a consequence of the necessity to cope with chemical damage which also confers resistance to radiation.     

Measurements of the energy spectra of charged particles within the vertical-10 rocket experiment

On December 21-st, 1981, at 18.35 hours UT from the territory of the USSR (coordinates - 49°N/L 2/) in implementation of scientific objectives and in accordance with the ‘INTERCOSMOS’ Programme, there was launched the heavy geophysical rocket ‘VERTICAL-10’. The scientific payload included a low-energy two-channel spectrometer for measuring the differential flows of electrons and protons within the energy range 0.1 to 10 keV, covered by 15 exponentially distributed energy levels.     

Organic solids produced from simple C/H/O/N ices by charged particles: applications to the outer solar system.

CH4, CO, and CO2 are all potential one-carbon molecular repositories in primitive icy objects. These molecules are all found in the Comet Halley coma, and are probable but, (except for CH4 detected on Triton and Pluto) undetected subsurface constituents in icy outer solar system objects. We have investigated the effects of charged particle irradiation by cold plasma discharge upon surfaces of H2O:CH4 clathrate having a 200:1 ratio, as well as upon ices composed of H2O plus C2H6 or C2H2 (sometimes plus NH3) which are also plausible constituents. These materials color and darken noticeably after a dose 10(9) - 10(10) erg cm-2, which is deposited rapidly (< or = 10(4) yr.) in solar system environments. The chromophore is a yellowish to tan organic material (a tholin) which we have studied by UV-VIS reflection and transmission, and IR transmission spectroscopy. Its yield, -1 C keV-1, implies substantial production of organic solids by the action of cosmic rays and radionuclides in cometary crusts and interiors, as well as rapid production in satellite surfaces. This material shows alkane bands which Chyba and Sagan have shown to well match the Halley infrared emission spectrum near 3.4 microns, and also bands due to aldehyde, alcohol and perhaps alkene/aromatic functional groups. We compare the IR spectral properties of these tholins with the spectra of others produced by irradiation of gases and ices containing simple hydrocarbons.     

Ionizing radiation-induced mutation of human cells with different DNA repair capacities.

We have observed significant differences in the response to ionizing radiation of two closely related human cells lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.     

Probability of cell hits in selected organs and tissues by high-LET particles at the ISS orbit.

The fluence of high-LET particles (HLP) with LET infinity H2O greater than 15 keV micrometers-1 in selected organs and tissues were measured with plastic nuclear track detectors using a life-size human phantom on the 9th Shuttle-Mir Mission (STS-91). The planar-track fluence of HLP during the 9.8-day mission ranged from 1.9 x 10(3) n cm-2 (bladder) to 5.1 x 10(3) n cm-2 (brain) by a factor of 2.7. Based on these data, a probability of HLP hits to a matured cell of each organ or tissue was roughly estimated for a 90-day ISS mission. In the calculation, all cells were assumed to be spheres with a geometric cross-sectional area of 500 micrometers2 and the cell-hit frequency from isotropic space radiation can be described by the Poisson-distribution function. As results, the probability of one or more than 1 hit to a single cell by HLP for 90 days ranged from 17% to 38%; that of two or more than 2 hits was estimated to be 1.3-8.2%.     

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.     

New approaches to biochemical radioprotection: antioxidants and DNA repair enhancement.

Chemical repair may be provided by radioprotective compounds present during exposure to ionizing radiation. Considering DNA as the most sensitive target it is feasible to biochemically improve protection by enhancing DNA repair mechanisms. Protection of DNA by reducing the amount of damage (by radical scavenging and chemical repair) followed by enhanced repair of DNA will provide much improved protection and recovery. Furthermore, in cases of prolonged exposure, such as is possible in prolonged space missions, or of unexpected variations in the intensity of radiation, as is possible when encountering solar flares, it is important to provide long-acting protection, and this may be provided by antioxidants and well functioning DNA repair systems. It has also become important to provide protection from the potentially damaging action of long-lived clastogenic factors which have been found in plasma of exposed persons from Hiroshima & Nagasaki, radiation accidents, radiotherapy patients and recently in "liquidators"--persons involved in salvage operations at the Chernobyl reactor. The clastogenic factor, which causes chromatid breaks in non-exposed plasma, might account for late effects and is posing a potential carcinogenic hazard. The enzyme superoxide dismutase (SOD) has been shown to eliminate the breakage factor from cultured plasma of exposed persons. Several compounds have been shown to enhance DNA repair: WR-2721, nicotinamide, glutathione monoester (Riklis et al., unpublished) and others. The right combination of such compounds may prove effective in providing protection from a wide range of radiation exposures over a long period of time.     

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.     

Modifying factors on repair phenomena.

Every step of radiation damage repair can be modified by several factors like temporal and local energy distribution (LET), physiological conditions, biochemical and chemical composition of the target environment. Most interesting in the case of space radiation is the possible influence of high LET and of microgravity which could change the repair i.e. fusion capacity as well as the phenomenon of self assembly.     

Radioprotection of DNA by biochemical mechanisms.

The title of this presentation should be understood as having two facets: the preservation of genetic information and function by protecting the DNA from the deleterious effects of radiation by reducing the degree of damage, is one important task. Yet another, which may be as important, is the utilization of biochemical entities whose function is to repair damages which have already been formed in DNA, thus enhancing the protection of living cells.     

Oncogenic transformation of mammalian cells by ultrasoft X-rays and alpha particles.

For a better understanding of oncogenic cell transformation by ionizing radiation, we conducted experiments with ultrasoft X rays and low energy alpha particles. Confluent C3H10T1/2 cells were irradiated by Al-K (1.5 keV) X rays or alpha particles from plutonium through a thin mylar sheet, on which the cells attached and grew. Our results indicated that Al-K X rays were more effective in causing cell inactivation and oncogenic transformation than 60Co gamma rays but less effective than 1.0 and 3.7 MeV alpha particles. There was no significant difference between 1.0 and 3.7 MeV alpha particles in transforming cells although the latter were slightly more effective than the former in producing lethal effect. These results indicated that track structure is important in causing biological effects by ionizing radiation.     

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.     

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.