S1-sensitive sites in DNA after gamma-irradiation

DNA from gamma-irradiated T1 bacteriophages was analyzed for "single-stranded" sites by cleavage with S1 nuclease from Aspergillus oryzae as lesion probe. The ratio of "S1-sensitive sites" to the amount of radiation-induced single-strand breaks was about one. Presumably these "denatured" sites were associated with single-strand breaks. The subsequent check for the persistence of "single-stranded" sites within the DNA molecule by thermokinetics demonstrated a strong affinity of the nuclease to its substrate, the single-stranded lesion, and a perfect excision. It is assumed that the direct absorption of radiation energy in the DNA gives rise to the formation of such bulky lesions.     

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.     

A Stochastic Model of DNA Fragments Rejoining

When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie''s stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation.     

Biological low-dose radiation effects.

It is theorized that biological responses to ionizing radiation in the low dose range are determined according to a doubly dichotomous pattern. Energy depositions fall into 2 categories: events at thermal energy levels where they may be experienced by cells as rates even at background exposure conditions, and events at energy levels of the order of 10-100 eV where damage to DNA may be caused. Variations in background exposure intensity may or may not lead preemptively to changes in the cell's capacity for response to radiation damage. High-level energy depositions lead post hoc to an initial stabilizing reaction largely leading to the fixation of the initial DNA damage, and to a subsequent restorative or palliative repair process. This model entails reinterpretation of some experimental results. The model has implications for the relationship between scientific analysis of low-dose effects and the regulatory needs for simplicity and homogeneity in risk evaluation. This represents a new challenge for the acceptability of radiation protection norms.     

Characteristics of DNA-binding proteins determine the biological sensitivity to high-linear energy transfer radiation

Non-homologous end-joining (NHEJ) and homologous recombination repair (HRR), contribute to repair ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Mre11 binding to DNA is the first step for activating HRR and Ku binding to DNA is the first step for initiating NHEJ. High-linear energy transfer (LET) IR (such as high energy charged particles) killing more cells at the same dose as compared with low-LET IR (such as X or γ rays) is due to inefficient NHEJ. However, these phenomena have not been demonstrated at the animal level and the mechanism by which high-LET IR does not affect the efficiency of HRR remains unclear. In this study, we showed that although wild-type and HRR-deficient mice or DT40 cells are more sensitive to high-LET IR than to low-LET IR, NHEJ deficient mice or DT40 cells are equally sensitive to high- and low-LET IR. We also showed that Mre11 and Ku respond differently to shorter DNA fragments in vitro and to the DNA from high-LET irradiated cells in vivo. These findings provide strong evidence that the different DNA DSB binding properties of Mre11 and Ku determine the different efficiencies of HRR and NHEJ to repair high-LET radiation induced DSBs.     

Neutron microscopy. The low-damage imaging of specialized organic materials

It is shown that, insofar as radiation damage is concerned, transmission neutron microscopy using neutrons in the energy range approximately 0.0001-1.0 eV is extremely attractive for the imaging of specialized organic materials. By "specialized organic materials" is meant organic specimens composed entirely of specific isotopes that have been selected on the basis of their favorable properties with regard to radiation damage. In connection with such specimens, it is demonstrated that at a resolution of, for example, 100 A, neutrons will have an advantage over soft X-rays in terms of radiation damage, provided that the inherent (neutron) bright field image contrast turns out to be greater than 10(-5). Suggestions relating to (a) the comprehensive calculation of the radiation damage sustained by specialized organic specimens under slow neutron irradiation, (b) the construction of a theory of image formation in the neutron microscope, (c) the development of neutron lenses/focusing devices, and (d) the development of a brighter neutron source (essential for neutron microscopy) are outlined in some detail. The paper concludes with two appendices, which provide important background material.     

Induction,repair and biological relevance of radiation-induced DNA lesions in eukaryotic cells

Summary This report summarizes data on the induction, repair and biological relevance of five types of radiation-induced DNA lesions for which repair kinetic studies have been performed in eukaryotic cells by various laboratories. These lesions are: DNA-protein crosslinks, base damage, single-strand breaks, double-strand breaks and bulky lesions (clustered base damage in the nm-range). The influence of various factors, such as oxia/anoxia, linear energy transfer of the radiation used, incubation medium, cell cycle stage, thiol content, hyperthermia, on the induction and repair of these lesions is described. Radiation-sensitive cell lines are also included.Paper given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Model for radial dependence of frequency distributions for energy imparted in nanometer volumes from HZE particles

This paper develops a deterministic model of frequency distributions for energy imparted (total energy deposition) in small volumes similar to DNA molecules from high-energy ions of interest for space radiation protection and cancer therapy. Frequency distributions for energy imparted are useful for considering radiation quality and for modeling biological damage produced by ionizing radiation. For high-energy ions, secondary electron (delta-ray) tracks originating from a primary ion track make dominant contributions to energy deposition events in small volumes. Our method uses the distribution of electrons produced about an ion's path and incorporates results from Monte Carlo simulation of electron tracks to predict frequency distributions for ions, including their dependence on radial distance. The contribution from primary ion events is treated using an impact parameter formalism of spatially restricted linear energy transfer (LET) and energy-transfer straggling. We validate our model by comparing it directly to results from Monte Carlo simulations for proton and alpha-particle tracks. We show for the first time frequency distributions of energy imparted in DNA structures by several high-energy ions such as cosmic-ray iron ions. Our comparison with results from Monte Carlo simulations at low energies indicates the accuracy of the method.     

Damage to cellular DNA from particulate radiations,the efficacy of its processing and the radiosensitivity of mammalian cells

Summary For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles.From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of direct and indirect action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a).Increases in linear energy transfer (LET) up to 100–200 keV µm^–1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make significant contributions to the relative biological effectiveness (RBE) of a given radiation. As the LET is raised above a few hundred keV µm^–1, the damage associated with DSBs continues to increase, but the efficiency of DSB induction declines to low values (0.1), as do RBE and the effective processing of DSBs and chromatin breaks, and the decline in RBE seems to mimic the overall decline in suitable processing of DSBs. Hence, the quality factor (Q) for a given radiation cannot be based solely upon the pattern of energy deposition, a fact attested to also by the quite different RBE responses exhibited by repair-deficient mutant (or variant) cells.Understanding of the biological effects of heavy ions is important not only for the welfare of astronauts who will undertake extended interplanetary missions in space but also for the facilitation of a rigorous scientific basis for conventional radiation therapy.Based on a review lecture delivered at the Fourth Workshop on Heavy Charged Particles in Biology and Medicine, GSl, Darmstadt, Germany, September 1991. A number of scientists, see Acknowledgements, also gave unreservedly of their time at the 40th Annual Meeting of the Radiation Research Society, Salt Lake City, Utah, March 1992, to discuss topics in this article with the author     

A model of interactions between radiation-induced oxidative stress, protein and DNA damage in Deinococcus radiodurans

Ionizing radiation triggers oxidative stress, which can have a variety of subtle and profound biological effects. Here we focus on mathematical modeling of potential synergistic interactions between radiation damage to DNA and oxidative stress-induced damage to proteins involved in DNA repair/replication. When sensitive sites on these proteins are attacked by radiation-induced radicals, correct repair of dangerous DNA lesions such as double strand breaks (DSBs) can be compromised. In contrast, if oxidation of important proteins is prevented by strong antioxidant defenses, DNA repair may function more efficiently. These processes probably occur to some extent even at low doses of radiation/oxidative stress, but they are easiest to investigate at high doses, where both DNA and protein damage are extensive. As an example, we use data on survival of Deinococcus radiodurans after high doses (thousands of Gy) of acute and chronic irradiation. Our model of radiogenic oxidative stress is consistent with these data and can potentially be generalized to other organisms and lower radiation doses.     

Detection of clustered DNA lesions: Biological and clinical applications

Humans are daily exposed to background radiation and various sources of oxidative stress. My research has focused in the last 12 years on the effects of ionizing radiation on DNA, which is considered as the key target of radiation in the cell. Ionizing radiation and endogenous cellular oxidative stress can also induce closely spaced oxidatively induced DNA lesions called "clusters" of DNA damage or locally multiply damage sites, as first introduced by John Ward. I am now interested in the repair mechanisms of clustered DNA damage, which is considered as the most difficult for the cell to repair. A main part of my research is devoted to evaluating the role of clustered DNA damage in the promotion of carcinogenesis in vitro and in vivo . Currently in my laboratory, there are two main ongoing projects. (1) Study of the role of BRCA1 and DNA-dependent protein kinase catalytic subunit repair proteins in the processing of clustered DNA damage in human cancer cells. For this project, we use several tumor cell lines, such as breast cancer cell lines MCF-7 and HCC1937 (BRCA1 deficient) and human glioblastoma cells MO59J/K; and (2) Possible use of DNA damage clusters as novel cancer biomarkers for prognostic and therapeutic applications related to modulation of oxidative stress. In this project human tumor and mice tissues are being used.     

Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition

Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF) per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from ⁶⁰Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This comparison allowed us to calculate a volume of 1.6 μm³ for which the spread of the specific energy distribution could explain the entire variability of RIF counts per cell in an exposed cell population. The definition of this volume may allow to use a microdosimetric quantity to predict heterogeneity in DNA damage. Moreover, this value is consistent with the order of magnitude of the volume occupied by the hydrated sugar-phosphate backbone of the DNA molecule, which is the part of the DNA molecule responsible for strand breaks.     

RNF8/RNF168信号通路在DNA损伤修复中的作用

细胞内DNA会受部分外界因素(如紫外辐射,电离辐射和化学毒素)和内部因素(如复制错误)的影响而发生损伤,包括DNA双链断裂、DNA错配和DNA交链等。DNA损伤发生后,损伤部位会被一些蛋白识别,进而招募一系列蛋白至损伤部位,形成一个修复系统。DNA双链断裂是最严重的一种DNA损伤,错误修复往往导致疾病的发生。DNA双链断裂(double strand break, DSB)后,细胞启动RNF8/RNF168信号通路进行修复。RNF8和RNF168是这条通路的枢纽蛋白;53BP和BRCA1是关键的效应蛋白,决定着DSB修复的方式;组蛋白泛素化、磷酸化和甲基化等翻译后修饰是这条通路顺利进行的基本条件;染色质重塑、泛素化酶/去泛素化酶平衡和蛋白稳定性是这条通路的主要调节方式。本综述对RNF8/RNF168信号通路进行了梳理总结,希望其能对相关研究者起到参考作用。     

Mutagenic and chromosomal events in radiation transformation

The transformation of a normal cell to a cancer cell is a complex multi-stage process. Data are presented from rodent cells which suggest that the initial radiation induced change does not represent a mutation in a specific structural gene or group of genes. Rather, DNA damage induced by radiation produces a heritable change which leads to the transformation of one or more of the progeny of the initial irradiated cells at some later time. This second rare event has certain characteristics of a mutation. Studies in human diploid cells indicate that radiation induces stable chromosomal rearrangements which persist throughout the lifespan of the cells in culture. Occasionally, such cells gain a selective growth advantage and are recognized as abnormal clones. These clones may expand to include the entire cell population and show a significantly prolonged lifespan in vitro. The hypothesis is presented that the transforming event occurs in such clones, possibly resulting from a mutational change which occurs at random during cellular proliferation.     

离子束生物效应及应用中值得研究的几个问题

重离子束生物效应及重离子束在生命科学中的应用研究,在国内外物理学与生命科学领域中得到了广泛的开展,但对出现的一些现象还没有深刻地揭露其本质,作出机理性解释。为了深入研究,本文提出一些值得研究的问题供参考,如:重离子径迹结构及能量沉积分布模型,DNA辐射敏感位点,质量沉积-分子改造,直接作用与间接作用,放射性核束的应用等。     

Radionuclide-induced evolution of DNA and the origin of life

Summary Artesian groundwaters of high radionuclide concentration are ubiquitous and may have provided the large, sustained energy sources that were required to drive the multistage process of DNA and primordial cell evolution. The rapid, early development of the genetic code as well as its degeneracy can be attributed to exceptionally high radiation-induced mutation rates in this unique environment. The ability of double-strand DNA to direct enzymatic repair of radiation damage to single strands contributed importantly to its selective evolution. It is postulated that the polymerization of nucleotides took place at elevated temperatures within -particle tracks of high ion and free-radical density, followed by rapid quenching to ambient conditions. It also is evident that radiation resistance and ploidy were important selection factors in cellular evolution.     

Effect of ionizing radiation on the DNA damage response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The DNA damage response (DDR) is induced by various DNA damaging factors and maintains genome stability in all organisms. The Chlamydomonas reinhardtii genome contains putative homologous genes involved in DDR; however, little is known about the functions and responses of these genes to DNA damage. In this study, DDR by gamma radiation was determined in C. reinhardtii. Irradiation with 80, and 200 Gy gamma radiation caused death in approximately 47 and 97 % of C. reinhardtii cells, respectively. The absolute lethality of cells was at 300 Gy. The rate of DNA breaks was also determined using comet assays after exposure to different doses of gamma radiation. Irradiation with 80 and 400 Gy resulted in 17 and 34 % of nuclear degradation in C. reinhardtii cells, respectively. To identify the major DDR pathway of C. reinhardtii induced by gamma radiation, 24 putative DDR genes were selected from the Joint Genome Institute (JGI) database. Gamma radiation significantly affected expression of 15 genes among these. Therefore, these genes displaying expressional changes by gamma radiation are involved in DDR, which indicate that C. reinhardtii may possess a fundamental conserved DDR pathway with higher plants. Furthermore, radiation responsive proteins were identified by proteomic analysis, which are involved in metabolisms of carbohydrate, energy, and photosynthesis. This is the first report to describe the responses of DDR homologous genes to gamma radiation and to identify gamma radiation-responsive proteins in C. reinhardtii. Our data should provide molecular insights into gamma radiation responses including DNA damage in green algae.     

DNA complex lesions induced by protons and ⋅-particles: track structure characteristics determining linear energy transfer and particle type dependence

The yield of DNA double-strand breaks (dsb) and DNA complex lesions induced by protons and α-particles of various energies was simulated using a Monte Carlo track structure code (MOCA15) and a simple model of the DNA molecule. DNA breaks of different complexity were analysed. The linear energy transfer (LET) and particle-type dependence of lesions of higher complexity seems to confirm the importance of clustered damage in DNA as a relevant step leading to biological endpoints such as cell inactivation. The detailed structure of proton and α-particle tracks was analysed to identify the main characteristics possibly responsible for such a dependence. The role of the primary ion and of its secondary electrons in inducing dsb and complex lesions is described, showing that the relative contribution of secondary electron tracks alone in inducing clustered lesions is almost negligible at high LET, but tends to dominate below ≈10 keV/μm. This is consistent with the observed similar effectiveness of low-LET fast particle radiation and sparsely ionizing radiation such as x-rays. The dependence on LET and particle type is mainly due to energy deposition events of the primary ion together with short range electrons surrounding the ion track; the yield of complex lesions due to secondary electron tracks alone is substantially LET independent. The radial distributions of the energy contributing to the induction of complex lesions were analyzed and compared with the radial distributions of energy deposition of the full tracks. The results suggest that the stochastic behaviour (i.e. cluster properties) of the energy deposition pattern within a radius of a few nanometers around the ion track plays a relevant role in determining the biological radiation effectiveness. Received: 20 December 1996 / Accepted in revised form: 5 March 1997