A New Standard DNA Damage (SDD) Data Format.

Our understanding of radiation-induced cellular damage has greatly improved over the past few decades. Despite this progress, there are still many obstacles to fully understand how radiation interacts with biologically relevant cellular components, such as DNA, to cause observable end points such as cell killing. Damage in DNA is identified as a major route of cell killing. One hurdle when modeling biological effects is the difficulty in directly comparing results generated by members of different research groups. Multiple Monte Carlo codes have been developed to simulate damage induction at the DNA scale, while at the same time various groups have developed models that describe DNA repair processes with varying levels of detail. These repair models are intrinsically linked to the damage model employed in their development, making it difficult to disentangle systematic effects in either part of the modeling chain. These modeling chains typically consist of track-structure Monte Carlo simulations of the physical interactions creating direct damages to DNA, followed by simulations of the production and initial reactions of chemical species causing so-called "indirect" damages. After the induction of DNA damage, DNA repair models combine the simulated damage patterns with biological models to determine the biological consequences of the damage. To date, the effect of the environment, such as molecular oxygen (normoxic vs. hypoxic), has been poorly considered. We propose a new standard DNA damage (SDD) data format to unify the interface between the simulation of damage induction in DNA and the biological modeling of DNA repair processes, and introduce the effect of the environment (molecular oxygen or other compounds) as a flexible parameter. Such a standard greatly facilitates inter-model comparisons, providing an ideal environment to tease out model assumptions and identify persistent, underlying mechanisms. Through inter-model comparisons, this unified standard has the potential to greatly advance our understanding of the underlying mechanisms of radiation-induced DNA damage and the resulting observable biological effects when radiation parameters and/or environmental conditions change.

BIOLOGICAL EFFECTIVENESS OF LOWER-ENERGY PHOTONS FOR CANCER RISK.

For risk assessment of photon exposures, the energy-dependence of relative effectiveness may be required. The NCRP has recently addressed this need by reviewing the available evidence from various fields of study and making recommendations for the effectiveness ratio of lower-energy photons, for the purposes of quantitative uncertainty analysis for specific cancer risk assessments where lower-energy photons and electrons are involved. The present paper provides a personal discussion of selected aspects of the evidence and analysis; it highlights the key role of low-energy electrons in determining biological effectiveness.

Classical approaches to microdosimetry, with example of use in radiation protection, medicine and mechanistic understanding.

Microdosimetry is the study and application of the microscopic features and stochastics of ionising radiations that cannot be described by the average macroscopic quantity absorbed dose. Microdosimetry is intimately related to radiation quality, but it also encompasses the inhomogeneities of interactions and energy depositions within a single radiation type. A variety of past and current approaches will be summarised and some examples given of implications to radiation protection and medicine, as well as to basic mechanistic studies of radiation effects.

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation.

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.

Chromosome breakpoint distribution of damage induced in peripheral blood lymphocytes by densely ionizing radiation.

PURPOSE: To assess the chromosomal breakpoint distribution in human peripheral blood lymphocytes (PBL) after exposure to a low dose of high linear energy transfer (LET) alpha-particles using the technique of multiplex fluorescence in situ hybridization (m-FISH). MATERIALS AND METHODS: Separated PBL were exposed in G0 to 0.5 Gy 238Pu alpha-particles, stimulated to divide and harvested approximately 48 - 50 hours after exposure. Metaphase cells were assayed by m-FISH and chromosome breaks identified. The observed distribution of breaks were then compared with expected distributions of breaks, calculated on the assumption that the distribution of breaks is random with regard to either chromosome volume or chromosome surface area. RESULTS: More breaks than expected were observed on chromosomes 2 and 11, however no particular region of either chromosome was identified as significantly contributing to this over-representation. The identification of hot or cold chromosome regions (pter,p,cen,q,qter) varied depending on whether the data were compared according to chromosome volume or surface area. CONCLUSIONS: A deviation from randomness in chromosome breakpoint distribution was observed, and this was greatest when data were compared according to the relative surface area of each individual chromosome (or region). The identification of breaks by m-FISH (i.e., more efficient observation of interchanges than intrachanges) and importance of territorial boundaries on interchange formation are thought to contribute to these differences. The significance of the observed non-random distribution of breaks on chromosomes 2 and 11 in relation to chromatin organization is unclear.

Increased complexity of radiation-induced chromosome aberrations consistent with a mechanism of sequential formation.

Complex chromosome aberrations (any exchange involving three or more breaks in two or more chromosomes) are effectively induced in peripheral blood lymphocytes (PBL) after exposure to low doses (mostly single particles) of densely ionising high-linear energy transfer (LET) alpha-particle radiation. The complexity, when observed by multiplex fluorescence in situ hybridisation (m-FISH), shows that commonly four but up to eight different chromosomes can be involved in each rearrangement. Given the territorial organisation of chromosomes in interphase and that only a very small fraction of the nucleus is irradiated by each alpha-particle traversal, the aim of this study is to address how aberrations of such complexity can be formed. To do this, we applied theoretical "cycle" analyses using m-FISH paint detail of PBL in their first cell division after exposure to high-LET alpha-particles. In brief, "cycle" analysis deconstructs the aberration "observed" by m-FISH to make predictions as to how it could have been formed in interphase. We propose from this that individual high-LET alpha-particle-induced complex aberrations may be formed by the misrepair of damaged chromatin in single physical "sites" within the nucleus, where each "site" is consistent with an "area" corresponding to the interface of two to three different chromosome territories. Limited migration of damaged chromatin is "allowed" within this "area". Complex aberrations of increased size, reflecting the path of alpha-particle nuclear intersection, are formed through the sequential linking of these individual sites by the involvement of common chromosomes.

Bound PCNA in nuclei of primary rat tracheal epithelial cells after exposure to very low doses of plutonium-238 alpha particles.

Bystander effects from ionizing radiation have been detailed for a number of cell systems and a number of end points. We wished to use a cell culture/ex vivo rat model of respiratory tissue to determine whether a bystander effect detected in culture could also be shown in a tissue. Examination by immunofluorescence techniques of tracheal cell cultures after exposure to very low doses of alpha particles revealed a large proportion of cells with proliferating cell nuclear antigen (PCNA) bound in their nuclei. PCNA was selected as an end point because it is involved in both DNA repair and the changes in cell cycle that are typical of many reported bystander effects. Maximum response can be detected in up to 28% of the cells in sub-confluent cultures with a dose of only 2 mGy. At this dose less than 2% of the cell nuclei have experienced a particle traversal and less than 6% of the cells have experienced an alpha-particle traversal through either their nucleus or some part of their cytoplasm. The hypothesis that this bystander response in nontargeted cells is mediated through secreted factor(s) is presented, and supporting evidence was found using partial irradiation and co-culture experiments. Examination of the effect with excised pieces of trachea demonstrated a response similar to that seen in culture.

Is the increased relative biological effectiveness of high LET particles due to spatial or temporal effects? Characterization and OER in V79-4 cells.

The efficiency of producing biological damage varies with radiation quality. Conventional explanations rely on spatial differences in the radiation track structure; generally however there are also very large temporal differences in delivery of the radiation at the cellular level. High-LET radiation normally deposits substantial amounts of energy by individual heavily ionizing tracks on a timescale of the order of picoseconds. By contrast each low-LET radiation track deposits a small amount of energy. Many of these tracks, distributed over the whole cell, are required to deliver an equivalent dose to a high-LET track and they are usually delivered over much longer timescales (typically seconds) during which chemical, biochemical and biological processes are occurring. In this paper the design, characterization and initial application of a high-brightness, laser-plasma ultrasoft x-ray source is described. This has been used to investigate the importance of the temporal differences by irradiating mammalian cells with an energy deposition with spatial properties of low-LET radiation and temporal properties similar to high-LET radiation. The present system delivers a typical dose, to the incident surface of the cells, of 0.12 Gy per pulse delivered in <10 ps. The capabilities of the x-ray source were tested by determining the survival of V79-4 hamster cells irradiated with picosecond pulses of ultrasoft x-rays under aerobic and anaerobic conditions, which were found to be consistent with previously published non pulsed data with x-rays of similar energy. These results support the expectation that the disappearance of an oxygen effect for high-LET radiation particles is due to their spatial properties rather than the very short timescale of each particle traversal. For other effects, particularly non-targeted phenomena such as induced genomic instability, expectations may be less clear cut.

Biological effectiveness of isolated short electron tracks: V79-4 cell inactivation following low dose-rate irradiation with Al(K) ultrasoft X-rays.

PURPOSE: To investigate the biological effect of single, isolated, short electron tracks (<70 nm) relevant to practical human exposures to low-linear energy transfer radiation. MATERIALS AND METHODS: An irradiation rig was constructed that allowed environmentally controlled, protracted irradiations with an individually prescribed dose to up to 20 samples over a period of days. Inactivation of V79-4 mammalian cells by Al(K) ultrasoft X-rays was studied at high and low dose-rates with a maximum exposure time of 42 h. RESULTS: A significant increase in clonogenic survival was observed at the higher doses when the exposure time was increased from <6 min to 21 h, with no further increase observed for 42-h exposures. Despite the short range of the low-energy electrons produced (<70 nm), significant cell inactivation was observed for these low dose-rate exposures. CONCLUSIONS: The results are consistent with the hypothesis that even individual tracks can be biologically effective.

Modelling of DNA damage induced by energetic electrons (100 eV to 100 keV).

Modelling and calculations are presented for the spectrum of initial DNA damage produced by 100 eV to 100 keV energetic electrons. Analysis of the initial spectrum of damage, based upon the source (direct energy deposition and reactions with diffusing OH radicals) and complexity of damage, indicates that the majority of the interactions cause no damage to DNA and any damage that does occur is most likely to be a simple single strand break (SSB). The fraction of complex damage for energetic electrons is lower than that induced by low energy electrons and ultrasoft X rays but still represents an appreciable fraction (20-30%) of the total double strand breaks (DSBs). Relative yields of strand breaks are investigated for dependence on the assumed energy deposition threshold and on the probability of the hydroxyl radicals to produce a single strand break. The ratio of direct to indirect damage does not change significantly across the electron energy range investigated and the values lie well within the experimental data. The direct energy deposition in DNA represents a larger proportion of the damage although the contribution from the hydroxyl radicals is also substantial, both in terms of the absolute yield of the breaks and the complexity of the damage.

Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays.

The large RBE (approximately 7) measured for the killing of Chinese hamster V79 cells by 340 eV ultrasoft X rays, which preferentially ionize the K shell of carbon atoms (Hervé du Penhoat et al., Radiat. Res. 151, 649-658, 1999), was used to investigate the location of sensitive sites for cell inactivation and the physical modes of action of radiation. The enhancement of the RBE above the carbon K-shell edge either may indicate a high intrinsic efficiency of carbon K-shell ionizations (due, for example, to a specific physical or chemical effect) or may be related to the preferential localization of these ionizations on the DNA. The second interpretation would indicate a strong local (within 3 nm) action of K-shell ionizations and consequently the importance of a direct mechanism for radiation lethality (without excluding an action in conjunction with an indirect component). To distinguish between these two hypotheses, the efficiencies of core ionizations in DNA atoms (phosphorus L-shell, carbon K-shell, and oxygen K-shell ionizations) to induce damages were investigated by measuring their capacities to produce DNA double-strand breaks (DSBs). The effect of photoionizations in isolated DNA was studied using pBS plasmids in a partially hydrated state. No enhancement of the efficiency of DSB induction by carbon K-shell ionizations compared to oxygen K-shell ionizations was found, supporting the hypothesis that it is the localization of these carbon K-shell events on DNA which gives to the 340 eV photons their high killing efficiency. In agreement with this interpretation, cell inactivation and DSB induction, which do not appear to be correlated when expressed in terms of yields per unit dose in the sample, exhibit a rather good correlation when expressed in terms of efficiencies per core event in the DNA. These results suggest that core ionizations in DNA, through core-hole relaxation in conjunction with localized effects of spatially correlated secondary and Auger electrons, may be the major critical events for cell inactivation, and that the resulting DSBs (or a constant fraction of these DSBs) may be a major class of unrepairable lesions.

Yields of SSB and DSB induced in DNA by Al(K) ultrasoft X-rays and alpha-particles: comparison of experimental and simulated yields.

PURPOSE: To compare experimental yields of single strand breaks (SSB) and double strand breaks (DSB) induced in plasmid DNA in aqueous solution by alpha-particles and Al(K) ultrasoft X-rays (USX) with the corresponding yields, generated via computer simulations, for a range of mean diffusion distances of the hydroxyl radical (*OH). MATERIALS AND METHODS: Aerobic, aqueous solutions of plasmid DNA were irradiated at 277K with 238Pu alpha-particles or USX in the presence of 10(-4) to 0.33 mol dm(-3) Tris and the yields of SSB and DSB determined by gel electrophoresis. Computer simulations, using Monte Carlo track-structure codes for 1.5keV electrons (CPA100) and 3.2MeV alpha-particle track segments (PITS), were used to obtain yields of DNA SSB and DSB at different *OH scavenger conditions. RESULTS: The experimental yield of SSB and DSB induced by AlK USX and SSB induced by alpha-particles and the dependences on the mean diffusion distance of the *OH are in reasonable agreement with the corresponding simulated yields and their corresponding dependences. However, for DSB induced by alpha-particles, a significant systematic difference exists between the simulated and experimental yields over the full *OH scavenging range, with the simulated yields being a factor of two to three greater than the experimental values. CONCLUSION: That the simulated yields of strand breaks are generally in reasonable agreement with those determined experimentally over a wide range of *OH scavenging capacities, increases confidence in the use of these simulations as a valuable source of quantitative, mechanistic information on DNA damage induced at very low radiation doses.

Computational approach for determining the spectrum of DNA damage induced by ionizing radiation.

To study the characteristics of molecular damage induced by ionizing radiation at the DNA level, Monte Carlo track simulation of energetic electrons and ions in liquid water, a canonical model of B-DNA, and a comprehensive classification of DNA damage in terms of the origin and complexity of damage were used to calculate the frequencies of simple and complex strand breaks. A threshold energy of 17.5 eV was used to model the damage by direct energy deposition, and a probability of 0.13 was applied to model the induction of a single-strand break produced in DNA by OH radical reactions. For preliminary estimates, base damage was assumed to be induced by the same direct energy threshold deposition or by the reaction of an OH radical with the base, with a probability of 0.8. Computational data are given on the complexity of damage, including base damage by electrons with energies of 100-4500 eV and ions with energies of 0.3-4.0 MeV/nucleon (59-9 keV microm(-1) protons and 170-55 keV microm(-1) alpha particles). Computational data are presented on the frequencies of single- and double-strand breaks induced as a function of the LET of the particles, and on the relative frequencies of complex single- and double-strand breaks for electrons. The modeling and calculations of strand breaks show that: (1) The yield of strand breaks per unit absorbed dose is nearly constant over a wide range of LET. (2) The majority of DNA damage is of a simple type, but the majority of the simple single-strand breaks are accompanied by at least one base damage. (3) For low-energy electrons, nearly 20-30% of the double-strand breaks are of a complex type by virtue of additional breaks. The proportion of this locally clustered damage increases with LET, reaching about 70% for the highest-LET alpha particles modeled, with the complexity of damage increasing further, to about 90%, when base damage is considered. (4) The extent of damage in the local hit region of the DNA duplex is mostly limited to a length of a few base pairs. (5) The frequency of base damage when no strand breaks are present in the hit segment of DNA varies between 20-40% as a function of LET for protons and alpha particles.

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA.

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.

Long-term genomic instability in human lymphocytes induced by single-particle irradiation.

Recent evidence suggests that genomic instability, which is an important step in carcinogenesis, may be important in the effectiveness of radiation as a carcinogen, particularly for high-LET radiations. Understanding the biological effects underpinning the risks associated with low doses of densely ionizing radiations is complicated in experimental systems by the Poisson distribution of particles that can be delivered. In this study, we report an approach to determine the effect of the lowest possible cellular radiation dose of densely ionizing alpha particles, that of a single particle traversal. Using microbeam technology and an approach for immobilizing human T-lymphocytes, we have measured the effects of single alpha-particle traversals on the surviving progeny of cells. A significant increase in the proportion of aberrant cells is observed 12-13 population doublings after exposure, with a high level of chromatid-type aberrations, indicative of an instability phenotype. These data suggest that instability may be important in situations where even a single particle traverses human cells.

Monte Carlo track structure for radiation biology and space applications.

Over the past two decades event by event Monte Carlo track structure codes have increasingly been used for biophysical modelling and radiotherapy. Advent of these codes has helped to shed light on many aspects of microdosimetry and mechanism of damage by ionising radiation in the cell. These codes have continuously been modified to include new improved cross sections and computational techniques. This paper provides a summary of input data for ionizations, excitations and elastic scattering cross sections for event by event Monte Carlo track structure simulations for electrons and ions in the form of parametric equations, which makes it easy to reproduce the data. Stopping power and radial distribution of dose are presented for ions and compared with experimental data. A model is described for simulation of full slowing down of proton tracks in water in the range 1 keV to 1 MeV. Modelling and calculations are presented for the response of a TEPC proportional counter irradiated with 5 MeV alpha-particles. Distributions are presented for the wall and wall-less counters. Data shows contribution of indirect effects to the lineal energy distribution for the wall counters responses even at such a low ion energy.

Kinetics of DSB rejoining and formation of simple chromosome exchange aberrations.

PURPOSE: To investigate the role of kinetics in the processing of DNA double strand breaks (DSB), and the formation of simple chromosome exchange aberrations following X-ray exposures to mammalian cells based on an enzymatic approach. METHODS: Using computer simulations based on a biochemical approach, rate-equations that describe the processing of DSB through the formation of a DNA-enzyme complex were formulated. A second model that allows for competition between two processing pathways was also formulated. The formation of simple exchange aberrations was modelled as misrepair during the recombination of single DSB with undamaged DNA. Non-linear coupled differential equations corresponding to biochemical pathways were solved numerically by fitting to experimental data. RESULTS: When mediated by a DSB repair enzyme complex, the processing of single DSB showed a complex behaviour that gives the appearance of fast and slow components of rejoining. This is due to the time-delay caused by the action time of enzymes in biomolecular reactions. It is shown that the kinetic- and dose-responses of simple chromosome exchange aberrations are well described by a recombination model of DSB interacting with undamaged DNA when aberration formation increases with linear dose-dependence. Competition between two or more recombination processes is shown to lead to the formation of simple exchange aberrations with a dose-dependence similar to that of a linear quadratic model. CONCLUSIONS: Using a minimal number of assumptions, the kinetics and dose response observed experimentally for DSB rejoining and the formation of simple chromosome exchange aberrations are shown to be consistent with kinetic models based on enzymatic reaction approaches. A non-linear dose response for simple exchange aberrations is possible in a model of recombination of DNA containing a DSB with undamaged DNA when two or more pathways compete for DSB repair.

Calculation of the neutron W value for neutron dosimetry below the MeV energy region.

The effective neutron W value for tissue-equivalent gas in the energy region from 5 keV to 5.7 MeV has been calculated using W values for recoil particles (protons, alpha particles, oxygen, carbon and nitrogen ions), which are produced by incident neutrons. The W value is assumed to be an energy-fluence-average over the W values of the recoil particles. The energy fluence spectra for the recoil particles are calculated by using a continuous slowing down approximation (CSDA). For the W values of recoil particles in the low-energy region, the recently evaluated data by Siebert et al and Taylor et al were used. Results are presented which show that the effective neutron W value depends strongly on energy in the low-energy region. This result indicates that neutron dose measurements using ionization chambers need a considerable correction of the W value in the low-energy region.