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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

一个控制超强电离辐射抗性开关基因的研究进展

电离辐射广泛存在于地球和空间,会引起生物体内的DNA损伤,导致机体突变甚至死亡.生物体的DNA损伤响应对于稳定基因组的完整性至关重要.耐辐射奇球菌因其超强的DNA修复能力成为研究DNA损伤修复的模式生物之一.PprI-DdrO系统是近年来发现的一种新型且高效的损伤响应途径,PprI作为响应损伤的重要开关蛋白,通过酶切DdrO调控DNA损伤响应基因的表达.本文从功能、结构、激活机制和潜在应用价值几方面描述了PprI的研究进展.      

Radiosensitivity to high energy iron ions is influenced by heterozygosity for Atm, Rad9 and Brca1

Loss of function of DNA repair genes has been implicated in the development of many types of cancer. In the last several years, heterozygosity leading to haploinsufficiency for proteins involved in DNA repair was shown to play a role in genomic instability and carcinogenesis after DNA damage is induced, for example by ionizing radiation. Since the effect of heterozygosity for one gene is relatively small, we hypothesize that predisposition to cancer could be a result of the additive effect of heterozygosity for two or more genes critical to pathways that control DNA damage signaling, repair or apoptosis. We investigated the role of heterozygosity for Atm, Rad9 and Brca1 on cell oncogenic transformation and cell survival induced by 1 GeV/n⁵⁶Fe ions. Our results show that cells heterozygous for both Atm and Rad9 or Atm and Brca1 have high survival rates and are more sensitive to transformation by high energy iron ions when compared with wild-type controls or cells haploinsufficient for only one of these proteins. Since mutations or polymorphisms for similar genes exist in a small percentage of the human population, we have identified a radiosensitive sub-population. This finding has several implications. First, the existence of a radiosensitive sub-population may distort the shape of the dose–response relationship. Second, it would not be ethical to put exceptionally radiosensitive individuals into a setting where they may potentially be exposed to substantial doses of radiation.     

Hematopoietic tissue repair under chronic low daily dose irradiation.

The capacity of the hematopoietic system to repair constantly accruing cellular damage under chronic, low daily dose gamma irradiation is essential for the maintenance of a functional hematopoietic system, and, in turn, long term survival. In certain individuals, however, such continuous cycles of damage and repair provide an essential inductive environment for selected types of hematopathologies, e.g., myeloid leukemia (ML). In our laboratory we have been studying temporal and causal relationships between hematopoietic capacity, associated repair functions, and propensities for hematologic disease in canines under variable levels of chronic radiation stress (0.3-26.3 cGy d-1). Results indicate that the maximum exposure rate tolerated by the hematopoietic system is highly individual-specific (three major responding subgroups identified) and is based largely on the degree to which repair capacity, and, in turn, hematopoietic restoration, is augmented under chronic exposure. In low-tolerance individuals (prone to aplastic anemia, subgroup 1), the failure to augment basic repair functions seemingly results in a progressive accumulation of genetic and cellular damage within vital progenitorial marrow compartments (particularly marked within erythroid compartments) that results in loss of reproductive capacity and ultimately in collapse of the hematopoietic system. The high-tolerance individuals (radioaccomodated and either prone- or not prone to ML, subgroup 2 & 3) appear to minimize the accumulating damage effect of daily exposures by extending repair functions, which preserves reproductive integrity and fosters regenerative hematopoietic responses. As the strength of the regenerative response manifests the extent of repair augmentation, the relatively strong response of high- tolerance individuals progressing to patent ML suggests an insufficiency of repair quality rather than repair quantity. The kinetics of these repair-mediated, regenerative hematopoietic responses within the major subgroups are under study and should provide useful insights into the nature of hematopoietic accommodation (or its failure) under greatly extended periods of chronic, low-daily-dose ionizing radiation exposure.     

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.     

Preliminary results of space experiment: Implications for the effects of space radiation and microgravity on survival and mutation induction in human cells

In view of the concern for the health of astronauts that may one day journey to Mars or the Moon, we investigated the effect that space radiation and microgravity might have on DNA damage and repair. We sent frozen human lymphoblastoid TK6 cells to the International Space Station where they were maintained under frozen conditions during a 134-day mission (14 November 2008 to 28 March 2009) except for an incubation period of 8 days under 1G or μG conditions in a CO₂ incubator. The incubation period started after 100 days during which the cells had been exposed to 54 mSv of space radiation. The incubated cells were then refrozen, returned to Earth, and compared to ground control samples for the determination of the influence of microgravity on cell survival and mutation induction. The results for both varied from experiment to experiment, yielding a large SD, but the μG sample results differed significantly from the 1G sample results for each of 2 experiments, with the mean ratio of μG to 1G being 0.55 for the concentration of viable cells and 0.59 for the fraction of thymidine kinase deficient (TK⁻) mutants. Among the mutants, non-loss of zygosity events (point mutations) were less frequent (31%) after μG incubation than after 1G incubation, which might be explained by the influence of μG on cellular metabolic or physiological function. Additional experiments are needed to clarify the effect of μG interferes on DNA repair.     

Heavy-ion induced genetic changes and evolution processes.

On Moon and Mars, there will be more galactic cosmic rays and higher radiation doses than on earth. Our experimental studies showed that heavy ion radiation can effectively cause mutation and chromosome aberrations and that high-LET heavy-ion induced mutants can be irreversible. Chromosome translocations and deletions are common in cells irradiated by heavy particles, and ionizing radiations are effective in causing hyperploidy. The importance of the genetic changes in the evolution of life is an interesting question. Through evolution, there is an increase of DNA content in cells from lower forms of life to higher organisms. The DNA content, however, reached a plateau in vertebrates. By increasing DNA content, there can be an increase of information in the cell. For a given DNA content, the quality of information can be changed by rearranging the DNA. Because radiation can cause hyperploidy, an increase of DNA content in cells, and can induce DNA rearrangement, it is likely that the evolution of life on Mars will be effected by its radiation environment. A simple analysis shows that the radiation level on Mars may cause a mutation frequency comparable to that of the spontaneous mutation rate on Earth. To the extent that mutation plays a role in adaptation, radiation alone on Mars may thus provide sufficient mutation for the evolution of life.     

Mechanisms underlying cellular radiosensitivity and R.B.E.: introductory remarks.

A general outline of the symposium titled "Mechanisms underlying cellular radiosensitivity and R.B.E." will be given in the introduction. The essential topics of molecular radiation biology are described with respect to the damage, repair and mutagenesis caused by high-LET irradiation to cellular DNA. The importance of clustered DNA lesions (locally multiply damaged sites) formed in vivo is discussed. This symposium is devoted to the mechanisms of the biological effects of radiation with high LET, especially with regard to the effects of heavy ions and neutrons which may cause possible risks in space flight, (e.g. carcinogenesis and mutagenesis). Detailed understanding of these risks, however, demands knowledge of the molecular mechanisms involved in the biological effects of high-LET radiations. Thus, it was the organizers' idea to hold a symposium dealing with primary physical and chemical events caused in cellular deoxyribonucleoproteins by densely-ionizing radiations and to relate them to track structures and energy transfer processes. The mechanisms of DNA damage were regarded from different points of view including those considering DNA repair and mutagenesis. Problems associated with cell survival and radiation protection were discussed as well. Our knowledge of the molecular mechanisms of high-LET radiation actions, however, is limited compared to what we know about low-LET radiation effects (e.g. from gamma-rays or X-rays). To emphasize this statement, I would like to summarize briefly the open questions in molecular radiation biology, what we know already about low-LET effects and what is lacking describing the effect of high-LET radiation.     

Survival of microorganisms in space: A review

Spores of $\text{Bacillus subtilis}$ were exposed to selected factors of space (vacuum, solar UV radiation, heavy ions of cosmic radiation), and their response was studied after recovery. These investigations were supplemented by ground-based studies under simulated space conditions. The vacuum of space did not inactivate the spores. However, vacuum-induced structural changes in the DNA, and probably in the proteins, caused a supersensitivity to solar UV radiation. This phenomenon is caused by the production of specific photoproducts in DNA and protein, which cannot be removed by normal cellular repair processes. In vegetative bacterial cells, exposed to vacuum, cell dehydration led to damage of the cell membrane, which could be partly repaired during subsequent incubation. The high local effectiveness of the cosmic heavy ions further decreases the chance that spores can survive for any length of time in space. Nonetheless, a spore travelling through space and protected from ultraviolet radiation could possibly survive an interplanetary journey. Such a situation favors panspermia as a possible explanation for the origin of life.     

DNA structures and radiation injury.

In the present paper experimental results from radiobiological investigations of the sedimentation behaviour of damaged and restored DNA-subunits attached to the nuclear membrane have been summarized. The studies were carried out preferably with Chinese Hamster cells V79-4 irradiated with different kinds of radiation (gamma-rays, neutrons and carbon ions) using the nucleoid sedimentation technique. Single-strand breaks relax the supercoiled DNA in the subunits resulting in a decreased sedimentation velocity. Rejoining leads to a correct restoration of the structure as can be studied by means of postincubation irradiation. Double-strand breaks release DNA fragments, again leading to an increased sedimentation velocity. If the average number of the induced double-strand breaks per subunit increases to a number higher than one, the measured results suggest that the structures should not be restored completely. The results are compatible with a new repair model developed in our laboratory on the assumption that, firstly, the single DNA subunits are the sensitive target rather than the whole DNA and, secondly, the repair of DNA damage takes place independently in each subunit.