Clustered DNA Damage and its Complexity: Tracking the History.

The concept of radiation-induced clustered damage in DNA has grown over the past several decades to become a topic of considerable interest across the scientific disciplines involved in studies of the biological effects of ionizing radiation. This paper, prepared for the 70th anniversary issue of Radiation Research, traces historical development of the three main threads of physics, chemistry, and biochemical/cellular responses that led to the hypothesis and demonstration that a key component of the biological effectiveness of ionizing radiation is its characteristic of producing clustered DNA damage of varying complexities. The physics thread has roots that started as early as the 1920s, grew to identify critical nanometre-scale clusterings of ionizations relevant to biological effectiveness, and then, by the turn of the century, had produced an extensive array of quantitative predictions on the complexity of clustered DNA damage from different radiations. Monte Carlo track structure simulation techniques played a key role through these developments, and they are now incorporated into many recent and ongoing studies modelling the effects of radiation. The chemistry thread was seeded by water-radiolysis descriptions of events in water as radical-containing 'spurs,' demonstration of the important role of the hydroxyl radical in radiation-inactivation of cells and the difficulty of protection by radical scavengers. This led to the concept and description of locally multiply damaged sites (LMDS) for DNA double-strand breaks and other combinations of DNA base damage and strand breakage that could arise from a spur overlapping, or created in very close proximity to, the DNA. In these ways, both the physics and the chemistry threads, largely in parallel, put out the challenge to the experimental research community to verify these predictions of clustered DNA damage from ionizing radiations and to investigate their relevance to DNA repair and subsequent cellular effects. The third thread, biochemical and cell-based research, responded strongly to the challenge by demonstrating the existence and biological importance of clustered DNA damage. Investigations have included repair of a wide variety of defined constructs of clustered damage, evaluation of mutagenic consequences, identification of clustered base-damage within irradiated cells, and identification of co-localization of repair complexes indicative of complex clustered damage after high-LET irradiation, as well as extensive studies of the repair pathways involved in repair of simple double-strand breaks. There remains, however, a great deal more to be learned because of the diversity of clustered DNA damage and of the biological responses.

Neutrons are forever! Historical perspectives.

Purpose: Neutrons were an active field of radiobiology at the time of publication of the first issues of the International Journal of Radiation Biology in 1959. Three back-to-back papers published by Neary and his colleagues contain key elements of interest at the time. The present article aims to put these papers into context with the discovery of the neutron 27 years previously and then give a feel for how the field has progressed to the present day. It does not intend to provide a comprehensive review of this enormous field, but rather to provide selective summaries of main driving forces and developments. Conclusions: Neutron radiobiology has continued as a vigorous field of study throughout the past 84 years. Main driving forces have included concern for protection from the harmful effects of neutrons, exploitation and optimization for cancer therapy (fast beam therapy, brachytherapy and boron capture therapy), and scientific curiosity about the mechanisms of radiation action. Effort has fluctuated as the emphasis has shifted from time to time, but all three areas remain active today. Whatever the future holds for the various types of neutron therapy, the health protection aspects will remain with us permanently because of natural environmental exposure to neutrons as well as increased additional exposures from a variety of human activities.

A restatement of the natural science evidence base concerning the health effects of low-level ionizing radiation.

Exposure to ionizing radiation is ubiquitous, and it is well established that moderate and high doses cause ill-health and can be lethal. The health effects of low doses or low dose-rates of ionizing radiation are not so clear. This paper describes a project which sets out to summarize, as a restatement, the natural science evidence base concerning the human health effects of exposure to low-level ionizing radiation. A novel feature, compared to other reviews, is that a series of statements are listed and categorized according to the nature and strength of the evidence that underpins them. The purpose of this restatement is to provide a concise entrée into this vibrant field, pointing the interested reader deeper into the literature when more detail is needed. It is not our purpose to reach conclusions on whether the legal limits on radiation exposures are too high, too low or just right. Our aim is to provide an introduction so that non-specialist individuals in this area (be they policy-makers, disputers of policy, health professionals or students) have a straightforward place to start. The summary restatement of the evidence and an extensively annotated bibliography are provided as appendices in the electronic supplementary material.

Dependence and independence of survival parameters on linear energy transfer in cells and tissues.

Carbon-ion radiotherapy has been used to treat more than 9000 cancer patients in the world since 1994. Spreading of the Bragg peak is necessary for carbon-ion radiotherapy, and is designed based on the linear-quadratic model that is commonly used for photon therapy. Our recent analysis using in vitro cell kills and in vivo mouse tissue reaction indicates that radiation quality affects mainly the alpha terms, but much less the beta terms, which raises the question of whether this is true in other biological systems. Survival parameters alpha and beta for 45 in vitro mammalian cell lines were obtained by colony formation after irradiation with carbon ions, fast neutrons and X-rays. Relationships between survival parameters and linear energy transfer (LET) below 100 keV/µm were obtained for 4 mammalian cell lines. Mouse skin reaction and tumor growth delay were measured after fractionated irradiation. The Fe-plot provided survival parameters of the tissue reactions. A clear separation between X-rays and high-LET radiation was observed for alpha values, but not for beta values. Alpha values/terms increased with increasing LET in any cells and tissues studied, while beta did not show a systematic change. We have found a puzzle or contradiction in common interpretations of the linear-quadratic model that causes us to question whether the model is appropriate for interpreting biological effectiveness of high-LET radiation up to 500 keV/µm, probably because of inconsistency in the concept of damage interaction. A repair saturation model proposed here was good enough to fit cell kill efficiency by radiation of wide-ranged LET. A model incorporating damage complexity and repair saturation would be suitable for heavy-ion radiotherapy.

Galactic cosmic ray simulation at the NASA Space Radiation Laboratory.

Most accelerator-based space radiation experiments have been performed with single ion beams at fixed energies. However, the space radiation environment consists of a wide variety of ion species with a continuous range of energies. Due to recent developments in beam switching technology implemented at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), it is now possible to rapidly switch ion species and energies, allowing for the possibility to more realistically simulate the actual radiation environment found in space. The present paper discusses a variety of issues related to implementation of galactic cosmic ray (GCR) simulation at NSRL, especially for experiments in radiobiology. Advantages and disadvantages of different approaches to developing a GCR simulator are presented. In addition, issues common to both GCR simulation and single beam experiments are compared to issues unique to GCR simulation studies. A set of conclusions is presented as well as a discussion of the technical implementation of GCR simulation.

Alpha-Particle-Induced Complex Chromosome Exchanges Transmitted through Extra-Thymic Lymphopoiesis In Vitro Show Evidence of Emerging Genomic Instability.

Human exposure to high-linear energy transfer α-particles includes environmental (e.g. radon gas and its decay progeny), medical (e.g. radiopharmaceuticals) and occupational (nuclear industry) sources. The associated health risks of α-particle exposure for lung cancer are well documented however the risk estimates for leukaemia remain uncertain. To further our understanding of α-particle effects in target cells for leukaemogenesis and also to seek general markers of individual exposure to α-particles, this study assessed the transmission of chromosomal damage initially-induced in human haemopoietic stem and progenitor cells after exposure to high-LET α-particles. Cells surviving exposure were differentiated into mature T-cells by extra-thymic T-cell differentiation in vitro. Multiplex fluorescence in situ hybridisation (M-FISH) analysis of naïve T-cell populations showed the occurrence of stable (clonal) complex chromosome aberrations consistent with those that are characteristically induced in spherical cells by the traversal of a single α-particle track. Additionally, complex chromosome exchanges were observed in the progeny of irradiated mature T-cell populations. In addition to this, newly arising de novo chromosome aberrations were detected in cells which possessed clonal markers of α-particle exposure and also in cells which did not show any evidence of previous exposure, suggesting ongoing genomic instability in these populations. Our findings support the usefulness and reliability of employing complex chromosome exchanges as indicators of past or ongoing exposure to high-LET radiation and demonstrate the potential applicability to evaluate health risks associated with α-particle exposure.

Variation in RBE for Survival of V79-4 Cells as a Function of Alpha-Particle (Helium Ion) Energy.

High linear energy transfer (LET) α particles are important with respect to the carcinogenic risk associated with human exposure to ionizing radiation, most notably to radon and its progeny. Additionally, the potential use of alpha-particle-emitting radionuclides in radiotherapy is increasingly being explored. Within the body the emitted alpha particles slow down, traversing a number of cells with a range of energies and therefore with varying efficiencies at inducing biological response. The LET of the particle typically rises from between ~70-90 keV µm(-1) at the start of the track (depending on initial energy) to a peak of ~237 keV µm(-1) towards the end of the track, before falling again at the very end of its range. To investigate the variation in biological response with incident energy, a plutonium-238 alpha-particle irradiator was calibrated to enable studies with incident energies ranging from 4.0 MeV down to 1.1 MeV. The variation in clonogenic survival of V79-4 cells was determined as a function of incident energy, along with the relative variation in the initial yields of DNA double-strand breaks (DSB) measured using the FAR assay. The clonogenic survival data also extends previously published data obtained at the Medical Research Council (MRC), Harwell using the same cells irradiated with helium ions, with energies ranging from 34.9 MeV to 5.85 MeV. These studies were performed in conjunction with cell morphology measurements on live cells enabling the determination of absorbed dose and calculation of the average LET in the cell. The results show an increase in relative biological effectiveness (RBE) for cell inactivation with decreasing helium ion energy (increasing LET), reaching a maximum for incident energies of ~3.2 MeV and corresponding average LET of 131 keV µm(-1), above which the RBE is observed to fall at lower energies (higher LETs). The effectiveness of single alpha-particle traversals (relevant to low-dose exposure) at inducing cell inactivation was observed to increase with decreasing energy to a peak of ~68% survival probability for incident energies of ~1.8 MeV (average LET of 190 keV µm(-1)) producing ~0.39 lethal lesions per track. However, the efficiency of a single traversal will also vary significantly with cell morphology and angle of incidence, as well as cell type.

The influence of dose rate on the induction of chromosome aberrations and gene mutation after exposure of plateau phase V79-4 cells with high-LET alpha particles.

While protracting exposures of low-LET radiations usually leads to a reduction in their effectiveness for a given dose, for high-LET radiation there is now substantial evidence for what has been called an inverse dose-rate effect, where under certain circumstances there is an increase in carcinogenesis or other biological effects, with decreasing dose rate. This study investigates the influence of dose rate on the induction of chromosome aberrations and gene mutations after irradiation of plateau phase V79-4 cells with high-LET alpha particles. The induction of chromosomal aberrations exhibited a linear relationship with dose and showed evidence of a small but significant conventional dose-rate dependence, with low-dose-rate exposures (0.28 Gy h(-1)) being less effective by about 20% (ratio 0.82 ± 0.04) compared to acute exposures. However no significant dose-rate effect was observed for cell survival or the induction of mutations in the HPRT gene for low-dose-rate exposure (8.0 × 10(-5) and 1.5 × 10(-2) Gy h(-1) for exposure of 0.36 and 0.69 Gy, respectively) when compared to acute exposures.

Evidence relevant to untargeted and transgenerational effects in the offspring of irradiated parents.

In this article we review health effects in offspring of human populations exposed as a result of radiotherapy and some groups exposed to chemotherapy. We also assess risks in offspring of other radiation-exposed groups, in particular those of the Japanese atomic bomb survivors and occupationally and environmentally exposed groups. Experimental findings are also briefly surveyed. Animal and cellular studies tend to suggest that the irradiation of males, at least at high doses (mostly 1Gy and above), can lead to observable effects (including both genetic and epigenetic) in the somatic cells of their offspring over several generations that are not attributable to the inheritance of a simple mutation through the parental germline. However, studies of disease in the offspring of irradiated humans have not identified any effects on health. The available evidence therefore suggests that human health has not been significantly affected by transgenerational effects of radiation. It is possible that transgenerational effects are restricted to relatively short times post-exposure and in humans conception at short times after exposure is likely to be rare. Further research that may help resolve the apparent discrepancies between cellular/animal studies and studies of human health are outlined.

Panel discussion: Do non-targeted effects impact the relation between microdosimetry and risk?

An invited panel discussed with the participants of the Symposium their views on the impact of non-targeted effects on the relationship between microdosimetry and risk. The discussion was wide ranging and lively, with divergences of opinion. At one end was the view that understanding microscopic features and distributions of radiation was an essential component to understanding risk, especially at low doses, and additionally that substantial questions and uncertainties arose from lack of information on the mechanisms and consequences of non-targeted effects. At the other end, it was held that microdosimetry and non-targeted effects are irrelevant to practical radiation protection because protection practice is already securely founded on macroscopic organ doses and direct epidemiological observations of risk. A summary of the discussion is provided in the sequence in which it took place. The session illustrated the underlying mixture of fundamental and practical issues and the need for increased scientific knowledge.

New radiobiological, radiation risk and radiation protection paradigms.

The long-standing conventional paradigm for radiobiology has formed a logical basis for the standard paradigm for radiation risk of cancer and heritable effects and, from these paradigms, has developed the internationally applied system for radiation protection, but with many simplifications, assumptions and generalizations. A variety of additional radiobiological phenomena that do not conform to the standard paradigm for radiobiology may have potential implications for radiation risk and radiation protection. It is suggested, however, that the current state of knowledge is still insufficient for these phenomena, individually or collectively, to be formulated systematically into a new paradigm for radiobiology. Additionally, there is at present lack of direct evidence of their relevance to risk for human health, despite attractive hypotheses as to how they might be involved. Finally, it remains to be shown how incorporation of such phenomena into the paradigm for radiation protection would provide sufficient added value to offset disruption to the present widely applied system. Further research should aim for better mechanistic understanding of processes such as radiation-induced genomic instability (for all radiation types) and bystander effects (particularly for low-fluence high-LET particles) and also priority should be given to confirmation, or negation, of the relevance of the processes to human health risks from radiation.

Fifth Warren K. Sinclair Keynote Address: Issues in quantifying the effects of low-level radiation.

Health risks from exposure to high doses of ionizing radiation are well characterized from epidemiological studies. Uncertainty and controversy remain for extension of these risks to the low doses and low dose rates of particular relevance in the workplace, in medical diagnostics and screening, and from background radiations. In order to make such extrapolations, a number of concepts have been developed for radiation protection, partly on the basis of assumed processes in the mechanisms of radiation carcinogenesis. Included amongst these are the assumptions of a linear no-threshold dose response and simple scaling factors for dose rate and radiation quality. With a progressive reduction in recommended dose limits over the past half century, these approaches have had considerable success in protecting humans. But do they go far enough or, conversely, are they overprotective? Four selected underlying aspects are considered. It is concluded that (1) even the lowest dose of radiation has the capability to cause complex DNA damage that can lead to a variety of permanent cellular changes; (2) the unique clustered characteristics of radiation damage, even at very low doses, enable it to stand out above the much larger quantity of endogenous DNA damage; (3) although a chromosome aberration may represent the rate-limiting initiating event for carcinogenesis, as is often assumed, direct evidence is still lacking; and (4) the extensive influence that dicentric aberrations have had on guiding extrapolations for radiation protection may be substantially misleading. Finally, some comments are offered on aspects that lie outside the current paradigm.

Understanding and characterisation of the risks to human health from exposure to low levels of radiation.

Exposure to ionising radiation can lead to a wide variety of health effects. Cancer is judged to be the main risk from radiation at low doses and low dose rates, and controlling this risk has been the main factor in developing radiation protection practice. Conventional paradigms of radiobiology and radiation carcinogenesis have served to guide extrapolations of epidemiological data on exposed human populations, so as to estimate risks at low doses and low dose rates, to other types of ionising radiation and to non-uniform exposures. These paradigms are founded on a century of experimental and theoretical studies, but nevertheless there remain many uncertainties. Major assumptions and simplifications have been introduced to achieve a practical system of additive doses (and implied risks) for radiation protection. Advancing epidemiological studies and experimental research continue to reduce uncertainties in some areas while, in others, they raise new challenges to the generality and applicability of the conventional paradigms.

The relevance of dose for low-energy beta emitters.

Specific issues in risk assessment for low-energy beta emitters include specification of the radiation weighting factor, values of relative biological effectiveness for specific or accurate risk estimates, non-uniformities of dose within tissues and cells, and use of standard tissue weighting factors for non-uniform situations. Unusual features of low-energy beta emitters include: increased average ionisation density on subcellular (and cellular) scales; short ranges of the beta electrons; non-uniformity of the absorbed dose over subcellular, cellular, and tissue dimensions; reduced hit frequencies; nuclear transmutations; different chemical forms, influencing biokinetics and dose distributions; and large isotopic mass differences, particularly in the case of tritium and hydrogen. Many of these features are not included explicitly in conventional radiation protection dosimetry, although they may be partly included in experimental determinations of relative biological effectiveness. Theoretical and experimental studies have shown low-energy electrons to be particularly efficient in producing double-strand breaks in DNA, including complex double-strand breaks. Hence, on fundamental grounds, tritium beta particles should be expected to have greater biological effectiveness per unit absorbed dose than (60)Co gamma-rays or orthovoltage x-rays. For practical purposes, and in view of the paucity of epidemiological estimates of risk from low-energy electrons, consideration should be given to applying a raised relative biological effectiveness, say of value 2, to all low-energy internal emitters, including beta particles and soft x-ray emissions.

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells.

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine.

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.

Energy deposition stochastics and track structure: what about the target?

The broad field of microdosimetry, as reflected in the proceedings of the 13 previous symposia in this series, has been largely concerned with the microscopic stochastics of energy deposition from ionising radiations of different qualities, the ways in which these can be described and the information that they can provide towards mechanistic understanding of the biological effects of radiation and for practical applications. Directions of the research have been strongly influenced by technical developments at particular times, most notably the tissue-equivalent proportional counter and later Monte Carlo track-structure simulation methods. Essential to the research have been evolving notions as to characteristics of the relevant biological targets, and in particular their sizes and structures in relation to the microscopic features of the radiation. Over the decades since the first Symposium on Microdosimetry, in 1967, emphasis has fluctuated from key targets being assumed to be of nanometre dimensions, then up to one micrometer, ten micrometers, and then back again to a few nanometres. Some of these historical threads are traced through the successive symposia, culminating in current emphasis on the predominant importance of clustered damage in DNA, first revealed by track-structure simulations, but tempered by recognition also of the contribution that novel 'non-targeted' effects may play in the overall biological consequences of radiation.

Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells.

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. 222Ra), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of 3He2+ ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups (> or = two-fold over controls) and was comparable whether cells were traversed by one or two 3He2+ ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer.

mFISH analysis reveals complexity of chromosome aberrations in individuals occupationally exposed to internal plutonium: a pilot study to assess the relevance of complex aberrations as biomarkers of exposure to high-LET alpha particles.

We recently demonstrated that a significant proportion of apparently stable insertions induced after exposure to a mean of one alpha particle/cell, detected using three-color FISH, were part of larger unstable complexes when visualized by 24-color FISH. Interestingly, regardless of the long-term persistence capability of the cell, the complexity of each alpha-particle-induced complex appeared to be specific to the nuclear traversal of a single alpha particle. To assess whether aberrations of a similar complexity are observed in vivo and also to examine the usefulness of detecting such aberrations as a biomarker of chronic exposure to alpha particles, we have carried out a limited pilot study of Russian workers with large body burdens of alpha-particle-emitting plutonium. We found unstable cells containing non-transmissible complex aberrations in all of the plutonium-exposed subjects analyzed by mFISH. In addition, all of the complexes seen were consistent with those previously observed in vitro. Non-transmissible complex aberrations were more common than transmissible-type complexes, consistent with ongoing/chronic exposure, and insertions were dominant features of both types of complex. Accordingly, this preliminary study supports the proposal that aberration complexity and non-transmissibility are the major cytogenetic features of alpha-particle exposure that could potentially be exploited as a specific indicator of chronic exposures to high-LET alpha particles.