The Central Role of Cytogenetics in Radiation Biology.

Radiation cytogenetics has a rich history seldom appreciated by those outside the field. Early radiobiology was dominated by physics and biophysical concepts that borrowed heavily from the study of radiation-induced chromosome aberrations. From such studies, quantitative relationships between biological effect and changes in absorbed dose, dose rate and ionization density were codified into key concepts of radiobiological theory that have persisted for nearly a century. This review aims to provide a historical perspective of some of these concepts, including evidence supporting the contention that chromosome aberrations underlie development of many, if not most, of the biological effects of concern for humans exposed to ionizing radiations including cancer induction, on the one hand, and tumor eradication on the other. The significance of discoveries originating from these studies has widened and extended far beyond their original scope. Chromosome structural rearrangements viewed in mitotic cells were first attributed to the production of breaks by the radiations during interphase, followed by the rejoining or mis-rejoining among ends of other nearby breaks. These relatively modest beginnings eventually led to the discovery and characterization of DNA repair of double-strand breaks by non-homologous end joining, whose importance to various biological processes is now widely appreciated. Two examples, among many, are V(D)J recombination and speciation. Rapid technological advancements in cytogenetics, the burgeoning fields of molecular radiobiology and third-generation sequencing served as a point of confluence between the old and new. As a result, the emergent field of "cytogenomics" now becomes uniquely positioned for the purpose of more fully understanding mechanisms underlying the biological effects of ionizing radiation exposure.

Dose-dependent Transmissibility of Chromosome Aberrations at First Mitosis after Exposure to Gamma Rays. I. Modeling and Implications Related to Risk Assessment.

The relationship between certain chromosomal aberration (CA) types and cell lethality is well established. On that basis we used multi-fluor in situ hybridization (mFISH) to tally the number of mitotic human lymphocytes exposed to graded doses of gamma rays that carried either lethal or nonlethal CA types. Despite the fact that a number of nonlethal complex exchanges were observed, the cells containing them were seldom deemed viable, due to coincident lethal chromosome damage. We considered two model variants for describing the dose responses. The first assumes independent linear-quadratic (LQ) dose response shapes for the yields of both lethal and nonlethal CAs. The second (simplified) variant assumes that the mean number of nonlethal CAs per cell is proportional to the mean number of lethal CAs per cell, meaning that the shapes and magnitudes of both aberration types differ only by a multiplicative proportionality constant. Using these models allowed us to assemble dose response curves for the frequency of aberration-bearing cells that would be expected to survive. This took the form of a joint probability distribution for cells containing ≥1 nonlethal CAs but having zero lethal CAs. The simplified second model variant turned out to be marginally better supported than the first, and the joint probability distribution based on this model yielded a crescent-shaped dose response reminiscent of those observed for mutagenesis and transformation for cells "at risk" (i.e. not corrected for survival). Among the implications of these findings is the suggestion that similarly shaped curves form the basis for deriving metrics associated with radiation risk models.

Destabilizing Effects of Ionizing Radiation on Chromosomes: Sizing up the Damage.

For long-term survival and evolution, all organisms have depended on a delicate balance between processes involved in maintaining stability of their genomes and opposing processes that lead toward destabilization. At the level of mammalian somatic cells in renewal tissues, events or conditions that can tip this balance toward instability have attracted special interest in connection with carcinogenesis. Mutations affecting DNA (and its subsequent repair) would, of course, be a major consideration here. These may occur spontaneously through endogenous cellular processes or as a result of exposure to mutagenic environmental agents. It is in this context that we discuss the rather unique destabilizing effects of ionizing radiation (IR) in terms of its ability to cause large-scale structural rearrangements to the genome. We present arguments supporting the conclusion that these and other important effects of IR originate largely from microscopically visible chromosome aberrations.

Robbing Peter to Pay Paul: Competition for Radiogenic Breaks During Rejoining Diminishes Curvature in the Dose Response for Simple Chromosome Exchanges.

The large majority of chromosome damage produced by ionizing radiations takes the form of exchange aberrations. For simple exchanges between two chromosomes, multi-fluor fluorescence in situ hybridization (mFISH) studies confirm that the dose response to X rays or gamma rays is quasilinear with dose. This result is in seeming conflict with generalized theories of radiation action that depend on the interaction of lesions as the source of curvature in dose-response relationships. A qualitative explanation for such "linearization" had been previously proposed but lacked quantitative support. The essence of this explanation is that during the rejoining of radiogenic chromosome breaks, competition for breaks (CFB) between different aberration types often results in formation of complex exchange aberrations at the expense of simple reciprocal exchange events. This process becomes more likely at high radiation doses, where the number of contemporaneous breaks is high and complex exchanges involving multiple breaks become possible. Here we provide mathematical support for this CFB concept under the assumption that the mean and variance for exchange complexity increase with radiation dose.

Occam's broom and the dirty DSB: cytogenetic perspectives on cellular response to changes in track structure and ionization density.

Given equal doses, it is well-known that densely ionizing radiations are more potent in causing a number of biological effects compared to sparsely ionizing radiations, such as x- or gamma rays. According to classical models of radiation action, this results from differences in the spatial distribution of lesions along charged particle tracks. In recent years investigators have been barraged with the alternative narrative that this is instead due to 'qualitative' differences in the types of molecular lesions that each type of radiation produces. The present review discusses, mainly from a cytogenetic perspective, the merits and shortcomings of these seemingly contradictory viewpoints. There may be a kernel of truth to the idea that qualitative differences in the types of molecular lesions produced at the nanometer level affect RBE/LET relationships, but to ignore the fact that such differences result from longer-range spatial distributions of lesions produced along charged particle tracks is an unjustifiably narrow stance tantamount to employing Occam's Broom. Not only are such spatial considerations indispensable in explaining the impact of ionization density upon higher-order biological endpoints, particularly chromosome aberrations, the explanations they provide render arguments based principally on the quality of IR damage largely superfluous.

Accounting for overdispersion of lethal lesions in the linear quadratic model improves performance at both high and low radiation doses.

PURPOSE: The linear-quadratic (LQ) model represents a simple and robust approximation for many mechanistically-motivated models of radiation effects. We believe its tendency to overestimate cell killing at high doses derives from the usual assumption that radiogenic lesions are distributed according to Poisson statistics. MATERIALS AND METHODS: In that context, we investigated the effects of overdispersed lesion distributions, such as might occur from considerations of microdosimetric energy deposition patterns, differences in DNA damage complexities and repair pathways, and/or heterogeneity of cell responses to radiation. Such overdispersion has the potential to reduce dose response curvature at high doses, while still retaining LQ dose dependence in terms of the number of mean lethal lesions per cell. Here we analyze several irradiated mammalian cell and yeast survival data sets, using the LQ model with Poisson errors, two LQ model variants with customized negative binomial (NB) error distributions, the Padé-linear-quadratic, and Two-component models. We compared the performances of all models on each data set by information-theoretic analysis, and assessed the ability of each to predict survival at high doses, based on fits to low/intermediate doses. RESULTS: Changing the error distribution, while keeping the LQ dose dependence for the mean, enables the NB LQ model variants to outperform the standard LQ model, often providing better fits to experimental data than alternative models. CONCLUSIONS: The NB error distribution approach maintains the core mechanistic assumptions of the LQ formalism, while providing superior estimates of cell survival following high doses used in radiotherapy. Importantly, it could also be useful in improving the predictions of low dose/dose rate effects that are of major concern to the field of radiation protection.

Directional Genomic Hybridization (dGH) for Detection of Intrachromosomal Rearrangements.

Fluorescence in situ Hybridization (FISH) techniques, including whole chromosome painting (WCP), spectral karyotyping (SKY), and multicolor FISH (mFISH), are used extensively to characterize and enumerate inter-chromosomal rearrangements (e.g., translocations). Directional genomic hybridization (dGH) is a relatively new cytogenomics-based methodology that combines the strand-specific strategy of Chromosome Orientation-FISH (CO-FISH) with bioinformatics-driven design of single-stranded DNA probe sets that are unique and of like orientation. Such a strategy produces directional probe sets that hybridize to one-and only one-chromatid of prepared (single-stranded) metaphase chromosomes, thereby facilitating high-resolution visualization of intra-chromosomal rearrangements, specifically inversions, and greatly improving our ability to detect such otherwise cryptic structural variants within the genome. In addition to its usefulness in the study of various disease states, including cancer, relevant applications of dGH include monitoring cytogenetic damage caused by exposure to clastogenic agents (e.g., ionizing radiation). dGH can be applied as a discovery tool to globally assess the integrity of the genome, but it can also be used in a more targeted fashion to interrogate fine structural changes at the kilobase level. Consequently, dGH is capable of providing significant mechanistic insight and information not easily obtainable by other approaches.

Estimation of Radiation Doses to U.S. Military Test Participants from Nuclear Testing: A Comparison of Historical Film-Badge Measurements, Dose Reconstruction and Retrospective Biodosimetry.

Retrospective radiation dose estimations, whether based on physical or biological measurements, or on theoretical dose reconstruction, are limited in their precision and reliability, particularly for exposures that occurred many decades ago. Here, we studied living U.S. military test participants, believed to have received high-dose radiation exposures during nuclear testing-related activities approximately six decades ago, with two primary goals in mind. The first was to compare three different approaches of assessing past radiation exposures: 1. Historical personnel monitoring data alone; 2. Dose reconstruction based on varying levels of completeness of individual information, which can include film badge data; and 3. Retrospective biodosimetry using chromosome aberrations in peripheral blood lymphocytes. The second goal was to use the collected data to make the best possible estimates of bone marrow dose received by a group with the highest military recorded radiation doses of any currently living military test participants. Six nuclear test participants studied had been on Rongerik Atoll during the 1954 CASTLE Bravo nuclear test. Another six were present at the Nevada Test Site (NTS) and/or Pacific Proving Ground (PPG) and were believed to have received relatively high-dose exposures at those locations. All were interviewed, and all provided a blood sample for cytogenetic analysis. Military dose records for each test participant, as recorded in the Defense Threat Reduction Agency's Nuclear Test Review and Information System, were used as the basis for historical film badge records and provided exposure scenario information to estimate dose via dose reconstruction. Dose to bone marrow was also estimated utilizing directional genomic hybridization (dGH) for high-resolution detection of radiation-induced chromosomal translocations and inversions, the latter being demonstrated for the first time for the purpose of retrospective biodosimetry. As the true dose for each test participant is not known these many decades after exposure, this study gauged the congruence of different methods by assessing the degree of correlation and degree of systematic differences. Overall, the best agreement between methods, defined by statistically significant correlations and small systematic differences, was between doses estimated by a dose reconstruction methodology that exploited all the available individual detail and the biodosimetry methodology derived from a weighted average dose determined from chromosomal translocation and inversion rates. Employing such a strategy, we found that the Rongerik veterans who participated in this study appear to have received, on average, bone marrow equivalent doses on the order of 300-400 mSv, while the NTS/ PPG participants appear to have received approximately 250-300 mSv. The results show that even for nuclear events that occurred six decades in the past, biological signatures of exposure are still present, and when taken together, chromosomal translocations and inversions can serve as reliable retrospective biodosimeters, particularly on a group-average basis, when doses received are greater than statistically-determined detection limits for the biological assays used.

Chromosome Translocations, Inversions and Telomere Length for Retrospective Biodosimetry on Exposed U.S. Atomic Veterans.

It has now been over 60 years since U.S. nuclear testing was conducted in the Pacific islands and Nevada, exposing military personnel to varying levels of ionizing radiation. Actual doses are not well-established, as film badges in the 1950s had many limitations. We sought a means of independently assessing dose for comparison with historical film badge records and dose reconstruction conducted in parallel. For the purpose of quantitative retrospective biodosimetry, peripheral blood samples from 12 exposed veterans and 12 age-matched (>80 years) veteran controls were collected and evaluated for radiation-induced chromosome damage utilizing directional genomic hybridization (dGH), a cytogenomics-based methodology that facilitates simultaneous detection of translocations and inversions. Standard calibration curves were constructed from six male volunteers in their mid-20s to reflect the age range of the veterans at time of exposure. Doses were estimated for each veteran using translocation and inversion rates independently; however, combining them by a weighted-average generally improved the accuracy of dose estimations. Various confounding factors were also evaluated for potential effects on chromosome aberration frequencies. Perhaps not surprisingly, smoking and age-associated increases in background frequencies of inversions were observed. Telomere length was also measured, and inverse relationships with both age and combined weighted dose estimates were observed. Interestingly, smokers in the non-exposed control veteran cohort displayed similar telomere lengths as those in the never-smoker exposed veteran group, suggesting that chronic smoking had as much effect on telomere length as a single exposure to radioactive fallout. Taken together, we find that our approach of combined chromosome aberration-based retrospective biodosimetry provided reliable dose estimation capability, particularly on a group average basis, for exposures above statistical detection limits.

A Cytogenetic Profile of Radiation Damage.

Most of the important biological effects associated with the exposure to ionizing radiations are mirrored at the chromosomal level. In all cases, changes in the levels of cytogenetic effects are associated with changes in absorbed dose, dose rate and radiation quality. Some of the complexities associated with the quantitative description of such changes in response can be circumvented by appealing to concepts embodied in what has been called the "mean inactivation dose". Additional metrics designed to provide LET-dependent "signatures" of damage have been employed with moderate degrees of success. These, along with some alternative approaches, are discussed in an effort to stimulate discussion, and to further work leading to a better understanding of mechanisms involved in the production and significance of chromosome aberrations after exposure to ionizing radiations.

Radiation quality and intra-chromosomal aberrations: Size matters.

The shift from plant to mammalian cell models in radiation cytogenetics hastened the development of methods suitable for the analysis of chromosome-type aberrations. These included methods to detect interchanges that take place between different chromosomes (dicentrics and translocations), and intrachanges occurring within a given chromosome (rings, interstitial deletions and inversions). In this review we consider the relationship between chromosome-type interchanges and intrachanges in response to changes in ionization density (linear energy transfer; LET). In that context, we discuss advantages and disadvantages of more modern methods used to measure intrachanges, and the implications that their increased resolution of measurement may have on the inter-to-intrachange fraction (i.e., the F-ratio). We conclude that the premise of the F-ratio is supported by its biophysical assumptions, but its intended use as an LET-dependent measure of prior radiation exposure is hampered mainly by our inability to accurately assess, on a cell-by-cell basis, inversions and interstitial deletions whose small sizes are below the detection limits of conventional cytogenetic techniques.

Molecular Cytogenetics Guides Massively Parallel Sequencing of a Radiation-Induced Chromosome Translocation in Human Cells.

Chromosome rearrangements are large-scale structural variants that are recognized drivers of oncogenic events in cancers of all types. Cytogenetics allows for their rapid, genome-wide detection, but does not provide gene-level resolution. Massively parallel sequencing (MPS) promises DNA sequence-level characterization of the specific breakpoints involved, but is strongly influenced by bioinformatics filters that affect detection efficiency. We sought to characterize the breakpoint junctions of chromosomal translocations and inversions in the clonal derivatives of human cells exposed to ionizing radiation. Here, we describe the first successful use of DNA paired-end analysis to locate and sequence across the breakpoint junctions of a radiation-induced reciprocal translocation. The analyses employed, with varying degrees of success, several well-known bioinformatics algorithms, a task made difficult by the involvement of repetitive DNA sequences. As for underlying mechanisms, the results of Sanger sequencing suggested that the translocation in question was likely formed via microhomology-mediated non-homologous end joining (mmNHEJ). To our knowledge, this represents the first use of MPS to characterize the breakpoint junctions of a radiation-induced chromosomal translocation in human cells. Curiously, these same approaches were unsuccessful when applied to the analysis of inversions previously identified by directional genomic hybridization (dGH). We conclude that molecular cytogenetics continues to provide critical guidance for structural variant discovery, validation and in "tuning" analysis filters to enable robust breakpoint identification at the base pair level.

Telomeres and NextGen CO-FISH: Directional Genomic Hybridization (Telo-dGH™).

The cytogenomics-based methodology of Directional Genomic Hybridization (dGH™) emerged from the concept of strand-specific hybridization, first made possible by Chromosome Orientation FISH (CO-FISH), the utility of which was demonstrated in a variety of early applications, often involving telomeres. Similar to standard whole chromosome painting (FISH), dGH™ is capable of identifying inter-chromosomal rearrangements (translocations between chromosomes), but its distinctive strength stems from its ability to detect intra-chromosomal rearrangements (inversions within chromosomes), and to do so at higher resolution than previously possible. dGH™ brings together the strand specificity and directionality of CO-FISH with sophisticated bioinformatics-based oligonucleotide probe design to unique sequences. dGH™ serves not only as a powerful discovery tool-capable of interrogating the entire genome at the megabase level-it can also be used for high-resolution targeted detection of known inversions, a valuable attribute in both research and clinical settings. Detection of chromosomal inversions, particularly small ones, poses a formidable challenge for more traditional cytogenetic approaches, especially when they occur near the ends or telomeric regions. Here, we describe Telo-dGH™, a strand-specific scheme that utilizes dGH™ in combination with telomere CO-FISH to differentiate between terminal exchange events, specifically terminal inversions, and an altogether different form of genetic recombination that often occurs near the telomere, namely sister chromatid exchange (SCE).

Seeking Beta: Experimental Considerations and Theoretical Implications Regarding the Detection of Curvature in Dose-Response Relationships for Chromosome Aberrations.

The concept of curvature in dose-response relationships figures prominently in radiation biology, encompassing a wide range of interests including radiation protection, radiotherapy and fundamental models of radiation action. In this context, the ability to detect even small amounts of curvature becomes important. Standard (ST) statistical approaches used for this purpose typically involve least-squares regression, followed by a test on sums of squares. Because we have found that these methods are not particularly robust, we investigated an alternative information theoretic (IT) approach, which involves Poisson regression followed by information-theoretic model selection. Our first objective was to compare the performances of the ST and IT methods by using them to analyze mFISH data on gamma-ray-induced simple interchanges in human lymphocytes, and on Monte Carlo simulated data. Real and simulated data sets that contained small-to-moderate curvature were deliberately selected for this exercise. The IT method tended to detect curvature with higher confidence than the ST method. The finding of curvature in the dose response for true simple interchanges is discussed in the context of fundamental models of radiation action. Our second objective was to optimize the design of experiments aimed specifically at detecting curvature. We used Monte Carlo simulation to investigate the following parameters. Constrained by available resources (i.e., the total number of cells to be scored) these include: the optimal number of dose points to use; the best way to apportion the total number of cells among these dose points; and the spacing of dose intervals. Counterintuitively, our simulation results suggest that 4-5 radiation doses were typically optimal, whereas adding more dose points may actually prove detrimental. Superior results were also obtained by implementing unequal dose spacing and unequal distributions in the number of cells scored at each dose.

Straightening Beta: Overdispersion of Lethal Chromosome Aberrations following Radiotherapeutic Doses Leads to Terminal Linearity in the Alpha-Beta Model.

Recent technological advances allow precise radiation delivery to tumor targets. As opposed to more conventional radiotherapy-where multiple small fractions are given-in some cases, the preferred course of treatment may involve only a few (or even one) large dose(s) per fraction. Under these conditions, the choice of appropriate radiobiological model complicates the tasks of predicting radiotherapy outcomes and designing new treatment regimens. The most commonly used model for this purpose is the venerable linear-quadratic (LQ) formalism as it applies to cell survival. However, predictions based on the LQ model are frequently at odds with data following very high acute doses. In particular, although the LQ predicts a continuously bending dose-response relationship for the logarithm of cell survival, empirical evidence over the high-dose region suggests that the survival response is instead log-linear with dose. Here, we show that the distribution of lethal chromosomal lesions among individual human cells (lymphocytes and fibroblasts) exposed to gamma rays and X rays is somewhat overdispersed, compared with the Poisson distribution. Further, we show that such overdispersion affects the predicted dose response for cell survival (the fraction of cells with zero lethal lesions). This causes the dose response to approximate log-linear behavior at high doses, even when the mean number of lethal lesions per cell is well fitted by the continuously curving LQ model. Accounting for overdispersion of lethal lesions provides a novel, mechanistically based explanation for the observed shapes of cell survival dose responses that, in principle, may offer a tractable and clinically useful approach for modeling the effects of high doses per fraction.

Three-Color Chromosome Painting as Seen through the Eyes of mFISH: Another Look at Radiation-Induced Exchanges and Their Conversion to Whole-Genome Equivalency.

Whole-chromosome painting (WCP) typically involves the fluorescent staining of a small number of chromosomes. Consequently, it is capable of detecting only a fraction of exchanges that occur among the full complement of chromosomes in a genome. Mathematical corrections are commonly applied to WCP data in order to extrapolate the frequency of exchanges occurring in the entire genome [whole-genome equivalency (WGE)]. However, the reliability of WCP to WGE extrapolations depends on underlying assumptions whose conditions are seldom met in actual experimental situations, in particular the presumed absence of complex exchanges. Using multi-fluor fluorescence in situ hybridization (mFISH), we analyzed the induction of simple exchanges produced by graded doses of (137)Cs gamma rays (0-4 Gy), and also 1.1 GeV (56)Fe ions (0-1.5 Gy). In order to represent cytogenetic damage as it would have appeared to the observer following standard three-color WCP, all mFISH information pertaining to exchanges that did not specifically involve chromosomes 1, 2, or 4 was ignored. This allowed us to reconstruct dose-responses for three-color apparently simple (AS) exchanges. Using extrapolation methods similar to those derived elsewhere, these were expressed in terms of WGE for comparison to mFISH data. Based on AS events, the extrapolated frequencies systematically overestimated those actually observed by mFISH. For gamma rays, these errors were practically independent of dose. When constrained to a relatively narrow range of doses, the WGE corrections applied to both (56)Fe and gamma rays predicted genome-equivalent damage with a level of accuracy likely sufficient for most applications. However, the apparent accuracy associated with WCP to WGE corrections is both fortuitous and misleading. This is because (in normal practice) such corrections can only be applied to AS exchanges, which are known to include complex aberrations in the form of pseudosimple exchanges. When WCP to WGE corrections are applied to true simple exchanges, the results are less than satisfactory, leading to extrapolated values that underestimate the true WGE response by unacceptably large margins. Likely explanations for these results are discussed, as well as their implications for radiation protection. Thus, in seeming contradiction to notion that complex aberrations be avoided altogether in WGE corrections - and in violation of assumptions upon which these corrections are based - their inadvertent inclusion in three-color WCP data is actually required in order for them to yield even marginally acceptable results.

Directional genomic hybridization: inversions as a potential biodosimeter for retrospective radiation exposure.

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short half-life (roughly 1-2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space.

Directional genomic hybridization for chromosomal inversion discovery and detection.

Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions-reversals in the orientation of DNA sequences within a chromosome-should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting-a whole new way of looking at structural features of the genome-that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility.

The LET dependence of unrepaired chromosome damage in human cells: a break too far?

Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to "open breaks". This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with ¹³7Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from ²³8Pu and 1 GeV/amu 56Fe ions. In agreement with previous studies, our results show that irrespective of radiation quality, the overall level of misrepaired damage exceeds that of unrepaired damage by a large margin. The unrepaired component of damage produced by γ rays and α particles was remarkably similar, about 5%. On that basis it is difficult to justify the popular notion that the strong LET-dependence for aberration formation is due to unrepaired DNA double-strand breaks (DSBs) that, by virtue of their complexity at the nanometer scale, are qualitatively different in nature. In marked contrast, this unrejoined component rose to about 14% after exposure to Fe ions. A closer look at the unrepaired component revealed that most of this roughly threefold difference was derived from incomplete exchanges. Despite vast differences in LET, unrejoined breaks from incomplete exchanges were far more likely to occur among exchanges that involved more than two breakpoints. We attempted to reconcile these observations in the form of a hypothesis that predicts that exchanges, irrespective of LET, should exhibit an increasing tendency for incompleteness as the number of initial breaks destined to take part in the exchange increases. This effect, we argue is not caused by the number of initial breaks per se, but instead reflects the maximum distance over which proximate breaks can interact. This adds a spatial aspect to multi-break interactions that we call "A Break Too Far".

The LET Dependence of Unrepaired Chromosome Damage in Human Cells: A Break Too Far?

Cytogenetic damage is among the few radiobiological end points that allow a precise distinction to be made between misrepaired damage, represented by exchange-type aberrations such as dicentrics and translocations, and unrepaired damage that leads to "open breaks". This latter category includes both terminal deletions and incomplete exchanges, whose different mechanisms of formation can be recognized by multicolor fluorescence in situ fluorescence hybridization (mFISH). mFISH was used to examine the yields of chromosome aberrations at the first postirradiation mitosis in human fibroblasts and lymphocytes irradiated with (137)Cs γ rays, a radiation of low-linear energy transfer (LET), and two sources of high-LET radiation: α particles from (238)Pu and 1 GeV/amu (56)Fe ions. In agreement with previous studies, our results show that irrespective of radiation quality, the overall level of misrepaired damage exceeds that of unrepaired damage by a large margin. The unrepaired component of damage produced by γ rays and α particles was remarkably similar, about 5%. On that basis it is difficult to justify the popular notion that the strong LET-dependence for aberration formation is due to unrepaired DNA double-strand breaks (DSBs) that, by virtue of their complexity at the nanometer scale, are qualitatively different in nature. In marked contrast, this unrejoined component rose to about 14% after exposure to Fe ions. A closer look at the unrepaired component revealed that most of this roughly threefold difference was derived from incomplete exchanges. Despite vast differences in LET, unrejoined breaks from incomplete exchanges were far more likely to occur among exchanges that involved more than two breakpoints. We attempted to reconcile these observations in the form of a hypothesis that predicts that exchanges, irrespective of LET, should exhibit an increasing tendency for incompleteness as the number of initial breaks destined to take part in the exchange increases. This effect, we argue is not caused by the number of initial breaks per se, but instead reflects the maximum distance over which proximate breaks can interact. This adds a spatial aspect to multi-break interactions that we call "A Break Too Far".